BACKGROUND: The distribution of protein intake throughout the day is frequently skewed, with breakfast having the lowest protein intake across all age groups. There is no review that addresses the association between breakfast protein intake and muscle mass and strength. OBJECTIVE: This scoping review aims to summarize the literature on the relationship between protein intake during breakfast and muscle mass and strength in adults. METHODS: This review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews. Two independent reviewers screened and appraised the articles identified from 3 electronic databases (Medline, Web of Science, and Scopus) that focused on protein intake per meal and its impact on muscle-related outcomes. RESULTS: A total of 14 763 articles were retrieved from 3 databases, and after the screening process, 15 articles were included for analysis. Approximately 58.8% of findings from 11 studies examining the association between high protein intake during breakfast and muscle mass indicated an increase in muscle mass among participants. Half of 6 studies (50.0%) exploring the relationship between high protein intake at breakfast and muscle strength demonstrated an increase in muscle strength. Consuming high amounts of protein at breakfast or more protein in the morning than in the evening was associated with an increase in the skeletal muscle index and lean body mass. CONCLUSION: Protein consumption at breakfast revealed potential benefits in increasing muscle mass across 5 studies involving an elderly population and 2 studies encompassing middle-aged women and young men in our review. However, the relationship between protein intake at breakfast and muscle strength remains unclear. Further high-quality randomized controlled trials are required to examine whether adults can preserve skeletal muscle health outcomes by consuming higher amounts of protein at breakfast.

The consumption of resistant dextrin improves constipation, while its fermentation and degradation by the intestinal microbiota produce short-chain fatty acids (SCFA) and lactic acid, which have beneficial effects on host metabolism and immunity. Mg oxide (MgO) is an important mineral that is used to treat constipation. Therefore, resistant dextrin and MgO are often administered together to improve constipation. However, limited information is available regarding the effect of this combination on SCFA and lactic acid production. Crl:CD1(ICR) mice were fed a Mg-free diet with 5% resistant dextrin, followed by oral administration of MgO. We collected the cecum contents and measured SCFA and lactic acid levels. Additionally, the human subjects received resistant dextrin and Mg supplements as part of their habitual diet. The results of this study demonstrate that intestinal microbiota cannot promote SCFA and lactic acid production in the absence of Mg. In a mouse model, low doses of MgO promoted the production of SCFA and lactic acid, whereas high doses decreased their production. In humans, the combined consumption of resistant dextrin and Mg supplements increased the production of SCFA and lactic acid. The production of SCFA and lactic acid from dietary fiber may be augmented by the presence of MgO.

AIM: Aging decreases muscle mass and bone mineral density (BMD), especially in older women. It has been reported that rowing and inulin intake positively affect muscle and bone, respectively. We examined the synergistic effect of rowing and functional food intake, including inulin, on lean body mass, BMD, and physical function parameters in older Japanese women. METHODS: Fifty women aged 65-79 years were divided into four groups with or without inulin intake and rowing. The interventions were carried out for 12 weeks in each group. We assessed lean body mass and BMD using dual-energy X-ray absorptiometry at baseline and after the intervention and examined the changes in the values in each group. RESULTS: Lean body mass in all groups decreased, and the change in lean body mass in the group with rowing and inulin intake was significantly smaller than that in the group without them (-0.05 ± 0.61; -0.83 ± 0.59 kg; P = 0.030). The BMD in the three intervention groups increased after the 12-week intervention. The change in BMD in each of the three intervention groups showed significant differences compared with the control group (Rowing + Inulin: P = 0.03; Rowing + No inulin: P = 0.01; No rowing + Inulin: P < 0.01). CONCLUSIONS: Rowing and the intake of functional foods, including inulin, synergistically prevented a decrease in lean body mass. These factors, individually and additively, might increase BMD in older Japanese women. Geriatr Gerontol Int 2023; ••: ••-••.

Ageing is associated with a decline in circadian clock systems, which correlates with the development of ageing-associated diseases. Chrononutrition is a field of chronobiology that examines the relationship between the timing of meal/nutrition and circadian clock systems. Although there is growing evidence regarding the role of chrononutrition in the prevention of lifestyle and ageing-related diseases, the optimal timing of meal intake to regulate the circadian clock in humans remains unknown. In this study, we investigated the relationship between clock gene expression and meal timing in young and older adults. In this cross-sectional study, we enrolled 51 healthy young men and 35 healthy older men (age, mean±standard deviation: 24 ± 4 and 70 ± 4 y, respectively). Under daily living conditions, beard follicle cells were collected at 4-h intervals over a 24-h period to evaluate clock gene expression. Participants were asked to record the timing of habitual sleep and wake-up, breakfast, lunch, and dinner. From these data, we calculated "From bedtime to breakfast time," "From wake up to first meal time," and "From dinner to bed time." NR1D1 and PER3 expressions in older adults at 06:00 h were significantly higher than those in young adults (P = 0.001). There were significant differences in the peak time for NR1D2 (P = 0.003) and PER3 (P = 0.049) expression between young and older adults. "From bedtime to breakfast time" was significantly longer in older adults than in young adults. In contrast, "From dinner to bed time" was significantly shorter in older adults than in young adults. Moreover, higher rhythmicity of NR1D1 correlated with longer "From bedtime to breakfast time" (r =  -0.470, P = 0.002) and shorter "From wake up to first meal time" in young adults (r = 0.302, P = 0.032). Higher rhythmicity of PER3 correlated with longer "From bedtime to breakfast time" in older adults (r =  -0.342, P = 0.045). These results suggest that the peak time of clock gene expression in older adults may be phase-advanced compared to that in young adults. In addition, a longer fasting duration from bedtime to breakfast in both young and older adults and earlier intake of meals after waking up in young adults may correlate with robust clock gene expression rhythms.

Abstract

Background

Psychological wellbeing during pregnancy is imperative for optimal maternal outcomes. The present study aimed to determine the association between sleep quality, light exposure at night, and psychological wellbeing in the 2^nd and 3^rd trimesters of pregnancy.

Methods

This prospective study was conducted in 9 randomly selected government maternity clinics in Kuala Lumpur, Malaysia. Healthy women aged 20–48 years old with single pregnancy were recruited using convenience sampling (n = 169). Sleep quality, light exposure at night, and psychological wellbeing were self-reported using the Pittsburgh Sleep Quality Index (PSQI), Harvard Light Exposure Assessment (H-LEA), and Depression, Anxiety, and Stress Scale (DASS-21) in the 2^nd trimester and followed-up at the 3^rd trimester.

Results

During the 2^nd and 3^rd trimesters of pregnancy, mild to severe symptoms of stress (10.7 and 11.3%), anxiety (42 and 44.3%), and depression (9.6 and 16.6%) were observed among the participants. Adjusted multiple linear regression revealed that poor sleep quality and higher light exposure at night were attributed to greater stress and depression symptoms in the 3^rd trimester. Higher lux level exposed from 10 pm to &lt; 1 am was associated with increased stress (β = 0.212, p = 0.037) and depression (β = 0.228, p = 0.024). Only poor sleep quality was observed to adversely affect anxiety (β = 0.243, p = 0.002) and depression levels (β = 0.259, p = 0.001) in the 2^nd trimester.

Conclusions

Present study provided preliminary findings on the association between sleep quality, light at night, and psychological wellbeing of pregnant women. As a recommendation, future research could investigate whether public health interventions aimed at decreasing artificial light at night can benefit sleep quality and the psychological health of pregnant women.

Abstract

Chrononutrition emerges as a novel approach to promote circadian alignment and metabolic health by means of time-of-the-day dietary intake. However, the relationship between maternal circadian rhythm and temporal dietary intake during pregnancy remains understudied. This study aimed to determine the change in melatonin levels in pregnant women across gestation and its association with temporal energy and macronutrient intake. This was a prospective cohort involving 70 healthy primigravidas. During the second and third trimesters, pregnant women provided salivary samples collected at 9:00, 15:00, 21:00, and 3:00 h over a 24 h day for melatonin assay. Data on chrononutrition characteristics were collected using a 3-day food record. Parameters derived from melatonin measurements including mean, amplitude, maximal level, area under the curve with respect to increase (AUC_I), and area under the curve with respect to ground (AUC_G) were computed. A rhythmic melatonin secretion over the day that remained stable across trimesters was observed among the pregnant women. There was no significant elevation in salivary melatonin levels as pregnancy advanced. In the second trimester, higher energy intake during 12:00–15:59 h and 19:00–06:59 h predicted a steeper melatonin AUC_I (β=-0.32, p = 0.034) and higher AUC_G (β = 0.26, p = 0.042), respectively. Macronutrient intake within 12:00–15:59 h was negatively associated with mean melatonin (Fat: β=-0.28, p = 0.041) and AUC_G (Carbohydrate: β=-0.37, p = 0.003; Protein: β=-0.27, p = 0.036; Fat: β=-0.32, p = 0.014). As pregnant women progressed from the second to the third trimester, a flatter AUC_I was associated with a reduced carbohydrate intake during 12:00–15:59 h (β=-0.40, p = 0.026). No significant association was detected during the third trimester. Our findings show that higher energy and macronutrient intakes particularly during 12:00–15:59 h and 19:00–06:59 h are associated with the disparities in maternal melatonin levels. Findings suggest the potential of time-based dietary approaches to entrain circadian rhythm in pregnant women.

The contribution of breakfast to daily nutrient intake is low, particularly among children, at only about 20%, and it is difficult to determine whether children are receiving adequate nutrients at breakfast. Although alterations in breakfast content are considered to affect lifestyle habits such as sleep and defecation, there have been few intervention studies in children. The relationship between nutritional balance, dietary intake, and lifestyle habits in children remains unclear. We conducted an intervention study on elementary school children's breakfasts and observed the effects of improving the nutritional balance of breakfast on sleep parameters and defecation status. An intervention study was conducted with 26 elementary school students in Tokyo. The study design was an open-label randomized cross-over trial. Subjects consumed their usual breakfast during the control period and a granola snack containing soy protein in addition to their usual breakfast during the intervention period. Questionnaires regarding breakfast, sleep, and bowel movements were administered during each period. Based on the answers to these questionnaires, we compared the nutritional sufficiency of macronutrients, vitamins, and minerals (29 in total), as well as changes in sleep parameters and defecation status. The additional consumption of granola snacks increased the breakfast intake of 15 nutrients. The changes were particularly significant for iron, vitamin B1, vitamin D, and dietary fiber. During the intervention, sleep duration was decreased and wake-up time became earlier. In terms of defecation, the intervention did not change stool characteristics, but the frequency of defecations per week increased on average by 1.2 per week. These results suggest that the nutritional balance and the amount of breakfast are linked to sleep and defecation and that improving breakfast content can lead to lifestyle improvements in children.

PURPOSE: It has been reported that the consumption of fruit granola (FG), mulberry leaves, and barley cookies as an afternoon snack suppresses the postprandial increase in glucose levels at dinner. However, there have been no reports on the second-meal effect of snacking on popular snacks, such as potato chips (PC), roasted sweet potato (SP), and black beans (BB), or on the interval between snacking and dinner. METHOD: The present study was an open-label randomized crossover trial of five study groups (PC, SP, BB, FG, and no snack) regarding the second-meal effects with different intervals between snacks and dinner. The subjects consumed prescribed meals for lunch and dinner at 12:00 and 19:00, and a snack fixed at 838 kJ (= 200 kcal) at 15:00 or 17:00. RESULTS: When the participants snacked at 15:00, the postprandial glucose elevation at dinner was suppressed in the FG and SP groups, and the area under the curve (AUC) was also low. When they snacked at 17:00, the postprandial glucose elevation was suppressed in all the groups. The AUCs for PC, FG, and SP were lower than those for no snacking. On the other hand, carbohydrate intake increased with snacking, but the total AUC of snacks and dinner did not differ in any of the groups. The duration of hyperglycemia decreased with snack intake, as did the glucose amplitude. CONCLUSION: We believe that the intake of carbohydrates and soluble fiber in snacks is an important factor in the second-meal effect at dinner. These results will contribute to the development of snacking and research into the second-meal effect.

Excess sodium intake and insufficient potassium intake are a prominent global issue because of their influence on high blood pressure. Supplementation of potassium induces kaliuresis and natriuresis, which partially explains its antihypertensive effect. Balancing of minerals takes place in the kidney and is controlled by the circadian clock; in fact, various renal functions exhibit circadian rhythms. In our previous research, higher intake of potassium at lunch time was negatively associated with blood pressure, suggesting the importance of timing for sodium and potassium intake. However, the effects of intake timing on urinary excretion remain unclear. In this study, we investigated the effect of 24 h urinary sodium and potassium excretion after acute sodium and potassium load with different timings in mice. Compared to other timings, the middle of the active phase resulted in higher urinary sodium and potassium excretion. In Clock mutant mice, in which the circadian clock is genetically disrupted, urinary excretion differences from intake timings were not observed. Restricted feeding during the inactive phase reversed the excretion timing difference, suggesting that a feeding-induced signal may cause this timing difference. Our results indicate that salt intake timing is important for urinary sodium and potassium excretion and provide new perspectives regarding hypertension prevention.

Abstract

Background/Objectives

Glucose tolerance is controlled by the internal clock and is worse in the evening. From a chrononutrition perspective, diabetes prevention requires evaluating the antidiabetic effects of the timing of functional ingredients and nutrient intake. The purpose of this study was to investigate the timing effects of acute mulberry leaf extract (MLE) intake on postprandial glucose levels in young adults.

Subjects/Methods

Twelve young adults underwent four trials. Blood samples were collected in a fasting state and at 30, 60, 120, and 180 min after eating a mixed meal. The study had a randomised, placebo-controlled, double-blind trial design involving: (1) morning placebo trial (08:00 h; MP trial), (2) evening placebo trial (18:00 h; EP trial), (3) morning MLE trial (08:00 h; MM trial), and (4) evening MLE trial (18:00 h; EM trial).

Results

The incremental area under the blood glucose curve (iAUC) in the EM trials was significantly lower than that in the EP trials (P = 0.010). The postprandial glucose concentrations 120 min after the meal were significantly lower in the EM trials than those in the EP trials (P = 0.006). The postprandial insulin concentrations at 120 min were significantly lower in the MM trials than those in the MP trials (P = 0.034). Moreover, the postprandial insulin concentrations 180 min after the meal were significantly lower in the EM trials than those in the EP trials (P = 0.034).

Conclusions

MLE intake in the evening, but not in the morning, was effective in improving glucose tolerance.

Trial registration

Clinical trial reference: UMIN 000045301; website of trial registry: https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000051340.

Chrononutrition has been suggested to have an entrainment effect on circadian rhythm which is crucial for metabolic health. Investigating how chrononutrition affects maternal circadian rhythm can shed light on its role during pregnancy. This study aims to determine chrononutrition characteristics of healthy primigravida during pregnancy and its association with melatonin and cortisol rhythm across gestation. A total of 70 healthy primigravidas were recruited from ten randomly selected government maternal and child clinics in Kuala Lumpur, Malaysia. During the second and third trimesters, chrononutrition characteristics including meal timing, frequency, eating window, breakfast skipping, and late-night eating were determined using a 3-day food record. Pregnant women provided salivary samples at five time-points over a 24 h period for melatonin and cortisol assay. Consistently across the second and third trimesters, both melatonin and cortisol showed a rhythmic change over the day. Melatonin levels displayed an increment toward the night whilst cortisol levels declined over the day. Majority observed a shorter eating window (≤12 h) during the second and third trimesters (66 and 55%, respectively). Results showed 23 and 28% skipped breakfast whereas 45 and 37% ate within 2 h pre-bedtime. During the third trimester, a longer eating window was associated with lower melatonin mean (β = –0.40, p = 0.006), peak (β = –0.42, p = 0.006), and AUC_G (β = –0.44, p = 0.003). During both trimesters, a lower awakening cortisol level was observed in pregnant women who skipped breakfast (β = –0.33, p = 0.029; β = –0.29, p = 0.044). Only during the second trimester, breakfast-skipping was significantly associated with a greater cortisol amplitude (β = 0.43, p = 0.003). Findings suggest that certain chrononutrition components, particularly eating window and breakfast skipping have a significant influence on maternal melatonin and cortisol rhythm. Dietary intervention targeting these characteristics may be useful in maintaining maternal circadian rhythm.

INTRODUCTION: In Japan, breakfast styles are categorized into five groups; Japanese breakfast (JB; rice and miso soup), Western breakfast (WB; bread and milk), Japanese-Western breakfast (J-WB; alternative daily serving), cereal breakfast (CB), and breakfast skipping. In our recent studies, breakfast style was highly associated with the daily sleep-wake phase (chronotype), and healthy eating habits. In contrast with other breakfast style consumers, JB-consumers were positively associated with the morning chronotype and healthy eating habits such as a high consumption of a variety of protein sources, vegetables, and dietary fibers, and low consumption of sweetened juices. These previous studies included only adult participants; hence, in the current study, we investigated whether similar observations can be made in children. METHODS: Preschool (aged 3-5 years) and elementary school children (6-8 years) (N = 6,104, 49.87% boys, 50.13% girls, mean body mass index 15.39 ± 0.03 kg/m2 for preschoolers and percentage of overweight -2.73 ± 0.22 for elementary school children) participated in this cross-sectional online survey on lifestyle, including eating and sleep habits, through their mother's responses. RESULTS: The results showed that the morning-evening type index values (chronotype indicator, smaller indicates morning type) were negatively correlated with JB intake (-0.05, p < 0.01) and positively correlated with WB (0.03, p < 0.05) and CB intake (0.06, p < 0.01), suggesting that the JB group exhibited the morning chronotype and the WB and CB groups exhibited the evening chronotype. The JB group consumed a variety of protein sources (mean ± SE; days/week) with more frequency (fish 2.95 ± 0.038 p < 0.001, soy 3.55 ± 0.043 p < 0.001, egg 3.82 ± 0.044 p < 0.001) compared with the WB group (fish 2.58 ± 0.033, soy 3.00 ± 0.038, egg 3.49 ± 0.039). On the other hand, the JB group consumed snacks (5.48 ± 0.042 p < 0.001) and sweetened juice (2.50 ± 0.050 p < 0.001) less frequently than the WB group (snacks; 5.80 ± 0.037 and sweetened juice; 2.74 ± 0.049). DISCUSSION: JB-eating children with a morning chronotype exhibited better sleep and eating habits than WB-eating children with an evening type pattern. The results suggest that JB eating habits may be associated with good eating and sleeping lifestyles, even among preschool and elementary school children.

Paramylon, a β-1,3-glucan storage polysaccharide derived from Euglena gracilis, has various health benefits, such as anti-obesity effects and modulation of immune function. However, whether paramylon intake affects the circadian clock remains unknown. In this study, we examined the effect of paramylon intake on the circadian clock. The results showed that the paramylon intake regulated peripheral clocks in mice. Furthermore, cecal pH and short-chain fatty acid concentrations after paramylon intake were measured. The correlation between changes in the expression of clock-related genes and alterations in the intestinal environment was confirmed. In addition, peripheral clock entrainment by paramylon intake was not observed in antibiotic-treated mice whose gut microbiota was weakened. These findings suggest that the regulation of the circadian clock by paramylon intake was mediated by changes in gut microbiota. In addition, the entraining effect of paramylon intake was also confirmed in mice bred under conditions mimicking social jetlag, which implies that paramylon intake may contribute to recovery from social jetlag. Thus, the appropriate consumption of paramylon may have a beneficial effect on health from a chrono-nutritional perspective.

Dietary calcium supplementation has been shown to be an effective adjunct therapy in an inflammatory bowel disease model. Soluble dietary fiber reduces intestinal pH and is known to enhance calcium absorption. Although many circadian clock regulations of nutrient absorption in the intestinal tract have been reported, the effects of clock regulation on calcium absorption have yet to be understood. In this study, we investigated the timing of efficient calcium intake by measuring urinary calcium excretion in mice. The diurnal variations in channel-forming tight junctions (claudins) were detected in both the jejunum and ileum. Following 2 days of feeding with a Ca2+-free diet, Ca2+-containing diets with or without soluble fiber (inulin) were fed at specific timings, and urine was subsequently examined every 4 hr. There was an evident increase in urinary calcium concentration when the inulin diet was fed at the beginning of the resting period. The Claudin 2 (Cldn2) expression level also showed a significant day-night change, which seemed to be a mechanism for the increased calcium excretion after inulin intake. This diurnal rhythm and enhanced Cldn2 expression were abolished by disruption of the suprachiasmatic nucleus, the central clock in the hypothalamus. This study suggests that intestinal calcium absorption might be modulated by the circadian clock and that the intake of inulin is more effective at the beginning of the resting period in mice.

This study aimed to examine the effect of high protein breakfast diet with or without lunch on the postprandial glucose level during the day. A randomized, crossover design that recruited 12 healthy young participants (three men and nine women) was performed and four trials (normal breakfast + skipped lunch, high protein breakfast + skipped lunch, normal breakfast + lunch, and high protein breakfast + lunch) were conducted in two weeks. During each trial, breakfast, lunch, and dinner on the trial day, and dinner before the trial day, were provided as test meals, and the meal timing was fixed. Continuous glucose monitoring (CGM) was used to assess the blood glucose level during the whole experiment. Incremental area under the curve (iAUC) of the postprandial glucose level was calculated. The results suggested that compared with normal breakfast, high protein breakfast suppressed the 3 h iAUC of postprandial glucose level after breakfast (p &lt; 0.05 or p &lt; 0.0001) and 1.5 h iAUC after lunch (p &lt; 0.01). During lunch, high protein breakfast diet suppressed the dinner and overall day postprandial glucose level (p &lt; 0.05 vs. normal breakfast), but no significant difference was observed when skipping lunch. Our findings indicate that high protein breakfast could suppress the breakfast postprandial glucose level, as well as following lunch and dinner, but this effect on dinner was attenuated when skipping lunch.

A balanced diet and protein source intake are reportedly good for health. However, many people skip breakfast or have a light breakfast. Thus, this study aimed to examine the influence of breakfast styles on eating habits among Japanese workers, including traditional Japanese-style breakfast (JB), a pattern in which Japanese foods are eaten; Japanese-Western-style breakfast (J-W B), a pattern in which Japanese and Western foods are eaten alternately; Western-style breakfast (WB), a pattern in which Western foods are eaten; and cereal-style breakfast (CB), a pattern in which cereal is eaten. We hypothesized that breakfast style may be related to good eating habits. Data from 4274 respondents (67.97% male, 33.03% female, age 48.12 ± 0.19 years), excluding night shift workers and breakfast absentees out of a total of 5535 respondents, were analyzed. The results suggest that Japanese food is linked to the intake of protein sources such as fish, eggs, and soy. Furthermore, it was suggested that Japanese food breakfast is effective for good eating habits, such as not eating irregular amounts of food, not eating snacks, not drinking sweet juices, and having a balanced diet.

CONTEXT: The mammalian circadian clock system regulates physiological function. Crude drugs, containing Polygalae Radix, and Kampō, combining multiple crude drugs, have been used to treat various diseases, but few studies have focussed on the circadian clock. OBJECTIVE: We examine effective crude drugs, which cover at least one or two of Kampō, for the shortening effects on period length of clock gene expression rhythm, and reveal the mechanism of shortening effects. MATERIALS AND METHODS: We prepared 40 crude drugs. In the in vitro experiments, we used mouse embryonic fibroblasts from PERIOD2::LUCIFERASE knock-in mice (background; C57BL/6J mice) to evaluate the effect of crude drugs on the period length of core clock gene, Per2, expression rhythm by chronic treatment (six days) with distilled water or crude drugs (100 μg/mL). In the in vivo experiments, we evaluated the free-running period length of C57BL/6J mice fed AIN-93M or AIN-93M supplemented with 1% crude drug (6 weeks) that shortened the period length of the PERIOD2::LUCIFERASE expression rhythm in the in vitro experiments. RESULTS: We found that Polygalae Radix (ED50: 24.01 μg/mL) had the most shortened PERIOD2::LUCIFERASE rhythm period length in 40 crude drugs and that the CaMKII pathway was involved in this effect. Moreover, long-term feeding with AIN-93M+Polygalae Radix slightly shortened the free-running period of the mouse locomotor activity rhythm. DISCUSSION AND CONCLUSIONS: Our results indicate that Polygalae Radix may be regarded as a new therapy for circadian rhythm disorder and that the CaMKII pathway may be regarded as a target pathway for circadian rhythm disorders.

Abstract

Aim

This study aimed to examine the effect of lunches with different caloric contents (Study 1) and nutrient balances (Study 2) on dinner-induced postprandial glucose fluctuation.

Methods

Energy trial (Study 1): Thirteen healthy young participants (n = 10 men, n = 3 women) were investigated to determine the effects of different caloric intakes at lunch on glucose level variability. The study was comprised of four trials (no lunch, low lunch, standard lunch, and high-energy lunch). Energy balance trial (Study 2): Fourteen healthy young adults (n = 8 men, n = 6 women) were investigated to determine the effect of different nutrient balances during lunch on glucose level variability. The study consisted of four trials (standard, protein-rich, fat-rich, and carbohydrate-rich). In studies 1 and 2, each trial was spaced at least 24 full hours apart, and breakfast and dinner were tested as meals. The mealtimes for each trial were then aligned. Continuous glucose monitoring was used to assess the blood glucose fluctuations.

Results

Study 1: The no-lunch (95% CI 95.5–149.7) and low-energy lunch (95% CI 90.8–143.1) trials had significantly higher values in the incremental area under the curve (iAUC) of postprandial blood glucose at dinner compared to the standard (95% CI 55.4–90.0) and high-energy lunch (95% CI 29.3–54.6) trials (P = 0.006, P = 0.001 vs. none), (P = 0.004, P = 0.001 vs. low-energy trial). Study 2: A significantly higher postprandial blood glucose iAUC for dinner was found in the fat-rich trial (95% CI 58.5–114.0) than that in the protein-rich (95% CI 25.6–63.9) and standard (95% CI 25.6–112.4) trials, (P = 0.006, P = 0.035 vs. fat-rich trial).

Conclusions

Our findings indicate that skipping lunch and low-calorie or high-lipid intake increased postprandial blood glucose levels after dinner.

Abstract

Study objectives

Sleep problems, such as accumulated sleep loss and social jetlag (SJL), which is characterized by a discrepancy in a person's sleep pattern between the weekday and the weekend, are associated with physical and mental health problems, and academic performance in young ages. However, sex differences in these associations are not fully understood. The purpose of this study was to investigate the effect of sex on sleep-related factors, mental health (negative mood), and academic performance in Japanese children and adolescents.

Methods

A cross-sectional online survey was conducted with 9,270 students (boys: N = 4,635, girls: N = 4,635) ranging from the fourth grade of elementary school to the third grade of high school, which typically includes ages 9 to 18 years in Japan. Participants completed the Munich ChronoType Questionnaire, the Athens Insomnia Scale, self-reported academic performance, and negative mood-related questions.

Results

School grade-related changes in sleep behavior (e.g., delayed bedtime, shortened sleep duration, and increased SJL) were detected. Girls had greater sleep loss on weekdays and SJL on weekends than boys. Multiple regression analysis revealed that sleep loss and SJL were more associated with negative mood and higher insomnia scores in girls than in boys, but not with academic performance.

Conclusion

Sleep loss and SJL in Japanese girls had a higher correlation to their negative mood and tendency to insomnia than in boys. These results suggest the importance of sex-dependent sleep maintenance for children and adolescents.

Postprandial hyperglycemia increases the risk of mortality among patients with type 2 diabetes and cardiovascular diseases. Additionally, the gut microbiota and type 2 diabetes and cardio-vascular disease are known to be correlated. Currently, fasting blood glucose is the primary in-dex for the clinical diagnosis of diabetes; however, postprandial blood glucose is associated with the risk of developing type 2 diabetes and cardiovascular disease and mortality. Therefore, the dynamic change in blood glucose levels under free-living conditions is considered an important and better marker than fasting glucose levels to study the relationship between glucose levels and microbiota. Here, we investigated the relationship between fasting and postprandial glucose levels and microbiota under free-living conditions for one week in older adults. In addition, in order to clarify the relationship between blood glucose level and intestinal bacteria, postprandial 4-h AUC was calculated and the correlation with gut bacteria was investigated. As a result of the present study, we observed many of the most significant correlations between the gut bacteria and the peak glucose levels after dinner and the 4-h AUC after dinner. Together, these findings suggest that the individual pattern of microbiota may help to predict post-dinner hyperglycemia and the risk of abnormal glucose metabolism, such as diabetes.

Background

Glucose and lipid tolerance reportedly exhibit diurnal variations, being lower in the evening than in the morning. Therefore, the effects of exercise on glucose and blood lipid levels at different times of the day may differ. This study aimed to investigate the effects of short-term endurance exercise intervention in the morning versus late afternoon on 24-h blood glucose variability and blood lipid levels.

Methods

Twelve healthy young men participated in a randomized crossover trial. The participants were assigned to morning (09:00–11:00) or late afternoon (16:00–18:00) endurance exercise for a week, consisting of supervised exercise sessions on Mondays, Wednesdays, and Fridays. In the morning and evening trials, the participants walked for 60 min on a treadmill at approximately 60% of maximal oxygen uptake (VO_2max). Following a 2-week wash-out period, the participants performed the exercise training regimen at another time point. Continuous glucose monitoring was used to evaluate blood glucose fluctuations during each 24-h trial period. Blood samples were collected before and after each intervention to examine blood lipid and hormonal responses.

Results

Examination of the area under the curve (AUC) of the glucose level changes for 24 h after the late afternoon versus morning exercise intervention revealed significantly lower values for the former versus the latter (P &amp;lt; 0.01). The AUC of glucose level changes after each meal was also lower after the late afternoon versus morning intervention, and significantly lower values were observed in the late afternoon versus morning trial for breakfast and dinner (P &amp;lt; 0.05, P &amp;lt; 0.01). In addition, a significant decrease in triglycerides (TG) and TG/high-density lipoprotein cholesterol (HDL-C) was noted after versus before the late afternoon intervention (P &amp;lt; 0.05).

Conclusions

These results suggest that late afternoon endurance exercise is more effective than morning endurance exercise at improving 24-h glucose and triglyceride levels.

Voluntary training and food modulate the fecal microbiota in humans and mice. Although there are some reports of the timing effects of voluntary training and feeding on metabolism, the timing effects of these factors on microbiota have not been investigated. Therefore, we investigated the effects of the timing of voluntary training and feeding on the gut microbiota. The ICR mice were housed under conditions with an early (in the morning) or late (evening) active phase of increased physical activity. Furthermore, to investigate why voluntary training affects the gut microbiota, mice were housed in a cold environment and received propranolol administration with increased physical activity. After that, we collected cecal contents and feces and measured cecal pH. Short-chain fatty acids (SCFA) were measured from cecal contents. Microbiota was determined using sequencing of the V3-V4 region of the 16S rDNA gene. This study found that increased evening physical activity rather than morning activity decreases cecal pH, increases SCFA, and changes the microbiota. It is especially important that increased evening physical activity is induced under the post-prandial voluntary training condition. Also, we found that cold room housing, sympathetic blockade, or both suppressed the increased physical activity-induced changes in cecal pH, SCFA, and microbiota. Allobaculum responded to increased physical activity through body temperature increases and sympathetic activation. Post-prandial increased physical activity, rather than pre-prandial increased physical activity by evening voluntary wheel training, altered the microbiota composition, which may be related to the increase in body temperature and sympathetic nervous system activation.

Okara is a major by-product of soymilk and tofu production. Despite retaining abundant nutrients after the process, okara is often under-utilized. In this study, solid-state fermentation (SSF) of okara was carried out using a koji starter (containing both Aspergillus oryzae and Aspergillus sojae) with the intention of releasing its untapped nutrients. Its effects on lipid metabolism in diet-induced obesity (DIO) were observed. The nutritional profile of fermented okara was elucidated using the following parameters: total phenolic content (TPC), pH, protein content, dietary fiber, amino acid content, and free sugar content. In vivo experiments were conducted using high-fat diets supplemented with unfermented okara and fermented okara over three weeks. Supplementation with fermented okara reduced body weight gain, adipose tissue weight, the serum triglyceride profile, and lipid accumulation in the liver, and altered the mRNA expression levels related to lipid metabolism; however, it did not affect pH and short-chain fatty acid (SCFA) production in this study. In conclusion, high-fat diets supplemented using okara fermented with Aspergillus spp. improved the lipid metabolism in mice, due to their high nutritional value, such as TPC, soy protein, and amino acids, and their synergistic effects without altering the gut microbiota.

The circadian clock maintains our health by controlling physiological functions. Social jet lag is one factor that can disrupt the body clock. This is caused by the difference in sleeping hours between weekdays when we live according to social time and holidays when we live according to our body clock. The body clock can be altered by exercise, nutrition, and stress, and several studies have reported that these factors can be used to improve a disturbed body clock. Here we focused on exercise and examined whether continuous wheel-running could improve the disordered body clock in a mouse model that mimics social jet lag. The results showed that the wheel-running exercise group showed faster synchronization of the onset of activities on weekdays which had been delayed by social jet lag and the results were even more pronounced in the high-fat diet feeding condition. Also, when the expression rhythms of the clock genes were examined, they experienced a sudden time shift in the advance light condition or social jet lag condition, it was found that the wheel-running group had a higher ability to adapt to the advance direction. Thus, it is possible that the effective inclusion of exercise in human, especially those who eat high-fat foods, life can improve the disordered body clock in terms of social jet lag.

There are 40 million people worldwide living with Alzheimer's Disease (AD), and by 2050 the prevalence is expected to increase to 150 million people, thus resulting in a 1 in 2 chance of having AD by the age of 85. An increase in amyloid-b plaques (Ab) and hyperphosphorylated tau are widely accepted as the core indicators of the disease. These indicators of AD onset and progression are becoming increasingly attributed to the disturbance of optimal sleep/wake cycles. Additionally, AD progression exacerbates normal sleep/wake cycles, resulting in a cyclical worsening of AD pathology, circadian rhythm, and cognitive dysfunction. By using an Ab intracerebroventricular (ICV) injection mouse model, mice can present an AD-like pathology within days to weeks after injection, allowing for the expedited examination of the disease. This model can serve as a tool to investigate the mechanism behind the disturbance of the biological clock and AD progression, and therefore find therapeutic targets to delay, prevent or cure the disease.

It is indicated that the intestinal environment affects the brain, which is known as the gut-brain axis. In fact, improvement of intestinal environment is reported to suppress anxiety-like behavior in mice model of depression and schizophrenia. However, its detailed mechanisms remain unclear, particularly in context of intestinal environment effects on emotion in non-disease mice. The object of this experiment is therefore to verify the effect of intestinal environment on anxiety-like behavior and its detailed physiological mechanisms.

We previously found that cellulose rich food (AIN-93M) suppresses the production of short-chain fatty acid and exacerbate the intestinal condition. In the present study, we fed mice with AIN-93M to modify the intestinal environment, or the mouse chow that can maintain a favorable intestinal environment (MF). After 8-weeks feeding, AIN-93M-fed animals displayed the significant increase of marble-burying behavior compared to MF-fed group, suggesting that dysfunction of intestine lead by AIN-93M enhanced anxiety-like behavior. Furthermore, dopamine release as well as dopamine receptor expression has been increased in amygdala of AIN-93M fed mice but not in MF animals, indicating that enhanced anxiety-like behavior in AIN-93M animals is due to such modification of amygdalar dopaminergic system. 

These results suggest that cellulose rich food may exacerbate intestinal environment, which may enhance the anxiety-like behavior and overactivation of dopaminergic system.

Cardiovascular diseases (CVDs) are one of the leading causes of death worldwide, and one of the most significant risk factors for CVDs is high blood pressure. Blood pressure is associated with various nutrients, such as sodium, potassium, and cholesterol. However, research focusing on the timing of intake of these nutrients and blood pressure has not been conducted. In this study, we used dietary data and a questionnaire asking about the sleep, physical activity, and blood pressure, collected from the food-log app "Asken" (total N = 2,402), to investigate the relationship between the dietary data of nutrient intake in the breakfast, lunch, and dinner and blood pressure. Daily total intake of various nutrients such as sodium, sodium-to-potassium ratio, total energy, lipid, carbohydrate, and saturated fat showed a significant association with blood pressure depending on the meal timing. From multiple regression analysis, eliminating the confounding factors, lunch sodium-to-potassium ratio, dinner energy, lipid, cholesterol, saturated fat, and alcohol intake were positively associated with blood pressure, whereas breakfast protein and lunch fiber intake showed a negative association with blood pressure. Our results suggest that nutrient intake timing is also an important factor in the prevention of high blood pressure. Our study provides possibilities to prevent hypertension by changing the timing of nutrient intake, especially sodium, together with potassium and lipids. However, because our research was limited to food-log app users, broader research regarding the general population needs to be conducted.

Conductive olfactory dysfunction (COD) is caused by an obstruction in the nasal cavity and is characterized by changeable olfaction. COD can occur even when the olfactory cleft is anatomically normal, and therefore, the cause in these cases remains unclear. Herein, we used computational fluid dynamics to examine olfactory cleft airflow with a retrospective cohort study utilizing the cone beam computed tomography scan data of COD patients. By measuring nasal-nasopharynx pressure at maximum flow, we established a cut-off value at which nasal breathing can be differentiated from combined mouth breathing in COD patients. We found that increased nasal resistance led to mouth breathing and that the velocity and flow rate in the olfactory cleft at maximum flow were significantly reduced in COD patients with nasal breathing only compared to healthy olfactory subjects. In addition, we performed a detailed analysis of common morphological abnormalities associated with concha bullosa. Our study provides novel insights into the causes of COD, and therefore, it has important implications for surgical planning of COD, sleep apnea research, assessment of adenoid hyperplasia in children, and sports respiratory physiology.

Chronic or acute ambient temperature change alter the gut microbiota and the metabolites, regulating metabolic functions. Short-chain fatty acids (SCFAs) produced by gut bacteria reduce the risk of disease. Feeding patterns and gut microbiota that are involved in SCFAs production are controlled by the circadian clock. Hence, the effect of environmental temperature change on SCFAs production is expected depending on the exposure timing. In addition, there is limited research on effects of habitual cold exposure on the gut microbiota and SCFAs production compared to chronic or acute exposure. Therefore, the aim was to examine the effect of cold or heat exposure timing on SCFAs production. After exposing mice to 7 or 37 °C for 3 h a day at each point for 10 days, samples were collected, and cecal pH, SCFA concentration, and BAT weight was measured. As a result, cold exposure at ZT18 increased cecal pH and decreased SCFAs. Intestinal peristalsis was suppressed due to the cold exposure at ZT18. The results reveal differing effects of intermittent cold exposure on the gut environment depending on exposure timing. In particular, ZT18 (active phase) is the timing to be the most detrimental to the gut environment of mice.

Longer durations for near-work activities, such as studying, worsen eyesight. In contrast, outdoor exercise is effective in reducing the risk of developing myopia. Despite these findings, however, the interaction between studying and exercise in eyesight has not been quantitatively evaluated. Moreover, since there is a culture of attending lessons in Japan, it is important to investigate the relationship between elementary school activities, such as cram schools or sports clubs, and vision. Therefore, in this study, we examined whether attending cram schools and/or sports clubs is associated with the use of glasses among elementary school students. We conducted a survey among 7419 elementary school students in Tokyo, Japan using a food education questionnaire. A logistic regression analysis was used to evaluate the relationship between wearing glasses, an objective variable, and attending sports clubs and cram schools. Sex and school year were considered confounding factors. The results of this study showed that students who attended only sports clubs were more likely to be categorized into the "not wearing glasses" group (p = 0.03, OR = 1.45), whereas those who attended only cram schools were more likely to be categorized into the "wearing glasses" group (p = 0.008, OR = 0.67). In addition, students who attended both cram schools and sports clubs were more likely to be categorized into the "not wearing glasses" group than those who only attended cram schools (p = 0.28, OR = 0.85). Our findings indicate that attending not only cram schools but also sports clubs may prevent deterioration of eyesight. Parents and health care providers need to take these findings into account in order to prevent visual impairment in children.

OBJECTIVE AND METHOD: The stay-at-home order during the COVID-19 pandemic has restricted individuals' social behaviors, and therefore, effected their lifestyle including sleep, diet, and physical activity. Using the cross-sectional study design with a large sample size (N = 30,275) from the mobile health App users in Japan, we show age-dependent lifestyle changes during a nonpunitive "mild lockdown" (from April to May 2020). RESULTS: Sleep onset and offset were delayed on work-days but not on free-days with increased sleep duration and decreased social jetlag, and the changes were more evident in the younger population. Although average weight change was close to none because of the users' characteristic (95% of App users try to lose weight), we investigated an association between lifestyle change and body-weight change. Participants who reported advanced sleep phase during mild lockdown described a weight decrease. In contrast, the delayed sleep phase reported a weight gain. The results were significant after adjustment of confounding factors including physical activity and meal changes. CONCLUSIONS: Although there is cumulative evidence showing a relationship between late chronotype and obesity, it is still unclear about the potential benefit of the chronotype management to control body weight. Thus, to the best of our knowledge, this is the first study investigating the association between chronotype and weight changes by leveraging a large cohort.

Breakfast skipping and nighttime snacking have been identified as risk factors for obesity, diabetes, and cardiovascular diseases. However, the effects of irregularity of meal timing on health and daily quality of life are still unclear. In this study, a web-based self-administered questionnaire survey was conducted involving 4490 workers (73.3% males; average age = 47.4 ± 0.1 years) in Japan to investigate the association between meal habits, health, and social relationships. This study identified that irregular meal timing was correlated with higher neuroticism (one of the Big Five personality traits), lower physical activity levels, and higher productivity loss. Irregular meal timing was also associated with a higher incidence of sleep problems and lower subjective health conditions. Among health outcomes, a high correlation of irregular meal timing with mental health factors was observed. This study showed that irregularity of meal timing can be explained by unbalanced diets, frequent breakfast skipping, increased snacking frequency, and insufficient latency from the last meal to sleep onset. Finally, logistic regression analysis was conducted, and a significant contribution of meal timing irregularity to subjective mental health was found under adjustment for other confounding factors. These results suggest that irregular meal timing is a good marker of subjective mental health issues.

Psychological distress during pregnancy may increase the risk of adverse maternal and infant outcomes. Past studies have demonstrated the association between circadian disturbances with psychological health. However, the roles of chronotype and social jetlag on psychological state during pregnancy are yet to be identified. We aimed to examine the psychological state in pregnant women and its relations to chronotype, social jetlag (SJL), sleep quality and cortisol rhythm. The current study included a subsample of participants from an ongoing cohort study. A total of 179 primigravidas (mean age 28.4 ± 4.0 years) were recruited. Chronotype and sleep quality during the second trimester were assessed using the Morning-Eveningness Questionnaire (MEQ) and Pittsburgh Sleep Quality Index (PSQI), respectively. SJL was calculated based on the difference between mid-sleep on workdays and free days. Psychological state of participants was evaluated using the Depression, Anxiety, and Stress Scale-21 (DASS-21). Subsamples (n = 70) provided salivary samples at 5 time points over a 24 h period during the second trimester for cortisol assay. A higher proportion of pregnant women experienced moderate to severe anxiety symptoms (n = 77, 43.0%), followed by depressive (n = 17, 9.5%) and stress (n = 14, 7.8%) symptoms. No association was observed between chronotype and psychological distress during pregnancy. There was no significant difference in cortisol rhythms in relation to psychological distress. SJL and sleep quality were significantly associated with stress symptoms among pregnant women in the second trimester. Poor sleep quality, particularly daytime dysfunction (β = 0.37, p = .006) and sleep disturbances (β = 0.23, p = .047), were significantly associated with psychological distress (depressive, anxiety and stress symptoms) during the second trimester. The findings suggest that sleep is a potential modifiable lifestyle factor that can be targeted to improve psychological health among pregnant women.

The aim of the present study was to investigate the association among lunar cycle, menstrual cycle onset, and subjective sleep quality. Menstrual cycle onset data from the six most recent menstrual cycles were obtained for 529 women (aged 25-39 years) using the smartphone app Luna Luna. We also collected questionnaire survey data on sleep quality from each participant. Overall, there was no association between the onset of menstrual cycle and lunar phase. Interestingly, the proportion of good sleepers with menstrual cycle beginning during the light period was significantly higher than that during the dark period, while the proportion of poor sleepers with menstrual cycle beginning during the dark period was significantly higher than that during the light period. When participants were categorized by the combination of lunar phases (light, dark, neutral periods) in the two most recent menstrual cycle onsets, the "both dark period" group and the "other (light and dark) period" group showed the lowest proportion of good sleepers. Menstrual cycle onset in the dark period was associated with a deterioration in subsequent subjective sleep quality, which was more apparent with consecutive onsets in the dark period or at a rapidly changing lunar phase.

This study investigated (a) site- and direction-dependent variations of passive triceps surae aponeurosis stiffness and (b) the relationships between aponeurosis stiffness and muscle strength and walking performance in older individuals. Seventy-nine healthy older adults participated in this study. Shear wave velocities of the triceps surae aponeuroses at different sites and in two orthogonal directions were obtained in a prone position at rest using supersonic shear imaging. The maximal voluntary isometric contraction torque of the plantar flexors and normal (preferred) and fast (fastest possible) walking speeds (5-m distance) were also measured. The shear wave velocities of the adjoining aponeuroses were weakly associated with plantar flexion torque (r = .23-.34), normal (r = .26), and fast walking speed (r = .25). The results show clear spatial variations and anisotropy of the triceps surae aponeuroses stiffness in vivo, and the aponeurosis stiffness was associated with physical ability in older adults.

Constipation is a common condition that occurs in many people worldwide. While magnesium oxide (MgO) is often used as the first-line drug for chronic constipation in Japan, dietary fiber intake is also recommended. Dietary fiber is fermented by microbiota to produce short-chain fatty acids (SCFAs). SCFAs are involved in regulating systemic physiological functions and circadian rhythm. We examined the effect of combining MgO and the water-soluble dietary fiber, inulin, on cecal SCFA concentration and microbiota in mice. We also examined the MgO administration timing effect on cecal SCFAs. The cecal SCFA concentrations were measured by gas chromatography, and the microbiota was determined using next-generation sequencing. Inulin intake decreased cecal pH and increased cecal SCFA concentrations while combining MgO increased the cecal pH lowered by inulin and decreased the cecal SCFA concentrations elevated by inulin. When inulin and MgO were combined, significant changes in the microbiota composition were observed compared with inulin alone. The MgO effect on the cecal acetic acid concentration was less when administered at ZT12 than at ZT0. In conclusion, this study suggests that MgO affects cecal SCFA and microbiota during inulin feeding, and the effect on acetic acid concentration is time-dependent.

Background: The effects of different intake patterns of meal protein on muscle mass have not been clarified. We cross-sectionally and longitudinally examined the effect of different timing of protein intake on sarcopenia-related factors in older adults. Methods: This cross-sectional study 1 included 219 (male, n = 69, female, n = 150) elderly subjects aged ≥65 years. Subjects who consumed more protein at breakfast than at dinner were grouped into the morning group (MG, n = 76; male, n = 26; female, n = 50), and those who consumed more protein at dinner than at breakfast were grouped into the evening group (EG, n = 143; male, n = 43; female, n = 100). In cross-sectional study 2-1 (female, n = 125), the subjects were classified into four groups according to the number of meals with sufficient protein intake. In cross-sectional studies 2-2 (female, n = 125) and 2-3 (female, n = 27), the subjects were classified into eight groups and three groups according to whether they had consumed sufficient protein at three meals; sarcopenia-related factors were compared. The intervention study was a placebo-controlled, double-blind, randomized controlled trial that included 40 elderly women with low daily breakfast protein intake. The subjects were divided into four groups: morning protein and placebo intake groups and evening protein and placebo intake groups. Each group consumed the test food (containing 10 g milk protein) or placebo in the morning or evening for 12 weeks. Blood indices and physical function were assessed before and after the intervention. Results: Comparing all subjects, MG showed significantly higher handgrip strength than did EG (P < 0.05). The higher ratio of morning protein intake relative to the total protein intake, the better the muscle mass (r = 0.452, P < 0.05) and handgrip strength (r = 0.383, P < 0.05). The intervention study showed an increase in muscle mass with the intake of milk protein in the morning rather than in the evening (P < 0.05). Conclusions: Protein intake at breakfast might have relatively stronger effects on skeletal muscle mass than at lunch and dinner.

Social jetlag (SJL) is defined as the discrepancy between social and biological rhythms and calculated by the difference between the midpoint of sleep time on working-days and free-days. Previous human and mouse studies showed SJL is positively related to evening chronotype and significantly related to smoking habit, cardiovascular risk, cognitive ability, and that SJL-mimicking conditions, simulating the real lifestyle situation of SJL in many humans, disrupt the regularity of estrous cycles of female animals. The effects of SJL-mimicking conditions on circadian rhythms and cognitive function and the reasons why the discrepancy between social and biological rhythms is involved in SJL have not yet been investigated. Therefore, in this study, we utilized a mouse model of SJL-mimicking conditions - 6-hour delayed-light/dark (LD) conditions for 2 days and normal-LD conditions for the following 5 days - applied for several weeks during which biological rhythms were monitored. Circadian rhythms of central and peripheral clocks and metabolism of the mice under the SJL-mimicking condition were always delayed for 2-3 hours compared with those under the normal-LD condition. Moreover, SJL-mimicking conditions impaired their cognitive function using a novel object recognition test. Only the delayed timing of either the light phase of the LD or of feeding for 2 days, comparable to the free-days situation of humans, delayed the circadian staging of rhythms the following 5 days. Furthermore, sleep deprivation during the early mornings for 5 days, which is comparable to early rise times experienced by humans on working-days and does affect the staging of circadian rhythms (circadian misalignment schedule), delayed the locomotor activity rhythms the next 2 days, comparable to free-days in humans, which is similar to the lifestyle rhythm of the evening chronotype. Our results demonstrated that the circadian misalignment schedule for 5 days changed the locomotor activity rhythms the following 2 days to the evening chronotype, that light- and/or feeding-shift conditions for 2 days exacerbate SJL, and that SJL-mimicking conditions delay the metabolic rhythm and cause cognitive impairment.

To examine the effects of the intake of a snack containing dietary fiber under free-living conditions on postprandial glucose levels in older adults, nine healthy older adults aged 76.9 ± 1.6 years (mean ± standard error) completed two crossover trials: 1) regular snack (BISCUIT) intake and 2) intake of snacks with a high dietary fiber content (DF-BISCUIT). In both trials, each participant consumed either BISCUIT or DF-BISCUIT between lunch and dinner time for 1 week. During the intervention, the blood glucose levels of all the subjects were observed using a continuous glucose monitoring system. Lower 24 h blood glucose levels were yielded in the DF-BISCUIT than the BISCUIT trials. Moreover, compared to the BISCUIT trials, the blood glucose levels after dinner and areas under the curve (AUCs) were significantly decreased in the DF-BISCUIT treatments. The blood glucose levels and AUCs after the intake of the next day’s breakfast were suppressed in the DF-BISCUIT treatments compared to those in the BISCUIT trials. Our data indicate that the intake of snacks with a high dietary fiber content under free-living conditions is an effective way to restrain postprandial glucose levels and that the effect lasts until breakfast the next day.

<title>Abstract</title>
<bold>Background: </bold>As internet use becomes more widespread, the screen time (ST) of elementary school students increases yearly. It is known that longer durations of ST can affect obesity, physical activity, dry eye disease, and learning ability. However, the effects of ST just before bedtime have not been clarified. Therefore, we examined ST duration and timing effects on elementary school children. <bold>Methods: </bold>We conducted a survey of 7,419 elementary school students in Tokyo, Japan using a questionnaire on food education. ST duration and timing (just before bedtime) served as the explanatory variables, and the relationship between obesity, physical activity, dry eyes, and learning ability was analyzed using logistic regression analysis. Gender, school year, height, and weight were considered confounding factors. First, we examined whether ST duration and timing were related to each objective variable, using a univariate model to examine all variables. Thereafter, we performed multivariate logistic regression analyses for all variables showing a significant difference in the univariate models.<bold>Results:</bold> The results showed that the relationship between the ST duration and obesity, physical activity, and learning ability was statistically significant. The relationship between ST timing and obesity, dry eyes, and learning ability was also statistically significant. Therefore, ST timing has a greater effect on dry eyes, and ST duration has a greater effect on academic performance. <bold>Conclusion: </bold>Our findings indicate that ST in school children is related to obesity, physical activity, dry eyes, and learning ability, and they suggest that not only the duration but also the timing of ST is important.

To date, nutritional studies have focused on the total intake of dietary fiber rather than intake timing. In this study, we examined the effect of the timing of daily Helianthustuberosus ingestion on postprandial and 24 h glucose levels, as well as on intestinal microbiota in older adults. In total, 37 healthy older adults (age = 74.9 ± 0.8 years) were recruited. The participants were randomly assigned to either a morning group (MG, n = 18) or an evening group (EG, n = 17). The MG and EG groups were instructed to take Helianthustuberosus powder (5 g/day) just before breakfast or dinner, respectively, for 1 week after the 1-week control period. The glucose levels of all participants were monitored using a continuous glucose monitoring system throughout the 2 weeks. The intestinal microbiota was analyzed by sequencing 16S rRNA genes from feces before and after the intervention. There were no significant differences in the physical characteristics or energy intake between groups. Helianthustuberosus intake led to decreases in tissue glucose levels throughout the day in both groups (p < 0.01, respectively). As a result of examining the fluctuations in tissue glucose levels up to 4 hours after each meal, significant decreases in the areas under the curves (AUCs) were observed for all three meals after intervention, but only in the MG (breakfast: p = 0.012, lunch: p = 0.002, dinner: p = 0.005). On the other hand, in the EG, there was a strong decrease in the AUC after dinner, but only slight decreases after breakfast and lunch (breakfast: p = 0.017, lunch: p = 0.427, dinner: p = 0.002). Moreover, the rate of change in the peak tissue glucose level at breakfast was significantly decreased in the MG compared to the EG (p = 0.027). A greater decrease was observed in the change in the blood glucose level after the ingestion of Helianthustuberosus in the MG than in the EG. Furthermore, the relative abundance of Ruminococcus in the MG at the genus level was significantly higher at baseline than in the EG (p = 0.016) and it was also significantly lower after the intervention (p = 0.013). Our findings indicate that Helianthustuberosus intake in the morning might have relatively stronger effects on the intestinal microbiota and suppress postprandial glucose levels to a greater extent than when taken in the evening.

This study examined the effects of the time of conducting a combined physical and cognitive exercise program on cognitive functions and the mood of older adults. We randomly assigned 17 participants (8 men, 9 women, age 73,24±3,75) to the morning group (AM Group), 18 (8 men, 10 women, age 73,11±4,84) to the afternoon group (PM Group), and they completed a 12-week, dual-task exercise program in the morning or the afternoon, respectively. Moreover, the waiting-list control group consisted of 12 participants (6 men, 6 women, age 73,25±5,93). The cognitive functions and the mood of the participants were assessed before and after the program by using the Neurobehavioral Cognitive Status Examination, and the short version of the Profile of Mood States, respectively. As a result, the exercise program conducted in the afternoon had broader effects on cognitive functions (increased Repetition and Memory) than in the morning (increased Repetition), or the waiting-list control group (increased Judgment). Moreover, the program in the afternoon tended to reduce Anger-Hostility, and Fatigue. In conclusion, conducting the exercise program in the afternoon might be more effective for improving cognitive functions and the mood of older adults than in the morning. However, there was heterogeneity between the groups despite the randomization. Therefore, we suggest conducting a more rigorously controlled study using a larger sample to validate these findings.

Adolescent alcohol exposure may increase anxiety-like behaviors by altering central monoaminergic functions and other important neuronal pathways. The present study was designed to investigate the anxiolytic effect of 0.5% γ-oryzanol (GORZ) and its neurochemical and molecular mechanisms under chronic 10% ethanol consumption. Five-week-old ICR male mice received either control (14% casein, AIN 93 M) or GORZ (14% casein, AIN 93 M + 0.5% GORZ) diets in this study. We showed that GORZ could potentially attenuate alcohol-induced anxiety-like behaviors by significantly improving the main behavioral parameters measured by the elevated plus maze test. Moreover, GORZ treatment significantly restored the alcohol-induced downregulation of 5-hydroxytryptophan and 5-hydroxyindole acetic acid in the hippocampus and improved homovanillic acid levels in the cerebral cortex. Furthermore, a recovery increase in the level of 3-methoxy-4-hydroxyphenylglycol both in the hippocampus and cerebral cortex supported the anxiolytic effect of GORZ. The significant elevation and reduction in the hippocampus of relative mRNA levels of brain-derived neurotrophic factor and interleukin 1β, respectively, also showed the neuroprotective role of GORZ in ethanol-induced anxiety. Altogether, these results suggest that 0.5% GORZ is a promising neuroprotective drug candidate with potential anxiolytic, neurogenic, and anti-neuroinflammatory properties for treating adolescent alcohol exposure.

Jerusalem artichokes contain high amounts of inulin, which is a prebiotic that supports digestive health, as well as a variety of insoluble fibers and caffeoylquinic acid. The individual impact of these components on gut microbiota is well known; however, the combinatorial effects are less clear. In this investigation, we fractionated Jerusalem artichokes into three parts (water-soluble extract, insoluble extract, and organic extract) and powdered them. Mice were fed a high-fat diet that included one or more of these extracts for 10 days, and then their cecal pH, cecal short-chain fatty acids (SCFAs), and fecal microbiota were evaluated. The combination of the water-soluble and organic extract decreased cecal pH and increased the concentration of SCFAs and led to dynamic changes in the composition of the gut microbiota. These results demonstrate that both the water-soluble and organic extracts in Jerusalem artichokes are bioactive substances that are capable of changing SCFA production and the composition of gut microbiota. Powdered Jerusalem artichokes, rather than inulin supplements, may be superior for promoting a healthy gut.

We examined the impact of consuming biscuits with a beverage of powdered mulberry or barley leaves in the afternoon on postprandial glucose levels at dinnertime among young adults. A total of 18 young adults participated in a partially double-blinded, randomized crossover trial over 2 weeks, consuming either: (1) no biscuits; (2) a biscuit; (3) a biscuit with a beverage of powdered mulberry leaves; or (4) a biscuit with a beverage of powdered barley leaves, as an afternoon snack followed by a standardized test dinner. Glucose levels were recorded after each meal. Results showed intake of biscuits with a beverage of mulberry and barley leaves significantly reduced postprandial rises in glucose after their immediate consumption and dinner, though there was no direct relationship between the glucose levels at the two meals. Compared to those with low glucose levels, participants with high glucose levels at dinner showed a stronger second meal effect, that was attributed to the mulberry or barley leaves, and were also more likely to have lean body weights and prefer evenings. Our findings indicate that eating snacks alongside mulberry or barley leaves is an effective way to suppress postprandial glucose levels in young adults with high glucose levels who prefer evenings.

The circadian clock system works not only as a cellular time-keeper but also as a coordinator for almost all physiological functions essential to maintaining human health. Therefore, disruptions or malfunctions of this system can cause many diseases and pre-symptomatic conditions. Indeed, previous studies have indicated that disrupted clock gene expression rhythm is closely related to obesity, and that allergic diseases can be regulated by controlling peripheral clocks in organs and tissues. Moreover, recent studies have found that obesity can lead to immune disorders. Accordingly, in this review, we assess the connection between obesity and allergy from the point of view of the circadian clock system anew and summarize the relationships among the circadian clock system, obesity, and allergy.

We examined the effects of the timing of acute and consecutive epigallocatechin gallate (EGCG) and catechin-rich green tea ingestion on postprandial glucose in mice and human adults. In mouse experiments, we compared the effects of EGCG administration early (morning) and late (evening) in the active period on postprandial glucose. In human experiments, participants were randomly assigned to the morning-placebo (MP, n = 10), morning-green tea (MGT, n = 10), evening-placebo (EP, n = 9), and evening-green tea (EGT, n = 9) groups, and consumed either catechin-rich green tea or a placebo beverage for 1 week. At baseline and after 1 week, participants consumed their designated beverages with breakfast (MP and MGT) or supper (EP and EGT). Venous blood samples were collected in the fasted state and 30, 60, 120, and 180 min after each meal. Consecutive administration of EGCG in the evening, but not in the morning, reduced postprandial glucose at 30 (p = 0.006) and 60 (p = 0.037) min in the evening trials in mice. In humans, ingestion of catechin-rich green tea in the evening decreased postprandial glucose (three-factor analysis of variance, p < 0.05). Thus, catechin intake in the evening more effectively suppressed elevation of postprandial glucose.

BACKGROUND: Circadian rhythm plays an important role as our internal body's clock that synchronizes behavior and physiology according to the external 24-h light-dark cycle. Past studies have associated disrupted circadian rhythm with higher risk of miscarriages, preterm birth and low birth weights. This paper described the protocol of a prospective cohort study which aims to determine the circadian rhythm in pregnant women, identify its association with maternal factors during pregnancy, gestational weight gain, birth and infant outcomes. METHODS: Ten government maternal and child health clinics in Kuala Lumpur, Malaysia will be randomly selected. Sample size of 438 first-trimester pregnant women will be followed-up until the birth of their infant. Salivary melatonin and cortisol concentration among subsample will be determined using enzyme-linked immunosorbent assay. Data on sleep quality, psychological distress and morningness/eveningness chronotype of pregnant women will be collected using validated questionnaires. Pedometer will be used to measure 5-day physical activity data. Total gestational weight gain will be determined at the end of pregnancy. Utilization of 3-day food record is to capture meal timing and nutrient intake. All measurements will be done in 2nd and 3rd trimester. Birth outcomes will be collected through clinic records and Centers for Disease Control and Prevention (CDC) Neonatal questionnaire. Infants will be followed-up at 6 and 12 months old to obtain anthropometric measurements. DISCUSSION: There is a growing recognition of the role of maternal circadian rhythm, which entrains fetal circadian rhythms that may subsequently have long-term health consequences. The present study will identify the effect of circadian rhythm on pregnancy outcomes and infant growth in the first year of life.

Water-soluble dietary fiber such as inulin improves the beta diversity of the intestinal microbiota of mice fed with a high-fat diet (HFD). The circadian clock is the system that regulates the internal daily rhythm, and it affects the pattern of beta diversity in mouse intestinal microbiota. Burdock (Arctium lappa) root contains a high concentration of inulin/fructan (approximately 50%) and is a very popular vegetable in Japan. Arctium lappa also contains functional substances that may affect intestinal microbiota, such as polyphenols. We compared the effects of inulin and A. lappa powder on the diversity of the intestinal microbiota of HFD-fed mice. 16S rDNA from the intestinal microbiota obtained from feces was analyzed by 16S Metagenomic Sequencing Library Preparation. It was found to have a stronger effect on microbiota than inulin alone, suggesting that inulin has an additive and/or synergic action with other molecules in A. lappa root. We examined the effects of intake timing (breakfast or dinner) of A. lappa on intestinal microbiota. The intake of A. lappa root in the evening had a stronger effect on microbiota diversity in comparison to morning intake. Therefore, it is suggested that habitual consumption of A. lappa root in the evening may aid the maintenance of healthy intestinal microbiota.

Chronic Kidney Disease (CKD) is increasing in incidence and has become a worldwide health problem. Sleep disorders are prevalent in patients with CKD raising the possibility that these patients have a disorganized circadian timing system. Here, we examined the effect of adenine-induced tubulointerstitial nephropathy on the circadian system in mice. Compared to controls, adenine-treated mice showed serum biochemistry evidence of CKD as well as increased kidney expression of inflammation and fibrosis markers. Mice with CKD exhibited fragmented sleep behavior and locomotor activity, with lower degrees of cage activity compared to mice without CKD. On a molecular level, mice with CKD exhibited low amplitude rhythms in their central circadian clock as measured by bioluminescence in slices of the suprachiasmatic nucleus of PERIOD 2::LUCIFERASE mice. Whole animal imaging indicated that adenine treated mice also exhibited dampened oscillations in intact kidney, liver, and submandibular gland. Consistently, dampened circadian oscillations were observed in several circadian clock genes and clock-controlled genes in the kidney of the mice with CKD. Finally, mice with a genetically disrupted circadian clock (Clock mutants) were treated with adenine and compared to wild type control mice. The treatment evoked worse kidney damage as indicated by higher deposition of gelatinases (matrix metalloproteinase-2 and 9) and adenine metabolites in the kidney. Adenine also caused non-dipping hypertension and lower heart rate. Thus, our data indicate that central and peripheral circadian clocks are disrupted in the adenine-treated mice, and suggest that the disruption of the circadian clock accelerates CKD progression.

<p>In mammals, the circadian rhythms have been shown to regulate several physiological functions, including body temperature, sleep-wake behavior, physical activity, hormonal secretions, and metabolism. These processes are controlled by circadian clock genes, and abnormal circadian rhythms are associated with the development of obesity, diabetes, and lifestyle-related diseases. In addition, the timing of behaviors such as food intake, exercise, and stress influence circadian rhythms, including clock gene expression in peripheral tissues. Therefore, the interaction between nutrition and the circadian clock is so-called "chrono-nutrition" is poised to become an important research field of chronobiology. In this review, we review the effects of a timed-nutrition on circadian clocks and their timing-dependent effects on physiological functions.</p>

Background: A high-fat diet (HFD) can induce obesity and metabolic disorders that are closely associated with cognitive impairments, and the progression of several psychiatric disorders such as anxiety. We have previously demonstrated the anxiolytic-like effect of Gamma oryzanol (GORZ) in chronic restraint stressed mice. Objective: We studied the neurochemical and molecular mechanisms that underlie the preventive effect of GORZ in HFD-induced anxiety-like behaviors, monoaminergic dysfunction, and inflammation. Methods: Eight-week-old Institute of Cancer (ICR) male mice weighing 33-34 g were divided into the following groups and free-fed for 8 weeks: control (14% casein, AIN 93M); HFD; HFD + GORZ (0.5% GORZ). Body weight gain was checked weekly. The anxiolytic-like effects of GORZ were examined via open-field test (OFT) and elevated plus maze (EPM) test. Brain levels of monoamines [5-hydroxy tryptamine (5-HT), dopamine (DA), and norepinephrine (NE)] and their metabolites [5-hydroxyindole acetic acid (5-HIAA), homovanillic acid (HVA), and 3-methoxy-4-hydroxyphenylglycol (MHPG)], proinflammatory cytokines such as tumor necrosis factor-αα (Tnf-α) mRNA levels, and interleukin 1-β (Il-1β) mRNA levels in the cerebral cortex and amygdala were examined using high-performance liquid chromatography-electrochemical detection (HPLC-ECD), and real-time reverse transcription-polymerase chain reaction (RT-PCR), respectively. Results: Mice fed a HFD for eight weeks showed anxiety-like behaviors in association with HFD-induced body weight gain. GORZ potentially blocked HFD-induced anxiety-like behaviors via significant improvement of the primary behavioral parameters in behavioral tests, with a minor reduction in HFD-induced body weight gain. Furthermore, GORZ treatment significantly downregulated HFD-induced upregulation of dopamine levels in the brain's amygdala. Significant reduction of the relative mRNA expression of Tnf-α and Il-1 β was also observed in the amygdala of HFD + GORZ mice, compared to HFD mice. Conclusions: Our findings strongly suggest that 0.5% GORZ exerts anxiolytic-like effects, possibly through downregulation of dopamine, and via expression of proinflammatory cytokines Tnf-α and Il-1 β in the case of chronic HFD exposure.

The mammalian circadian clock drives the temporal coordination in cellular homeostasis and it leads the day-night fluctuation of physiological functions, such as sleep/wake cycle, hormonal secretion, and body temperature. The mammalian circadian clock system in the body is classified hierarchically into two classes, the central clock in the suprachiasmatic nucleus (SCN) of the hypothalamus and the peripheral clocks in peripheral tissues such as the intestine and liver, as well as other brain areas outside the SCN. The circadian rhythm of various tissue-specific functions is mainly controlled by each peripheral clock and partially by the central clock as well. The digestive, absorptive, and metabolic capacities of nutrients also show the day-night variations in several peripheral tissues such as small intestine and liver. It is therefore indicated that the bioavailability or metabolic capacity of nutrients depends on the time of day. In fact, the postprandial response of blood triacylglycerol to a specific diet and glucose tolerance exhibit clear time-of-day effects. Meal frequency and distribution within a day are highly related to metabolic functions, and optimal time-restricted feeding has the potential to prevent several metabolic dysfunctions. In this review, we summarize the time-of-day-dependent postprandial response of macronutrients to each meal and the involvement of circadian clock system in the time-of-day effect. Furthermore, the chronic beneficial and adverse effects of meal time and eating pattern on metabolism and its related diseases are discussed. Finally, we discuss the timing-dependent effects of exercise on the day-night variation of exercise performance and therapeutic potential of time-controlled-exercise for promoting general health.

Dietary protein intake is important for skeletal muscle protein synthesis. In this study, we investigated the differential effect of protein sources on hypertrophy of plantaris muscle induced by surgical ablation of gastrocnemius and soleus muscles. Six-week old mice were fed diets containing caseinate, whey, or soy as protein sources for 2 weeks. Plantaris muscle hypertrophy was induced by a unilateral ablation of synergistic muscles after a week. Food intake of soy protein-fed mice was higher than that of caseinate and whey-fed mice, resulting in higher body and fat weights. Plantaris muscle weight in sham-operated mice was not different across the groups. Overload-operated plantaris muscle weight and increased ratio of overloaded muscle to sham-operated muscle weights were higher in caseinate-fed mice than in whey- and soy protein-fed mice, suggesting caseinate as a promising protein source for muscle hypertrophy.

Metabolism of lipids such as cholesterol and triglycerides has daily variations and is controlled by a circadian clock. Sesamin isomers, a mixture of sesamin and episesamin (SE), are types of lignans in sesame seed that have shown the improvement of lipid metabolism with various diseases in an animal model. We therefore tested whether the effects of SE on lipid metabolism are influenced by timing of administration. High-fat diet (HFD)-loaded rat was administered SE in the ZT13 or 14 (at the beginning of the active phase) or ZT23 or 22 (at the end of the active phase) every day for 7 or 28 days, and the effects on lipid metabolism were evaluated. The effects of SE were enhanced by duration-dependency: 28-day administration of SE strongly affected some parameters related to lipid metabolism, particularly cholesterol metabolism, as compared to 7-day administration. In particular, in 28-day administration, the analysis of serum and liver cholesterol levels revealed that SE administration decreases more effectively at the beginning of the active phase when compared to at the end of that. Furthermore, quantitative real-time polymerase chain reaction (QRT-PCR) and functional analysis indicated that suppression of cholesterol synthesis in the liver and promotion of cholesterol excretion from the liver, as well as inhibition of the functional activity and gene expression of sterol response element-binding protein 2 (Srebp2), which is a transcriptional factor and controls the gene expression involved in cholesterol-metabolism enzymes, contribute to enhancement of SE's effects at this administration timing. No significant differences were observed in triglyceride metabolism with regard to timing of SE administration. After 28-day administration of SE, administration at the beginning of the active phase only affected the expression of clock genes in the liver with phase-advance. In the pharmacokinetic study, administration time had no effect on the level of sesamin, episesamin or their metabolites in the liver after administration of SE for 28 days. The present results suggest that continuous long administration of SE at the beginning of the active phase is preferable for obtaining beneficial effects on lipid metabolism.

Soy protein intake is known to cause microbiota changes. While there are some reports about the effect of soy protein intake on gut microbiota and lipid metabolism, effective timing of soy protein intake has not been investigated. In this study, we examined the effect of soy protein intake timing on microbiota. Mice were fed twice a day, in the morning and evening, to compare the effect of soy protein intake in the morning with that in the evening. Mice were divided into three groups: mice fed only casein protein, mice fed soy protein in the morning, and mice fed soy protein in the evening under high-fat diet conditions. They were kept under the experimental condition for two weeks and were sacrificed afterward. We measured cecal pH and collected cecal contents and feces. Short-chain fatty acids (SCFAs) from cecal contents were measured by gas chromatography. The microbiota was analyzed by sequencing 16S rRNA genes from feces. Soy protein intake whether in the morning or evening led to a greater microbiota diversity and a decrease in cecal pH resulting from SCFA production compared to casein intake. In addition, these effects were relatively stronger by morning soy protein intake. Therefore, soy protein intake in the morning may have relatively stronger effects on microbiota than that in the evening.

Type 2 diabetes mellitus (T2DM) is a chronic disease, and dietary modification is a crucial part of disease management. Okara is a sustainable source of fibre-rich food. Most of the valorization research on okara focused more on the physical attributes instead of the possible health attributes. The fermentation of okara using microbes originated from food source, such as tea, sake, sufu and yoghurt, were explored here. The aim of this study is to investigate fermented okara as a functional food ingredient to reduce blood glucose levels. Fermented and non-fermented okara extracts were analyzed using the metabolomic approach with UHPLC-QTof-MSE. Statistical analysis demonstrated that the anthraquinones, emodin and physcion, served as potential markers and differentiated Eurotium cristatum fermented okara (ECO) over other choices of microbes. The in-vitro α-glucosidase activity assays and in-vivo mice studies showed that ECO can reduce postprandial blood glucose levels. A 20% ECO loading crispy snack prototype revealed a good nutrition composition and could serve as a fundamental formulation for future antidiabetes recipe development, strengthening the hypothesis that ECO can be used as a novel food ingredient for diabetic management.

Green tea polyphenols, particularly catechins, decrease fasting and postprandial glucose. However, no studies have compared the timing of green tea ingestion on glucose metabolism and changes in catechin concentrations. Here, we examined the effects of timing of acute catechin-rich green tea ingestion on postprandial glucose metabolism in young men. Seventeen healthy young men completed four trials involving blood collection in a fasting state and at 30, 60, 120, and 180 min after meal consumption in a random order: 1) morning placebo trial (09:00 h; MP trial), 2) evening placebo trial (17:00 h; EP trial), 3) morning catechin-rich green tea trial (09:00 h; MGT trial), and 4) evening catechin-rich green tea trial (17:00 h; EGT trial). The concentrations of glucose at 120 min (P=.031) and 180 min (P=.013) after meal intake were significantly higher in the MGT trials than in the MP trials. Additionally, the concentration of glucose was significantly lower in EGT trials than in the EP trials at 60 min (P=.014). Moreover, the concentrations of glucose-dependent insulinotropic polypeptide were significantly lower in the green tea trials than in the placebo trials at 30 min (morning: P=.010, evening: P=.006) and 60 min (morning: P=.001, evening: P=.006) after meal intake in both the morning and evening trials. Our study demonstrated that acute ingestion of catechin-rich green tea in the evening reduced postprandial plasma glucose concentrations.

Water-soluble dietary fiber is known to modulate fecal microbiota. Although there are a few reports investigating the effects of fiber intake timing on metabolism, there are none on the effect of intake timing on microbiota. Therefore, in this study, we examined the timing effects of inulin-containing food on fecal microbiota. Mice were housed under conditions with a two-meals-per-day schedule, with a long fasting period in the morning and a short fasting period in the evening. Then, 10-14 days after inulin intake, cecal content and feces were collected, and cecal pH and short-chain fatty acids (SCFAs) were measured. The microbiome was determined using 16S rDNA sequencing. Inulin feeding in the morning rather than the evening decreased the cecal pH, increased SCFAs, and changed the microbiome composition. These data suggest that inulin is more easily digested by fecal microbiota during the active period than the inactive period. Furthermore, to confirm the effect of fasting length, mice were housed under a one-meal-per-day schedule. When the duration of fasting was equal, the difference between morning and evening nearly disappeared. Thus, our study demonstrates that consuming inulin at breakfast, which is generally after a longer fasting period, has a greater effect on the microbiota.

Introduction: To detect congenital hearing loss in mice, an objective measure is needed other than mouse behavioral observation. This study aimed to refine the methodology of auditory brainstem response measurement and identify potential congenital hearing loss models in laboratory mouse strains. Methods: Mice were anesthetized and fitted with head electrodes. Each mouse underwent four ABR measurements according to four testing conditions: A - no chamber; conventional tone; B - chamber; conventional tone; C - no chamber; short tone; D - chamber; short tone. Potential congenital hearing loss models were identified using 10 mice from each strain (C57BL, BALB/c, CH3, ICR, and ddY) through sound-attenuated ABR measurements with short-tone bursts. Potential congenital hearing loss models exhibited hearing thresholds ≥30 dB in both ears. Data were analyzed for normal distribution and variance homogeneity using the D'Agostino-Pearson/Kolmogorov-Smirnov and F value tests, respectively. One-way analysis of variance (ANOVA), with the Tukey-Kramer test, was used to conduct parametric analysis, and the Kruskal-Wallis/Friedman test, with a Dunn's test for post hoc analysis, was used to perform non-parametric analysis. Results: The simultaneous use of a sound-attenuating chamber and short-tone bursts provided clearly defined wave patterns, even at lower sound intensities. Inbred strains, especially C57BL/6 sub-strains, constitute suitable congenital hearing loss models. Conclusions: Our study shows that environmental factors should be addressed in animal studies of hearing function. Potential congenital hearing loss models may be found amongst commercially available inbred strains.

Clock genes that comprise the circadian clock system control various physiological functions. Delayed sleep-wake phase disorder (DSWPD) and night eating syndrome (NES) are characterized by delayed sleep and meal timing, respectively. We estimated that clock gene expression rhythms in DSWPD patients may be delayed in comparison with the healthy subjects due to delayed melatonin secretion rhythms, producing eveningness chronotype in these individuals. However, it was difficult to estimate which clock gene expression rhythms were delayed or not in NES patients, because previous studies revealed that melatonin secretion rhythm was a little delayed compared with healthy individuals and that chronotype of NES patients depended on the individuals. Therefore, we examined expression rhythms of clock genes such as Period3 (Per3), nuclear receptor subfamily 1, group D, member 1 (Nr1d1) and Nr1d2 in these patients. Further, we expected sleep and meal patterns in DSWPD and NES patients may be more diverse than patterns observed in healthy subjects, and thus analyzed relationships among clock gene expression rhythms, sleep quality, sleep midpoint time, and meal times. We enrolled healthy male participants along with DSWPD and NES male patients, and asked all participants to answer questionnaires and to keep diaries to record information on their sleep and meals. Further, we asked them to collect 5-10 beard follicle samples, 6 times every 4 h. We measured clock gene expression rhythms using total RNA extracted from beard follicle cells. Peak time of clock gene expression in the NES group showed more diversity than the other groups, and that in the DSWPD group was delayed compared with the control group. In addition, the peak time of clock gene expression was negatively correlated with sleep quality and positively correlated with meal time after a long fast. Amplitudes of clock gene expression, especially Per3, positively responded to better mental and physical conditions as well as with better sleep quality. Results of this study suggest that peak times of clock gene expression in NES patients depended on the individuals; some patients with NES showed similar clock gene expression rhythm to healthy subjects, and other patients with NES showed similar to DSWPD patients. Moreover, this study suggests that meal time after a long fast may influence more determination in clock gene expression rhythms than the time of breakfast. Therefore, this study also indicates that Per3 clock gene may be one of the parameters that will help us understand sleep and meal rhythm disturbances.

The expression rhythms of clock genes, such as Per1, Per2, Bmal1, and Rev-erb α, in mouse peripheral clocks, are entrained by a scheduled feeding paradigm. In terms of food composition, a carbohydrate-containing diet is reported to cause strong entrainment through insulin secretion. However, it is unknown whether human diets entrain peripheral circadian clocks. In this study, we used freeze-dried diets for type 2 diabetes (DB) and chronic kidney disease (CKD), as well as low-carbohydrate diets. After 24 h of fasting, PER2::LUC knock-in mice were given access to food for 2 days during inactive periods, and bioluminescence rhythm was then measured using an in vivo imaging system. AIN-93M, the control mouse diet with a protein:fat:carbohydrate (PFC) ratio of 14.7:9.5:75.8, caused a significant phase advance (7.3 h) in the liver clock compared with that in 24 h fasted mice, whereas human diets caused significant but smaller phase advances (4.7-6.2 h). Compared with healthy and high fat/sucrose-induced DB mice, adenine-induced CKD mice showed attenuation of a phase-advance with a normal diet. There were no significant differences in phase-advance values between human diets (normal, DB, and CKD). In addition, a normal-carbohydrate diet (PFC ratio of 20.3:23.3:56.4) and a low-carbohydrate diet (PFC ratio of 36.4:42.9:20.7) caused similar phase advances in peripheral clocks. The present results strongly suggest that scheduled feeding with human diets can cause phase advances in the peripheral clocks of not only healthy, but also DB and CKD mice. This discovery provides support to the food-induced entrainment of peripheral clocks in human clinical trials.

Gluconeogenesis is de novo glucose synthesis from substrates such as amino acids and is vital when glucose is lacking in the diurnal nutritional fluctuation. Accordingly, genes for hepatic gluconeogenic enzymes exhibit daily expression rhythms, whose detailed regulations under nutritional variations remain elusive. As a first step, we performed general systematic characterization of daily expression profiles of gluconeogenic enzyme genes for phosphoenolpyruvate carboxykinase (PEPCK), cytosolic form (Pck1), glucose-6-phosphatase (G6Pase), catalytic subunit (G6pc), and tyrosine aminotransferase (TAT) (Tat) in the mouse liver. On a standard diet fed ad libitum, mRNA levels of these genes showed robust daily rhythms with a peak or an elevation phase during the late sleep-fasting period in the diurnal feeding/fasting (wake/sleep) cycle. The rhythmicity was preserved in constant darkness, modulated with prolonged fasting, attenuated by Clock mutation, and entrained to varied photoperiods and time-restricted feedings. These results are concordant with the notion that gluconeogenic enzyme genes are under the control of the intrinsic circadian oscillator, which is entrained by the light/dark cycle, and which in turn entrains the feeding/fasting cycle and also drives systemic signaling pathways such as the hypothalamic-pituitary-adrenal axis. On the other hand, time-restricted feedings also showed that the ingestion schedule, when separated from the light/dark cycle, can serve as an independent entrainer to daily expression rhythms of gluconeogenic enzyme genes. Moreover, nutritional changes dramatically modified expression profiles of the genes. In addition to prolonged fasting, a high-fat diet and a high-carbohydrate (no-protein) diet caused modification of daily expression rhythms of the genes, with characteristic changes in profiles of glucoregulatory hormones such as corticosterone, glucagon, and insulin, as well as their modulators including ghrelin, leptin, resistin, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide-1 (GLP-1). Remarkably, high-protein (60% casein or soy-protein) diets activated the gluconeogenic enzyme genes atypically during the wake-feeding period, with paradoxical up-regulation of glucagon, which frequently formed correlation networks with other humoral factors. Based on these results, we propose that daily expression rhythms of gluconeogenic enzyme genes are under the control of systemic oscillator-driven and nutrient-responsive hormones.

OBJECTIVE: Shift work encompasses a broad range of work time arrangements. However, how shift work affects the circadian expression of clock genes remains to be explored. The objective of this study was to evaluate the pattern of clock gene expression in shift workers in the field. METHODS: We examined clock gene expression in Japanese men who work: (1) one night shift followed by a day off (caregivers: nurses and doctors; the one-night group); (2) three or more consecutive night shifts (factory workers; the consecutive-night group); or (3) daytime only (the daytime group), using beard follicle samples. The expression of Period3, Nuclear Receptor Subfamily 1 Group D Member 1 (Nr1d1), and Nuclear Receptor Subfamily 1 Group D Member 2 (Nr1d2) was examined by real-time polymerase chain reaction. RESULTS: Period3 expression in the daytime and one-night groups together with Nr1d2 expression in the one-night group fitted a 24-h-period cosine curve better than in the consecutive-night group (p = 0.004, 0.012, and 0.001, respectively). The level of overall Period3 gene expression, calibrated with that of 18S-rRNA, was decreased in the consecutive-night group compared with that in the daytime group (p = 0.006). The patterns of Period3 and Nr1d2 expression in the daytime and one-night groups were more coherent than those in the consecutive-night group. CONCLUSIONS: These results suggest that night shift work affects the rhythms and levels of circadian Period3 and Nr1d2 expression dependent on the shift schedule or type of the shift; however, there is substantial variation between individuals.

[Purpose] Little is known about the effectiveness of daily physical activity on depression biomarkers in older adults. This study aimed to investigate the effects of increased daily physical activity for 8 weeks on depression biomarkers in postmenopausal women. [Participants and Methods] Thirty-eight postmenopausal females were randomly assigned into a control or an active group and were asked to wear a uniaxial accelerometer for 8 weeks. Blood samples were obtained at baseline and at the end of the intervention. During the intervention, the active group was asked to increase their physical activity level above their usual lifestyle whereas those in the control group maintained their daily lifestyle. [Results] After the 8-week intervention, the step counts of the participants in the active group increased. The serum concentration of the brain-derived neurotrophic factor and serotonin increased significantly in the active group, but not in the control group, as compared with baseline values. The serum concentration of derivatives of reactive oxygen metabolites and biological antioxidant potential did not change after the intervention in either group. [Conclusion] These findings may suggest that promotion of daily physical activity in postmenopausal women has a positive impact on depression without any change in oxidative stress.

AIMS: The present study was aimed to investigate the anxiolytic effect of γ-oryzanol (GORZ) during chronic restraint stress treatment (CRST), which is a well-documented model of stress-related disorders, like anxiety, and its potential molecular mechanisms. MATERIALS AND METHODS: In this experiment, 5-week-old male ICR mice were used and the concentration of GORZ was fixed at 0.5% in the mouse standard diet (14% casein, AIN 93 M). Mice were immobilized daily for 3 h from ZT 2.5 to 5.5 (ZT0 was designated as light-on time) for 20 consecutive days, followed by behavioral testing, including the open field test (OFT) and elevated plus maze (EPM) test. The concentration of serum corticosterone (CORT) was measured. In addition, the expression of central monoamine neurotransmitters with their metabolites in the hippocampus, cerebral cortex, and amygdala of the brain were examined. KEY FINDINGS: 0.5% GORZ partially blocked stress-induced reduction of body weight gain while stressed mice had significantly lower body weights during the entire experimental period. Further, 0.5% GORZ treatment could significantly improve the main behavioral parameters even in CRST situations. The significant increase in serum CORT levels indicated CRST-induced stress, which was almost unaffected by 0.5% GORZ treatment. Moreover, 0.5% GORZ also supported the anxiolytic mechanism with enhancement of 5-HIAA and NE levels in the amygdala of brain after CRST. SIGNIFICANCE: Taken together, our studies suggested that 0.5% GORZ is a potential therapeutic drug candidate against anxiety under chronic stress conditions.

We examined the effects of meal timing on postprandial glucose metabolism, including the incretin response and metabolites in healthy adults. Nineteen healthy young men completed two trials involving blood collection in a fasting state and at 30, 60 and 120 min after meal provision in a random order: (1) morning (~0900 h) and (2) evening (~1700 h). The blood metabolome of eight participants was analyzed using capillary electrophoresis-mass spectrometry. Postprandial glucose concentrations at 120 min (p = 0.030) and glucose-dependent insulinotropic polypeptide concentrations (p = 0.005) at 60 min in the evening trials were higher than those in the morning trials. The incremental area under the curve values of five glycolysis, tricarboxylic acid cycle and nucleotide-related metabolites and 18 amino acid-related metabolites were higher in the morning trials than those in the evening trials (p < 0.05). Partial least-squares analysis revealed that the total metabolic change was higher in the morning. Our study demonstrates that a meal in the evening exacerbates the state of postprandial hyperglycemia in healthy adults. In addition, this study provides insight into the difference of incretion and blood metabolites between breakfast and dinner, indicating that the total metabolic responses tends to be higher in the morning.

Bioelectrical impedance spectroscopy (BIS) can assess intracellular water (ICW) and total water (TW) in limbs. This study aimed to examine whether BIS can explain a part of the inter-individual variation of the muscle size-strength relationship in older adults. We analyzed the data of 79 participants aged 64-86 years. The maximal voluntary isometric torques of dorsiflexion and plantar flexion on the right side were measured. The anterior and posterior muscle thickness (MT) in the right lower leg was assessed using ultrasonography. The length of the right lower leg (L) was measured, and the ICW-to-TW ratio (ICW/TW) in the right lower leg was obtained using BIS. The MT was multiplied by L to represent an index of muscle volume (MV). Correlation and stepwise regression analyses were performed. The anterior and posterior MT × L significantly and positively correlated with the muscle torque of dorsiflexion and plantar flexion (r = 0.710 and 0.649, respectively, P < 0.001). In the stepwise regression analyses, ICW/TW was selected as a significant predictor of muscle torque independent of MT × L (P < 0.05) for both dorsiflexion and plantar flexion. Electrical parameters of BIS (membrane capacitance, characteristics frequency, and phase angle) in the lower leg also significantly correlated with muscle torques. In addition, the skeletal muscle mass index (appendicular lean mass/height2) was also associated with ICW/TW (P < 0.001). The present results suggest that ICW/TW explains the interindividual variations of the muscle size-strength relationship.

BACKGROUND: Atrogin1, which is one of the key genes for the promotion of muscle atrophy, exhibits day-night variation. However, its mechanism and the role of its day-night variation are largely unknown in a muscle atrophic context. METHODS: The mice were induced a muscle atrophy by hindlimb-unloading (HU). To examine a role of circadian clock, Wild-type (WT) and Clock mutant mice were used. To test the effects of a neuronal effects, an unilateral ablation of sciatic nerve was performed in HU mice. To test a timing-dependent effects of weight-bearing, mice were released from HU for 4 h in a day at early or late active phase (W-EAP and W-LAP groups, respectively). FINDINGS: We found that the day-night oscillation of Atrogin1 expression was not observed in Clock mutant mice or in the sciatic denervated muscle. In addition, the therapeutic effects of weight-bearing were dependent on its timing with a better effect in the early active phase. INTERPRETATION: These findings suggest that the circadian clock controls the day-night oscillation of Atrogin1 expression and the therapeutic effects of weight-bearing are dependent on its timing. FUND: Council for Science, Technology, and Innovation, SIP, "Technologies for creating next-generation agriculture, forestry, and fisheries".

Menstrual symptoms may have a significant impact on women's lives. Many women experience menses-related health problems, such as menstrual pain, heavy menstrual bleeding, and premenstrual syndrome, during their reproductively fertile years. Circadian misalignment in shift workers has been reported to contribute to menstrual cycle irregularity and/or painful menstruation. However, the relationship between social jetlag (SJL) and menstrual symptoms/menstrual cycle has not been elucidated. In this study, we aimed to elucidate this relationship among female university students. One-hundred and fifty female university students (mean [SD]: 18.8 [0.71]-years old) completed self-reported questionnaires consisting of menstrual symptoms and menstrual cycle, sleep quality and sleep habits, quality of life, and demographic variables. The average menstrual cycle was 32.0 [5.4] days. The percentage of students who showed menstrual cycle irregularity, having less than 25 days or more than 39 days of menstrual cycle during the previous four menstrual cycles, was 60.6%. SJL, the difference between mid-sleep time on free days and mid-sleep time on school days, was categorized into small (absolute SJL < 1 h) or large (≥1 h). Overall, 78.0% of participants had SJL ≥ 1 h. Among the menstrual symptoms, pain, behavioral change, and water retention subscale scores were significantly higher in the SJL ≥ 1 h group than in the SJL < 1 h group. However, no significant differences were found in concentration, autonomic reaction, or negative affect subscale scores between the two groups. The menstrual cycle was 31.2 [5.5] days in the SJL < 1 h group and 32.2 [5.4] days in the SJL ≥ 1 h group, without significant difference. Logistic regression analysis showed that more than 1 h of SJL was a significant associated factor with severe menstrual symptom, independently of sleep duration and late chronotype. This study indicated that SJL was a significant factor associated with severe menstrual symptoms, suggesting the possibilities of association between circadian system and reproductive function among humans.

Huntington's disease (HD) patients suffer from progressive neurodegeneration that results in cognitive, psychiatric, cardiovascular, and motor dysfunction. Disturbances in sleep-wake cycles are common among HD patients with reports of delayed sleep onset, frequent bedtime awakenings, and excessive fatigue. The BACHD mouse model exhibits many HD core symptoms including circadian dysfunction. Because circadian dysfunction manifests early in the disease in both patients and mouse models, we sought to determine if early interventions that improve circadian rhythmicity could benefit HD symptoms and delay disease progression. We evaluated the effects of time-restricted feeding (TRF) on the BACHD mouse model. At 3 months of age, the animals were divided into 2 groups: ad lib and TRF. The TRF-treated BACHD mice were exposed to a 6-h feeding/18-h fasting regimen that was designed to be aligned with the middle (ZT 15-21) of the period when mice are normally active (ZT 12-24). Following 3 months of treatment (when mice reached the early disease stage), the TRF-treated BACHD mice showed improvements in their locomotor activity and sleep behavioral rhythms. Furthermore, we found improved heart rate variability, suggesting that their autonomic nervous system dysfunction was improved. On a molecular level, TRF altered the phase but not the amplitude of the PER2::LUC rhythms measured in vivo and in vitro. Importantly, treated BACHD mice exhibited improved motor performance compared with untreated BACHD controls, and the motor improvements were correlated with improved circadian output. It is worth emphasizing that HD is a genetically caused disease with no known cure. Lifestyle changes that not only improve the quality of life but also delay disease progression for HD patients are greatly needed. Our study demonstrates the therapeutic potential of circadian-based treatment strategies in a preclinical model of HD.

Insulin is a key molecule that synchronizes peripheral clocks, such as that in the liver. Although we previously reported that mice fed a low-protein diet showed altered expression of lipid-related genes in the liver and induction of hepatic steatosis, it is unknown whether a low-protein diet impairs insulin secretion and modifies the hepatic circadian rhythm. Therefore, we investigated the effects of the intake of a low-protein diet on the circadian rhythm of insulin secretion and hepatic lipid metabolism in mice. Under 12-h light/12-h dark cycle, mice fed a low-protein diet for 7 days displayed enhanced food intake at the end of the light phase, although central and peripheral PER2 expression rhythm was maintained. Serum insulin levels in mice fed a low-protein diet remained low during the day, and the insulin secretion in OGTT was also markedly lower than in normal mice. In mice fed low-protein diet, hepatic TG accumulation was observed during the nighttime, with relatively high levels of ACC1 mRNA and total ACC proteins. Although there were no differences in the activity rhythm of hepatic mTOR between mice fed a normal or low-protein diet, hepatic IRS-2 expression in mice fed a low-protein diet remained low during the day, with no increase at the beginning of the light period. These results suggested that the low-protein diet eliminated the circadian rhythm of serum insulin and hepatic lipid metabolism in mice, providing insights into our understanding of the mechanisms of hepatic disorders of lipid metabolism.

A short photoperiod is reported to promote body weight gain in quail, and might therefore influence lipid metabolism and induce liver steatosis. Protein deficiency or malnutrition can also cause hepatic steatosis in mammals, but the effect in birds has not previously been investigated. In order to determine the influence of a shortened photoperiod and low crude protein diet on avian hepatic lipid metabolism, 10-day-old male Japanese quail (Coturnix coturnix japonica) were assigned to a 2 × 2 factorial arrangement of treatments in four groups, each of eight birds, and fed for three days, after which liver triglyceride (TG) accumulation and lipid-related gene expression were measured. Experimental factors included two dietary protein levels of 24% or 10% crude protein (CP), and two photoperiod conditions of a 12:12 h light/dark cycle (12L:12D) or a 1:23 h light/dark cycle (1L:23D). The liver TG level in quail fed the 10% CP diet under 1L:23D was higher than in birds maintained on either the 10% CP diet under 12L:12D (15.86 vs. 10.56 mg/g liver, P &lt
0.01) or 24% CP diet under 1L:23D (7.95 mg/g liver, P &lt
0.01). The sterol regulatory element-binding protein 1 mRNA, which is involved in TG synthesis, was more highly expressed in quail on the 10% CP diet under 1L:23D than in those maintained on the 24% CP diet under 12L:12D (2.2 vs. 7.9, P &lt
0.01), whereas the mRNA level of very low-density apolipoprotein II (apoVLDLII), which encodes a VLDL structural protein, was lower (157.3 vs. 12.5, P &lt
0.01). To our knowledge, this is the first report of a selective protein restriction and shorter photoperiod, alone and in combination, inducing a fatty liver state in an avian species. Our study provides insight into factors that govern lipid metabolism in birds, and the efficiency of domestic fowl production.

We examined the relationships between chronotype or social jetlag and clock gene expression. Twenty-four young men [Chronotype: morningness, n = 8; intermediate, n = 8, eveningness, n = 8], aged 27 ± 2 years old (mean ± SE), completed two trials in a randomized order: (1) a Friday trial and (2) a Monday trial. In both trials, hair follicle cells were collected to evaluate the expression of clock genes over a 24-hour period at 4-hour intervals. There was a significant main effect of time on the expression of NR1D1, NR1D2, and PER3 (P < 0.001) in the morningness group, but not in the eveningness group. Changes in the peak time of expression of NR1D1 (r = 0.434, P = 0.034), NR1D2 (r = 0.481, P = 0.017), and PER3 (r = 0.457, P = 0.025) from the Friday to Monday trials were positively correlated with social jetlag (SJL) time. Our findings indicate that there was no change in the patterns of clock gene expression between workdays and the day after the holiday in the morningness group, and that SJL time influences the peak time of clock gene expression, moving it from the early to late workday, after a holiday.

A number of diverse cell-surface proteins are anchored to the cytoskeleton via scaffold proteins. Na+/H+ exchanger regulatory factor-1 (NHERF1), encoded by the Slc9a3r1 gene, functions as a scaffold protein, which is implicated in the regulation of membrane expression of various cell-surface proteins. Here, we demonstrate that the circadian clock component PERIOD2 (PER2) modulates transcription of the mouse Slc9a3r1 gene, generating diurnal accumulation of NHERF1 in the mouse liver. Basal expression of Slc9a3r1 was dependent on transcriptional activation by p65/p50. PER2 bound to p65 protein and prevented p65/p50-mediated transactivation of Slc9a3r1. The time-dependent interaction between PER2 and p65 underlay diurnal oscillation in the hepatic expression of Slc9a3r1/NHERF1. The results of immunoprecipitation experiments and liquid chromatography-mass spectrometry analysis of mouse liver revealed that NHERF1 time-dependently interacted with fatty acid transport protein-5 (FATP5). Temporary accumulation of NHERF1 protein stabilized plasmalemmal localization of FATP5, thereby enhancing hepatic uptake of fatty acids at certain times of the day. Our results suggest an unacknowledged role for PER2 in regulating the diurnal expression of NHERF1 in mouse liver. This machinery also contributed to diurnal changes in the ability of hepatic cells to uptake fatty acids.

We evaluated the anxiolytic effect of γ-oryzanol (GORZ) and elucidated the molecular mechanisms involved in its inhibition of behavioral test-induced anxiety. Behavioral tests were conducted on day 13, and mice were subjected to 30 min of acute restraint stress treatment (ARST) before sacrifice on day 16. In other group, behavioral tests were conducted on day 13 and 14 after ARST. 0.5% GORZ significantly weakened the effect of behavioral stress, but not the effect of strong ARST. GORZ downregulated ARST-induced cFos levels in the cerebral cortex. In conclusion, GORZ has potential ant-anxiety effect in the treatment of weak behavioral test-induced stress.

The circadian clock system in mammals plays a fundamental role in maintaining homeostasis. Entrainment is an important characteristic of the internal clock, by which appropriate timing is maintained according to external daily stimuli, such as light, stress, exercise, and/or food. Disorganized entrainment or a misaligned clock time, such as jet lag, increases health disturbances. The central clock in the suprachiasmatic nuclei, located in the hypothalamus, receives information about arousal stimuli, such as physical stress or exercise, and changes the clock time by modifying neural activity or the expression of circadian clock genes. Although feeding stimuli cannot entrain the central clock in a normal light-dark cycle, the central clock can partially detect the metabolic status. Local clocks in the peripheral tissues, including liver and kidney, have a strong direct response to the external stimuli of stress, exercise, and/or food that is independent of the central clock. The mechanism underlying entrainment by stress/exercise is mediated by glucocorticoids, sympathetic nerves, oxidative stress, hypoxia, pH, cytokines, and temperature. Food/nutrition-induced entrainment is mediated by fasting-induced hormonal or metabolic changes and re-feeding-induced insulin or oxyntomodulin secretion. Chrono-nutrition is a clinical application based on chronobiology research. Future studies are required to elucidate the effects of eating and nutrient composition on the human circadian clock. Here, we focus on the central and peripheral clocks mostly in rodents' studies and review the findings of recent investigations of the effects of stress, exercise, and food on the entrainment system.

The circadian system controls the behavior and multiple physiological functions. In mammals, the suprachiasmatic nucleus (SCN) acts as the master pacemaker and regulates the circadian clocks of peripheral tissues. The SCN receives information regarding the light-dark cycle and is thus synchronized to the external 24-hour environment. In contrast, peripheral clocks, such as the liver clock, receive information from the SCN and other factors; in particular, food intake which leads to insulin secretion induces strong entrainment of the liver clock. On the other hand, the liver clock of insulin-depleted mice treated with streptozotocin (STZ) has been shown to be entrained by scheduled feeding, suggesting that insulin is not necessary for entrainment of the liver clock by feeding. In this study, we aimed to elucidate additional mechanism on entraining liver clock by feeding a protein-only diet and/or amino-acid administration which does not increase insulin levels. We demonstrated that protein-only diet and cysteine administration elicit entrainment of the liver clock via glucagon secretion and/or insulin-like growth factors (IGF-1) production. Our findings suggest that glucagon and/or IGF-1 production are additional key factors in food-induced entrainment.

Microbiota-derived short-chain fatty acids (SCFAs) and organic acids produced by the fermentation of non-digestible fibre can communicate from the microbiome to host tissues and modulate homeostasis in mammals. The microbiome has circadian rhythmicity and helps the host circadian clock function. We investigated the effect of SCFA or fibre-containing diets on circadian clock phase adjustment in mouse peripheral tissues (liver, kidney, and submandibular gland). Initially, caecal SCFA concentrations, particularly acetate and butyrate, induced significant day-night differences at high concentrations during the active period, which were correlated with lower caecal pH. By monitoring luciferase activity correlated with the clock gene Period2 in vivo, we found that oral administration of mixed SCFA (acetate, butyrate, and propionate) and an organic acid (lactate), or single administration of each SCFA or lactate for three days, caused phase changes in the peripheral clocks with stimulation timing dependency. However, this effect was not detected in cultured fibroblasts or cultured liver slices with SCFA applied to the culture medium, suggesting SCFA-induced indirect modulation of circadian clocks in vivo. Finally, cellobiose-containing diets facilitated SCFA production and refeeding-induced peripheral clock entrainment. SCFA oral gavage and prebiotic supplementation can facilitate peripheral clock adjustment, suggesting prebiotics as novel therapeutic candidates for misalignment.

The circadian clock system is associated with feeding and mood. Patients with night eating syndrome (NES) delay their eating rhythm and their mood declines during the evening and night, manifesting as time-specific depression. Therefore, we hypothesized that the NES feeding pattern might cause time-specific depression. We established new NES model by restricted feeding with high-fat diet during the inactive period under normal-fat diet ad libitum. The FST (forced swimming test) immobility time in the NES model group was prolonged only after lights-on, corresponding to evening and early night for humans. We examined the effect of the NES feeding pattern on peripheral clocks using PER2::LUCIFERASE knock-in mice and an in vivo monitoring system. Caloric intake during the inactive period would shift the peripheral clock, and might be an important factor in causing the time-specific depression-like behavior. In the NES model group, synthesis of serotonin and norepinephrine were increased, but utilization and metabolism of these monoamines were decreased under stress. Desipramine shortened some mice's FST immobility time in the NES model group. The present study suggests that the NES feeding pattern causes phase shift of peripheral clocks and malfunction of the monoamine system, which may contribute to the development of time-specific depression.

Circadian clock system has been widely maintained in many spices from prokaryote to mammals. “Circadian” means “approximately day” in Latin, thus circadian rhythm means about 24 hour rhythms. The earth revolves once every 24 hours, and our circadian system has been developed for adjusting to this 24 hour cycles, to get sun light information for getting their foods or for alive in birds or mammals. We have two different circadian systems so-called main oscillator located in the suprachiasmatic nucleus (SCN) of the hypothalamus, and local oscillator located in the various peripheral organ tissues such as liver, kidney and skeletal muscle. The SCN is directly entrained by light-dark information through retinal-hypothalamic tract, and then organizes local clock in peripheral tissues via many pathways including neural and hormonal functions. On the other hand, peripheral local clocks are entrained by feeding, exercise and stress stimuli through several cell signaling. Foods (protein, carbohydrate, and lipid) are important regulator of circadian clocks in peripheral tissues. Thus, controlling the timing of food consumption and food composition, a concept known as chrononutrition, is one area of active research to aid recovery from many physiological dysfunctions. In this review, we focus on molecular mechanisms of entrainment and the relationships between circadian clock systems and n-3 polyunsaturated fatty acid. We concentrate on experimental data obtained from cells or animals and humans and discuss how these findings translate into clinical research, and we highlight the latest developments in chrononutritional studies.

Objective Obesity is a risk factor for psychiatric diseases. Recently, a number of single nucleotide polymorphisms (SNPs) have been shown to be related to body mass index (BMI). In this study, we investigated the association of BMI-related SNPs with psychiatric diseases and one of their endophenotypes, memory performance, in a Japanese population. Methods The subjects were 1624 patients with one of three psychiatric diseases (799 patients with major depressive disorder, 594 with schizophrenia, and 231 with bipolar disorder) and 1189 healthy controls. Memory performance was assessed using the Wechsler Memory Scale-Revised (WMS-R). Genomic DNA was prepared from venous blood and used to genotype 23 BMI-related SNPs using the TaqMan 5′-exonuclease allelic discrimination assay. We then analysed the relationships between the SNPs and psychiatric disease and various subscales of the WMS-R. Results Three SNPs (rs11142387, rs12597579, and rs6548238) showed significant differences in the genotype or allele frequency between patients with any psychiatric diseases and controls. Furthermore, six SNPs (rs11142387, rs12597579, rs2815752, rs2074356, rs4776970, and rs2287019) showed significant differences in at least one subscale of the WMS-R depending on the genotypes of the healthy controls. Interestingly, rs11142387 near the Kruppel-like factor 9 (KLF9) was significantly associated with psychiatric disease and poor memory function. Conclusions We identified three and six BMI-related SNPs associated with psychiatric disease and memory performance, respectively. In particular, carrying the A allele of rs11142387 near KLF9 was found to be associated with psychiatric disease and poor memory performance, which warrants further investigations.

Objective: Obesity is a risk factor for psychiatric diseases. Recently, a number of single nucleotide polymorphisms (SNPs) have been shown to be related to body mass index (BMI). In this study, we investigated the association of BMI-related SNPs with psychiatric diseases and one of their endophenotypes, memory performance, in a Japanese population.
Methods: The subjects were 1624 patients with one of three psychiatric diseases (799 patients with major depressive disorder, 594 with schizophrenia, and 231 with bipolar disorder) and 1189 healthy controls. Memory performance was assessed using the Wechsler Memory Scale Revised (WMS-R). Genomic DNA was prepared from venous blood and used to genotype 23 BMI-related SNPs using the TaqMan 5'-exonuclease allelic discrimination assay. We then analysed the relationships between the SNPs and psychiatric disease and various subscales of the WMS-R.
Results: Three SNPs (rs11142387, rs12597579, and rs6548238) showed significant differences in the genotype or allele frequency between patients with any psychiatric diseases and controls. Furthermore, six SNPs (rs11142387, rs12597579, rs2815752, rs2074356, rs4776970, and rs2287019) showed significant differences in at least one subscale of the WMS-R depending on the genotypes of the healthy controls. Interestingly, rs11142387 near the Kruppel-like factor 9 (KLF9) was significantly associated with psychiatric disease and poor memory function.
Conclusions: We identified three and six BMI-related SNPs associated with psychiatric disease and memory performance, respectively. In particular, carrying the A allele of rs11142387 near KLF9 was found to be associated with psychiatric disease and poor memory performance, which warrants further investigations.

c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase family and controls various physiological processes including apoptosis. A specific upstream activator of JNKs is the mitogen-activated protein kinase kinase 7 (MKK7). It has been reported that MKK7-JNK signaling plays an important regulatory role in neural development, however, post-developmental functions in the nervous system have not been elucidated. In this study, we generated neuron-specific Mkk7 knockout mice (MKK7 cKO), which impaired constitutive activation of JNK in the nervous system. MKK7 cKO mice displayed impaired circadian behavioral rhythms and decreased locomotor activity. MKK7 cKO mice at 8 months showed motor dysfunctions such as weakness of hind-limb and gait abnormality in an age-dependent manner. Axonal degeneration in the spinal cord and muscle atrophy were also observed, along with accumulation of the axonal transport proteins JNK-interacting protein 1 and amyloid beta precursor protein in the brains and spinal cords of MKK7 cKO mice. Thus, the MKK7-JNK signaling pathway plays important roles in regulating circadian rhythms and neuronal maintenance in the adult nervous system.

The salivary gland is rhythmically controlled by sympathetic nerve activation from the suprachiasmatic nucleus (SCN), which functions as the main oscillator of circadian rhythms. In humans, salivary IgA concentrations reflect circadian rhythmicity, which peak during sleep. However, the mechanisms controlling this rhythmicity are not well understood. Therefore, we examined whether the timing of parasympathetic (pilocarpine) or sympathetic (norepinephrine; NE) activation affects IgA secretion in the saliva. The concentrations of saliva IgA modulated by pilocarpine activation or by a combination of pilocarpine and NE activation were the highest in the middle of the light period, independent of saliva flow rate. The circadian rhythm of IgA secretion was weakened by an SCN lesion and Clock gene mutation, suggesting the importance of the SCN and Clock gene on this rhythm. Adrenoceptor antagonists blocked both NE- and pilocarpine-induced basal secretion of IgA. Dimeric IgA binds to the polymeric immunoglobulin receptor (pIgR) on the basolateral surface of epithelial cells and forms the IgA-pIgR complex. The circadian rhythm of Pigr abundance peaked during the light period, suggesting pIgR expression upon rhythmic secretion of IgA. We speculate that activation of sympathetic nerves during sleep may protect from bacterial access to the epithelial surface through enhanced secretion of IgA.

Background: Interleukin-33 (IL-33) is an alarmin cytokine that binds to the interleukin 1 receptor-like 1 protein ST2. Clock is a key circadian gene that is essential for endogenous clockworks in mammals. This study investigated whether Clock temporally regulated IL-33-mediated responses in mast cells.
Methods: The kinetics of IL-33-mediated IL-6, IL-13, and TNF-alpha productions were compared between bone marrow-derived mast cells (BMMCs) from wild-type and Clock-mutated mice (Clock(Delta 19/Delta 19) mice). The kinetics of the neutrophil influx into the peritoneal cavity or expression of IL-13 and Gob-5 in the lung in response to IL-33 were compared between wild-type and Clock(Delta 19/Delta 19) mice. We also examined the kinetics of ST2 expression in mast cells and its association with Clock expression.
Results: There was a time-of-day-dependent variation in IL-33-mediated IL-6, IL-13, and TNF-alpha production in wild-type BMMCs, which was absent in Clock-mutated BMMCs. IL-33-induced neutrophil infiltration into the peritoneal cavity also showed a time-of-day-dependent variation in wild-type mice, which was absent in Clock(Delta 19/Delta 19) mice. Furthermore, IL-33-induced IL-13 and Gob-5 expression in the lung exhibited a time-of-day-dependent variation in wild-type mice. These temporal variations in IL-33 mediated mast cell responses were associated with temporal variations of ST2 expression in mast cells. In addition, CLOCK bound to the promoter region of ST2 and Clock deletion resulted in down-regulation of ST2 expression in mast cells.
Conclusions: CLOCK temporally gates mast cell responses to IL-33 via regulation of ST2 expression. Our findings provide novel insights into IL-33/mast cell-associated physiology and pathologies. Copyright (C) 2017, Japanese Society of Allergology. Production and hosting by Elsevier B.V.

In mammals, daily physiological events are precisely regulated by an internal circadian clock system. An important function of this system is to readjust the phase of the clock daily. In Japan, traditional herb medicines, so-called crude drugs (Shoyaku), are widely used for many diseases, and some are reported to affect circadian clock impairment, suggesting that some of them might have an ability to modify clock gene expression rhythms. Therefore, from selected 40 crude drugs, finding candidates that control the circadian clock phases was the first purpose of this study. As there are several crude drugs used for liver- and/or kidney-related diseases, the second aim of the present study was to find some crude drugs affecting liver/kidney circadian clock in vivo. To assess phase changes in the daily circadian rhythm, bioluminescence from the core clock gene product Period 2 was continuously monitored in mouse embryonic fibroblasts in vitro and in some peripheral tissues (kidney, liver, and submandibular gland) of PERIOD2::LUCIFERASE knock-in mice in vivo. In our screening, Polyporus and Bupleuri radix were found to be good candidates to effectively manipulate the peripheral circadian clock phase acutely, with stimulation time-of-day dependency in vitro as well as in vivo. Interestingly, Polyporus and Bupleuri radix are traditional herb medicines use for treating edema and promoting diuresis, and for chronic hepatitis, respectively. These crude drugs may be therefore good modulators of the circadian peripheral clocks including liver and kidney, and circadian clock genes become new molecular targets for these crude drugs.

The circadian clock regulates many physiological functions including physical activity and feeding patterns. In addition, scheduled exercise and feeding themselves can affect the circadian clock. The purpose of the present study was to investigate the relationship between physical/feeding activity and expression of clock genes in hair follicle cells in older adults. Twenty adult men (age, 68 ± 7 years, mean ± SE) were examined in this cross-sectional study. Prior to hair follicle cell collection, the participants were asked to wear a uniaxial accelerometer for one week. The timings of breakfast, lunch, and dinner were also recorded. Hair follicle cells were then collected over a 24 h period at 4 h intervals. The amplitude of PER3 expression was positively correlated with moderate and vigorous physical activity (r = 0.582, p = 0.007) and peak oxygen uptake (r = 0.481, p = 0.032), but these correlations were not observed for NR1D1 or NR1D2. No association was noted between meal times and the amplitude or the acrophase for any of these three clock genes. These findings suggest that rhythmic expression of the circadian clock gene PER3 is associated with the amount of daily physical activity and physical fitness in older adults.

The mammalian circadian clock regulates day-night fluctuations in various physiological processes. The circadian clock consists of the central clock in the suprachiasmatic nucleus of the hypothalamus and peripheral clocks in peripheral tissues. External environmental cues, including light/dark cycles, food intake, stress, and exercise, provide important information for adjusting clock phases. This review focuses on stress and exercise as potent entrainment signals for both central and peripheral clocks, especially in regard to the timing of stimuli, types of stressors/exercises, and differences in the responses of rodents and humans. We suggest that the common signaling pathways of clock entrainment by stress and exercise involve sympathetic nervous activation and glucocorticoid release. Furthermore, we demonstrate that physiological responses to stress and exercise depend on time of day. Therefore, using exercise to maintain the circadian clock at an appropriate phase and amplitude might be effective for preventing obesity, diabetes, and cardiovascular disease.

At steady state, plasma histamine levels exhibit circadian variations with nocturnal peaks, which is implicated in the nighttime exacerbation of allergic symptoms. However, the regulatory mechanisms are largely unexplored. This study determined how steady-state plasma histamine levels are regulated and affected by environmental factors. We found that plasma histamine levels decreased in mast cell-deficient mice and their circadian variations were lost in mast cell-deficient mice reconstituted with bone marrow-derived mast cells (BMMCs) harboring a mutation in the circadian gene Clock. Clock temporally regulates expression of organic cation transporter 3 (OCT3), which is involved in histamine transport, in mast cells; OCT inhibition abolished circadian variations in plasma histamine levels. Mice housed under aberrant light/dark conditions or suffering from restraint stress exhibited desynchronization of the mast cell clockwork, concomitant with the loss of circadian variations in OCT3 expression and plasma histamine levels. The degree of compound 48/80-induced plasma extravasation in mice was correlated with plasma histamine levels. Collectively, the mast cell clock mediates circadian regulation of plasma histamine levels at steady state, in part by controlling OCT3 expression, which can be modulated by stress. Additionally, we propose that plasma histamine levels potentiate mast cell-mediated allergic reactions.

Flavonoids are natural polyphenols that are widely found in plants. The effects of flavonoids on obesity and numerous diseases such as cancer, diabetes, and Alzheimer's have been well studied. However, little is known about the relationships between flavonoids and the circadian clock. In this study, we show that continuous or transient application of flavonoids to the culture medium of embryonic fibroblasts from PER2::LUCIFERASE (PER2::LUC) mice induced various modifications in the circadian clock amplitude, period, and phase. Transient application of some of the tested flavonoids to cultured cells induced a phase delay of the PER2::LUC rhythm at the down slope phase. In addition, continuous application of the polymethoxy flavonoids nobiletin and tangeretin increased the amplitude and lengthened the period of the PER2::LUC rhythm. The nobiletin-induced phase delay was blocked by co-treatment with U0126, an ERK inhibitor. In summary, among the tested flavonoids, polymethoxy flavones increased the amplitude, lengthened the period, and delayed the phase of the PER2::LUC circadian rhythm. Therefore, foods that contain polymethoxy flavones may have beneficial effects on circadian rhythm disorders and jet lag.

The mammalian circadian clock regulates the day and night cycles of various physiological functions. The circadian clock system consists of a central clock in the suprachiasmatic nucleus (SCN) of the hypothalamus and peripheral clocks in peripheral tissues. According to the results of circadian transcriptomic studies in several tissues, the majority of rhythmic genes are expressed in a tissue-specific manner and are influenced by tissue-specific circadian rhythms. Here we review the diurnal variations of musculoskeletal functions and discuss the impact of the circadian clock on homeostasis in skeletal muscle and bone. Peripheral clocks are controlled by not only photic stimulation from the central clock in the SCN but also by external cues, such as feeding and exercise. In this review, we discuss the effects of feeding and exercise on the circadian clock and diurnal variation of musculoskeletal functions. We also discuss the therapeutic potential of chrono-nutrition and chrono-exercise on circadian disturbances and the failure of homeostasis in skeletal muscle and bone.

In mammals, the central clock (the suprachiasmatic nuclei, SCN) is entrained mainly by the light-dark cycle, whereas peripheral clocks in the peripheral tissues are entrained/synchronized by multiple factors, including feeding patterns and endocrine hormones such as glucocorticoids. Clock-mutant mice (Clock/Clock), which have a mutation in a core clock gene, show potent phase resetting in response to light pulses compared with wild-type (WT) mice, owing to the damped and flexible oscillator in the SCN. However, the phase resetting of the peripheral clocks in Clock/Clock mice has not been elucidated. Here, we characterized the peripheral clock gene synchronization in Clock/Clock mice by daily injections of a synthetic glucocorticoid (dexamethasone, DEX) by monitoring in vivo PER2::LUCIFERASE bioluminescence. Compared with WT mice, the Clock/Clock mice showed significantly decreased bioluminescence and peripheral clock rhythms with decreased amplitudes and delayed phases. In addition, the DEX injections increased the amplitudes and advanced the phases. In order to examine the robustness of the internal oscillator, T-cycle experiments involving DEX stimulations with 24- or 30-h intervals were performed. The Clock/Clock mice synchronized to the 30-h T-cycle stimulation, which suggested that the peripheral clocks in the Clock/Clock mice had increased synchronizing ability upon DEX stimulation, to that of circadian and hour-glass type oscillations, because of weak internal clock oscillators.

The ability of the circadian clock to adapt to environmental changes is critical for maintaining homeostasis, preventing disease, and limiting the detrimental effects of aging. To date, little is known about age-related changes in the entrainment of peripheral clocks to external cues. We therefore evaluated the ability of the peripheral clocks of the kidney, liver, and submandibular gland to be entrained by external stimuli including light, food, stress, and exercise in young versus aged mice using in vivo bioluminescence monitoring. Despite a decline in locomotor activity, peripheral clocks in aged mice exhibited normal oscillation amplitudes under light-dark, constant darkness, and simulated jet lag conditions, with some abnormal phase alterations. However, age-related impairments were observed in peripheral clock entrainment to stress and exercise stimuli. Conversely, age-related enhancements were observed in peripheral clock entrainment to food stimuli and in the display of food anticipatory behaviors. Finally, we evaluated the hypothesis that deficits in sympathetic input from the central clock located in the suprachiasmatic nucleus of the hypothalamus were in part responsible for age-related differences in the entrainment. Aged animals showed an attenuated entrainment response to noradrenergic stimulation as well as decreased adrenergic receptor mRNA expression in target peripheral organs. Taken together, the present findings indicate that age-related circadian disorganization in entrainment to light, stress, and exercise is due to sympathetic dysfunctions in peripheral organs, while meal timing produces effective entrainment of aged peripheral circadian clocks.

The peripheral circadian clock is entrained by factors in the external environment such as scheduled feeding, exercise, and mental and physical stresses. In addition, recent studies in mice demonstrated that some food components have the potential to control the peripheral circadian clock during scheduled feeding, although information about these components remains limited. L-Ornithine is a type of non-protein amino acid that is present in foods and has been reported to have various physiological functions. In human trials, for example, L-ornithine intake improved a subjective index of sleep quality. Here we demonstrate, using an in vivo monitoring system, that repeated oral administration of L-ornithine at an early inactive period in mice induced a phase advance in the rhythm of PER2 expression. By contrast, L-ornithine administration to mouse embryonic fibroblasts did not affect the expression of PER2, indicating that L-ornithine indirectly alters the phase of PER2. L-Ornithine also increased plasma levels of insulin, glucose and glucagon-like peptide-1 alongside mPer2 expression, suggesting that it exerts its effects probably via insulin secretion. Collectively, these findings demonstrate that L-ornithine affects peripheral clock gene expression and may expand the possibilities of L-ornithine as a health food.

Exercise during the inactive period can entrain locomotor activity and peripheral circadian clock rhythm in mice; however, mechanisms underlying this entrainment are yet to be elucidated. Here, we showed that the bioluminescence rhythm of peripheral clocks in PER2::LUC mice was strongly entrained by forced treadmill and forced wheel-running exercise rather than by voluntary wheel-running exercise at middle time during the inactivity period. Exercise-induced entrainment was accompanied by increased levels of serum corticosterone and norepinephrine in peripheral tissues, similar to the physical stress-induced response. Adrenalectomy with norepinephrine receptor blockers completely blocked the treadmill exercise-induced entrainment. The entrainment of the peripheral clock by exercise is independent of the suprachiasmatic nucleus clock, the main oscillator in mammals. The present results suggest that the response of forced exercise, but not voluntary exercise, may be similar to that of stress, and possesses the entrainment ability of peripheral clocks through the activation of the adrenal gland and the sympathetic nervous system.

Background: Although the sit-to-stand (STS) test score has been shown to relate to the strength and size of the quadriceps femoris (QF) for elderly population, it is unknown whether this relationship is influenced by a posture (i.e., the trunk being allowed to stoop or not) during the STS test. The present study investigated the relationship between STS test score and QF anatomical cross-sectional area (ACSA) in the middle-aged and elderly population with regard to the difference in the posture during STS test, and aimed to develop an accurate predicting equation of the QF ACSA from the STS test score.
Methods: 105 males (40-81 years) and 113 females (41-79 years) participated in the present study, then the subjects were divided at random as validation and cross-validation groups. Mid-thigh QF ACSA was determined by magnetic resonance imaging. Subjects performed a 10-repeated STS as fast as possible in two conditions: (1) with the trunk being allowed to stoop during the sitting phases, and (2) kept upright throughout the test. A power index of the STS test score was calculated based on an equation obtained in a previous study using the time taken for each test condition, the thigh and shank lengths, and body mass. In the validation group (n = 109), a stepwise multiple linear regression analysis was performed to create a predictive model of the ACSA with sex, age, the STS time, and power for both conditions as independent variables. The formulated predictive equation was examined in the cross-validation group (n = 109).
Results: In the validation group, a stepwise regression analysis revealed that STS power with upright trunk condition, sex, and age but not with the stooping condition, were selected as variables to predict QF ACSA (R-2 = 0.64, P &lt; 0.001). There was no systematic error for the relationship between predicted and measured values in the cross-validation group.
Conclusions: These results indicate that STS test score with upright trunk condition is one of the indices of QF muscle size of the middle-aged and elderly population. The estimated predicting equation should be useful in clinical and practical settings for the health promotion.

In mammals, circadian rhythms in physiological function are generated by a molecular oscillator driven by transcriptional-translational feedback loop consisting of negative and positive regulators. Disruption of this circadian clock machinery is thought to increase the risk of cancer development, but the potential contributions of each component of circadian clock to oncogenesis have been little explored. Here we reported that negative and positive transcriptional regulators of circadian feedback loop had different roles in oncogene-induced neoplastic transformation. Mouse embryonic fibroblasts prepared from animals deficient in negative circadian clock regulators, Period2 (Per2) or Cryptochrome1/2 (Cry1/2), were prone to transformation induced by co-expression of H-ras(V12) and SV40 large T antigen (SV40LT). In contrast, mouse embryonic fibroblasts prepared from mice deficient in positive circadian clock regulators, Bmal1 or Clock, showed resistance to oncogene-induced transformation. In Per2 mutant and Cry1/2-null cells, the introduction of oncogenes induced expression of ATF4, a potent repressor of cell senescence-associated proteins p16INK4a and p19ARF. Elevated levels of ATF4 were sufficient to suppress expression of these proteins and drive oncogenic transformation. Conversely, in Bmal1-null and Clock mutant cells, the expression of ATF4 was not induced by oncogene introduction, which allowed constitutive expression of p16INK4a and p19ARF triggering cellular senescence. Although genetic ablation of either negative or positive transcriptional regulators of the circadian clock leads to disrupted rhythms in physiological functions, our findings define their different contributions to neoplastic cellular transformation.

Background: The circadian clock temporally gates signaling through the high-affinity IgE receptor (Fc epsilon RI) in mast cells, thereby generating a marked day/night variation in allergic reactions. Thus manipulation of the molecular clock in mast cells might have therapeutic potential for IgE-mediated allergic reactions.
Objective: We determined whether pharmacologically resetting the molecular clock in mast cells or basophils to times when FceRI signaling was reduced (ie, when core circadian protein period 2 [PER2] is upregulated) resulted in suppression of IgE-mediated allergic reactions.
Methods: We examined the effects of PF670462, a selective inhibitor of the key clock component casein kinase 1 delta/epsilon, or glucocorticoid, both of which upregulated PER2 in mast cells, on IgE-mediated allergic reactions both in vitro and in vivo.
Results: PF670462 or corticosterone (or dexamethasone) suppressed IgE-mediated allergic reactions in mouse bone marrow-derived mast cells or basophils and passive cutaneous anaphylactic reactions in mice in association with increased PER2 levels in mast cells or basophils. PF670462 or dexamethasone also ameliorated allergic symptoms in a mouse model of allergic rhinitis and downregulated allergen-specific basophil reactivity in patients with allergic rhinitis.
Conclusion: Pharmacologically resetting the molecular clock in mast cells or basophils to times when Fc epsilon RI signaling is reduced can inhibit IgE-mediated allergic reactions. The results suggest a new strategy for controlling IgE-mediated allergic diseases. Additionally, this study suggests a novel mechanism underlying the antiallergic actions of glucocorticoids that relies on the circadian clock, which might provide a novel insight into the pharmacology of this drug in allergic patients.

Although it is known that a low-protein diet induces hepatic triglyceride (TG) accumulation in both rodents and humans, little is known about the underlying mechanism. In the present study, we modeled hepatic TG accumulation by inducing dietary protein deficiency in mice and aimed to determine whether certain amino acids could prevent low-protein diet-induced TG accumulation in the mouse liver. Mice fed a diet consisting of 3 % casein (3C diet) for 7 days showed hepatic TG accumulation with up-regulation of TG synthesis for the Acc gene and down-regulation of TG-rich lipoprotein secretion from hepatocytes for Mttp genes. Supplementing the 3 % casein diet with essential amino acids, branched-chain amino acids, or the single amino acid leucine rescued hepatic TG accumulation. In the livers of mice fed the 3 % casein diet, we observed a decrease in the levels of the autophagy substrate p62, an increase in the expression levels of the autophagy marker LC3-II, and an increase in the splicing of the endoplasmic reticulum (ER) stress-dependent Xbp1 gene. Leucine supplementation to the 3 % casein diet did not affect genes related to lipid metabolism, but inhibited the decrease in p62, the increase in LC3-II, and the increase in Xbp1 splicing levels in the liver. Our results suggest that ER stress responses and activated autophagy play critical roles in low-protein diet-induced hepatic TG accumulation in mice, and that leucine suppresses these two major protein degradation systems. This study contributes to understanding the mechanisms of hepatic disorders of lipid metabolism.

The circadian clock system consists of a central clock located in the suprachiasmatic nucleus in the hypothalamus and peripheral clocks in peripheral tissues. Peripheral clocks in the liver have fundamental roles in maintaining liver homeostasis, including the regulation of energy metabolism and the expression of enzymes controlling the absorption and metabolism of xenobiotics. Over the past two decades, research has investigated the molecular mechanisms linking circadian clock genes with the regulation of hepatic physiological functions, using global clock-gene-knockout mice, or mice with liver-specific knockout of clock genes or clock-controlled genes. Clock dysfunction accelerates the development of liver diseases such as fatty liver diseases, cirrhosis, hepatitis and liver cancer, and these disorders also disrupt clock function. Food is an important regulator of circadian clocks in peripheral tissues. Thus, controlling the timing of food consumption and food composition, a concept known as chrononutrition, is one area of active research to aid recovery from many physiological dysfunctions. In this Review, we focus on the molecular mechanisms of hepatic circadian gene regulation and the relationships between hepatic circadian clock systems and liver physiology and disease. We concentrate on experimental data obtained from cell or mice and rat models and discuss how these findings translate into clinical research, and we highlight the latest developments in chrononutritional studies.

Circadian rhythms are regulated by the suprachiasmatic nucleus (SCN) clock, which is the main oscillator and peripheral clock. SCN clock can be entrained by both photic and non-photic stimuli, and an interaction exists between photic and non-photic entrainment. Moreover, peripheral circadian clocks can be entrained not only by scheduled restricted feeding, but also by scheduled exercise. Thus, the entrainment of peripheral circadian clocks may be the result of an interaction between the entrainment caused by feeding and exercise. In this study, we examined the effect of wheel-running exercise on the phase of the peripheral clocks (kidney, liver and submandibular gland) in PER2::LUC mice under various feeding schedules. Phase and waveforms of the peripheral clocks were not affected by voluntary wheel-running exercise. Exercise for a period of 4 h during the early dark period (morning) delayed the peripheral clocks, while exercise for the same duration during the late dark period (evening) advanced the peripheral clocks. The feeding phase was advanced and delayed by evening and morning exercise, respectively, suggesting that the feeding pattern elicited by the scheduled exercise may entrain the peripheral clocks. Exercise did not affect the phase of the peripheral clock under the 1 meal per day schedule. When the phase of the peripheral clocks was advanced by the feeding schedule of 2 or 4 meals per day during light and/or dark periods, wheel-running exercise during the morning period significantly and equally shifted the phase of all organs back to the original positions observed in mice maintained under free-feeding conditions and with no exercise. When the schedule of 2 meals per day during the dark period failed to affect the phase of peripheral clock, morning exercise did not affect the phase. Wheel-running exercise increased the levels of serum corticosterone, and the injection of dexamethasone/corticosterone instead of exercise shifted a phase that had advanced under the feeding schedule of 2 meals per day, back to the normal position. The liver and submandibular glands exhibit higher sensitivity to dexamethasone than the kidneys. In adrenalectomized mice, treadmill-induced normalization of the advanced phase under a feeding schedule of 2 meals per day was not observed. In summary, scheduled exercise-induced phase shifts were weaker compared to scheduled feeding-induced phase shifts. The phase advance caused by the feeding schedule of 2 or 4 meals per day was suppressed by wheel-running, treadmill exercise or dexamethasone/corticosterone injection in the early dark period (morning). Corticosterone release may be involved in exercise-induced phase shift of peripheral clocks. These results suggest that there is an interaction between the phase shifts caused by feeding and exercise schedules in peripheral clocks.

Mammalian circadian rhythms are governed by an endogenous circadian clock system, including the molecular clock works in each cell and tissue. Adaptation of the circadian clock to different environmental stimuli such as light, food, and stress is essential for homeostasis maintenance. However, the influence of oxidative stress on the circadian clock phase is not fully understood in vitro and in vivo. Here, we examined the effects of hydrogen peroxide (H2O2)-induced oxidative stress on the PERIOD2::LUCIFERASE bioluminescence rhythm in mouse embryonic fibroblasts in vitro and in mouse peripheral tissues in vivo. The circadian clock phase changed with the dose of H2O2 and time of day in vitro
similar phase changes were observed in vivo in the circadian clocks of the peripheral tissues. In addition, mice treated with hemin-induced oxidative stress also showed phase changes of peripheral clocks, similarly as H2O2 treatment. Thus, oxidative stress can entrain circadian clock systems.

There are several reports suggesting that the pathophysiology of psoriasis may be associated with aberrant circadian rhythms. However, the mechanistic link between psoriasis and the circadian time-keeping system, "the circadian clock," remains unclear. This study determined whether the core circadian gene, Clock, had a regulatory role in the development of psoriasis. For this purpose, we compared the development of psoriasis-like skin inflammation induced by the Toll-like receptor 7 ligand imiquimod (IMQ) between wild-type mice and mice with a loss-of-function mutation of Clock. We also compared the development of IMQ-induced dermatitis between wild-type mice and mice with a loss-of-function mutation of Period2 (Per2), another key circadian gene that inhibits CLOCK activity. We found that Clock mutation ameliorated IMQ-induced dermatitis, whereas the Per2 mutation exaggerated IMQ-induced dermatitis, when compared with wild-type mice associated with decreased or increased IL-23 receptor (IL-23R) expression in gamma/delta(+) T cells, respectively. In addition, CLOCK directly bound to the promoter of IL-23R in gamma/delta(+) T cells, and IL-23R expression in the mouse skin was under circadian control. These findings suggest that Clock is a novel regulator of psoriasis-like skin inflammation in mice via direct modulation of IL-23R expression in gamma/delta(+) T cells, establishing a mechanistic link between psoriasis and the circadian clock.

In mammals, circadian rhythms in peripheral organs are impaired when animals are maintained in abnormal environmental light-dark cycles such as constant light (LL). This conclusion is based on averaged data from groups of experimental animals sacrificed at each time point. To investigate the effect of LL housing on the peripheral clocks of individual mice, an in vivo imaging system was used to observe the circadian bioluminescence rhythm in peripheral tissues of the liver, kidney, and submandibular salivary gland in PER2::LUCIFERASE knock-in mice. Using this technique, we demonstrated that the majority of individual peripheral tissues still had rhythmic oscillations of their circadian clocks in LL conditions. However, LL housing caused decreased amplitudes and a broad distribution of peak phases in PER2::LUCIFERASE oscillations irrespective of the state of the animals' behavioral rhythmicity. Because both scheduled feeding and scheduled exercise are effective recovery stimuli for circadian clock deficits, we examined whether scheduled feeding or scheduled exercise could reverse this impairment. The results showed that scheduled feeding or exercise could not restore the amplitude of peripheral clocks in LL. On the other hand, the LL-induced broad phase distribution was reversed, and peak phases were entrained to a specific time point by scheduled feeding but only slightly by scheduled exercise. The present results demonstrate that LL housing impairs peripheral circadian clock oscillations by altering both amplitude and phase in individual mice. The broad distribution of clock phases was clearly reversed by scheduled feeding, suggesting the importance of scheduled feeding as an entraining stimulus for impaired peripheral clocks.

OBJECTIVE: A number of animal studies have implicated circadian clock genes in the regulation of mood, anxiety, and reward. However, the effect of misalignment of the environmental light-dark and internal circadian clock on the monoamine system is not fully understood. In the present study, we examined whether an abnormal light-dark schedule would affect behavior-, circadian clock-, and monoamine-related gene expressions, along with monoamine contents in the amygdala and hippocampus of mice. METHODS: Mice were subjected to an 8-hour phase delay in the light-dark cycle (Shift) every two days for four weeks, and locomotor activity was continuously measured. We examined the circadian expression of clock genes (Per1, Per2, and Bmal1) and genes of the NE/5HT uptake transporters (Net and Sert). In addition, the levels of NE/5HT and their metabolites MHPG/5HIAA were analyzed in the amygdala and hippocampus. RESULTS: Locomotor activity showed a free-running phenotype with a longer period (>24 hours) and showed misalignment between the light-dark and inactive-active cycles. The amplitude of the day-night fluctuation of Bmal1 expression was reduced in the amygdala and hippocampus of light-dark-shifted mice. Net gene expression in the Shift group showed different profiles compared with the Control group. In addition, NE and 5HT levels in the amygdala of the Shift group increased during the active period. CONCLUSIONS: The present results suggest that misalignment of the internal and external clocks by continuous shifting of the light-dark cycle affects the circadian clocks and monoamine metabolism in the amygdala and hippocampus of mice.

Although the results of previous studies have suggested that disruptions in circadian rhythms are involved in the pathogenesis of depression, no studies have examined the interaction of clock gene expression deficit and depression state. In this study, we examined clock gene expression levels and depressive-like behavior in mice housed under 3.5h light, 3.5h dark (T = 7) conditions to investigate the association between clock gene expression and depressive state. C57BL/6J mice were housed under a T = 24 cycle (12h light, 12h dark) or a T = 7 cycle and clock gene expression levels in the hippocampus and the amygdala were measured by real-time RT-PCR. Depressive state was evaluated by the forced swim test (FST). Although circadian rhythms of Per1 and Per2 clock gene expression in the hippocampus and amygdala were still detected under T = 7 conditions, rhythmicity and expression levels of both significantly decreased. Mice housed with a T = 7 cycle showed increased immobile time in the FST than those with a T = 24 cycle. The present results suggest that the presence of a depressive state around the early active phase of activity may be related to impairment of rhythmicity and expression levels of Per1 and Per2 genes under abnormal light-dark conditions.

Pre-eclampsia affects approximately 5% of all pregnant women and remains a major cause of maternal and fetal morbidity and mortality. The hypertension associated with pre-eclampsia develops during pregnancy and remits after delivery, suggesting that the placenta is the most likely origin of this disease. The pathophysiology involves insufficient trophoblast invasion, resulting in incomplete narrow placental spiral artery remodeling. Placental insufficiency, which limits the maternal-fetal exchange of gas and nutrients, leads to fetal intrauterine growth restriction. In this study, in our attempt to develop a new therapy for pre-eclampsia, we directly rescued placental and fetal hypoxia with nano-scale size artificial oxygen carriers (hemoglobin vesicles). The present study is the first to demonstrate that artificial oxygen carriers successfully treat placental hypoxia, decrease maternal plasma levels of anti-angiogenic proteins and ameliorate fetal growth restriction in the pre-eclampsia rat model.

The aim of this study was to examine the effect of exposure to different types of television displays at habitual bedtime on human melatonin and cortisol secretion. Thirteen male participants (mean age: 22.7 +/- 0.85 years) were tested over three nights in one baseline and two experimental sessions. Participants were instructed to watch a movie on four different luminance- and wavelength-controlled television displays: normal luminance (450 candela [cd]/m(2)) or high luminance (1200 cd/m(2)) and normal blue light or half blue light. Salivary melatonin and cortisol levels were measured at two time points before and after television viewing. There was no significant difference in cortisol secretion due to the different displays. Melatonin suppression was significantly lower following the exposure to the half-blue light display compared with the normal blue light display. These results suggest that the use of half-blue light displays during night time may prevent circadian rhythm dysfunction.

The mammalian circadian clock controls many physiological processes that include immune responses and allergic reactions. Several studies have investigated the circadian regulation of intestinal permeability and tight junctions known to be affected by cytokines. However, the contribution of circadian clock to food allergy symptoms remains unclear. Therefore, we investigated the role of the circadian clock in determining the severity of food allergies. We prepared an ovalbumin food allergy mouse model, and orally administered ovalbumin either late in the light or late in the dark period under light-dark cycle. The light period group showed higher allergic diarrhea and weight loss than the dark period group. The production of type 2 cytokines, IL-13 and IL-5, from the mesenteric lymph nodes and ovalbumin absorption was higher in the light period group than in the dark period group. Compared to the dark period group, the mRNA expression levels of the tight junction proteins were lower in the light period group. We have demonstrated that increased production of type 2 cytokines and intestinal permeability in the light period induced severe food allergy symptoms. Our results suggest that the time of food antigen intake might affect the determination of the severity of food allergy symptoms.

The circadian clock system in peripheral tissues can endogenously oscillate and is entrained by the light-dark and fasting-feeding cycles in mammals. Although the system's range of entrainment to light-dark cycles with a non-24 h (<24 h) interval has been studied, the range of entrainment to fasting-feeding cycles with shorter periods (<24 h) has not been investigated in peripheral molecular clocks. In the present study, we measured this range by monitoring the mouse peripheral PER2::LUCIFERASE rhythm in vivo at different periods under each feeding cycle (Tau (T) = 15-24 h) under normal light-dark conditions. Peripheral clocks could be entrained to the feeding cycle with T = 22-24 h, but not to that with T = 15-21 h. Under the feeding cycle with T = 15-18 h, the peripheral clocks oscillated at near the 24-h period, suggesting that they were entrained to the light-dark cycle. Thus, for the first time, we demonstrated the range of entrainment to the non-24 h feeding cycle, and that the circadian range (T = 22-24 h) of feeding stimulus is necessary for peripheral molecular clock entrainment under light-dark cycles.

In mammals, both circadian rhythm and aging play important roles in regulating time-dependent homeostasis. We previously discovered an age-related increase element binding protein, hnRNP A3, which binds to the 3'-untranslated region (UTR) of blood coagulation factor IX (FIX). Here, we describe other members of this protein family, hnRNP C and hnRNP H, which bind to the 3'-UTR of the mouse circadian clock gene Period 2 (mPer2). RNA electrophoretic mobility shift assays using a P-32-labeled Per2 RNA probe coupled with two-dimensional gel electrophoresis followed by MALDI-TOF/MS peptide mass fingerprint analysis was used to analyze these proteins. Western blotting suggested that the total expression of these proteins in mouse liver cell nuclei does not increase with age. Two-dimensional gel electrophoresis analysis of age-related protein expression showed that many isoforms of these proteins exist in the liver and that each protein exhibits a complex age-related expression pattern. These results suggest that many isoforms of proteins are regulated by different aging systems and that many age regulation systems function in the liver.

The effects of acute stress on the peripheral circadian system are not well understood in vivo. Here, we show that sub-acute stress caused by restraint or social defeat potently altered clock gene expression in the peripheral tissues of mice. In these peripheral tissues, as well as the hippocampus and cortex, stressful stimuli induced time-of-day-dependent phase-advances or -delays in rhythmic clock gene expression patterns; however, such changes were not observed in the suprachiasmatic nucleus, i.e. the central circadian clock. Moreover, several days of stress exposure at the beginning of the light period abolished circadian oscillations and caused internal desynchronisation of peripheral clocks. Stress-induced changes in circadian rhythmicity showed habituation and disappeared with long-term exposure to repeated stress. These findings suggest that sub-acute physical/psychological stress potently entrains peripheral clocks and causes transient dysregulation of circadian clocks in vivo.

Mice that exercise after meals gain less body weight and visceral fat compared to those that exercised before meals under a one meal/exercise time per day schedule. Humans generally eat two or three meals per day, and rarely have only one meal. To extend our previous observations, we examined here whether a "two meals, two exercise sessions per day'' schedule was optimal in terms of maintaining a healthy body weight. In this experiment, "morning'' refers to the beginning of the active phase (the "morning'' for nocturnal animals). We found that 2-h feeding before 2-h exercise in the morning and evening (F-Ex/F-Ex) resulted in greater attenuation of high fat diet (HFD)-induced weight gain compared to other combinations of feeding and exercise under two daily meals and two daily exercise periods. There were no significant differences in total food intake and total wheel counts, but feeding before exercise in the morning groups (F-Ex/F-Ex and F-Ex/Ex-F) increased the morning wheel counts. These results suggest that habitual exercise after feeding in the morning and evening is more effective for preventing HFD-induced weight gain. We also determined whether there were any correlations between food intake, wheel rotation, visceral fat volume and skeletal muscle volumes. We found positive associations between gastrocnemius muscle volumes and morning wheel counts, as well as negative associations between morning food intake volumes/body weight and morning wheel counts. These results suggest that morning exercise-induced increase of muscle volume may refer to anti-obesity. Evening exercise is negatively associated with fat volume increases, suggesting that this practice may counteract fat deposition. Our multifactorial analysis revealed that morning food intake helps to increase exercise, and that evening exercise reduced fat volumes. Thus, exercise in the morning or evening is important for preventing the onset of obesity.

This chapter centers on the new keyword "chrono-nutrition", which helps to understand the importance of mutual interactions between circadian rhythms and nutrition/diet. The timing of food can have a major impact on the circadian system. The "food entrainable oscillator" (FEO) senses food timing and/or nutritional factors, and then organizes the circadian system independent of suprachiasmatic nucleus (SCN) regulation. To understand the mechanism of FEO, many types of food have been used for food-induced phase entrainment in the circadian system. Food-induced insulin secretion is an important direct pathway for food entrainment in the liver circadian clock. The chapter also highlights the interactions between food, circadian system, and disease. Regular meal time has the possibility to improve the deficit of the circadian system, independent of the master clock function. Food can become a "zeitgeber" to entrain the circadian system and it is hoped that eating times function as a "zeitgeber" in humans.

IF:6.335

The mammalian circadian rhythm is entrained by multiple factors, including the light-dark cycle, the organism's feeding pattern and endocrine hormones such as glucocorticoids. Both a central clock (the suprachiasmatic nucleus, or SCN) and peripheral clocks (i.e. in the liver and lungs) in mice are entrained by photoperiod. However, the factors underlying entrainment signals from the SCN to peripheral clocks are not well known. To elucidate the role of entrainment factors such as corticosterone and feeding, we examined whether peripheral clock rhythms were impaired by adrenalectomy (ADX) and/or feeding of 6 meals per day at equal intervals under short-day, medium-day and long-day photoperiods (SP, MP and LP, respectively). We evaluated the waveform and phase of circadian rhythms in the liver, kidney and salivary gland by in vivo imaging of PER2::LUCIFERASE knock-in mice. In intact mice, the waveforms of the peripheral clocks were similar among all photoperiods. The phases of peripheral clocks were well adjusted by the timing of the "lights-off"-operated evening (E) oscillator but not the "lights-on"-operated morning (M) oscillator. ADX had almost no effect on the rhythmicity and phase of peripheral clocks, regardless of photoperiod. To reduce the feeding-induced signal, we placed mice on a restricted feeding regimen with 6 meals per day (6 meals RF). This caused advances of the peripheral clock phase in LP-housed mice (2-5 h) and MP-housed mice (1-2 h) but not SP-housed mice. Thus, feeding pattern may affect the phase of peripheral clocks, depending on photoperiod. More specifically, ADX + 6 meals RF mice showed impairment of circadian rhythms in the kidney and liver but not in the salivary gland, regardless of photoperiod. However, the impairment of peripheral clocks observed in ADX + 6 meals RF mice was reversed by administration of dexamethasone for 3 days. The phase differences in the salivary gland clock among SP-, MP- and LP-housed mice became very small following treatment with ADX + 6 meals RF, suggesting that the effect of photoperiod was reduced by ADX and 6 meals RF. Because the SCN rhythm (as evaluated by PER2 immunohistochemistry) was not disrupted by ADX + 6 meals RF, impairment of peripheral clocks in these mice was not because of impaired SCN clock function. In addition, locomotor activity rhythm and modifications of the feeding pattern may not be completely responsible for determining the phase of peripheral clocks. Thus, this study demonstrates that the phase of peripheral clocks responds to a photoperiodic lights-off signal, and suggests that signals from normal feeding patterns and the adrenal gland are necessary to maintain the oscillation and phase of peripheral clocks under various photoperiods.

The circadian peripheral clock is entrained by restricted feeding (RF) at a fixed time of day, and insulin secretion regulates RF-induced entrainment of the peripheral clock in mice. Thus, carbohydrate-rich food may be ideal for facilitating RF-induced entrainment, although the role of dietary oils in insulin secretion and RF-induced entrainment has not been described. The soybean oil component of standard mouse chow was substituted with fish or soybean oil containing docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA). Tuna oil (high DHA/EPA), menhaden oil (standard), and DHA/EPA dissolved in soybean oil increased insulin secretion and facilitated RF-induced phase shifts of the liver clock as represented by the bioluminescence rhythms of PER2::LUCIFERASE knock-in mice. In this model, insulin depletion blocked the effect of tuna oil and fish oil had no effect on mice deficient for GPR120, a polyunsaturated fatty acid receptor. These results suggest food containing fish oil or DHA/EPA is ideal for adjusting the peripheral clock.

The mammalian circadian system is controlled not only by the suprachiasmatic nucleus (SCN), but also by the peripheral clocks located in tissues such as liver, kidney, small intestine, and colon, mediated through signals such as hormones. Peripheral clocks, but not the SCN, can be entrained by food intake schedules. While it is known that cell proliferation exhibits a circadian rhythm in the colon epithelium, it is unclear how this rhythm is influenced by food intake schedules. Here, we aimed to determine the relationships between feeding schedules and cell proliferation in the colon epithelium by means of immunochemical analysis, using 5-bromo-2'-deoxyuridine (BrdU), as well as to elucidate how feeding schedules influence the colonic expression of clock and cell cycle genes, using real-time reverse-transcription PCR (qRT-PCR). Cell proliferation in the colonic epithelium of normal mice exhibited a daily fluctuation, which was abrogated in Clock mutant mice. The day/night pattern of cellular proliferation and clock gene expression under daytime and nighttime restricted feeding (RF) schedules showed opposite tendencies. While daytime RF for every 4 h attenuated the day/night pattern of cell proliferation, this was restored to normal in the Clock mutant mice under the nighttime RF schedule. These results suggest that feeding schedules contribute to the establishment of a daily fluctuation of cell proliferation and RF can recover it in Clock mutant mice. Thus, this study demonstrates that the daily fluctuation of cell proliferation in the murine colon is controlled by a circadian feeding rhythm, suggesting that feeding schedules are important for rhythmicity in the proliferation of colon cells.

Scheduled food access during the daytime for nocturnal mice or rats entrains the food-entrainable oscillator (FEO) in the brain and the food-entrainable peripheral oscillator (FEPO) in the peripheral tissues. FEO and FEPO are not regulated by the central clock, which is in the suprachiasmatic nucleus. FEO produces food anticipatory activity (FAA) 2-3 h before the scheduled feeding time initiates. FEPO produces entrainment in the rhythm of peripheral clock gene expression and in the rhythm of food-metabolic functions in peripheral organs. At present, the mechanisms of the FEO and FEPO are not completely understood, despite many studies that have been performed in this field. In addition, circadian clocks affect metabolism of nutrition (absorption, distribution, metabolism, excretion). In this review, we describe and review the characteristics and biological implications of FEO and FEPO and the mechanism of metabolism of nutrition with day-night differences. We call this relationship between nutrition and chronobiology –Chrono-nutrition,— which is an important study field to understand how our body clocks contribute to our health throughout the day.

BACKGROUND AND PURPOSE: Caffeine is one of the most commonly used psychoactive substances. Circadian rhythms consist of the main suprachiasmatic nucleus (SCN) clocks and peripheral clocks. Although caffeine lengthens circadian rhythms and modifies phase changes in SCN-operated rhythms, the effects on caffeine on the phase, period and amplitude of peripheral organ clocks are not known. In addition, the role of cAMP/Ca(2+) signalling in effects of caffeine on rhythm has not been fully elucidated. EXPERIMENTAL APPROACH: We examined whether chronic or transient application of caffeine affects circadian period/amplitude and phase by evaluating bioluminescence rhythm in PER2::LUCIFERASE knock-in mice. Circadian rhythms were monitored in vitro using fibroblasts and ex vivo and in vivo for monitoring of peripheral clocks. KEY RESULTS: Chronic application of caffeine (0.1-10 mM) increased period and amplitude in vitro. Transient application of caffeine (10 mM) near the bottom of the decreasing phase of bioluminescence rhythm caused phase advance in vitro. Caffeine (0.1%) intake caused a phase delay under light-dark or constant dark conditions, suggesting a period-lengthening effect in vivo. Caffeine (20 mg·kg(-1) ) at daytime or at late night-time caused phase advance or delay in bioluminescence rhythm in the liver and kidney respectively. The complicated roles of cAMP/Ca(2+) signalling may be involved in the caffeine-induced increase of period and amplitude in vitro. CONCLUSIONS AND IMPLICATIONS: Caffeine affects circadian rhythm in mice by lengthening the period and causing a phase shift of peripheral clocks. These results suggest that caffeine intake with food/drink may help with food-induced resetting of peripheral circadian clocks.

In mice, obesity has been observed not only in those freely fed a high-fat diet (HFD) but also in those fed while physically inactive. In contrast, a HFD during physically active periods protects against obesity and the impairments in the circadian rhythm induced by free feeding of a HFD. Although exercise is known to be effective for obesity prevention and management, the optimal timing of exercise has not yet been determined. In the present experiments, we aimed to determine the best combination of daily timing of HFD consumption and exercise for the prevention of HFD-induced weight gain in mice. In this experiment, "morning" refers to the beginning of the active phase (the "morning" for nocturnal animals). Increases in body weight related to free feeding of a HFD was significantly reduced with 4 h of exercise during the late (evening) or middle (noon) active period compared to 4 h of exercise during the early (morning) active period or free access to exercise, which resulted in hours of exercise similar to that of morning exercise. These results suggested that eating in the morning or at noon followed by exercise in the evening could prevent weight gain more effectively than exercise in the morning followed by eating at noon or in the evening. The group fed a HFD for 4 h in the morning had lower body weight than the group fed a HFD for 4 h in the evening without exercise. The last group of experiments tested the hypothesis that there would be an interaction between mealtime and exercise time (i.e. time of day) versus order (i.e. which comes first) effects. We compared groups that exercised for 4 h at noon and were fed either in the morning or evening and groups that were fed for 4 h at noon and either exercised in the morning or evening. We found that the groups that were fed before exercise gained less body and fat weight and more skeletal muscle weight compared to the groups that exercised before eating. Corresponding to the body and fat weight changes, the respiratory exchange ratio (RER) was lower and energy expenditure was higher in the groups fed before exercise than in the groups fed after exercise, and these effects on energy metabolism were also observed in the early stage of HFD feeding before obesity. When obese mice fed a HFD for 12 weeks were exposed to a combination of feeding and exercise timing in an effort to reduce body weight, eating followed by exercise resulted in greater weight loss, similar to the experiments conducted to prevent weight gain. These results demonstrate that a combination of daily timing of eating and exercise may influence weight gain and that eating followed by exercise may be effective for minimizing increases in body and fat weight as well as maximizing increases in skeletal muscle weight.

Free feeding (FF) with a high fat diet (HFD) causes excessive body weight gain, whereas restricted feeding (RF) with a HFD attenuates body weight gain. The effects of timing of feeding with a HFD (day vs. night) and feeding duration on energy homeostasis have not yet been investigated. In this study, we fed mice a HFD or a normal diet (ND) twice a day, during their active and inactive periods, on a schedule. The amount of food was regulated by feeding duration (2, 4 or 8 h). First, we investigated the effects of 4-h RF during active-inactive periods (ND-ND, HFD-HFD, ND-HFD or HFD-ND). Among all the 4-h RF groups, mice consumed almost the same amount of calories as those in the FF[ND] group, even those fed a HFD. Body weight and visceral fat in these three groups were lower than that in the FF[HFD] group. Second, we investigated the effects of RF duration. Body weight and visceral fat were higher in the 8-h groups than in the 4-h groups. Body weight and visceral fat were higher in the 2-h groups than in the 4-h groups even though the 2-h groups had less food. Third, we investigated the effects of eating a HFD during the inactive period, when RF duration was extended (2, 6 or 12 h). Mice were fed with a HFD during the inactive period for 2 h and fed with a ND during the active period for 2, 6 or 12 h. Body weight and visceral fat in these mice were comparable to those in the FF[ND] mice. The results of our first set of experiments suggest that 4-h RF was an adequate feeding duration to control the effect of a HFD on obesity. The results of our second set of experiments suggest 2-h RF (such as speed-eating) and 8-h RF, representative of eating disorders, are unhealthy feeding patterns related to obesity. The results of our third set of experiments suggest that eating a HFD for a short period during the night does not affect body weight and visceral fat. Taken together, these results indicate that consideration to feeding with a HFD during the inactive period and restricting eating habits relieve the risks of body weight gain and visceral fat accumulation.

Digested proteins are mainly absorbed as small peptides composed of two or three amino acids. The intestinal absorption of small peptides is mediated via only one transport system: the proton-coupled peptide transporter-1 (PepT1) encoded from the soluble carrier protein Slc15a1. In mammals, intestinal expression of PepT1/Slc15a1 oscillates during the daily feeding cycle. Although the oscillation in the intestinal expression of PepT1/Slc15a1 is suggested to be controlled by molecular components of circadian clock, we demonstrated here that bile acids regulated the oscillation of PepT1/Slc15a1 expression through modulating the activity of peroxisome proliferator-activated receptor alpha (PPAR alpha). Nocturnally active mice mainly consumed their food during the dark phase. PPAR alpha activated the intestinal expression of Slc15a1 mRNA during the light period, and protein levels of PepT1 peaked before the start of the dark phase. After food intake, bile acids accumulated in intestinal epithelial cells. Intestinal accumulated bile acids interfered with recruitment of co-transcriptional activator CREB-binding protein/p300 on the promoter region of Slc15a1 gene, thereby suppressing PPAR alpha-mediated transactivation of Slc15a1. The time-dependent suppression of PPAR alpha-mediated transactivation by bile acids caused an oscillation in the intestinal expression of PepT1/Slc15a1 during the daily feeding cycle that led to circadian changes in the intestinal absorption of small peptides. These findings suggest a molecular clock-independent mechanism by which bile acid-regulated PPAR alpha activity governs the circadian expression of intestinal peptide transporter.

Food deprivation (FD) induces hepatic steatosis in both rodents and humans. Although body composition, age, and sex influence hepatic triglyceride (TG) levels after FD, whether feeding patterns affect FD-induced liver TG increases is unknown. We hypothesized that restricted feeding (RF) of 1 meal per day during the active or inactive period (especially the inactive period) augments FD-induced elevation of liver TGs because RF in the inactive period impairs the circadian rhythm. Triglyceride levels and the expression of genes related to TG metabolism in the liver were examined by a bioassay and real-time reverse transcription-polymerase chain reaction, respectively. In the first experiment, when compared to nonfasted mice, mice that fasted for 24 hours showed a 1.5-fold (FD starting during the inactive period) to 3-fold (FD started during the active period) increase in liver TG levels. This experiment showed that TG levels depend upon the starting time of FD. In the second experiment, mice were given free access to food for 3 hours at the beginning of either the inactive ("supper-only") or the active ("breakfast-only") period for 2 weeks. Restricted feeding inhibited the FD-induced increases in liver and serum TG levels, serum free fatty acids, and the expression of genes related to fatty acid uptake in the liver, including fatty acid transport protein 1 (Fatp1) and 4 (Fatp4). Unexpectedly, compared to free feeding, RF during the active or inactive period resulted in resistance to FD-induced fatty liver. This is the first study to demonstrate that feeding patterns affect FD-induced TG accumulation in the mouse liver.

Background & Aims: The circadian clock drives daily rhythms in behavior and physiology. A recent study suggests that intestinal permeability is also under control of the circadian clock. However, the precise mechanisms remain largely unknown. Because intestinal permeability depends on tight junction (TJ) that regulates the epithelial paracellular pathway, this study investigated whether the circadian clock regulates the expression levels of TJ proteins in the intestine.
Methods: The expression levels of TJ proteins in the large intestinal epithelium and colonic permeability were analyzed every 4, 6, or 12 hours between wild-type mice and mice with a mutation of a key clock gene Period2 (Per2; mPer2(m/m)). In addition, the susceptibility to dextran sodium sulfate (DSS)-induced colitis was compared between wild-type mice and mPer2(m/m) mice.
Results: The mRNA and protein expression levels of Occludin and Claudin-1 exhibited daily variations in the colonic epithelium in wild-type mice, whereas they were constitutively high in mPer2(m/m) mice. Colonic permeability in wild-type mice exhibited daily variations, which was inversely associated with the expression levels of Occludin and Claudin-1 proteins, whereas it was constitutively low in mPer2(m/m) mice. mPer2(m/m) mice were more resistant to the colonic injury induced by DSS than wild-type mice.
Conclusions: Occludin and Claudin-1 expressions in the large intestine are under the circadian control, which is associated with temporal regulation of colonic permeability and also susceptibility to colitis.

BACKGROUND: It remains elusive how allergic symptoms exhibit prominent 24-hour variations. In mammals the circadian clocks present in nearly all cells, including mast cells, drive the daily rhythms of physiology. Recently, we have shown that the circadian clocks drive the daily rhythms in IgE/mast cell-mediated allergic reactions. However, the precise mechanisms, particularly the specific roles of the mast cell-intrinsic clockwork in temporal regulation, remain unclear. OBJECTIVE: We determined whether the mast cell clockwork contributes to the temporal regulation of IgE/mast cell-mediated allergic reaction. METHODS: The kinetics of a time of day-dependent variation in passive cutaneous anaphylactic reactions were compared between mast cell-deficient mice reconstituted with bone marrow-derived cultured mast cells generated from mice with a wild-type allele and a dominant negative type mutation of the key clock gene Clock. We also examined the temporal responses of wild-type and Clock-mutated bone marrow-derived cultured mast cells to IgE stimulation in vitro. Furthermore, factors influencing the mast cell clockwork were determined by using in vivo imaging. RESULTS: The Clock mutation in mast cells resulted in the absence of temporal variations in IgE-mediated degranulation in mast cells both in vivo and in vitro associated with the loss of temporal regulation of FcεRI expression and signaling. Additionally, adrenalectomy abolished the mast cell clockwork in vivo. CONCLUSION: The mast cell-intrinsic clockwork, entrained by humoral factors from the adrenal gland, primarily contributes to the temporal regulation of IgE/mast cell-mediated allergic reactions. Our results reveal a novel regulatory mechanism for IgE-mediated mast cell responses that might underlie the circadian pathophysiology in patients with allergic diseases.

Several lines of evidence suggest that 5-HT receptors play a critical role in the expression of clock genes in the suprachiasmatic nucleus, the main circadian oscillator in hamsters. The contributions of 5-HT-receptor subtypes in the intestine, where they are expressed at high concentrations, are however not yet clarified. The 5-HT synthesis inhibitor,p-chlorophenylalanine, attenuated the daily rhythm of Per1 and Per2 gene expression in the intestine. Injection of 5-HT and agonists of the 5-HT3 and 5-HT4 receptors increased Per1/Per2 expression and decreased Bmall expression in a dose-dependent manner Although treatment with antagonists of 5-HT3 and 5-HT4 alone did not affect clock gene expression, co-injection of these antagonists with 5-HT blocked the 5-HT-induced changes in clock gene expression. Increased tissue levels of 5-HT due to treatment with the antidepressants clomipramine and fluvoxamine did not affect clock gene expression. The present results suggest that the 5-HT system in the small intestine may play a critical role in regulating circadian rhythms through 5-HT3/5-HT4-receptor activation.

The circadian clock system in mammals drives many physiological processes including the daily rhythms of sleep-wake behavior, hormonal secretion, and metabolism. This system responds to daily environmental changes, such as the light-dark cycle, food intake, and drug administration. In this review, we focus on the central and peripheral circadian clock systems in response to drugs, food, and nutrition. We also discuss the adaptation and anticipation mechanisms of our body with regard to clock system regulation of various kinetic and dynamic pathways, including absorption, distribution, metabolism, and excretion of drugs and nutrients. "Chrono-pharmacology" and "chrono-nutrition" are likely to become important research fields in chrono-biological studies.

Anaphylaxis is a severe systemic allergic reaction which is rapid in onset and potentially fatal, caused by excessive release of mediators including histamine and cytokines/chemokines from mast cells and basophils upon allergen/IgE stimulation. Increased prevalence of anaphylaxis in industrialized countries requires urgent needs for better understanding of anaphylaxis. However, the pathophysiology of the disease is not fully understood. Here we report that the circadian clock may be an important regulator of anaphylaxis. In mammals, the central clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus synchronizes and entrains peripheral circadian clock present in virtually all cell types via neural and endocrine pathways, thereby driving the daily rhythms in behavior and physiology. We found that mechanical disruption of the SCN resulted in the absence of a time of day-dependent variation in passive systemic anaphylactic (PSA) reaction in mice, associated with loss of daily variations in serum histamine, MCP-1 (CCL2), and IL-6 levels. These results suggest that the central SCN clock controls the time of day-dependent variation in IgE-mediated systemic anaphylactic reaction, which may provide a novel insight into the pathophysiology of anaphylaxis.

Circadian clocks in the peripheral tissues of mice are known to be entrained by pulse stimuli such as restricted feeding, novel wheel running, and several other agents. However, there are no reports on high temperature pulse-mediated entrainment on the phase-shift of peripheral clocks in vivo. Here we show that temperature treatment of mice for two days at 41°C, instead of 37°C, for 1-2 h during the inactive period, using a temperature controlled water bath stimulated phase-advance of peripheral clocks in the kidney, liver, and submandibular gland of PER2::LUCIFERASE mice. On the other hand, treatment for 2 days at 35°C ambient room temperature for 2 h did not cause a phase-advance. Maintenance of mice at 41°C in a water bath, sustained the core body temperature at 40-41°C. However, the use of 37°C water bath or the 35°C ambient room temperature elevated the core body temperature to 38.5°C, suggesting that at least a core body temperature of 40-41°C is necessary to cause phase-advance under light-dark cycle conditions. The temperature pulse stimulation at 41°C, instead of 37°C water bath for 2 h led to the elevated expression of Per1 and Hsp70 in the peripheral tissue of mice. In summary, the present study demonstrates that transient high temperature pulse using water bath during daytime causes phase-advance in mouse peripheral clocks in vivo. The present results suggest that hot water bath may affect the phase of peripheral clocks.

Circadian rhythms are related to various psychiatric disorders. Recently, antipsychotics, including quetiapine (QTP), have been accepted as potential therapeutic agents for the treatment of depression, but its mechanism remains poorly understood. In this study, we examined clock gene fluctuation patterns in QTP-treated mice. QTP significantly increased Per2 mRNA at ZT12 and Per1 and Per2 expression at ZT18 in the amygdala. There were significant differences between the control and QTP groups in the cross-time effects of Per2 mRNA expression in the amygdala. Our findings suggest that QTP possibly acts on the circadian system, which then induces changes in mood symptoms.

Although fasting induces hepatic triglyceride (TG) accumulation in both rodents and humans, little is known about the underlying mechanism. Because parasympathetic nervous system activity tends to attenuate the secretion of very-low-density-lipoprotein-triglyceride (VLDL-TG) and increase TG stores in the liver, and serum cholinesterase activity is elevated in fatty liver disease, the inhibition of the parasympathetic neurotransmitter acetylcholinesterase (AChE) may have some influence on hepatic lipid metabolism. To assess the influence of AChE inhibition on lipid metabolism, the effect of physostigmine, an AChE inhibitor, on fasting-induced increase in liver TG was investigated in mice. In comparison with ad libitum-fed mice, 30 h fasting increased liver TG accumulation accompanied by a downregulation of sterol regulatory element-binding protein 1 (SREBP-1) and liver-fatty acid binding-protein (L-FABP). Physostigmine promoted the 30 h fasting-induced increase in liver TG levels in a dose-dependent manner, accompanied by a significant fall in plasma insulin levels, without a fall in plasma TG. Furthermore, physostigmine significantly attenuated the fasting-induced decrease of both mRNA and protein levels of SREBP-1 and L-FABP, and increased IRS-2 protein levels in the liver. The muscarinic receptor antagonist atropine blocked these effects of physostigmine on liver TG, serum insulin, and hepatic protein levels of SREBP-1 and L-FABP. These results demonstrate that AChE inhibition facilitated fasting-induced TG accumulation with up regulation of the hepatic L-FABP and SREBP-1 in mice, at least in part via the activation of muscarinic acetylcholine receptors. Our studies highlight the crucial role of parasympathetic regulation in fasting-induced TG accumulation, and may be an important source of information on the mechanism of hepatic disorders of lipid metabolism. (C) 2014 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

In this paper, we study the response of a rat to a rat-like robot capable of generating different types of behaviour (stressful, friendly, neutral). Experiments are conducted in an open-field where a rat-like robot called WR-4 is put together with live rats. The activity level of each rat subject is evaluated by scoring its locomotor activity and frequencies of performing rearing (rising up on its hind limbs) and body grooming (body cuddling and head curling) actions, whereas the degree of preference of that is indicated by the robot-rat distance and the frequency of contacting WR-4. The moving speed and behaviour of WR-4 are controlled in real-time based on the feedback from rat motion. The activity level and degree of preference of rats for each experimental condition are analysed and compared to understand the influence of robot behaviour. The results of this study show that the activity level and degree of preference of the rat decrease when exposed to a stressful robot, and increase when the robot exhibit friendly behaviour, suggesting that a rat-like robot can modulate rat behaviour in a controllable, predictable way. © 2013 IOP Publishing Ltd.

Several lines of evidence indicate that serotonin type 7 (5-HT7) receptors play a critical role for non-photic resetting of the mammalian circadian clock; however, the contributions of other types of 5-HT receptors to non-photic entrainment are not yet clarified. The present study demonstrates that MKC-242, a selective 5-HT1A receptor agonist, can evoke a non-photic-like phase-response in hamsters in vivo. This phase-shifting response to MKC-242 was antagonized not only by the selective 5-HT1A receptor blocker WAY100635 but also by the selective 5-HT7 receptor blocker DR4004. These suggest that synchronous activation of 5-HT1A and 5-HT7 receptors mediates non-photic signals to the hamster circadian clock.

The circadian rhythm controls many physiological functions, such as feeding, motor activity, endocrine secretion and autonomic nerve. Regular feeding pattern can entrain the peripheral circadian clock, whereas peripheral clock systems can control the absorption distribution, metabolism and excretion of nutrients, suggesting mutual interactions between circadian clocks and nutrition/food. The interactions were so-called by "chrono-nutrition", and bigger meals for breakfast were good for entrainment of peripheral clock and protection of obesity. Similar to chrono-nutrition the timing of exercise ("chrono-exercise") is important for both entrainment signals and energy expenditure. Evening exercise and/or feeding then exercise was good timing exercise for protection of obesity. Taken all, it is suggested that timing of feeding and exercise is now one of key factors for metabolic syndrome.

Restricting feeding to daytime can entrain circadian clocks in peripheral organs of rodents, and nutrients that rapidly increase the blood glucose level are suitable for inducing entrainment. However, dietetic issues, for example, whether or not the diet comprises heated food, have not been fully explored. We therefore hypothesized that rapidly digested starch causes stronger entrainment than slowly digested starch. The entrainment ability of the liver clock in PER2::LUCIFERASE knock-in mice, blood glucose levels, insulin levels, and acute changes in liver clock gene expression were compared between a β-starch (native)-substituted AIN-93M standard diet and an α-starch (gelatinized)-substituted diet. β-Corn and β-rice starch induced larger phase delays of the liver clock, larger blood glucose increases, and higher Per2 gene expression in the liver compared with β-potato starch. Starch granule size, as examined by electron microscopy, was larger for β-potato starch than for β-corn or β-rice starch. After heating, we obtained gelatinized α-potato, α-corn, and α-rice starch, which showed destruction of the crystal structure and a high level of gelatinization. No difference in the increase of blood glucose or insulin levels was observed between β-corn and α-corn starch, or between β-rice and α-rice starch. In contrast, α-potato starch caused higher levels of glucose and insulin compared with β-potato starch. An α-potato starch-substituted diet induced larger phase delays of the liver clock than did β-potato starch. Therefore, rapidly digested starch is appropriate for peripheral clock entrainment. Dietetic issues (heated vs unheated) are important when applying basic mouse data to humans. © 2013 Elsevier Inc.

Various features, components, and functions of the immune system present daily variations. Immunocompetent cell counts and cytokine levels present variations according to the time of day and the sleep-wake cycle. Moreover, different immune cell types, such as macrophages, natural killer cells, and lymphocytes, contain a circadian molecular clockwork. The biological clocks intrinsic to immune cells and lymphoid organs, together with inputs from the central pacemaker of the suprachiasmatic nuclei via humoral and neural pathways, regulate the function of cells of the immune system, including their response to signals and their effector functions. Consequences of this include, for example, the daily variation in the response to an immune challenge (e. g., bacterial endotoxin injection) and the circadian control of allergic reactions. The circadian-immune connection is bidirectional, because in addition to this circadian control of immune functions, immune challenges and immune mediators (e. g., cytokines) were shown to have strong effects on circadian rhythms at the molecular, cellular, and behavioral levels. This tight crosstalk between the circadian and immune systems has wide-ranging implications for disease, as shown by the higher incidence of cancer and the exacerbation of autoimmune symptoms upon circadian disruption.

The mammalian circadian system is comprised of a central clock in the suprachiasmatic nucleus (SCN) and a network of peripheral oscillators located in all of the major organ systems. The SCN is traditionally thought to be positioned at the top of the hierarchy, with SCN lesions resulting in an arrhythmic organism. However, recent work has demonstrated that the SCN and peripheral tissues generate independent circadian oscillations in Per1 clock gene expression in vitro. In the present study, we sought to clarify the role of the SCN in the intact system by recording rhythms in clock gene expression in vivo. A practical imaging protocol was developed that enables us to measure circadian rhythms easily, noninvasively, and longitudinally in individual mice. Circadian oscillations were detected in the kidney, liver, and submandibular gland studied in about half of the SCN-lesioned, behaviorally arrhythmic mice. However, their amplitude was decreased in these organs. Free-running periods of peripheral clocks were identical to those of activity rhythms recorded before the SCN lesion. Thus, we can report for the first time that many of the fundamental properties of circadian oscillations in peripheral clocks in vivo are maintained in the absence of SCN control.

BACKGROUND: Recent studies on humans and rodents have suggested that the timing of food intake plays an important role in circadian regulation and metabolic health. Consumption of high-fat foods during the inactive period or at the end of the awake period results in weight gain and metabolic syndrome in rodents. However, the distinct effects of breakfast size and the breakfast/dinner size ratio on metabolic health have not yet been fully examined in mice. METHODS: We examined whether the parameters of metabolic syndrome were differentially affected in mice that consumed a large meal at the beginning of the awake period (breakfast; one meal group) and a relatively smaller meal at end of the awake period (dinner; two meals group). The mice of each group were provided equal food volume per day. RESULTS: Mice on one meal exhibited an increase in body weight gain, hyperinsulinemia, hyperleptinemia, and a decrease of gene expression associated with β-oxidation in adipose tissue and liver compared with those on two meals. The circadian expression pattern of the Clock gene in mice on one meal was disturbed compared with those on two meals. CONCLUSIONS: In conclusion, a bigger breakfast with a smaller dinner (two meals per day) but not breakfast only (one meal per day) helps control body weight and fat accumulation in mice on a high-fat meals schedule. The findings of this study suggest that dietary recommendations for weight reduction and/or maintenance should include information on the timing and quantity of dietary intake.

Comprehensive gene expression profiling in mice in response to the inhalation of sevoflurane has revealed that circadian clock gene expression is affected strongly in the liver, heart, lung, and kidney, in this order, but moderately in the spleen and slightly in the brain. Therefore, we examined whether the administration of general anesthetics at different times of the day induces phase shifts of the liver clock in Per2::Luciferase knockin mice. One to 4 days of intraperitoneal injection of 2,2,2-tribromoethanol (240 mg/kg, anesthetic time 60 min) or 2,2,2-trichloroethanol (240 mg/kg, 60 min), common anesthetics in veterinary surgery, caused phase delays when injected during the daytime and phase advances when injected during the nighttime. Inhalation administration of isoflurane for 30 or 60 min during the daytime did not induce a phase delay. Injection of propofol (300 mg/kg, 17 min) during the daytime induced an insignificant phase delay of the Per2 bioluminescence rhythm. Injection of 2,2,2-tribromoethanol did not induce a phase shift in the suprachiasmatic nucleus, the main oscillator, or in behavioral locomotor rhythms, suggesting that 2,2,2-tribromoethanol induced phase shifts of the liver clock independent of the main suprachiasmatic clock. The expression of clock genes, such as Bmal1 and Clock, in mouse liver was decreased strongly 1 and 4 h after a single injection of 2,2,2-tribromoethanol. These results demonstrate that 2,2,2-tribromoethanol or 2,2,2-trichloroethanol produce phase shifts of the peripheral clock, independent of anesthetic activity. These anesthetics may cause circadian rhythm disorders in peripheral organs when administered as general anesthetics several times during the day.

The stress kinase mitogen-activated protein kinase kinase 7 (MKK7) is a specific activator of c-Jun N-terminal kinase (JNK), which controls various physiological processes, such as cell proliferation, apoptosis, differentiation, and migration. Here we show that genetic inactivation of MKK7 resulted in an extended period of oscillation in circadian gene expression in mouse embryonic fibroblasts. Exogenous expression in cultured mammalian cells of an MKK7-JNK fusion protein that functions as a constitutively active form of JNK induced phosphorylation of PER2, an essential circadian component. Furthermore, JNK interacted with PER2 at both the exogenous and endogenous levels, and MKK7-mediated JNK activation increased the half-life of PER2 protein by inhibiting its ubiquitination. Notably, the PER2 protein stabilization induced by MKK7-JNK fusion protein reduced the degradation of PER2 induced by casein kinase 1 epsilon. Taken together, our results support a novel function for the stress kinase MKK7 as a regulator of the circadian clock in mammalian cells at steady state.

Peripheral circadian clocks in mammals are strongly entrained by light-dark and eating cycles. Their physiological functions are maintained by the synchronization of the phase of organs via clock gene expression patterns. However, little is known about the adaptation of peripheral clocks to the timing of multiple daily meals. Here, we investigated the effect of irregular eating patterns, in terms of timing and volume, on their peripheral clocks in vivo. We found that the phase of the peripheral clocks was altered by the amount of food and the interval between feeding time points but was unaffected by the frequency of feeding, as long as the interval remained fixed. Moreover, our results suggest that a late dinner should be separated into 2 half-dinners in order to alleviate the effect of irregular phases of peripheral clocks.

The number of patients with mental disorders is increasing in advanced countries, hence more effective psychotropic drugs are recently desired. In process of development of psychotropic drugs, animal experiments have been playing a very important role. Mental disorder model animals which exhibit behavior disorder like patients with mental disorders are used in these experiments. These animals are normally developed by genetic manipulation, surgical operation in their brain or drug administration. A candidate for a new drug is administrated in these animals to evaluate its effect. However, we have some doubts about conventional mental disorder model animals because they are induced these disorders by using methods which are quite different from causes of mental disorder of human beings. Therefore, the purpose of this study is to develop an novel methodology to create mental disorder model animals. We then developed a small mobile robot and a control system for it. Using them, we have performed some experiments to create a mental disorder model rat. We had then succeeded in developing a mental disorder model rat by exposing stress using the robot during immature period. This rat exhibits low activity in some behavior tests during mature period. For better understanding of how stress exposure induces mental disorder in a rat, we conducted another experiment based on stress vulnerability hypothesis. In this experiment, stress was exposed during both immature and mature period while that had been exposed only during immature period. We prepared several conditions of stress exposure by changing robot behavior pattern to find the one to induce much stress in a rat. From a result of experiment, we found that a rat which received gentle chase by the robot during immature period was induced much stress when it received robot attack during mature period. Thus, we consider that this rat is more appropriate to the mental disorder model than that was developed in our past experiment. © 2012 IEEE.

The number of patients with mental disorders is increasing in advanced countries, hence more effective psychotropic drugs are recently desired. In process of development of psychotropic drugs, animal experiments have been playing a very important role. Mental disorder model animals which exhibit behavior disorder like patients with mental disorders are used in these experiments. These animals are normally developed by genetic manipulation, surgical operation in their brain or drug administration. A candidate for a new drug is administrated in these animals to evaluate its effect. However, we have some doubts about conventional mental disorder model animals because they are induced these disorders by using methods which are quite different from causes of mental disorder of human beings. Therefore, the purpose of this study is to develop an novel methodology to create mental disorder model animals. We then developed a small mobile robot and a control system for it. Using them, we have performed some experiments to create a mental disorder model rat. We had then succeeded in developing a mental disorder model rat by exposing stress using the robot during immature period. This rat exhibits low activity in some behavior tests during mature period. For better understanding of how stress exposure induces mental disorder in a rat, we conducted another experiment based on stress vulnerability hypothesis. In this experiment, stress was exposed during both immature and mature period while that had been exposed only during immature period. We prepared several conditions of stress exposure by changing robot behavior pattern to find the one to induce much stress in a rat. From a result of experiment, we found that a rat which received gentle chase by the robot during immature period was induced much stress when it received robot attack during mature period. Thus, we consider that this rat is more appropriate to the mental disorder model than that was developed in our past experiment.

To determine the effects of a [6]-gingerol analogue (6G), a major chemical component of the ginger rhizome, and its stable analogue after digestion in simulated gastric fluid, aza-[6]-gingerol (A6G), on diet-induced body fat accumulation, we synthesized 6G and A6G. Mice were fed either a control regular rodent chow, a high-fat diet (HFD), or a HFD supplemented with 6G and A6G. Magnetic resonance imaging adiposity parameters of the 6G- and A6G-treated mice were compared with those of control mice. Supplementation with 6G and A6G significantly reduced body weight gain, fat accumulation, and circulating levels of insulin and leptin. The mRNA levels of sterol regulatory element-binding protein 1c (SREBP-1c) and acetyl-CoA carboxylase 1 in the liver were significantly lower in mice fed A6G than in HFD control mice. Our findings indicate that A6G, rather than 6G, enhances energy metabolism and reduces the extent of lipogenesis by downregulating SREBP-1c and its related molecules, which leads to the suppression of body fat accumulation.

We analyzed the association between dietary intake and chronotype as assessed by both Morningness-Eveningness Questionnaire (MEQ) score and preferred midpoint of sleep in 112 young Japanese women. Dietary intake was assessed by a brief, self-administered diet history questionnaire. A lower MEQ score (evening-type tendency) showed a significant association with a lower energy-adjusted intake of protein, calcium, magnesium, zinc, vitamins (D, riboflavin, and B(6)), and vegetables, and with a higher intake of noodles. Furthermore, a later midpoint of sleep showed a significant association with a lower energy-adjusted intake of protein, cholesterol, potassium, calcium, magnesium, zinc, vitamins (D, riboflavin, B(6), and B(12)), soy, fish and shellfish, and eggs, and with a higher intake of noodles, bread, and confections. These data suggest that evening chronotype is associated with inadequate dietary habits such as low vitamin and mineral intakes.

The mammalian circadian clock is known to be entrained by both a daily light-dark cycle and daily feeding cycle. However, the mechanisms of feeding-induced entrainment are not as fully understood as those of light entrainment. To elucidate the first step of entrainment of the liver clock, we identified the circadian clock gene(s) that show both phase advance and acute change of gene expression during the early term of the daytime refeeding schedule in mice. The expressions of liver Per2 and Rev-erbα genes were phase-advanced within 1 day of refeeding. Additionally, the upregulation of Per2 mRNA and down-regulation of Rev-erbα mRNA were induced within 2 hours, not only by food intake but also by insulin injection in intact mice. These expression changes by food intake were not revealed in streptozotocin-treated insulin-deficient mice, but insulin injection was able to recover the impairment of Per2 and Rev-erbα gene expression. Furthermore, we demonstrated using an ex vivo luciferase monitoring system that insulin injection during the daytime causes a phase advance of liver Per2 expression rhythm in Per2::luciferase knock-in mice. In embryonic fibroblasts from Per2::luciferase knock-in mice, insulin infusion caused an acute increase of Per2 gene expression and a similar phase advance of Per2 expression rhythm. Our results indicate that an acute change of Per2 and Rev-erbα gene expression mediated by refeeding-induced insulin secretion is a critical step mediating the early phase of feeding-induced entrainment of the liver clock.

BACKGROUND: IgE-mediated immediate-type skin reaction shows a diurnal rhythm, although the precise mechanisms remain uncertain. Period2 (Per2) is a key circadian gene that is essential for endogenous clockworks in mammals. OBJECTIVE: This study investigated whether Per2 regulates a time-of-day-dependent variation in IgE-mediated immediate-type skin reaction. METHODS: The kinetics of a passive cutaneous anaphylactic reaction were compared between wild-type mice and mice with a loss-of-function mutation of Per2 (mPer2(m/m) mice). The effects of adrenalectomy, aging, and dexamethasone on the kinetics of a passive cutaneous anaphylactic reaction were also examined. In addition, the extent of IgE-mediated degranulation in bone marrow-derived mast cells (BMMCs) was compared between wild-type and mPer2(m/m) mice. RESULTS: A time-of-day-dependent variation in a passive cutaneous anaphylactic reaction observed in wild-type mice was absent in mPer2(m/m) mice and in adrenalectomized and aged mice associated with the loss of rhythmic secretion of corticosterone. In addition, mPer2(m/m) mice showed decreased sensitivity to the inhibitory effects of dexamethasone on the passive cutaneous anaphylactic reactions. IgE-mediated degranulation in BMMCs was comparable between wild-type and mPer2(m/m) mice, but Per2 mutation decreased sensitivity to the inhibitory effects of dexamethasone on IgE-mediated degranulation in BMMCs. CONCLUSION: A circadian oscillator, Per2, regulates a time-of-day-dependent variation in a passive cutaneous anaphylactic reaction in mice. Per2 may do so by controlling the rhythmic secretion of glucocorticoid from adrenal glands and/or by gating the glucocorticoid responses of mast cells to certain times of the day (possibly when Per2 levels are high in mast cells).

Regulators of G protein signaling (RGS) are a multi-functional protein family, which functions in part as GTPase-activating proteins (GAPs) of G protein alpha-subunits to terminate G protein signaling. Previous studies have demonstrated that the Rgs16 transcripts exhibit robust circadian rhythms both in the suprachiasmatic nucleus (SCN), the master circadian light-entrainable oscillator (LEO) of the hypothalamus, and in the liver. To investigate the role of RGS16 in the circadian clock in vivo, we generated two independent transgenic mouse lines using lentiviral vectors expressing short hairpin RNA (shRNA) targeting the Rgs16 mRNA. The knockdown mice demonstrated significantly shorter free-running period of locomotor activity rhythms and reduced total activity as compared to the wild-type siblings. In addition, when feeding was restricted during the daytime, food-entrainable oscillator (FEO)-driven elevated food-anticipatory activity (FAA) observed prior to the scheduled feeding time was significantly attenuated in the knockdown mice. Whereas the restricted feeding phase-advanced the rhythmic expression of the Per2 clock gene in liver and thalamus in the wild-type animals, the above phase shift was not observed in the knockdown mice. This is the first in vivo demonstration that a common regulator of G protein signaling is involved in the two separate, but interactive circadian timing systems, LEO and FEO. The present study also suggests that liver and/or thalamus regulate the food-entrained circadian behavior through G protein-mediated signal transduction pathway(s).

Objectives: How human chronotype is correlated to nutrient and food-group intakes and dietary behavior remains to be elucidated. We cross-sectionally examined the association between the midpoint of sleep and these dietary variables in young Japanese women. A calculated halfway point between bedtime and rise time was used as midpoint of sleep.
Methods: The subjects were 3304 female Japanese dietetics students aged 18-20 years from 53 institutions in Japan. Dietary intake during the previous month was assessed by a validated, self-administered diet history questionnaire. The midpoint of sleep was calculated using self-reported bedtimes and rise times.
Results: Late midpoint of sleep was significantly negatively associated with the percentage of energy from protein and carbohydrates, and the energy-adjusted intake of cholesterol, potassium, calcium, magnesium, iron, zinc, vitamin A, vitamin D, thiamin, riboflavin, vitamin B(6), folate, rice, vegetables, pulses, eggs, and milk and milk products. It was also significantly positively associated with the percentage of energy from alcohol and fat, and the energy-adjusted intake of noodles, confections, fat and oil, and meat. Furthermore, subjects with a later midpoint of sleep tended to begin meals later, eat for a longer time, skip meals more frequently, and watch TV at meals, not only at breakfast but also at lunch and dinner.
Conclusions: The midpoint of sleep is significantly associated with dietary intake of certain nutrients and foods and other dietary behaviors in young Japanese women. This finding may contribute to consider the relationships between chronotype and dietary intakes and behaviors. (C) 2011 Elsevier B.V. All rights reserved.

Although the circadian liver clock is entrained by the feeding cycle, factors such as food volume and starvation interval are poorly understood. Per2::Luc knock-in mice were given two meals per day with different food volume sizes and/or with different intervals of starvation between two mealtimes with the total food volume per day fixed at 3.6 g (80 food pellets, ∼75% of free feeding) per mouse. The bioluminescence rhythm in the liver produced a unimodal peak but not bimodal peak under the regimen of two meals per day over 14-15 days. Peak Per2 expression occurred concurrently with the mealtime of the larger food volume, when the first and second meal were given as different food volume ratios under a 12 h feeding interval. When an equal volume of food was given under different starvation interval (8 h:16 h), the peak of the Per2 rhythm was close to peak by mealtime after long starvation (16 h). When food volumes for each mealtime were changed under 8 h:16 h, the peak rhythm was influenced by combined factors of food volume and starvation interval. Food intake after the 16-h starvation caused a significant increase in liver Per2, Dec1, and Bmal1 gene expression compared with food intake after the 8-h starvation with 8 h:16 h feeding intervals. In conclusion, the present results clearly demonstrate that food-induced entrainment of the liver clock is dependent on both food volume and the starvation interval between two meals. Therefore, normal feeding habits may help to maintain normal clock function in the liver organ.

Sterol regulatory element-binding protein-1 (SREBP-1) plays a central role in transcriptional regulation of genes for hepatic lipid synthesis that utilizes diet-derived nutrients such as carbohydrates and amino acids, and expression of SREBP-1 exhibits daily rhythms with a peak in the nocturnal feeding period under standard housing conditions of mice. Here, we report that the Srebp-1 expression rhythm shows time cue-independent and Clock mutation-sensitive circadian nature, and is synchronized with varied photoperiods apparently through entrainment of locomotor activity and food intake. Fasting caused diminution of Srebp-1 expression, while diabetic db/db and ob/ob mice showed constantly high expression with loss of rhythmicity. Time-restricted feedings during mid-light and mid-dark periods exhibited differential effects, the latter causing more severe damping of the oscillation. Therefore, "when to eat in a day (the light/dark cycle)," rather than "whenever to eat in a day," is a critical determinant to shape the daily rhythm of Srebp-1 expression. We further found that a high-carbohydrate diet and a high-protein diet, as well as a high-fat diet, cause phase shifts of the oscillation peak into the light period, underlining the importance of "what to eat." Daily rhythms of SREBP-1 protein levels and Akt phosphorylation levels also exhibited nutrient-responsive changes. Taken together, these findings provide a model for mechanisms by which time of day and nutrients in feeding shape daily rhythms of the Srebp-1 expression and possibly a number of other physiological functions with interindividual and interdaily differences in human beings and wild animals subjected to day-by-day changes in dietary timing and nutrients.

Daily restricted feeding entrains the circadian rhythm of mouse clock gene expression in the central nervous system, excluding the suprachiasmatic nucleus (SCN), as well as in the peripheral tissues such as the liver, lung, and heart. In addition to entrainment of the clock genes, daily restricted feeding induces a locomotor activity increase 2-3h before the restricted feeding time initiates. The increase in activity is called the food-anticipatory activity (FAA). In addition to FAA, daily restricted feeding can also entrain peripheral circadian clocks in other organs such as liver, lung, and heart. This type of oscillator is called the food-entrainable peripheral oscillator (FEPO). At present, the mechanisms for restricted feeding-induced entrainment of locomotor activity (FAA) and/or peripheral clock (FEPO) are still unknown. In this review, we describe the role of the central nervous system and peripheral tissues in FAA performance and also in the entrainment of clock gene expression. In addition, the mechanism for entrainment of circadian oscillators by the abuse of drugs, such as methamphetamine, is discussed.

Intrauterine growth retardation (IUGR) is putatively involved in the pathophysiology of schizophrenia. The animal model of IUGR induced by synthetic thromboxane A2 (TXA2) is useful to clarify the effect of IUGR on pups' brains, however, analysis at the cellular level is still needed. Brain-derived neurotrophic factor (BDNF), which plays a role in neuronal survival and synaptic plasticity in the central nervous system (CNS), may also be associated with schizophrenia. However, the possible relationship between IUGR and BDNF function remains unclear. Here, we examined how IUGR by TXA2 impacts BDNF function by using dissociated cortical neurons. We found that, although BDNF levels in cultured neurons from the cerebral cortex of low birth weight pups with IUGR were unchanged, TrkB (BDNF receptor) was decreased compared with control-rats. BDNF-stimulated MAPK/ERK1/2 and PI3K/Akt pathways, which are downstream intracellular signaling pathways of TrkB, were repressed in IUGR-rat cultures. Expression of glutamate receptors such as GluA1 and GluN2A was also suppressed in IUGR-rat cultures. Furthermore, in IUGR-rat cultures, anti-apoptotic protein Bcl2 was decreased and BDNF failed to prevent neurons from cell death caused by serum-deprivation. Taken together, IUGR resulted in reductions in cell viability and in synaptic function following TrkB down-regulation, which may play a role in schizophrenia-like behaviors. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Disturbed circadian rhythmicity is associated with human diseases such as sleep and mood disorders. However, study of human endogenous circadian rhythm is laborious and time-consuming, which hampers the elucidation of diseases. It has been reported that peripheral tissues exhibit circadian rhythmicity as the suprachiasmatic nucleus-the center of the biological clock. We tried to study human circadian rhythm using cultured peripheral blood mononuclear cells (PBMCs) obtained from a single collection of venous blood. Activated human PBMCs showed self-sustained circadian rhythm of clock gene expression, which indicates that they are useful for investigating human endogenous circadian rhythm. (C) 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Restricted feeding induces anticipatory activity rhythm and also entrains the peripheral circadian clocks, although the underlying brain mechanisms have not been fully elucidated. The dorsomedial hypothalamus (DMH) has been implicated in the regulation of restricted feeding-induced anticipatory activity rhythms (FAA), but the role of the DMH in restricted feeding- induced entrainment of peripheral circadian clocks is still unknown. In the present study, the role of the DMH in entrainment of the peripheral circadian clock was examined using Per2::luciferase knock-in mice. The results indicate that lesions that destroy the large mediobasal hypothalamic (MBH) lesions destroying the DMH, ventrolateral hypothalamus (VMH), and arcuate nucleus (ARC) significantly reduce daily locomotor activity rhythms and FAA formation. In addition, these lesions phase advanced the peak of liver Per2 expression by 2 h when compared to sham-operated mice. Following the administration of MBH lesions, the animals run less and start later in the restricted feeding- induced FAA rhythm but do not have any alterations in the restricted feeding- induced phase shift of the liver Per2 rhythm. These results demonstrate that the hypothalamus, including the MBH, is an important brain area for maintaining the locomotor rhythm and FAA formation. However, it is not necessary for restricted feeding-induced entrainment of the liver clock.

Glucocorticoids are known to cause psychiatric disorders including depression. Prednisolone (PSL) is one of the most widely used synthetic glucocorticoids to treat various medical diseases; however, little is known about PSL-induced behavioral changes and its molecular basis in the brain. Growing evidence has implicated that hippocampal remodeling or damage play a role in the pathogenic effect of glucocorticoids. In this study, mice were administered PSL (50 or 100 mg/kg) or vehicle for 6 or 7 days and subjected to a series of behavioral tests, i.e., open field, elevated plus maze, prepulse inhibition, forced swim, and tail suspension tests. Hippocampal tissues were subject to microarray analysis using the GeneChip Mouse Genome 430 2.0 Array (Affymetrix) containing 45,101 probes of transcripts. Increased anxiety- and depression-like behaviors assessed with open field, elevated plus maze, and tail suspension tests were observed. Microarray analysis detected 108 transcripts with a fold change of &gt;2.0 or &lt;0.5 in which many cell-death-related genes were found. The microarray data was validated by quantitative reverse transcriptase-polymerase chain reaction analysis. Our results demonstrated that PSL causes anxiety- and depression-like behaviors, and suggest that altered gene expressions related to hippocampal remodeling or damage are involved in the effect of PSL on such behaviors. (C) 2009 Elsevier Inc. All rights reserved.

Gamma-aminobutyric acid (GABA), and its biosynthetic enzyme, glutamic decarboxylase, are widely distributed in the suprachiasmatic nucleus (SCN). In the present study, we examined the role of the GABA(A) receptor on in vitro SCN responses to photic-like signals. We found that 100 mu M GABA(A) receptor antagonist bicuculline partially blocked field potentials evoked by optic nerve stimulation. NMDA- and SP-induced phase shifts of SCN neuronal activity rhythms, were blocked with 10 mu M bicuculline. Application of 100 mu M bicuculline alone induced phase advance of SCN neuronal activity rhythm. These results show that NMDA- and SP-induced phase shifts are blocked by bicuculline and suggest GABA has an important role as neurotransmitter in the neuronal network regulating phase shifts of the circadian clock. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Daily restricted feeding entrains the circadian rhythm of mouse clock gene expression in the central nervous system excluding the suprachiasmatic nucleus, as well as in the peripheral tissues such as the liver, lungs, and heart. In addition to entrainment of the clock gene, daily restricted feeding induces a locomotor activity increase 2-3 h before the restricted feeding time. The increase of activity is called the food anticipatory activity (FAA). At present, the mechanisms for restricted feeding-induced entrainment are still unknown. In this review, we describe the role of the central nervous system and peripheral tissues in FAA performance and also in entrainment of clock gene expression.

Lipopolysaccharide (LPS) is a pathogen-associated large molecule responsible for sepsis-related endotoxic shock, and the heart is one of the most common organs adversely affected. LPS is reported to increase serum TNF alpha levels and reduce Per1 and Per2 gene expression. Therefore, in this experiment, we determined the time-dependent effects of LPS on heart rate (HR) and circadian gene expression in the mouse heart and liver. HR of the LPS group was significantly elevated 2 and 8 h after injection compared to the control group. A significant percent increase in HR was observed at ZT6, 12, and 18. LPS increased Tnf alpha mRNA expression in the heart and liver at ZT6, 18, and 24. A time-dependent effect of LPS on reduction of Per1 and Per2 gene expression was also observed in the heart and liver. In order to examine the effect of LPS on cell damage, we examined apoptosis-related gene expression after LPS injection. Bax mRNA expression level of the LPS group was higher than that of the control group 8 and 26 h after injection. On the other hand, Bcl2 mRNA expression level of the LPS group was lower than that of the control group 2 and 26 h after injection. Dexamethasone strongly attenuated the LPS-induced increase of serum TNF alpha without significant change in Per1 and Per2 gene expression in the heart. In conclusion, the present results demonstrated that LPS exerts a time-dependent inhibitory effect on Per1 and Per2 gene expression in the heart and liver. The chronopharmacological lethal effect of LPS may be related to the time-dependent increase of serum TNF alpha level and simultaneously high level of Per2 gene expression in the heart and liver between ZT12-18. Taken together, chronopharmacological effect of LPS may be related to not only sickness behavior syndrome and mortality, but also circadian rhythm systems. (Author correspondence: shibatas@waseda.jp).

BACKGROUND: The peripheral circadian clock in mice is entrained not only by light-dark cycles but also by daily restricted feeding schedules. Behavioral and cell culture experiments suggest an increase in glucose level as a factor in such feeding-induced entrainment. For application of feeding-induced entrainment in humans, nutrient content and dietary variations should be considered. PRINCIPAL FINDING: To elucidate the food composition necessary for dietary entrainment, we examined whether complete or partial substitution of dietary nutrients affected phase shifts in liver clocks of mice. Compared with fasting mice or ad libitum fed mice, the liver bioluminescence rhythm advanced by 3-4 h on the middle day in Per2::luciferase knock-in mice that were administered a standard mouse diet, i.e. AIN-93M formula [0.6-0.85 g/10 g mouse BW] (composition: 14% casein, 47% cornstarch, 15% gelatinized cornstarch, 10% sugar, 4% soybean oil, and 10% other [fiber, vitamins, minerals, etc.]), for 2 days. When each nutrient was tested alone (100% nutrient), an insignificant weak phase advance was found to be induced by cornstarch and soybean oil, but almost no phase advance was induced by gelatinized cornstarch, high-amylose cornstarch, glucose, sucrose, or casein. A combination of glucose and casein without oil, vitamin, or fiber caused a significant phase advance. When cornstarch in AIN-93M was substituted with glucose, sucrose, fructose, polydextrose, high-amylose cornstarch, or gelatinized cornstarch, the amplitude of phase advance paralleled the increase in blood glucose concentration. CONCLUSIONS: Our results strongly suggest the following: (1) balanced diets containing carbohydrates/sugars and proteins are good for restricted feeding-induced entrainment of the peripheral circadian clock and (2) a balanced diet that increases blood glucose, but not by sugar alone, is suitable for entrainment. These findings may assist in the development of dietary recommendations for on-board meals served to air travelers and shift workers to reduce jet lag-like symptoms.

Evidence that circadian food-anticipatory activity and temperature rhythms are absent in Bmal1 knockout mice and rescued by restoration of Bmal1 expression selectively in the dorsomedial hypothalamus was published in 2008 by Fuller et al and critiqued in 2009 by Mistlberger et al. Fuller et al have responded to the critique with new information. Here we update our critique in the light of this new information. We also identify and correct factual and conceptual errors in the Fuller et al response. We conclude that the original results of Fuller et al remain inconclusive and fail to clarify the role of Bmal1 or the dorsomedial hypothalamus in the generation of food-entrainable rhythms in mice. © 2009 Mistlberger et al
licensee BioMed Central Ltd.

Circadian rhythms in mammals are regulated by a light-entrainable circadian pacemaker in the hypothalamic suprachiasmatic nucleus and food-entrainable oscillators located elsewhere in the brain and body. The dorsomedial hypothalamic nucleus (DMH) has been proposed to be the site of oscillators driving food-anticipatory circadian rhythms, but this is controversial. To further evaluate this hypothesis, we measured clock gene, temperature and activity rhythms in intact and DMH-ablated mice. A single 4-h midday feeding after an overnight fast induced mPer1 and mPer2 mRNA expression in the DMH, arcuate nucleus, nucleus of the solitary tract and area postrema, and reset daily rhythms of mPer1, mPer2 and mBMAL1 in the DMH, arcuate and neocortex. These rhythms persisted during 2 days of food deprivation after 12 days of scheduled daytime feeding. Acute induction of DMH mPer1 and mPer2 was N-methyl-d-aspartate (NMDA) receptor-dependent, whereas rhythmic expression after 6 days of restricted feeding was not. Thermal DMH lesions did not affect acute induction or rhythmic expression of clock genes in other brain regions in response to scheduled daytime feeding. DMH lesions attenuated mean daily activity levels and nocturnality but did not affect food-anticipatory rhythms of activity and body temperature in either light-dark or constant darkness. These results confirm that the DMH and other brain regions express circadian clock gene rhythms sensitive to daytime feeding schedules, but do not support the hypothesis that DMH oscillations drive food-anticipatory behavioral or temperature rhythms.

In humans, chronic ethanol consumption leads to a characteristic set of changes to the metabolism of lipids in the liver that is referred to as an "alcoholic fatty liver (AFL)". In severe cases, these metabolic changes result in the enlargement and fibrillization of the liver and are considered risk factors for cirrhosis and liver cancer. Clock-mutant mice have been shown to display abnormal lipid metabolism and alcohol preferences. To further understand the potential interactions between ethanol consumption, lipid metabolism, and the circadian clock, we investigated the effect of chronic ethanol intake on the lipid metabolism of Clock-mutant mice. We found that ethanol treatment produced a number of changes in the liver of Clock-mutant mice without impacting the wild-type controls. First, we found that 8 weeks of exposure to ethanol in the drinking water increased the weight of the liver in Clock-mutant mice. Ethanol treatment also increased triglyceride content of liver in Clock-mutant and wild-type mice. This increase was larger in the mutant mice. Finally, ethanol treatment altered the expression of a number of genes related to lipid metabolism in the Clock-mutant mice. Interestingly, this treatment did not impact circadian clock gene expression in the liver of either genotype. Thus, ethanol produces a number of changes in the liver of Clock-mutant mice that are not seen in the wild-type mice. These changes are consistent with the possibility that disturbance of circadian rhythmicity associated with the Clock mutation could be a risk factor for the development of an alcoholic fatty liver. © 2009 Kudo et al
licensee BioMed Central Ltd.

Circadian rhythms in mammals are regulated by a light-entrainable circadian pacemaker in the hypothalamic suprachiasmatic nucleus and food-entrainable oscillators located elsewhere in the brain and body. The dorsomedial hypothalamic nucleus (DMH) has been proposed to be the site of oscillators driving food-anticipatory circadian rhythms, but this is controversial. To further evaluate this hypothesis, we measured clock gene, temperature and activity rhythms in intact and DMH-ablated mice. A single 4-h midday feeding after an overnight fast induced mPer1 and mPer2 mRNA expression in the DMH, arcuate nucleus, nucleus of the solitary tract and area postrema, and reset daily rhythms of mPer1, mPer2 and mBMAL1 in the DMH, arcuate and neocortex. These rhythms persisted during 2 days of food deprivation after 12 days of scheduled daytime feeding. Acute induction of DMH mPer1 and mPer2 was N-methyl-d-aspartate (NMDA) receptor-dependent, whereas rhythmic expression after 6 days of restricted feeding was not. Thermal DMH lesions did not affect acute induction or rhythmic expression of clock genes in other brain regions in response to scheduled daytime feeding. DMH lesions attenuated mean daily activity levels and nocturnality but did not affect food-anticipatory rhythms of activity and body temperature in either light-dark or constant darkness. These results confirm that the DMH and other brain regions express circadian clock gene rhythms sensitive to daytime feeding schedules, but do not support the hypothesis that DMH oscillations drive food-anticipatory behavioral or temperature rhythms.

Daily feeding schedules generate food anticipatory rhythms of behavior and physiology that exhibit canonical properties of circadian clock control. The molecular mechanisms and location of food-entrainable circadian oscillators hypothesized to control food anticipatory rhythms are unknown. In 2008, Fuller et al reported that food-entrainable circadian rhythms are absent in mice bearing a null mutation of the circadian clock gene Bmal1 and that these rhythms can be rescued by virally-mediated restoration of Bmal1 expression in the dorsomedial nucleus of the hypothalamus (DMH) but not in the suprachiasmatic nucleus (site of the master light-entrainable circadian pacemaker). These results, taken together with controversial DMH lesion results published by the same laboratory, appear to establish the DMH as the site of a Bmal1-dependent circadian mechanism necessary and sufficient for food anticipatory rhythms. However, careful examination of the manuscript reveals numerous weaknesses in the evidence as presented. These problems are grouped as follows and elaborated in detail: 1. data management issues (apparent misalignments of plotted data), 2. failure of evidence to support the major conclusions, and 3. missing data and methodological details. The Fuller et al results are therefore considered inconclusive, and fail to clarify the role of either the DMH or Bmal1 in the expression of food-entrainable circadian rhythms in rodents. © 2009 Mistlberger et al
licensee BioMed Central Ltd.

In mammals, it is well established that circadian rhythms in physiology and behavior, including the rhythmic secretion of hormones, are regulated by a brain clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. While SCN regulation of gonadal hormone secretion has been amply studied, the mechanisms whereby steroid hormones affect circadian functions are less well known. This is surprising considering substantial evidence that sex hormones affect many aspects of circadian responses, and that there are significant sex differences in rhythmicity. Our previous finding that "core" and "shell" regions of the SCN differ in their expression of clock genes prompted us to examine the possibility that steroid receptors are localized to a specific compartment of the brain clock, with the discovery that the androgen receptor (AR) is concentrated in the SCN core in mate mice. In the present study, we compare AR expression in female and male mice using Western blots and immunochemistry. Both of these methods indicate that ARs are more highly expressed in males than in females; gonadectomy eliminates and androgen treatment restores these sex differences. At the behavioral level, gonadectomy produces a dramatic loss of the evening activity onset bout in males, but has no such effect in females. Treatment with testosterone, or with the non-aromatizable androgen dihydrotestosterone, restores male locomotor activity and eliminates sex differences in the behavioral response. The results indicate that androgenic hormones regulate circadian responses, and suggest an SCN site of action. (c) 2007 Elsevier Inc. All rights reserved.

It is well known that clock genes such as Per1 and Per2 operate molecular clock works of circadian system in mammals. Clock gene expression is observed not only in the suprachiasmatic nucleus (SCN), a main oscillator, but several brain nuclei such as hippocampus, amygdala and cerebral cortex. Thus, clock genes that appeared in the brain must possess some role in brain function, for example clock system may play a critical role in clock function such as clock and time perception.<BR>There are two kinds of circadian clock; SCN-dependent and SCN-independent circadian rhythms. The former rhythm is reset by environmental light-dark signal, and the latter is reset by daily feeding with fixed time during daytime. Under restricted feeding conditions, the peak of circadian rhythm of Per1 and Per2 genes in the cerebral cortex and liver was advanced for 4-6hrs. This result strongly suggests that cerebral circadian rhythm of clock gene expression is sensitive to time cue induced by feeding schedule rather than light-dark schedule. Internal clock system governed by SCN-dependent or SCN-independent circadian rhythm may be used for reference of clock perception, time perception or time estimation.

Cholesterol (CH) homeostasis in the liver is regulated by enzymes of CH synthesis such as 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and catabolic enzymes such as cytochrome P-450, family 7, subfamily A, and polypeptide 1 (CYP7A1). Since a circadian clock controls the gene expression of these enzymes, these genes exhibit circadian rhythm in the liver. In this study, we examined the relationship between a diet containing CH and/or cholic acid (CA) and the circadian regulation of Hmgcr, low-density lipoprotein receptor (Ldlr), and Cyp7a1 gene expression in the mouse liver. A 4-wk CA diet lowered and eventually abolished the circadian expression of these genes. Not only clock genes such as period homolog 2 (Drosophila) (Per2) and brain and muscle arnt-like protein-1 (Bmal1) but also clock-controlled genes such as Hmgcr, Ldlr, and Cyp7a1 showed a reduced and arrhythmic expression pattern in the liver of Clock mutant mice. The reduced gene expression of Cyp7a1 in mice fed a diet containing CA or CH + CA was remarkable in the liver of Clock mutants compared with wild-type mice, and high liver CH accumulation was apparent in Clock mutant mice. In contrast, a CH diet without CA only elevated Cyp7a1 expression in both wild-type and Clock mutant mice. The present findings indicate that normal circadian clock function is important for the regulation of CH homeostasis in the mouse liver, especially in conjunction with a diet containing high CH and CA.

Spot14 is a putative transcriptional regulator for genes involved in fatty acid synthesis. The Spot14 gene is activated in response to lipogenic stimuli such as dietary carbohydrate and is also under circadian regulation. The author's investigated factors responsible for daily oscillation of Spot14 expression. If mice were kept under a 12-h light/ 12-h dark cycle with ad libitum feeding, Spot14 mRNA levels in the liver reached a peak at an early dark period when mice, as nocturnal animals, start feeding. Under fasting, while Spot14 mRNA levels were generally decreased, the rhythmicity was still maintained, suggesting contribution of both nutritional elements and circadian clock factors on robust rhythmicity of Spot14 expression. Effects of circadian clock factors were confirmed by the observations that the circadian rhythm of Spot14 expression was seen also under the constant darkness and that the rhythmicity was lost in Clock mutant mice. When mice were housed in short-photoperiod (6-h light/18-h dark) and long-photoperiod (18-h light/6-h dark) cycles, rhythms of Spot14 mRNA levels were phase advanced and phase delayed, respectively, being concordant with the notion that Spot14 expression is under the control of the light-entrainable oscillator. As for nutritional mediators, in the liver of db/db mice exhibiting hyperinsulinemia-accompanied hyperglycemia, Spot14 mRNA levels were constantly high without apparent rhythmicity, consistent with previous observations for strong activation of the Spot14 gene by glucose and insulin. Restricted feeding during the 4-h mid-light period caused a phase advance of the Spot14 expression rhythm. On the other hand, restricted feeding during the 4-h mid-dark period led to damping of the rhythmicity, apparently resulting from the separation of phases between effects of the light/dark cycle and feeding on Spot14 expression. Thus, the daily rhythm of Spot14 expression in the liver is under the control of the light-entrainable oscillator, food-entrainable oscillator, and food-derived nutrients, in a separate or cooperative manner.

Energy homeostasis is subjected to a circadian control that synchronizes energy intake and expenditure. The transcription factor CLOCK, a key component of the molecular circadian clock, controls many kinds of rhythms, such as those for locomotor activity, body temperature, and metabolic functions. The purpose of the present study is to understand the function of the Clock gene during lipid metabolism in the liver using Clock-mutant mice. Clock-mutant mice with an ICR background were fed a high-fat diet for 13 weeks, and liver triglyceride, serum triglyceride, and serum free fatty acid levels were examined. Triglyceride content in the liver was significantly less increased in Clock-mutant mice on a high-fat diet compared to wild-type mice on a high-fat diet. Acsl4 and Fabp1 mRNA levels in the liver showed daily rhythms in wild-type mice, In contrast, Clock-mutant mice had attenuated daily rhythms of Acs14 and Fabp1 gene expression in the liver under both normal and high-fat diet conditions compared to wild-type mice. In Clock-mutant mice, suppression of Acs14 and Fabp1 mRNA in the liver under high-fat diet conditions may have attenuated the accumulation of triglycerides in the liver compared to wild-type mice under the same conditions. In conclusion, the authors demonstrate that mice with a Clock mutation showed less triglyceride accumulation in the liver through the suppression of Acs14 and Fabp1 gene expression when fed a high-fat diet compared to wild-type mice fed the same diet.

Glucocorticoid receptor agonists such as dexamethasone ( DEXA) have been recommended for the treatment of asthma. An increased frequency of dosing with these drugs seems preferable for cases of severe or uncontrolled asthma. The purpose of this experiment was to find the appropriate dosing schedule ( frequency and timing) for DEXA inhalation based on chronotherapeutic dosing to minimize phase shifts of clock function in the lungs of the ovalbumin-treated asthmatic mouse. The daily rhythm of clock gene expression was similar between control and ovalbumin-treated mice. Acute inhalation of DEXA significantly increased mPer1 gene expression in the lungs but not the liver of mice. Daily exposure of DEXA at zeitgeber time 0 ( lights on) or at zeitgeber time 18 ( 6 h after lights off) for 6 d caused a phase advance or phase delay of bioluminescence rhythm in the lungs, respectively, similar to light-induced phase shifts in locomotor activity rhythm. Daily zeitgeber time 0 exposure to DEXA attenuated the expression level of the mClca3 gene, which is associated with mucus overproduction, and there was a phase-advancing peak time of the mClca3 rhythm. The present results denote the importance of selecting the most appropriate time of day for nebulizer administration of DEXA to minimize adverse effects such as the phase shifting of clock function in asthmatic lungs. This is the first report of a successful protocol that could obtain phase shifts of clock gene expression rhythm in isolated peripheral organs in vivo.

Recent progress at the molecular level has revealed that nuclear receptors play an important role in the generation of mammalian circadian rhythms. To examine whether peroxisome proliferator-activated receptor alpha (PPAR alpha) is involved in the regulation of circadian behavioral rhythms in mammals, we evaluated the locomotor activity of mice administered with the hypolipidemic PPAR alpha ligand, bezafibrate. Circadian locomotor activity was phase-advanced about 3 h in mice given bezafibrate under light-dark (LD) conditions. Transfer from LD to constant darkness did not change the onset of activity in these mice, suggesting that bezafibrate advanced the phase of the endogenous clock. Surprisingly, bezafibrate also advanced the phase in mice with lesions of the suprachiasmatic nucleus (SCN; the central clock in mammals). The circadian expression of clock genes such as period2, BMAL1, and Rev-erba was also phase-advanced in various tissues (cortex, liver, and fat) without affecting the SCN. Bezafibrate also phase-advanced the activity phase that is delayed in model mice with delayed sleep phase syndrome (DSPS) due to a Clock gene mutation. Our results indicated that PPARa is involved in circadian clock control independently of the SCN and that PPARa could be a potent target of drugs to treat circadian rhythm sleep disorders including DSPS. (c) 2007 Elsevier Inc. All rights reserved.

Delayed sleep phase syndrome (DSPS) is very often seen among patients with sleep-wake rhythm disorders. Humans with the 3111C allele of the human Clock gene tend to demonstrate a higher evening preference on the morningness-eveningness (ME) preference test. DSPS is thought to be an extreme form of this evening preference. Clock-mutant mice have been proposed as an animal model of evening preference. In this study, we looked at whether constant light (LL) housing of Clock-mutant mice during lactation would result in evening preference and/or DSPS. Housed under light-dark (LD) or constant dark (DD) conditions during the lactation period, both wild-type and Clock-mutant mice did not show a phase-delay in the locomotor activity measured under light-dark conditions, whereas constant light housing during lactation significantly caused a delayed onset. The magnitude of the delay during the light-dark cycle was positively associated with free-running period measured during constant darkness. Among wild, heterozygote, and homozygote pups born from heterozygous dams, only homozygote pups showed a delayed onset. Constant light-housed Clock-mutant mice exhibited a lower number and delayed peak of phospho-MAPK-immunoreactive cells in core regions of the suprachiasmatic nucleus (SCN) compared to light-dark housed wild-type or Clock-mutant mice. Activity onset returned to normal with daily melatonin injection at the lights-off time for 5 days. The present results demonstrate that Clock-mutant mice exposed to constant light during lactation can function as an animal model of DSPS and can be used to gain an understanding of the ethological aspects of DSPS as well as to find medication for its treatment.

The circadian pacemaker in the suprachiasmatic nucleus (SCN) generates the near 24-h period of the circadian rhythm and is entrained to the 24-h daily cycle by periodic environmental signals, such as the light/dark cycle (photic signal), and can be modulated by various drugs (non-photic signals). The mechanisms by which non-photic signals modulate the circadian clock are not well understood in mice. In mice, many reportedly non-photic stimuli have little effect on the circadian rhythm in vivo. Herein, we investigated the molecular mechanism in W-212393-induced phase advance using mice. W-212393 caused a significant phase advance of locomotor activity rhythm in mice at subjective day. Injection of W-212393 during subjective day elicited down-regulation of mPER2 protein in the SCN shell region, but not mPei-2 mRNA. Administration of W-212393 during subjective day failed to produce phase advance in mPet-2-mutant mice as well as in ORL1 receptor deficient mice. Furthermore, we show that such inhibition of mPER2 accelerates re-entrainment of the circadian clock following an abrupt shift in the environmental light/dark cycle, such as occurs with transmeridian flight. The present results suggest that post-translational down-regulation of mPER2 protein in the shell region of mouse SCN may be involved in W-212393-induced non-photic phase advance. (c) 2006 Elsevier Ltd. All rights reserved.

Lithium is one of the most commonly used drugs in the prophylaxis and treatment of bipolar disorder. It is also known to lengthen circadian period in several organisms. Previously, we reported that there was the association between lengthening circadian period by lithium and GSK-3 protein and its enzyme activity in the mouse suprachiasmatic nucleus (SCN). In this study, we show that lithium affects the circadian oscillator in young and old hamster SCN, in an age-dependent manner. We found that basal levels of phosphorylated GSK-3 (pGSK-3) protein expression in old hamsters are much lower than that in young hamsters. Furthermore, in the old hamsters, lithium did not affect the period of the locomotor activity rhythm or pGSK-3 expression, while changing period and pGSK-3 in the younger animals. These results indicate that the content of pGSK-3 in the SCN has an important role in age-dependent effects of lithium on the circadian oscillator. (c) 2007 Elsevier Inc. All rights reserved.

A circadian clock controls various physiological and behavioral rhythms. In mammals, a master circadian clock exists in the suprachiasmatic nucleus of the hypothalamus, and slave oscillators can be found in most tissues. These circadian oscillations are controlled by "clock genes". The negative feedback loop is thought to function as a molecular mechanism of the circadian clock. It is plausible that clock genes may control lipid metabolism through so-called clock-controlled genes and that lipid metabolism-related clock-controlled genes may play important roles in the circadian change of lipid metabolism. Recently research has focused on the relationships between the clock system and lipid metabolism. In this review, we discuss the following items: 1) circadian clock system, 2) effect of the diet on clock gene expression, 3) effect of clock mutation on lipid metabolism, and 4) effect of streptozotocin-induced diabetes and ob mutation on clock gene expression and lipid metabolism. In this review we have summarized how the circadian clock affects lipid metabolism through the expression of lipid metabolism-related clock-controlled genes and at the same time discussed how abnormal metabolism of lipid affects the expression of clock genes. Further experiments are needed to elucidate the detailed mechanism of interaction between clock genes and lipid metabolisms.

To understand the role of the circadian molecular clock in mouse reproduction, we investigated the daily rhythms associated with nursing and pup growth in Clock-mutant mice maintained under light-dark housing conditions. The daily rhythm associated with the maternal behavior of crouching had a strong diurnal peak and two weak nocturnal peaks in wild-type dams, whereas homozygotes (Clock/Clock) exhibited no significant peaks in activity. Wild-type, but not Clock-mutant, dams showed high, rhythmic levels of prolactin content in serum that corresponded with crouching. Pup body weight increased at a significantly slower rate in Clock-mutant dams compared with wild-type dams under all experimental conditions when the pups ranged from 10-15 in number. Heterozygote dams equally bred wild-type, heterozygote, or homozygote pups. The amount of milk secreted from dams, as calculated by the increase in pup body weight through suckling, was lower in Clock-mutant mothers vs. wild-type mice. When Clock-mutant dams gave birth to more than 10 pups, survival was poor for offspring until the time of weaning. The present results demonstrate that Clock mutation disrupts daily maternal behavior and the growth and survival rate of pups, especially with the breeding of more than 10 pups.

The ORL1 receptor (nociceptin receptor), the fourth member of the opioid receptor family, is widely distributed throughout the central nervous system and mediates diverse physiological functions including pain, emotion, learning and memory. Nociceptin receptors are densely expressed in the suprachiasmatic nucleus, the principal pacemaker of circadian rhythms, and nociceptin inhibits light-induced phase advances (Allen et al, 1999)
however, nociceptin's role in nonphotic entrainment has not been described. We used a small-molecule nociceptin receptor agonist capable of crossing the brain blood-brain barrier, and demonstrated that nociceptin acts as a nonphotic zeitgeiber. In this review, we describe a role for nociceptin in the biological clock and discuss the possibility of developing nociceptin receptor agonists for treatment of circadian rhythm disorders.

Precise, rhythmic, daily change of the internal milieu is a conspicuous feature of all living organisms. It affects temporal patterns of all kinds of behaviors during a day and deeply influences both the social structure and daily life of individual human beings. These daily variations arise from the internal circadian mechanisms. Three functions of the endogenous clock are discriminated as rhythm generation, entrainment to light-dark cycle and output from the clock. The endogenous clock is localized in the suprachiasmatic nucleus (SCN) in mammals. Recent papers demonstrated strong expression of clock genes such as Per1, Per2 and Per3 in the SCN. Circadian oscillation is basically regulated by the transcription/translation feedback system of the Per gene in mammals. As serotonin/antidepressant and GABA/benzodiazepine drugs affect the light and non-light-induced entrainment, these drugs can regulate the circadian oscillation of clock genes and environmental stimuli-induced change of Per gene expression in the SCN. There are two main stimuli that entrain circadian rhythm, the light-dark cycle (LD) and restricted feeding. Light resets the circadian clock with induction of Per1 and Per2 gene in the SCN, the locus of a main oscillator. Mice were allowed access to food for 4 h during daytime (7 h in advance of feeding time) under LD or constant darkness. The peaks of mPer1 and mPer2 mRNA in the cerebral cortex and liver were advanced 6-12 h after 6 days of RF, whereas those in SCN were unaffected. The increase of mPer expression by RF treatment was observed in SCN-lesioned mice. The present results suggest that RF strongly entrained the expression of mPer and clock-controlled genes in the cerebral cortex and liver without affecting light-dependent SCN clock function.

1 We synthesized a small-molecule opioid receptor-like 1 (ORL1) receptor agonist, 2-{3-[1-((1R)-acenaphthen- 1-yl)piperidin-4-yl]-2,3-dihydro-2-oxo-benzimidazol-1-yl}-N-methylacetamide (W-212393), and investigated its effect on the circadian body temperature rhythm of rats.
2 W-212393 has high affinity for ORL1 receptors in the rat cerebral cortex and human ORL1 receptors expressed in HEK293 cells with K-i values of 0.76 and 0.50 nM, respectively.
3 W-212393 concentration-dependently stimulated GTP gamma 35S binding and its efficacy was similar to nociceptin/orphanin FQ (N/OFQ), suggesting that W-212393 is a full agonist at ORL1 receptors.
4 W-212393 dose-dependently occupied ORL1 receptors following intraventricular or intraperitoneal administration, suggesting that W-212393 is a brain-penetrating compound.
5 W-212393 (100 nM) and N/OFQ (100 nM) significantly suppressed the activity of spontaneously firing rat suprachiasmatic nucleus neurons. These suppressive effects were blocked by an ORL1 receptor antagonist, J-113397 (1 mu M).
6 W-212393 (3 mg kg(-1), i.p.) induced a significant phase advance at circadian time 6 (CT6) and CT9, but not at other CTs. The magnitude of the W-212393 (0.3-3 mg kg(-1), i.p.)-induced phase advance was dose-dependent and greater than those produced by 8-hydroxy-2-(di-n-propylamino)tetralin (0.3-3 mg kg(-1), i.p.) or melatonin (0.3-3 mg kg(-1), i.p.). The W-212393 (3 mg kg(-1), i.p.)-induced phase advance was antagonized by J-113397 (10 mg kg(-1), i.p.).
7 W-212393 (3 mg kg(-1), i.p.) significantly accelerated the re-entrainment of the body temperature rhythm to a 6 h advanced light-dark cycle.
8 These results indicate that activation of ORL1 receptors contributes to the circadian entrainment and W-212393 may represent an interesting agent for the study of circadian rhythms.

In nocturnal rodents, restricted feeding to daytime (RF) causes feeding-associated diurnal locomotor activity that persists for the next 1-2 days when food is withheld. Along with this anticipatory behavior, the expression pattern of clock genes such as mPer1 and mPer2 changes from a nocturnal to diurnal pattern in the liver and cerebral cortex but not in the suprachiasmatic nucleus (SCN). Whether the molecular clockwork, in which mCry1 and mCry2 genes are essential components, is involved in food-anticipatory circadian rhythms is unknown.
In this study, we investigated the impact of the absence of mCRY products upon the locomotion pattern induced by RF. RF caused an increase in daytime activity that lasted even for 2 days after food was withheld, in wild-type and mCry1-1-mCry2-1-mice. However, RF-induced activity was less stable and appeared more gradually in mutant mice. Similar results were obtained with mice housed under constant darkness or with SCN-lesioned wild-type and mutant mice. Our data reveal that mCry proteins are basically dispensable for food-entrainable oscillation. However, it is also important to note that mCry deficiency affects the stability and development of RF-induced anticipatory locomotor activity. (c) 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Mammalian circadian rhythms can be entrained by photic and non-photic stimuli. Although we know that non-photic entrainment interferes with photic entrainment signals, there is no information about whether photic entrainment interferes with non-photic entraining signals. We examined whether triazolam (TRZ), a non-photic form of entrainment, could be attenuated by pre-treatment with (S)-5-[3-[(1,4-benzodioxan-2-ylmethyl)amino]propoxy]-1,3-benzodioxole hydrochloride (MKC-242), a photic enhancing drug. We found that TRZ-induced phase advances in hamster behavioral circadian rhythms and the increase of locomotor activity were both significantly attenuated by MKC-242. Thus, in hamsters, the photic enhancing drug MKC-242 seemed to hinder benzodiazepine-induced non-photic entrainment.

Restricted feeding-induced free-running oscillation of clock genes in the liver was studied in homozygous Clock-mutant (Clock/Clock) mice. Similar to wild-type mice, Clock/Clock mice showed robust food-anticipatory behavioral activity in accordance with a restricted feeding schedule. Also, the peak of all clock gene mRNAs tested was phase-advanced in the liver of Clock/Clock mice as well as wild-type mice, although the amplitude of clock gene expression was low in Clock/Clock mice. The food-anticipatory behavioral rhythm in Clock/Clock mice maintained a period similar to wild-type mice during 2-day fasting after the cessation of restricted feeding. However, during the fasting days after temporal feeding cues were removed, the oscillation of clock genes in the liver and heart, excluding the suprachiasmatic nuclei, appeared to result in arrhythmicity in Clock/Clock mice. Thus, although the CLOCK-based molecular mechanism is not required for the expression of food-anticipatory activity, intact CLOCK protein might be involved in sustaining several cycles of peripheral circadian oscillations after restricted feeding-induced resetting. (c) 2005 Published by Elsevier Ltd on behalf of IBRO.

Daily restricted feeding (RF) produces an anticipatory locomotor activity rhythm and entrains the peripheral molecular oscillator independently of the central pacemaker located in the suprachiasmatic nucleus (SCN). As orexins (hypocretins) are neuropeptides that coordinate sleep/wake patterns and motivated behaviours, such as food seeking, we studied the involvement of orexin in the food anticipatory activity (FAA) induced by RF. Daily RF shifted the mRNA rhythm of a clock-controlled gene mDbp in the cerebral cortex and caudate putamen but not in the SCN. Under these experimental conditions, prepro-orexin mRNA and orexin A immunoreactivity in the lateral hypothalamic area (LHA) did not show daily variation. Fasting increased the number of orexin A-ir cells, while RF did not. However, RF shifted the peak of Fos expression of the orexin neurons from night to day. Genetic ablation of orexin neurons in orexin/ataxin-3 transgenic mice severely reduced the formation of FAA under RF conditions. The expression of mNpas2 mRNA, a transcription factor thought to be involved in regulation of the food entrainable oscillator as well as mPer1 and mBmal1 mRNA, was reduced in the forebrain of orexin/ataxin-3 mice. Based on these results, we suggest that activity of the orexin neuron in the LHA contributes to the promotion and maintenance of FAA.

Myocardial infarction frequently occurs in the morning, a phenomenon in part resulting from the downregulation of fibrinolytic activity. Plasminogen activator inhibitor-1 (PAI-1) is a key factor behind fibrinolytic activity, and its gene expression is controlled under the circadian clock gene in the mouse heart and liver. Hypercholesterolemia has been associated with impaired fibrinolysis due to enhanced PAI-1 activity, which has also been implicated in atherosclerosis. The aim of this study was to decipher whether the Pai-1 gene is still expressed daily with hypercholesterolemia. Hypercholesterolemia (1% cholesterol diet) did not significantly affect the daily expression of clock genes (Per2 and Bmal1) and clock-controlled genes (Dbp and E4bp4) in the liver (P&gt;0.05); however, daily expression of the Pai-1 gene and Pai-1 promoter regulating factor genes such as Nr4a1 was significantly upregulated (P&lt;0.01). Daily restricted feeding for 4 h during the day reset the gene expression of Per2, Pai-1, Nr4a1, and Tnf-&alpha;. Lesion of the suprachiasmatic nucleus, the location of the main clock system, led to loss of Per2 and Pai-1 daily expression profiles. In the present experiments, we demonstrated that hypercholesterolemia enhanced daily expression of the Pai-1, Tnf-&alpha;, and Nr4a1 genes in the mouse liver without affecting clock and clock-controlled genes. Therefore, the risk or high frequency of acute atherothrombotic events in the morning still seems to be a factor that may be augmented under conditions of hypercholesterolemia.

Several lines of evidence indicate that 5-HT neuronal systems may play a critical role for the non-photic entrainment of the rodent circadian clock. Although it is well established that (+)8-hydroxy-2-(di-n-propylamino)tetralin [(+)8-OH-DPAT], a 5-HT1A/7 receptor agonist, causes a phase-advance of behavioral rhythm in hamsters, little is known whether this agent produces phase shifts of activity rhythm in mice. Therefore, we examined the effect of (+)8-OH-DPAT on the mouse locomotor activity rhythm. Systemic administration of this chemical at mid-subjective daytime induced a clear and dose-dependent phase advance, while there were no significant phase shifts at other times (early-subjective day, late-subjective day, or subjective night). Additionally, (+)8-OH-DPAT accelerated the re-entrainment of mouse behavioral rhythm to a 6-h advanced light-dark cycle. These results suggest that we can use mice for understanding the molecular mechanism of (+)8-OH-DPAT-induced phase shift because of availability of clock gene targeted mice. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

A microarray analysis experiment has revealed that there are many genes, including so-called clock genes, expressing a circadian rhythm in the liver. The clock genes mentioned above are expressed not only in the suprachiasmatic nucleus (SCN) of the hypothalamus, where the master clock exists, but also in other brain regions and various peripheral tissues. In the liver, clock genes are abundantly expressed and show a clear circadian rhythm. Thus, clock genes seem to play a critical role in the molecular clockworks of both the SCN and the liver. Although oscillation of clock genes in the liver is controlled under the circadian clock mechanism in the SCN, we do not know the resetting signals on liver clock function. Over the past few years, use of the pseudorabies virus, a transsynaptic tract tracer, has allowed us to map neural connections between the SCN and peripheral tissues in several physiological systems. Communication between the SCN and peripheral tissues occurs through autonomic nervous systems involving the sympathetic and parasympathetic neurons. This review mainly describes both anatomical and physiological experiments to reveal the sympathetic control over liver clock function. Although further study is necessary to produce the precise mechanism underlying neural control of liver clock systems, evolution of this mechanism will help our understanding of liver clock functions such as drug metabolism and energy metabolism. (C) 2004 Wiley-Liss, Inc.

Recent studies have shown a dampened amplitude of clock gene rhythm in the heart and liver of streptozotocin (STZ)-treated rats and mice, however it is unknown whether impairment is due to dysfunction of the suprachiasmatic nucleus (SCN) or not. Rhythmic expression of mPER2 was dampened in the STZ-treated mouse liver but not SCN and cerebral cortex. Injection of insulin could normalize an impairment of mPer2 and mPER2 expression rhythm in the liver, when it was injected at nighttime, but not at daytime. In the present study, we demonstrated that insulin-dependent diabetes impaired oscillation of the peripheral clock gene and its product. Insulin injection can recover dampened oscillation of the peripheral clock depending on its injection time. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Aims/hypothesis. An increase in PAI-1 activity is thought to be a key factor underlying myocardial infarction. Mouse Pai-1 (mPai-1) activity shows a daily rhythm in vivo, and its transcription seems to be controlled not only by clock genes but also by humoral factors such as insulin and triglycerides. Thus, we investigated daily clock genes and mPai-1 mRNA expression in the liver of db/db mice exhibiting high levels of glucose, insulin and triglycerides.
Methods. Locomotor activity was measured using an infrared detection system. RT-PCR or in situ hybridisation methods were applied to measure gene expression. Humoral factors were measured using measurement kits.
Results. The db/db mice showed attenuated locomotor activity rhythms. The rhythmic expression of mPer2 mRNA was severely diminished and the phase of mBmal1 oscillation was advanced in the db/db mouse liver, whereas mPai-1 mRNA was highly and constitutively expressed. Night-time restricted feeding led to a recovery not only from the diminished locomotor activity, but also from the diminished Per2 and advanced mBmal1 mRNA rhythms. Expression of mPai-1 mRNA in db/db mice was reduced to levels far below normal. Pioglitazone treatment slightly normalised glucose and insulin levels, with a slight reduction in mPai-1 gene expression.
Conclusions/interpretation. We demonstrated that Type 2 diabetes impairs the oscillation of the peripheral oscillator. Night-time restricted feeding rather than pioglitazone injection led to a recovery from the diminished locomotor activity, and altered oscillation of the peripheral clock and mPai-1 mRNA rhythm. Thus, we conclude that scheduled restricted food intake may be a useful form of treatment for diabetes.

The mammalian circadian clock lying in suprachiasmatic nucleus (SCN) is synchronized to about 24h by the environmental light-dark cycle (LD). The circadian clock exhibits limits of entrainment above and below 24h, beyond which it will not entrain. Little is known about the mechanisms regulating the limits of entrainment. In this study, we show that wild-type mice entrain to only an LD 24 h cycle, whereas Clock mutant mice can entrain to an LD 24, 28, and 32 h except for LD 20 h and LD 36 h cycle. Under an LD 28 h cycle, Clock mutant mice showed a clear rhythm in Per2 mRNA expression in the SCN and behavior. Light response was also increased. This is the first report to show that the Clock mutation makes it possible to adapt the circadian oscillator to a long period cycle and indicates that the clock gene may have an important role for the limits of entrainment of the SCN to LD cycle. (C) 2004 Elsevier Inc. All rights reserved.

A circadian clock located in the suprachiasmatic nucleus (SCN) regulates the period of physiological and behavioural rhythms to approximately 24 h. Lithium can lengthen the period of circadian rhythms in most organisms although little is known about the underlying mechanism. In the present study, we examined Drosophila shaggy ortholog glycogen synthase kinase-3 (GSK-3) protein expression in the SCN after lithium treatment. When locomotor activity was assessed, we found an association between the effect of lithium and the period of circadian oscillation as well as the level of GSK-3 protein expression. The decreased expression of GSK-3 and increased expression of phosphorylated GSK-3 (pGSK-3) resulted in an antiphasic circadian rhythm between the two in the SCN of lithium-treated mice housed under both light-dark and constant dark conditions. The enzyme activity of GSK-3 in the SCN was low when the level of pGSK-3 protein was high, as examined by immunoblotting analysis. Thus, GSK-3 enzyme activity has a correlation with the expression of GSK-3 protein in the SCN. Although both GSK-3 and pGSK-3 proteins are also expressed in the arcuate nucleus, lithium did not affect their expression. Based on the association that we found between lengthened circadian period and GSK-3 protein and GSK-3 activity in the SCN, we suggest that GSK-3 plays a role in regulating the period of the mammalian circadian pacemaker.

Genes expressed with day/night rhythms in the mouse liver were searched for by microarray analysis using an in-house array harboring mouse liver cDNAs. The rhythmic expression with a single peak and trough level was confirmed by RNA blot analysis for 3beta-Hsd and Gabarapl1 genes exhibiting a peak in the light phase and Spot14, Hspa8, Hspa5, and Hsp84-1 genes showing a peak in the dark phase. On the other hand, mRNA levels for all of the three fibrinogen subunits, Aalpha, Bbeta and gamma, exhibited two peaks each in the light and dark phases in a synchronized manner. This two-peaked rhythmic pattern of fibrinogen genes as well as the single peak-trough pattern of other genes was diminished or almost completely lost in the liver of Clock mutant mice, suggesting that the two-peaked expression is also under the control of oscillation-generating genes. In constant darkness, the first peak of the expression rhythm of fibrinogen genes was almost intact, but the second peak disappeared. Therefore, although the first peak in the subjective day is a component of the innate circadian rhythm, the second peak seems to require light stimuli. Fasting in constant darkness caused shifts of time phases of the circadian rhythms. Protein levels of the fibrinogen subunits in whole blood also exhibited circadian rhythms. In the mouse and human loci of the fibrinogen gene cluster, a number of sequence elements resembling circadian transcription factor-binding sites were found. The fibrinogen gene locus provides a unique system for the study of two-peaked day/night rhythms of gene expression in a synchronized form.

A main oscillator in the suprachiasmatic nucleus (SCN) conveys circadian information to the peripheral clock systems for the regulation of fundamental physiological functions. Although polysynaptic autonomic neural pathways between the SCN and the liver were observed in rats, whether activation of the sympathetic nervous system entrains clock gene expression in the liver has yet to be understood. To assess sympathetic innervation from the SCN to liver tissue, we investigated whether injection of adrenaline/noradrenaline (epinephrine/norepinephrine) or sympathetic nerve stimulation could induce mPer gene expression in mouse liver. Acute administration of adrenaline or noradrenaline increased mPer1 but not mPer2 expression in the liver of mice in vivo and in hepatic slices in vitro. Electrical stimulation of the sympathetic nerves or adrenaline injection caused an elevation of bioluminescence in the liver area of transgenic mice carrying mPer1 promoter-luciferase. Under a light-dark cycle, destruction of the SCN flattened the daily rhythms of not only mPer1, mPer2, and mBmal1 genes but also noradrenaline content in the liver. Daily injection of adrenaline, administered at a fixed time for 6 days, recovered oscillations of mPer2 and mBmal1 gene expression in the liver of mice with SCN lesion on day 7. Sympathetic nerve denervation by 6-hydroxydopamine flattened the daily rhythm of mPer1 and mPer2 gene expression. Thus, on the basis of the present results, activation of the sympathetic nerves through noradrenaline and/or adrenaline release was a factor controlling the peripheral clock.

While peripheral oscillators can be reset by Immoral factors such as glucocorticoid hormones, indirect neural communications involving sympathetic and parasympathetic neurons from the suprachiasmatic nucleus to various peripheral tissues suggest that autonomic nerve innervations also function in the resetting and synchronization of peripheral tissues. To study the role of sympathetic adrenergic signaling on clock gene expression, we constructed NIH3T3 cells that stably expressed each of three alpha(1)-adrenergic receptor subtypes (alpha(1A), alpha(1B) and alpha(1D)). We found that noradrenaline transiently induced the expression of mPer1, mPer2, and mE4bp4 1-2 h after alpha(1)-receptor activation. The extent and time course of clock gene mRNA induction by noradrenaline or the alpha(1)-receptor agonist phenylephrine (PE) was similar to that seen by 50% horse serum shock. Clock gene mRNA induction by PE was inhibited by U0126, a MEK inhibitor, suggesting involvement of the mitogen-activated protein kinase signaling pathway. We also found that both mPer1 and mPet-2 mRNAs were induced in the mouse liver 60 min after PE injection. These results suggest that although humoral factors are important for entrainment of the peripheral clock, the autonomic nervous system may also be involved in the process. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

This study was designed to test whether the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-facilitating drug, aniracetam, could potentiate photic responses of the biological clock in the suprachiasmatic nucleus (SCN) of rodents. Using the whole-cell patch technique, we first demonstrated that AMPA currents elicited by either local AMPA application or optic chiasm stimulation were augmented by aniracetam in the neurons of the SCN. The AMPA application-elicited increase of intracellular Ca2+ concentration in SCN slices was also enhanced by aniracetam treatment. The systemic injection of aniracetam dose-dependently (10-100 mg/kg) potentiated the phase delay in behavioral rhythm induced by brief light exposure of low intensity (3 lux) but not high intensity (10 or 60 lux) during early subjective night. Under the blockade of NMDA receptors by (+) MK801, aniracetam failed to potentiate a light (3 lux)-induced phase delay in behavioral rhythm. Aniracetam increased the photic induction of c-Fos protein in the SCN that was elicited by low intensity light exposure (3 lux). These results suggest that AMPA receptor-mediated responses facilitated by aniracetam can explain enhanced photic responses of the biological clock in the SCN of rodents.

In mammals, the autonomic nervous system mediates the central circadian clock oscillation from the suprachiasmatic nucleus (SCN) to the peripheral organs, and controls cardiovascular, respiratory and gastrointestinal functions. The present study was conducted in mice to address whether light signals conveyed to the SCN can control peripheral autonomic functions, and further examined the impact of centrally administered melatonin on peripheral autonomic functions via activation of melatonin receptor signalling. In vivo electrophysiological techniques were performed in anaesthetised, open-chest and artificially ventilated mice whilst monitoring the arterial blood pressure and heart rate. Light induced an increase of the renal sympathetic nerve activity, arterial blood pressure and heart rate immediately after lights on. Conversely, light rapidly suppressed the gastric vagal parasympathetic nerve activity, which was affected neither by hepatic vagotomy nor by total subdiaphragmatic vagotomy. These autonomic responses were mediated by the SCN since bilateral SCN lesion totally abolished the light-evoked neuronal and cardiovascular responses. Melatonin administered intracerebroventricularly (i.c.v.) attenuated the sympathetic and vagal nerve activities in a dose-dependent manner with a threshold of 0.1 ng and these effects were blocked by i.c.v. pre-treatment of the competitive melatonin receptor antagonist luzindole. These results suggest that light induces sympathoexcitation and vagal suppression through the SCN and that melatonin modulates the light-induced autonomic responses via activation of the central melatonin receptor signalling.

Constant light (LL) or constant dark (DD) environmental lighting conditions cause a free-running period and activity reduction in the rodent behavioral circadian rhythm. In order to understand the molecular process underlying behavioral rhythms in LL or DD housing conditions, we examined the circadian profile of mPer2 mRNA and mPER2 in the suprachiasmatic nucleus (SCN), a main oscillator, of free-running mice. The circadian expression rhythm of mPer2 in the SCN was dampened under 7-day LL conditions, whereas that of mPER2 protein was moderately attenuated and its expression peak delayed. The circadian expression of mPer2 and its product was slightly attenuated and advanced by 7-day DD conditions. With arrhythmic behavioral activity caused by long-term LL housing, mPER2 protein lost its rhythmicity in the SCN. On the other hand, ILL or DD housing did not affect the mPer2 gene and its product in the cerebral cortex. The present results suggest that mPER2 circadian expression in the SCN corresponds well with behavioral circadian oscillation under LL or DD conditions. Thus, the behavioral circadian rhythm seems to correlate with molecular clock works in the SCN. (C) 2003 IBRO. Published by Elsevier Ltd. All rights reserved.

We recently reported that Ca2+/calmodulin-dependent protein (CaM) kinase 11 is involved in light-induced phase delays and Per gene induction in the suprachiasmatic nucleus (SCN). To clarify the activation mechanisms of CaM kinase 11 by glutamate receptor stimulation in the SCN, we documented CaM kinase 11 activation following induction of long-term potentiation (LTP) in the rat SCN. High-frequency stimulation (100 Hz, 1 sec) applied to the optic nerve resulted in LTP of a postsynaptic field potential in the rat SCN. Unlike LTP in the hippocampal CA1 region, LTP onset in the SCN was slow and partly dependent on N-methyl-D-aspartate receptor activation. LTP induction in the SCN was completely inhibited by treatment with a nitric oxide synthase inhibitor or with a specific CaM kinase 11 inhibitor. Immunoblotting analysis using phosphospecific antibodies against autophosphorylated CaM kinase 11 revealed that LTP induction was accompanied by an increase in autophosphorylation. After high-frequency stimulation, we could visualize activation of CaM kinase 11 in vasoactive intestinal polypeptide-positive neurons in the SCN by immunohistochemistry. Treatment with cyclosporin A, a calcineurin inhibitor, potentiated LTP induction in the rat SCN. Interestingly, treatment with melatonin totally prevented LTP induction, without changes in basal synaptic transmission. Analyses of phosphorylation of CaM kinase 11, mitogen-activated protein kinase, and cAMP-responsive element binding protein revealed that stimulatory and inhibitory effects on CaM kinase 11 autophosphorylation underlie the effects of cyclosporin A and melatonin, respectively. These results suggest that CaM kinase 11 plays critical roles in LTP induction in the SCN and that melatonin has inhibitory effects on synaptic plasticity through CaM kinase 11. (C) 2002 Wiley-Liss, Inc.

The effect of glutamate on optic nerve stimulation-evoked field potentials in rat suprachiasmatic nucleus (SCN) was examined in vitro. Glutamate application for 20 min induced long-term potentiation (LTP) of the field potentials in the SCN at nighttime, whereas that induced a weak LTP at daytime. On the other hand, application for 40 min induced LP in the SCN during the daytime, whereas it induced a weak one at nighttime. These results indicate that the effect of glutamate is dependent on the application time and that the effect is influenced by the duration of glutamate exposure.

While the suprachiasmatic nucleus (SCN) coordinates the majority of daily rhythms, some circadian patterns of expression are controlled from outside of the SCN. These include responses to daily methamphetamine (MAP) injection, or daily restricted feeding. The mechanisms underlying these SCN-independent circadian rhythms are unknown. A circadian oscillation in the expression of mPer1 and/or mPer2 , mouse period, in the SCN is considered necessary to generate an SCN-dependent circadian rhythm. Therefore, in this experiment, we examined the association between mPer gene expression and the MAP-induced, SCN-independent circadian rhythm. Acute injection of MAP caused an elevation of mPer1, mBmal1, and mNpas2 gene expression in the striatum and mPer1 in the liver. Daily MAP injection at a fixed time for 6 days shifted the rhythmic mPer1 and mPer2 expression in the striatum from a nocturnal to a diurnal rhythm, but failed to affect that in the SCN. Although lesion of the SCN 'flattened'mPer gene oscillation in the striatum and liver, daily MAP injection caused both behavioural and mPer gene expression rhythms. Daily MAP injection at variable injection intervals (12-36 h) for 6 days, however, failed to produce mPer gene rhythm in the striatum. Daily repeated MAP signals may strengthen the oscillatory force of SCN-independent circadian behavioural and molecular rhythms. The present results suggest that daily oscillation of mPer genes outside the SCN is closely associated with the regulation of SCN-independent rhythms. Thus, the present experiment highlights strongly the important role of clock gene expression, in the brain, that underlies the circadian behavioural rhythm.

Plasminogen activator inhibitor-1 (PAI-1) is a key factor of fibrinolytic activity. The activity and mRNA abundance show a daily rhythm. To elucidate the mechanism of daily Pai-1 gene expression, the expression of Pai-1 and several clock genes was examined in the heart of homozygous Clock mutant (Clock/Clock) mice. Damping of the daily oscillation of Pai-1 gene expression in Clock/Clock mice was accompanied with damped or attenuated oscillations of mPer1, mPer2, mBmal1, and mNpas2 mRNA. Daily restricted feeding induced a daily mRNA rhythm of all clock genes and Pai-1 mRNA in Clock/Clock mice as well as wild-type mice. The peaks of clock genes and Pai-1 mRNA were phase-advanced in the heart of both genotypes after 6 days of restricted feeding. The present results demonstrate that daily Pai-1 gene expression depends on clock gene expression in the heart and that a functional Clock gene is not required for restricted feeding-induced resetting of the peripheral clock. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

We reported previously that (S)-5-[3-[(1,4-benzodioxan-2ylmethyl)amino]propoxy]-1,3-benzodioxole hydrochloride (MKC-242) (3 mg kg(-1), i.p.), a selective 5-HT1A receptor agonist, accelerated the re-entrainment of hamster wheel-running rhythms to a new 8 hr delayed or advanced light-dark cycle, and also potentiated the phase advance of the wheel-running rhythm produced by light pulses. The molecular mechanism underlying MKC-242-induced potentiation of this phase shift, however, has not yet been elucidated. We examined the effects of MKC-242 on light-induced mPer1 and mPer2 mRNA expression in the suprachiasmatic nucleus (SCN) of mice. MKC-242 (5 mg kg(-1), i.p.) potentiated light-induced mPer1 and mPer2 expression in the SCN of mice housed in constant darkness for 2 days, when mRNA levels were observed 3 hr after light-exposure. More potentiating action of MKC-242 on mPet2 expression in the SCN was observed in mice housed in constant darkness for 9-10 days. This facilitatory action of MKC-242 on mPer1 expression was antagonized by WAY100635, a selective 5-HT1A receptor blocker, indicating that MKC-242 activated 5-HT1A receptors. Other drugs such as 8-hydroxy-dipropylaminotetralin (10 mg kg(-1),p.), paroxetine (10 mg kg(-1), i.p.), buspirone (10 mg kg(-1) i.p.), and diazepam (10 mg kg(-1), i.p.) did not display a potentiating action on light-induced mPer1 and mPer2 expression in the SCN. In the behavioral experiments, we found that MKC-242 (5 mg kg(-1), i.p.) potentiated lightinduced phase delays of free-running rhythm in mice. The present results suggest that prolonged increase of mPer1 or mPer2 expression in the SCN by MKC-242 may be involved in the potentiation of photic entrainment by MKC242 in mice. (C) 2002 wiley-Liss, Inc.

Junctional complexes between the plasma membrane and endoplasmic reticulum (ER), often called "subsurface cisternae" or "peripheral coupling," are shared by excitable cells. These junctional membranes probably provide structural foundation for functional crosstalk between cell-surface and intracellular ionic channels. Our current studies have indicated that junctophilins (JPs) take part in the formation of functional membrane complexes by spanning the ER membrane and interacting with the plasma membrane. Of the JP subtypes defined, JP type 3 (JP-3) is specifically expressed in neurons in the brain. It has been currently reported that triplet repeat expansions in the JP-3 gene are associated with Huntington's disease-like symptoms including motor disorder in human. To survey the physiological role of JP-3, we generated the knockout mice. The JP-3-knockout mice grew and reproduced normally, and we did not observe any morphological abnormality in the mutant brain. In the behavioral study, the mutant mice showed impaired performance specifically in balance/motor coordination tasks. Although obvious defects could not be observed in excitatory transmission among cerebellar neurons from the mutant mice, the data indicate that JP-3 plays an active role in certain neurons involved in motor coordination. (C) 2002 Elsevier Science (USA).

The main mammalian circadian pacemaker is located in the suprachiasmatic, nucleus of the hypothalamus. Clock genes such as the mouse Period gene (mPer) play a role in this core clock mechanism in the mouse. With brief light exposure during the subjective night, the photic information, which is conveyed directly to the suprachiasmatic nucleus via the retinohypothalamic tract, results in mPer1 and mPer2 expression in the suprachiasmatic nucleus.
Glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP) are co-stored in the retinohypothalamic tract. Recent studies have suggested that not only glutamate but also PACAP are key players in the phase shift that occurs during subject night; however, research demonstrating a direct association between the PACAP-induced phase shift and mPer gene expression has yet to be conducted.
In the present study, PACAP (200 pmol) injected into the lateral ventricle during subjective night (circadian time 16; circadian time 12, onset of locomotor activity) caused a moderate phase delay associated with moderate expression of mPer1 and only slight expression of mPer2 in the mouse suprachiasmatic nucleus. PACAP-induced mPer1 expression was also observed in the paraventricular nucleus and periventricular area of the hypothalamus. (+)MK-801 (0.5 mg/kg), an N-methyl-D-aspartate (NMDA) receptor antagonist, suppressed both the PACAP-induced phase delay and mPer1 expression. From these results we suggest that PACAP induces phase delays in the mouse circadian rhythm in association with ;an increase of mPer expression in the suprachiasmatic nucleus via the activation of NMDA receptors. (C) 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved.

The suprachiasmatic nucleus (SCN), locus of the central circadian clock, consists of two neuronal populations (i.e., a light-recipient ventral SCN subpopulation directly entrained by light and a dorsal SCN subpopulation with an autonomous oscillatory function possessing an indirect or weak light response). However, the mechanism underlying the transmission of photic signals from the ventral to dorsal SCN remains unclear. Because gastrin-releasing peptide (GRP), expressed mainly in the ventral SCN, exerts phase-shifting actions, loss of the GRP receptor intuitively implies a reduction of photic information from the ventral to dorsal SCN. Therefore, using GRP receptor-deficient mice, we examined the involvement of GRP and the GRP receptor in light- and GRP-induced entrainment by the assessment of behavioral rhythm and induction of mouse-Period (mPer) gene in the SCN, which is believed to be a critical for photic entrainment. Administration of GRP during nighttime dose dependently produced a phase delay of behavior in wildtype but not GRP receptor-deficient mice. This phase-shift by GRP was closely associated with induction of mPer1 and mPer2 mRNA as well as c-Fos protein in the dorsal portion of the SCN, where the GRP receptor was also expressed abundantly. Both the light-induced phase shift in behavior and the induction of mPer mRNA and c-Fos protein in the dorsal SCN were attenuated in GRP receptor-deficient mice. Our present studies suggest that GRP neurons in the retinorecipient ventral area of the SCN convey the photic entrainable signals from the ventral SCN to the dorsal SCN via induction of the mPer gene.

Aging alters circadian components such as the free-running period, the day-to-night activity ratio and photic entrainment in behavioral rhythms, and 2-deoxyglucose uptakes and neuronal firing in the suprachiasmatic nucleus (SCN). A core clock mechanism in the mouse SCN appears to involve a transcriptional feedback loop in which Period (Per) and Cryptochrome (Cry) genes play a role in negative feedback. The circadian rhythm systems include photic entrainment, clock oscillation, and outputs of clock information such as melatonin production. In this experiment, we examined clock gene expression to determine whether circadian input, oscillation, and output are disrupted with aging. Circadian expression profiles of rPer1, rPer2, or rCry1 mRNA were very similar in the SCN, the paraventricular nucleus of the hypothalamus (PVN), and the pineal body of young and aged (22-26 months) rats. On the other hand, the photic stimulation-induced rapid expression of Per1 and Per2 in the SCN was reduced with aging. The present results suggest that the molecular mechanism of clock oscillation in the SCN, PVN, and pineal body is preserved against aging, whereas the impairment of Per1 induction in the SCN after light stimulation may result in impaired behavioral photic entrainment in aged rats. (C) 2001 Wiley-Liss, Inc.

Stress induces secretion of corticosterone through activation of hypothalamic-pituitary-adrenal axis. This corticosterone the hy secretion is thought to be controlled by a circadian clock in the suprachiasmatic nucleus (SCN). The hypothalamic paraventricular nucleus (PVN) receives convergent information from both stress and the circadian clock. Recent reports demonstrate that mammalian orthologs (Per1, Per2, and Per3) of the Drosophila clock gene Period are expressed in the SCN, PVN, and peripheral tissues. In this experiment, we examined the effect of physical and inflammatory stressors on mPer gene expression in the SCN, PVN, and liver. Forced swimming, immobilization, and lipopolysaccharide injection elevated mPer1 gene expression in the PVN but not in the SCN or liver. A stress-induced increase in mPer1 expression was observed in the corticotropin-releasing factor-positive cells of the PVN; however, the stressors used in this study did not affect mPer2 expression in the PVN, SCN, or liver. The present study suggests that a stress-induced disturbance of circadian corticosterone secretion may be associated with the stress-induced expression of mPer1 mRNA in the PVN.

Many physiological and behavioral phenomena are controlled by an internal, self-sustaining oscillator with a periodicity of approximately 24 hr. In mammals, the principal oscillator resides in the suprachiasmatic nucleus (SCN). A light pulse during the subjective night causes a phase shift of the circadian rhythm via direct glutamatergic retinal afferents to the SCN [1]. Along with the accepted theoretical models of the clock, it is suggested that behavioral resetting of mammals is completed within 2 hr [2]; however, the molecular mechanism has not been elucidated. Here, we show the realtime image of the transcription of the circadian-clock gene mPer1 in the cultured SCN by using the transgenic. mice that carry a luciferase reporter gene under the control of the mPer1 promoter [3]. The real-time image demonstrates that the mPer1 promoter activity oscillates robustly in a circadian manner and that this promoter activity is reset rapidly (within 2-3 hr) when a phase shift occurs.

Mammalian circadian clock genes Per1 and Per2 are rhythmically expressed not only in the suprachiasmatic nucleus where the mammalian circadian clock exists, but also in other brain regions and peripheral tissues. The induced circadian oscillation of Per genes after treatment with high concentrations of serum or various drugs in cultured cells suggests the ubiquitous existence of the oscillatory mechanism. These treatments also result in a rapid surge of expression of Per1. It has been shown that multiple signaling pathways are involved in Per1 gene induction in culture cells. We used a dispersed primary cell culture made up of mouse cerebellar granule cells to examine the stimuli inducing the mPer genes and their signaling pathways in neuronal tissues expressing mPer genes. We demonstrated that mPer1, but not mPer2, mRNA expression was dependent on the depolarization state controlled by extracellular KCI concentration in the granule cell culture. Nifedipine treatment reduced mPer1 induction, suggesting that mPer1 mRNA expression depends on intracellular calcium concentration regulated through a voltage-dependent Ca2+ channel. Transient mPer1 mRNA induction was observed after elevating KCI concentration in the medium from 5 mm to 25 mm. This increased expression was suppressed by a calmodulin antagonist, or CaMKII/IV inhibitor, but not by MEK inhibitors. Addition of pituitary adenylate cyclase-activating polypeptide-38 to the medium also induced transient Per1 gene expression. This induction was mimicked by dibutyryl-cAMP and suppressed by a protein kinase A (PKA) inhibitor, but not by MEK inhibitors. These results suggest that Ca2+/calmodulin-dependent protein kinase II/IV- and PKA-dependent pathways are involved in high-KCI and PACAP-induced mPer1 induction, respectively, and neural tissues use multiple signaling pathways for mPer1 induction similar to culture cells.

It is known that Ca2+-dependent phosphorylation of cAMP response element binding protein (CREB) and the rapid induction of mPer1 and mPer2, mouse period genes in the suprachiasmatic nucleus (SCN) are associated with light-induced phase shifting. The CREB/CRE transcriptional pathway has been shown to be activated by calcium/calmodulin dependent kinase II (CaMKII) and mitogen-activated protein kinase (MAPK); however, there is a lack of evidence concerning whether the activation of CaMKII and/or MAPK elicited by photic stimuli are associated with the change in Per gene expression and behavioral phase shifting. In this experiment, we found there was an inhibitory effect by KN93, CaMKII inhibitor, on hamster Per1 and Per2 expression in the SCN and on phase delays in wheel running rhythm induced by light pulses. PD98059 and U0126, MAPK kinase inhibitors, however, affected neither light-induced Per1 and Per2 expression nor behavioral phase delays, even though PD98059 attenuated the light-induced phosphorylation of MAPK in the SCN. The present findings demonstrate that the light-induced activation of CaMKII plays an important role in the induction of Per1 and Per2 mRNA in the hamster SCN as well as phase shifting. These results suggest that gated induction of Per1 and/or Per2 genes through CaMKII-CREB/CRE accompanied with photic stimuli may be a critical step in phase shifting.

Methamphetamine (MAP) causes the sensitization phenomena not only in MAP-induced locomotor activity, dopamine release, and Fos expression, but also in MAP-induced circadian rhythm. Cocaine-induced sensitization is reportedly impaired in Drosophila melanogaster mutant for the Period (Per) gene. Thus, sensitization may be related to induction of the Per gene. A rapid induction of mPer1 and/or mPer2 in the suprachiasmatic nucleus after light exposure is believed to be necessary for light-induced behavioral phase shifting. Although the caudate/putamen (CPu) expresses mPer1 and/or mPer2 mRNA, the function of these genes in this nucleus has not yet been elucidated. Therefore, we examined whether MAP affects the expression of mPer1 and/or mPer2 mRNA in the mouse CPu. Injection of MAP augmented the expression of mPer1 but not mPer2 or mPer3 in the CPu, and this MAP-induced increase in mPer1 expression lasted for 2 h. Also, the MAP-induced increase of mPer1 mRNA was strongly antagonized by pretreatment with a dopamine D1 receptor and N-methyl-D-aspartate (NMDA) receptor antagonist, but not by a D2 receptor antagonist. Interestingly, application of either the D1 or the D2 agonist alone did not cause mPer1 expression. The present results demonstrate that activation of both NMDA and D1 receptors is necessary to produce MAP-induced mPer1 expression in the CPu. Repeated injection of MAP caused a sensitization in not only the locomotor activity but also mPer1 expression in the CPu without affecting the level of mPer2, mPer3, or mTim mRNA. Thus, these results suggest that MAP-induced mPer1 gene expression may be related to the mechanism for MAP-induced sensitization in the mouse.

Background: There are two main stimuli that entrain the circadian rhythm, the light-dark cycle (LD) and restricted feeding (RF). Light-induced entrainment requires induction of the Per1 and Per2 genes in the suprachiasmatic nucleus (SCN), the locus of a main oscillator. In this experiment, we determined whether RF resets the expression of circadian clock genes in the mouse liver with or without participation of the SCN.
Results: Mice were allowed access to food for 4 h during the daytime (7 h advance of feeding time) under LD or constant darkness (DD). The peaks of mPer1, mPer2, D-site-binding protein (Dbp) and cholesterol 7 alpha -hydroxylase (Cyp7A) mRNA in the liver were advanced 6-12 h after 6 days of RF, whereas those in SCN were unaffected. The advance of mPer expression in the liver by RF was still observed in SCN-lesioned mice. A 7 h advance in the LD cycle advanced the peaks of clock gene expression in both the liver and SCN, whereas, a shift in the LD did not move the phase of the liver clock when the shift was carried out under a fixed RF schedule during the night-time.
Conclusions: These results suggest that restricted feeding strongly entrained the expression of circadian clock genes in the liver without the participation of an SCN clock function.

Daily restricted feeding (RF) can produce food-entrainable oscillations in both intact and suprachiasmatic nucleus (SCN)-lesioned animals. Thus, there are two circadian rhythms, one of which is SCN-dependent and the other SCN-independent. Recently, it has been established that several mouse clock genes, such as mPer1, mPer2 and mPer3 are expressed in the SCN and other brain tissues. Although the role of mPer genes expressed in the SCN has recently been evaluated in the SCN-dependent rhythm, their function in the SCN-independent rhythm is still poorly understood. In order to understand the role of these genes in SCN-independent rhythm, we examined the expression pattern of mPer1 and mPer2 mRNA in each brain area of mice under RF. Mice were allowed access to food for 4 h during either the daytime under a light-dark cycle or the subjective daytime under constant dark. After 6 days of scheduled RF, the night-time or subjective night-time peak of mPer mRNA changed to a daytime peak in the cerebral cortex and hippocampus, with moderate expression in the striatum, pyriform cortex and paraventricular nucleus, and no expression in the SCN. The daytime peak in the cerebral cortex returned to a night-time peak after the release of RF to a free-feeding schedule. Although the basal rhythm of mPer expression disappeared in SCN-lesioned mice, RF produced mPer mRNA rhythm in the cerebral cortex of these mice. The present results provide evidence of an association between food-entrainable oscillations and the expression of mPer1 and mPer2 in the cerebral cortex and hippocampus.

Recent progress in study an the molecular component of mammalian clocks has claimed that mammals and Drosophila share the similar fundamental clock oscillating system. In the present study, we investigated expression of Per1, the first gene of the mammalian homolog of the Drosophila clock gene period, in the hamster brain, and we also examined its circadian expression pattern in the mammalian clock center, the suprachiasmatic nucleus (SCN). In situ hybridization using isotope-labeled cRNA probes revealed a wide and region-specific distribution of Per1 in the hamster brain and spinal cord. High levels of Per1 were found in the internal granular layer of the granular cells of the olfactory bulb, anterior olfactory nuclei, tenia tecta, olfactory tubercle, piriform cortex,suprachiasmatic nucleus, and gyrus dentatus of hippocampus. Moderate levels of expression were detected in many brain regions including the granular layer of the cerebellum, anterior paraventricular thalamic nucleus, caudate-putamen, inferior colliculus, pontine nuclei, inferior olive, and nucleus of the solitary tract. We examined the circadian profile of hamster Per1 mRNA in the SCN in constant darkness and found that Per1 expression showed a peak at subjective day (circadian time [CT] 4) and formed a trough at subjective night (CT16-CT20). A brief exposure of light at CT16 could acutely induce large quantities of Per1 mRNA in the hamster SCN, except for its dorsomedial subdivision. These findings suggest that the characteristics of Per1 gene expression in the mammalian circadian center (showing a peak in the daytime and a trough in the nighttime and a rapid inducibility by light) are common among mammalian species. Lastly, in hamster brain, Per1 gene is also inducible in extra-SCN brain nuclei, since light at night also elicited Per1 mRNA in neurons of the hypothalamic paraventricular nucleus. (C) 2001 Wiley-Liss, Inc.

It is well known that there are circadian rhythms of 2-deoxyglucose uptake and neuronal firing in the rat suprachiasmatic nucleus (SCN) during fetal and early postnatal periods. A core clock mechanism in the mouse SCN appears to involve a transcriptional feedback loop in which CLOCK and BMAL1 function as positive regulators and three mPeriod (mPer) genes play a role in negative feedback. Per genes expression occurs not only in the adult SCN but also in the fetal SCN. However, the developmental change in these genes remains unclear. In this experiment, we examined the day-night pattern of expression of Per1 and Per2 mRNA in the mouse SCN and cerebral cortex on embryonic day 17, postnatal day 3, and in young adult mice under a light-dark cycle. Daily rhythms of mRNA content were observed in mPer1 but not mPer2 in the fetal SCN. Interestingly, the expression of mPer2 in the SCN was high throughout the entire day, and a significant daily rhythm of this gene was observed on postnatal day 6. The expression pattern of SCN mPer1 in constant darkness was similar to that seen in the light-dark cycle. The present results suggest that the daily oscillation of mPer1 but not of mPer2 in the SCN in fetal and early postnatal mice may be associated with the daily rhythms of 2-deoxyglucose uptake and neuronal firing.

It is well known that light induces both mPer1 and mPer2 mRNA in the suprachiasmatic nucleus. We have reported that mPer1 antisense oligonucleotides (ODNs) inhibited the light-induced phase delays of mouse locomotor rhythm. In this study, we asked whether both or either mPer1 or mPer2 expression is necessary to induce the phase shift. We examined the effects of inhibition of mRNA expression on light-induced phase delays of mouse circadian behavior rhythm. Light-induced phase delays were moderately attenuated by microinjection of mPer1 or mPer2 antisense ODN, but not by mPer3 antisense or mPer1, mPer2 scrambled ODNs, whereas following simultaneous injection of both mPer1 and mPer2 antisense ODNs they disappeared. The present results suggest that acute induction of mPer1 and mPer2 gene play an additive effect on photic entrainment. NeuroReport 12:127-131 (C) 2001 Lippincott Williams & Wilkins.

1 Triazolam reportedly causes phase advances in hamster wheel-running rhythm after injection during subjective daytime. However, it is unclear whether benzodiazepine affects the Per gene expression accompanying a behavioural phase shift.
2 Brotizolam (0.5-10 mg kg(-1)) induced large phase advances in hamster rhythm when injected during mid-subjective daytime (circadian time 6 or 9), but not at circadian time 0, 3 or 15.
3 Brotizolam (5 mg kg(-1)) significantly reduced the expression of Per1 and Per2 in the suprachiasmatic nucleus 1 and 2 h after injection at circadian time 6, and slightly reduced them at circadian time 20.
4 Injection of 8-OH-DPAT (5 mg kg(-1)) at subjective daytime induced similar phase advances with a reduction of Per1 and Per2 expression. Co-administration of brotizolam with 8-OH DPAT failed to potentiate the 8-OH DPAT-induced phase advances and reduced Per expression.
5 Both phase advance and rapid induction of Per1 and Per2 in the suprachiasmatic nucleus after light exposure (5 lux, 15 min) at circadian time 20 was strongly attenuated by co-treatment with brotizolam 5 mg kg(-1).
6 The present results strongly suggest that reduction of Per1 and/or Per2 expression during subjective daytime by brotizolam may be an important step in causing a behavioural phase advance. The co-administration experiment suggests that common mechanism(s) are involved in brotizolamor 8-OH DPAT-induced phase advances and the reduction of Per gene expression.
7 These results suggest that brotizolam is not only a good drug for insomnia but also a drug capable of facilitating re-entrainment like melatonin.

Because the rapid induction of Period (Per) genes is associated with the photic entrainment of the biological clock, we examined whether N-methyl-D-aspartate (NMDA) receptors were involved in the photic induction of Per genes in the hamster suprachiasmatic nucleus (SCN). In situ hybridization observation revealed that light during the early subjective night [circadian time (CT) 13.5] or the late subjective night (CT20) caused an induction of Per1 and Per2 but not Per3 mRNA in the SCN. Photic induction of Per mRNA at CT13.5 was observed especially in the ventrolateral SCN, whereas that at CT20 was more widespread from the ventrolateral to the dorsal SCN. A noncompetitive NMDA receptor antagonist, +MK801, dose-dependently (0.1-5.0 mg/kg) suppressed only the ventrolateral part of Per1 and Per2 mRNA induction by light at CT13.5 or CT20 in the SCN. The suppressive effects of +MK801 on Per mRNA strongly correlated with the attenuating action of this compound on phase shifts by light at both CT13.5 and CT20. A competitive NMDA receptor antagonist, D-2-amino-5-phosphonovalerate (D-APV), also exhibited inhibitory actions on light (CT20)-induced Per1 and Per2 mRNA expression in the ventrolateral SCN. Furthermore, local injection of NMDA into the SCN resulted in the induction of Per1 and Per2 mRNA in the SCN. Among NMDA receptors, NR2B and NR2C mRNA were expressed in the ventrolateral and dorsal SCN, respectively. These results suggest that the activation of NMDA receptor is a critical step for photic induction of Per1 and Per2 transcripts in the SCN, which are linked to a photic behavioral entrainment.

We analyzed effects of aging on behavioral rhythms in the mouse showing senescence acceleration, SAMP8 strains. The free-running rhythms had longer free-running periods (tau) in SAMP8 than in the control strain (SAMR1). Drinking of melatonin promoted the adaptation to advanced LD in SAMR1 but not in SAMP8, although both strains exhibited melatonin MTI and MT2, receptors. The present results suggest that melatonin promotes the adaptation to advanced LD cycles in normal aging mice. (C) 2000 Elsevier Science B.V. All rights reserved.

Ishida et at. [1994: Neurosci Lett 166: 211-215] reported the circadian change of N-methyl-D-aspartate (NMDA) receptor subtype 2C mRNA and photic induction of this receptor's mRNA in the suprachiasmatic nucleus (SCN). Therefore, we investigated the role of NMDA receptor subtypes in the biological clock using NMDA receptor 2A (NR2A)- or 2C (NR2C)-deficient mice. However, NR2C-/-mice showed normal light-dark (LD)-entrained locomotor activity rhythms and free-running rhythms under constant darkness and also exhibited normal reentrainment to 6-hr LD shifts and phase delays with single light pulses. Thus, present results demonstrated no significant NR2C contribution to circadian oscillation and photic entrainment, even though expression of NR2C mRNA was highly observed in the SCN. On the other hand, the period of the free-running activity rhythm in NR2A-/mice but not NR2C-/- mice was slightly longer than that in wild-type mice in spite of low expression of NR2A in the SCN, Furthermore, reentrainment to an LD advance in NR2A-/- mice was slower under low-intensity light conditions, Thus, we suggest that NR2A plays a role in determining the behavioral state that affects the circadian rhythm. in order to elucidate the role of NR2A and NR2C in the SCN, we examined NMDA-induced Ca2+ elevations in the SCN of mutant mice using a Ca2+ imaging method, A partial reduction in Ca2+ elevation was observed in both NR2A-/- and NR2C-/- mice when high concentrations (100 or 300 mu M) of NMDA were applied. The present results suggest that NR2A plays a weak role in oscillation or entrainment of the biological clock, and that NR2C does not participate in the functions of circadian oscillation and light entrainment, (C) 2000 Wiley-Liss, Inc.

The general mechanism underlying memory and learning is an area under intense investigation and debate, yet this mechanism still remains elusive. Auditory fear conditioning (when a tone is paired with a foot shock) is a simple associative form of learning for which many mechanistic details are known. Lesions of the lateral/basolateral nuclei of the amygdala result in the selective impairment of fear conditioning, indicating that this is a key region for this type of learning. Fear conditioning induces a lasting synaptic potentiation in the lateral nuclei of the amygdala. In addition, recent results from several laboratories suggest that N-methyl-D-aspartate (NMDA) receptor activation in the amygdala is required for the acquisition and expression of cue-conditioned fear responses using several kinds of antagonists. Little is known, however, about the signal transduction pathway and molecular substrate underlying fear conditioning. Here we use NMDA receptor-deficient mice to demonstrate that calmodulin-dependent kinase II, CaMKII beta, and CaMKII alpha. activation involves the NR2A subunit in the lateral/basolateral amygdala during memory retrieval following auditory fear conditioning. These results suggest that auditory fear conditioning involves a close linkage between NMDA2A receptors and the CaMKII cascade.

In mammals, the environmental light/dark cycle strongly synchronizes the circadian clock within the suprachiasmatic nuclei (SCN) to 24 hr. It is well known that not only photic but also nonphotic stimuli can entrain the SCN clock. Actually, many studies have shown that a daytime injection of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH DPAT), a serotonin 1A/7 receptor agonist, as a nonphotic stimulus induces phase advances in hamster behavioral circadian rhythms in vivo, as well as the neuron activity rhythm of the SCN in vitro. Recent reports suggest that mammalian homologs of the Drosophila clock gene, Period (Per), are involved in photic entrainment. Therefore, we examined whether phase advances elicited by 8-OH DPAT were associated with a change of Period mRNA levels in the SCN. In this experiment, we cloned partial cDNAs encoding hamster Per1, Per2, and Per3 and observed both circadian oscillation and the light responsiveness of Period. Furthermore, we found that the inhibitory effect of 8-OH DPAT on hamster Per1 and Per2 mRNA levels in the SCN occurred only during the hamster's mid-subjective day, but not during the early subjective day or subjective night. The present findings demonstrate that the acute and circadian time-dependent reduction of Per1 and/or Per2 mRNA in the hamster SCN by 8-OH DPAT is strongly correlated with the phase resetting in response to 8-OH DPAT.

The mPer1 gene is assumed to be a key molecule in the regulation and functioning of the mammalian circadian clock, which is based on the oscillation generated by a transcription-(post)translation feedback loop of a set of clock genes [1]. Robust circadian oscillation and acute light-elicited induction of mPer1 mRNA expression have been observed in the suprachiasmatic nucleus (SCN), the mammalian circadian center [2,3]. To Investigate the mechanism underlying the complex regulation of mPer1 expression, we isolated and characterized the 5' upstream region of the mPer1 gene. Unexpectedly, we identified two promoters, each followed by alternative first exons of mPer1, Consistent with the presence of multiple E-boxes in the promoters, exon-specific in site hybridization of the SCN established that both promoters function in circadian oscillation and in light-induction of mPer1 expression. Transgenic mice carrying the 5' upstream region of the mPer1 gene fused to the luciferase gene demonstrated that a DNA fragment carrying both promoter regions is sufficient to elicit striking circadian oscillation in the SCN and responsiveness to light. Moreover, luminescence in the SCN accurately mirrored the mPer1 transcriptional activity. These transgenic mice will be very useful for monitoring clock-specific mPer1 expression in intact organisms and to follow the circadian clock in real time.

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is believed to play an essential role for GABA synthesis in the central nervous system. Using mice with targeted disruption of the GAD65 gene (GAD65(-/-) mice) we investigated the contribution of GAD65 to GABA synthesis in different brain areas during postnatal development and in adulthood. In the amygdala, hypothalamus and parietal cortex of GAD65(+/+) mice an increase of GABA levels was observed during postnatal development, most prominently between the first ii- mice and and second month after birth. This increase appeared to be dependent on GAD65, as it was delayed by 2 months in GAD65 was not observed in GAD65(-/-) mice. Likely as a consequence of their GABA deficit, adult GAD65(-/-) mice showed a largely abnormal neural activity with frequent paroxysmal discharges and spontaneous seizures. They furthermore displayed increased anxiety-like behaviour in a light/dark avoidance test and reduced intermale aggression, as well as a reduced forced-swimming-induced immobility indicative of an antidepressant-like behavioural change. Adult GAD65(-/-) mice did not show behavioural disturbances except for a reduced aggressive behaviour that was comparable to that in GAD65(-/-) mice. We conclude that GAD65-mediated GABA synthesis may be crucially involved in control of emotional behaviour and indispensable fur a tonic inhibition that prevents the development of hyperexcitability in the maturating central nervous system. Aggressive, and possibly other social behaviour may be especially prone to regulation through GAD65-mediated GABA synthesis. (C) 2000 Elsevier Science B.V. All rights reserved.

Cholinergic regulation of the suprachiasmatic nucleus (SCN) has been extensively studied although the intracellular signaling mechanisms are not well understood. We examined immunostaining for phospholipase C-beta (PLC-beta) families that couple to muscarinic acetylcholine receptors (mAChR) and demonstrated the expression of PLC-beta 1 and beta 4 in the mouse SCN. Ca2+ imaging analysis indicated that the MI-mAChR antagonist, pirenzepine blocked carbachol-induced Ca2+ elevation in the SCN and the response was equivalent between the wild type and the PLC-beta 4-knockout mice. In addition. the knockout mice displayed locomotor and temperature rhythms coupling to 24 h light/dark cycles. Therefore, it was proposed that PLC-beta 1 but not PLC-beta 4 was involved in the mAChR-mediated Ca2+ signaling in the SCN. NeoroReport 11:907-912 (C) 2000 Lippincott Williams & Wilkins.

To clarify the role of glial fibrillary acidic protein (GFAP)expressed glial cells in the circadian clock, we examined GFAP expression in the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) under various lighting conditions in mice. We demonstrated that GFAP expression did not show daily change in the SCN under a light-dark cycle; however, long-term housing under constant lighting conditions led to dramatic changes in GFAP expression, i.e., a decrease in the SCN and an increase in the IGL. Furthermore, mice that had a targeted deletion in the GFAP gene (GFAP mutant mice) showed longer and more arrhythmic circadian activity rhythms in constant lighting conditions than wild-type mice, while GFAP mutant mice exhibited stable circadian rhythms both in a light-dark cycle and constant darkness, and showed normal entrainment to environmental light stimuli. These results suggest that the GFAP-expressed astroglial cells in the SCN and the IGL may have some role in circadian oscillation under constant lighting conditions. (C) 2000 Wiley-Liss, Inc.

As it is known that ryanodine receptor type 3 is expressed in the hippocampus, we examined the contribution of this receptor to contextual fear conditioning behavior and to the activation of Ca2+/calmodulin-dependent protein kinase II using mice lacking the receptor. Ryanodine receptor type 3-deficient mice exhibited impairments of performance in the contextual fear conditioning test, passive avoidance test, and Y-maze learning test. Both the activities of Ca2+/calmodulin-dependent protein kinase II beta and Ca2+/calmodulin-dependent protein kinase II alpha were significantly increased in the experimental group compared to the control group in the hippocampus, but not in the cingulate cortex on the testing day 24 h after contextual fear training. However, the activities of Ca2+/calmodulin-dependent protein kinase II beta and alpha were almost the same in the experimental and control groups in the hippocampus on the training day. Ryanodine receptor type 3-deficient mice did not show the increment of Ca2+/calmodulin-dependent protein kinase II beta and alpha activities in the hippocampus on the testing day. In addition, these mutant mice showed the reduction of fear response in the elevated plus-maze test. The present results suggest that calcium-induced calcium release through the activation of ryanodine receptor type 3 in the hippocampus is important to the expression of the performance of contextual learning through the elevation of Ca2+/calmodulin-dependent protein kinase II beta and alpha activities. (C) 2000 Elsevier Science B.V. All rights reserved.

Although it is known that ryanodine receptor type 3 is expressed in the striatum, the function of this receptor has not been elucidated. Therefore, we examined whether caffeine- and ryanodine-induced dopamine release in striatal slices is affected in mice lacking ryanodine receptor type 3, Pretreatment with thapsigargin, an inhibitor of the Ca2+ ATPase pump of the endoplasmic reticulum, abolished caffeine- or ryanodine-induced dopamine release in slices from normal mice. Dopamine concentration in the striatum and KCl-induced dopamine release were unaffected by a ryanodine receptor type 3 deficiency. Ryanodine-induced dopamine release was significantly attenuated in mice lacking ryanodine receptor type 3, whereas caffeine-induced dopamine release was partially attenuated. Caffeine produced a similar hypermotor activity in both wild and homozygous mice. The present results suggest the involvement of ryanodine receptor type 3 in dopamine release from the striatum. (C) 1999 Academic Press.

1 The mPer1 and mPer2 genes are putative mouse clock genes that regulate circadian oscillator present in the suprachiasmatic nucleus (SCN) neuron. While they are also expressed in the granular cell layer in the cerebellum, their function is unknown. In a first step to verify the physiological roles of mPer1 and mPer2 genes in the cerebellum, we examined the effects of benzodiazepines on the expression of the mPer1 and mPer2 genes.
2 mPer2 mRNA expression was higher at ZT16 than ZT4 in the mouse cerebellum.
3 High-dose administration of diazepam (10 mg kg(-1)) or triazolam (1 mg kg(-1)) reduced mPer1 mRNA level 1 h after treatment in the cerebellum.
4 Reduced expression of mPer1 by diazepam treatment was transient. No difference of mPer1 mRNA level between diazepam (10 mg kg(-1))- and vehicle-treated group was observed 6 h after treatment.
5 Administration of high doses of tandospirone (30 mg kg(-1)), a non-benzodiazepine anxiolytic also reduced mPer1 mRNA expression 1 h after treatment.
6 Administration of high doses of clozapine (5 mg kg(-1)) or haloperidol (1 mg kg(-1)) impaired the rota-rod performance without affecting on mPer1 mRNA level.
7 Diazepam and tandospirone inhibited the expression of Per1 mRNA in the primary cultured cerebellum granule cells.
8 Transient reductions of mPer1 mRNA levels by various benzodiazepines and tandospirone is associated with impairment of coordinated movement, such as rota-rod performance and equilibrium.

Methamphetamine (MAP) was administered to rats through drinking water repeatedly (three sessions, one session:administration for 60 days followed by withdrawal of 30 days) in order to examine whether or not MAP-induced disorganization of daily activity rhythm is sensitized. Each session (60 days) was divided into six blocks of 10 days. In the Ist session, daily locomotor activity rhythm of rats became disorganized around at 40 days (4th block) after the start of MAP drinking, However, MAP-induced disorganization of daily activity rhythm appeared at 20 days (2nd block) in the 2nd session and at 10 days (Ist block) in the 3rd session following re-start of MAP drinking. On the other hand, the amount of MAP intake was decreased on the 2nd and 3rd sessions as compared with the Ist session. These results indicate that the mechanism of MAP-induced disorganization of daily activity rhythm may involve sensitization. (C) 1999 Elsevier Science B.V. All rights reserved.

The suprachiasmatic nucleus (SCN) has been identified as a mammalian circadian rhythm clock. Treatment with substance P (SP) at zeitgeber time 13-14 produced phase delays of circadian rhythm in spontaneous neural activity in SCN neurons in vitro. SP-induced phase delays are blocked by treatment with not only SP receptor antagonist, spantide, but N-methyl-D-aspartate receptor antagonist, MK-801. In the biochemical experiment, we demonstrated that SP-induced glutamate release from the SCN slices was observed by the high-performance liquid chromatography assay. The present results suggest that glutamate release may be involved in SP-induced phase delays. (C) 1999 Elsevier Science B.V. All rights reserved.

We investigated the effects of the prolyl endopeptidase inhibitors 1-[1-(Benzyloxycarbonyl)-L-prolyl]prolinal (Z-Pro-Prolinal) and N-benzyloxycarbonyl-thioprolyl-thioprolinal-dimethylacetal (ZTTA) on delayed neuronal death induced by four-vessel-occlusion transient ischemia in rats. We also examined the effects of [pGlu(4), Cyt(6), Arg(8)]vasopressin (vasopressin-(4-9)) and thyrotropin-releasing hormone (TRH) on the delayed neuronal death. Furthermore, we investigated the role of vasopressin receptors in the effects of vasopressin and prolyl endopeptidase inhibitors. Z-Pro-Prolinal, vasopressin-(4-9) and TRH protected pyramidal cells in the CA1 subfield of the rat hippocampus from delayed neuronal death after 10-min ischemia, The effect of vasopressin-(4-9) was abolished by vasopressin receptor antagonists. The effect of Z-Pro-Prolinal was also abrogated by the antagonists. These results suggest that the neuroprotective effect of prolyl endopeptidase inhibitors is mediated by neuropeptides such as [Arg(8)]vasopressin and TRH, and indicate the involvement of vasopressin receptors in the neuroprotective effect of vasopressin-(4-9) and prolyl endopeptidase inhibitors. (C) 1999 Elsevier Science B.V. All rights reserved.

We previously reported the mammalian period repeat mRNA fluctuates during circadian time in the rat suprachiasmatic nucleus (SCN) which is considered to be a clock pacemaker in mammalian brain. Presently we discovered a period repeat sequence (PR) DNA-binding protein in the rat SCN nuclear extract. In the SCN, the binding activity of PR DNA-binding protein to (ACAGGC)(3) was most highest during the late day and most lowest during the late night by electro-mobility shift assay (EMSA). In the cortex nuclear extract, the binding of PR DNA-binding protein did not show a significant variation during a day. This is the first report to show the existence of diurnal regulated PR DNA-binding protein in the SCN. (C) 1999 Elsevier Science B.V. All rights reserved.

mPer1, a mouse gene, is a homolog of the Drosophila clock gene period and has been shown to be closely associated with the light-induced resetting of a mammalian circadian clock. To investigate whether the rapid induction of mPer1 after light exposure is necessary for light-induced phase shifting, we injected an antisense phosphotioate oligonucleotide (ODN) to mPer1 mRNA into the cerebral ventricle. Light-induced phase delay of locomotor activity at CT16 was significantly inhibited when the mice were pretreated with mPer1 antisense ODN 1 hr before light exposure. mPer1 sense ODN or random ODN treatment had little effect on phase delay induced by light pulses. In addition, glutamate-induced phase delay of suprachiasmatic nucleus (SCN) firing rhythm was attenuated by pretreatment with mPer1 antisense ODN, but not by random ODN. The present results demonstrate that induction of mPer1 mRNA is required for light- or glutamate-induced phase shifting, suggesting that the acute induction of mPer1 mRNA in the SCN after light exposure is involved in light-induced phase shifting of the overt rhythm.

The suprachiasmatic nucleus (SCN) has been identified as a pacemaker for mammalian circadian rhythms. Excitatory amino acid receptors, especially N-methyl-D-aspartate (NMDA) receptors, have been considered to play an important role in the transmission of light information from the retina to the circadian clocks in the SCN. In the present study, we showed that application of NMDA at circadian time (CT) 12-15 induced significant glutamate release from the SCN region in vitro. The NMDA-induced glutamate release was blocked by co-application of the NMDA receptor antagonist MK-801, but not by that of tetrodotoxin. These results suggested that glutamate stimulated its own release by activating NMDA receptors. This NMDA-induced glutamate release through NMDA receptor-mediated mechanisms might be involved in NMDA-induced potent phase shifts. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

1 Serotonergic projections from the midbrain raphe nuclei to the suprachiasmatic nuclei (SCN) are known to regulate the photic entrainment of circadian clocks. However, it is not known which 5-hydroxytryptamine (5-HT) receptor subtypes are involved in the circadian regulation. In order to verify the role of 5-HT1A receptors, we examined the effects of 5-{3-[((2S)-1,4-benzodioxan-2-ylmethyl)amino]propoxy}-1,3-benzodioxole HCl (MKC-242), a selective 5-HT1A receptor agonist, on photic entrainment of wheel-running circadian rhythms of hamsters.
2 MKC-242 (3 mg kg(-1), i.p.) significantly accelerated the re-entrainment of wheel-running rhythms to a new 8 h delayed or advanced light-dark cycle.
3 MKC-242 (3 mg kg(-1), i.p.) also potentiated the phase advance of the wheel-running rhythm produced by low (5 lux) or high (60 lux) intensity light pulses. In contrast, 8-hydroxydipropylaminotetralin (8-OH-DPAT)(5 mg kg(-1), i.p.), a well known 5-HT1A/5-HT7 receptor agonist, only suppressed low intensity (5 lux) light-induced phase advances.
4 The potentiating actions of MKC-242 on light pulse-induced phase advances were observed even when injected 20 or 60 min after the light exposure.
5 The potentiating action of MKC-242 was antagonized by WAY100635, a selective 5-HT1A receptor blocker, but not by ritanserin, a 5-HT2/5-HT7 receptor blocker, indicating that MKC-242 is activating 5-HT1A receptors.
6 Light pulse-induced c-fas expression in the SCN and the intergeniculate leaflet (IGL) were unaffected by MKC-242 (3 mg kg(-1), i.p.).
7 HPLC analysis demonstrated that MKC-242 (3 mg kg(-1), i.p.) decreased the 5-HIAA content in the SCN.
8 The present results suggest that presynaptic 5-HT,A receptor activation may be involved in the potentiation of photic entrainment by MKC-242 in hamsters.

We examined the influence of ischemia on methamphetamine (MAP)-induced behavioral sensitization and enhancement of dopamine (DA) release. After the recovery period of the ischemia operation, rats were treated with MAP (1 mg/kg, i.p.) once daily for 6 consecutive days. Re-administration of MAP (0.5 mg/kg, i.p.) potentiated the increase of locomotor activity after a 3-day withdrawal and the enhancement of DA release from striatal slices after a 6-day withdrawal. The MAP-induced sensitization was impaired by 5 min ischemia. On the other hand, the increase of locomotor activity induced by single MAP (1 mg/kg, i.p.) administration was impaired by 20 min of ischemia. Moreover, in saline-treated rats the increase of DA release from striatal slices induced by MAP (10 μM) application was also impaired by 20 min of ischemia. These results indicate that the neuronal plastic change may be very vulnerable to ischemia in MAP-induced sensitization.

Effects of methylcobalamin (methyl-B-12), a putative drug for treating human circadian rhythm disorders, on the melatonin-induced circadian phase shifts were examined in the rat. An intraperitoneal injection of 1-100 mu g/kg melatonin 2-h before the activity onset time (CT 10) induced phase advances of free-running activity rhythms in a dose-dependent manner (ED50 = 1.3 mu g/kg). Injection of methyl-B-12 (500 mu g/kg) prior to melatonin (1 mu g/kg) injection induced larger phase advances than saline preinjected controls, while the injection of methyl-B-12 in combination with saline did not induce a phase advance. These results indicate amplification of melatonin-induced sphase advances by methyl-B-12. Pinealectomy abolished the phase alternating effect of methyl-B-12, suggesting a site of action within the lineal gland. In fact, methyl-B-12 significantly increased the content of melatonin in the pineal collected 2-h after activity onset (CT 14). In contrast, no difference in melatonin content was found at CT 10, indicating that the effect of methyl-B-12 may be gated after the activity onset time when endogenous melatonin synthesis is known to increase. These results suggest that methyl-B-12 amplifies melatonin-induced phase advances via an increase in melatonin synthesis during the early subjective night at a point downstream from the clock regulation. (C) 1998 Elsevier Science B.V. All rights reserved.

Circadian rhythm is an endogenous rhythm that persists in constant conditions with a period of nearly but not identical to 24 hr. Under natural conditions, the circadian clock is precisely entrained to the daily (24 hr) cycle, because environmental stimulus (especially light) induces a phase shift of the clock. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus has been shown to be the primary pacemaker that drives daily rhythms of behavioral and physiological activity. Photic information is conveyed. from the retina to the SCN directly by the retinohypothalamic tract (RHT) and indirectly by the geniculohypothalamic tract (GHT). The transmitter of the RHT is glutamate, while the GHT is GABA and neuropeptide Y. Serotonergic innervation from the median raphe and melatonin from the pineal body are likely to provide non-photic information to the SCN. Single gene mutations that dramatically alter circadian phenotype were found in the hamster (tau) and mouse (clock). Moreover, the homologous genes of the Drosophila clock gene, per, were found in mammals and the homologue of the mammalian clock was found in Drosophila. These data suggest that the some constitutes of the biological clock may be conserved between Drosophila and mammals, and a transcription-translation feedback loop involving some clock gene products may be a oscillator itself.

Optic nerve (ON) stimulation caused a postsynaptic field potential in the suprachiasmatic nucleus (SCN) of rat hypothalamic slices. The postsynaptic field potential was suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, in a concentration-dependent manner, but not affected by D-amino-5-phosphonovaleric acid (APV), a competitive NMDA receptor antagonist. Tetanic stimulation to the ON induced long-term potentiation (LTP) in the SCN. Application of APV at 50 mu M inhibited the induction of LTP by tetanic stimulation but CNQX at lower dose (5 mu M) didn't inhibit it. These results suggest that NMDA receptors are indispensable for the induction of LTP after tetanic stimulation. (C) 1998 Elsevier Science B.V.

To understand how light might entrain a mammalian circadian clock, we examined the effects of light on mPer1, a sequence homolog of Drosophila per, that exhibits robust rhythmic expression in the SCN.mPer1 is rapidly induced by short duration exposure to light at levels sufficient to reset the clock, and dose-response curves reveal that mPer1 induction shows both reciprocity and a strong correlation with phase shifting of the overt rhythm. Thus, in both the phasing of dark expression and the response to light mPer1 is most similar to the Neurospora clock gene fro. Within the SCN there appears to be localization of the induction phenomenon, consistent with the localization of both light-sensitive and light-insensitive oscillators in this circadian center.

The muscarinic acetylcholine receptor antagonist scopolamine, but not the beta-adrenoceptor antagonist propranolol or atenolol, suppressed tetanus-induced long-term potentiation (LTP) of population spikes in the rat hippocampal CA1 region. When scopolamine was coapplied with propranolol or atenolol, a synergistic effect in preventing LTP generation was observed, On the other hand, the coapplication of scopolamine and atenolol failed to affect tetanus-induced LTP of field EPSP. These findings suggest that cooperative mechanisms via muscarinic and beta-adrenergic receptor activation might contribute to LTP induction in terms of the EPSP-spike potentiation, i.e., an increase in the excitability of hippocampal CA1 pyramidal cells after tetanic stimulation, but are independent of the tetanus-evoked potentiation of a synaptic component. (C) 1997 Elsevier Science B.V.

We assessed light-induced Fos-immunoreactive cells in the suprachiasmatic nucleus of diabetic rats. The number of Fos-immunoreactive cells significantly decreased in diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats as compared with control Long-Evans Tokushima Otsuka (LETO) rats. In contrast there was no decrease in the number of Fos-immunoreactive cells in young OLETF rats which have not yet developed diabetes. Two months after the administration of streptozotocin (STZ) to Wistar rats, the number of Fos-immunoreactive cells significantly decreased, although 1 week after the administration of STZ, the number had not yet changed in these STZ-induced diabetic rats. These results suggest that chronic diabetic (hyperglycemic) conditions may affect the light entraining responses in the suprachiasmatic nucleus (SCN). (C) 1997 Elsevier Science Ireland Ltd.

We investigated the involvement of calmodulin and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the photic entrainment of circadian rhythms using calmodulin inhibitors such as calmidazolium (CMZ) and trifluoperazine (TFP), and a CaMKII inhibitor, KN-62, in rats. Fos expression in the suprachiasmatic nucleus (SCN) of rats induced by photic stimulation (300 lux, 1 h) during the early subjective night of the rats was inhibited by treatment with CMZ (10 mg/kg i.p.) or TFP (20 mg/kg i.p.) 30 min before photic stimulation. With respect to the neuronal firing rate in the rat SCN slice, KN-62 and CMZ application during the early subjective night attenuated the glutamate (10 mu M)-induced phase shift. The present results suggest that calmodulin and CaMKII are involved in the photic entrainment mechanism in the rodent SCN. (C) 1997 Elsevier Science Ireland Ltd.

The present study investigated the effects of arginine-vasopressin (AVP) and (1-[3-(2-indanylacetyl)-L-thioprolyl] pyrrolidine (Z-321), an inhibitor of prolyl endopeptidase (PEP; (EC 3.4.21.26)) which degrades AVP in vitro, on the short-lasting potentiation of the field excitatory postsynaptic potentials (EPSP) coupled with a weak tetanus. The EPSP, after the electrical stimulation of the Schaffer collateral/commissural pathway, were recorded in the CA1 region of rat hippocampal slices. AVP at 10(-8) M and Z-321 at 10(-4) M augmented the potentiation induced by the weak tetanus; the magnitude of the post-tetanic potentiation of the EPSP was enhanced and the potentiation lasted for 60 min. In contrast, the racemic D-thioprolyl compound of Z-321, which virtually lacks any inhibitory effects on PEP, failed to affect the potentiation at 10(-4) M. The facilitatory effect of Z-321 was reversed by the application of [d(CH2)(5),Tyr(Me)(2)]AVP (10(-8) M), an antagonist of the AVP V-1 receptors, indicating that the effect of Z-321 was mediated through the V-1 receptors. These findings suggest that Z-321 augmented the potentiation due to its inhibitory influence on the AVP degradation by PEP.

We studied the roles of metabotropic glutamate receptors in methamphetamine (MAP)-induced sensitization of dopamine (DA) release from striatal slices. Rats were first treated with MAP (1 mg/kg, i.p.) once daily for 6 consecutive days. After a 6-day withdrawal, DA release from striatal slices evoked by ±-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) was measured. trans-ACPD-induced DA release was significantly enhanced in MAP-sensitized rats, but the inactive form of trans-ACPD (1R,3S-ACPD) did not enhance DA release. The active form of trans-ACPD (1S,3R-ACPD) (0.1 mM)-evoked DA release was attenuated by treatment with 0.4 mM RS-α-methyl-4-carboxyphenylglycine, a metabotropic glutamate receptor antagonist. The present results suggest that metabotropic glutamate receptors play an important role in expression of MAP-induced sensitization.

Optic nerve stimulation has been reported to evoke a field potential (FP) in rat suprachiasmatic nucleus (SCN) slices. Methylcobalamin,δ-(5,6-dymethylbenzimidazolyl)-Co-methyl-cobamide (Me-B12) enhanced this FP and the enhancement lasted more than 1 h after washing out. Maximal enhancement (143.6 ± 9.8%) was achieved at a concentration of 10 μM. By contrast, cyanocobalamin containing CN- instead of CH3- showed no enhancement of the amplitude in the FP. Me-B12 induced enhhncement of FP was strongly blocked by an N-methyl-D-aspartate (NMDA) receptor antagonist, DL-2-amino-5-phosphonovaleric acid (APV). These results indicate that CH3- in the Me-B12 is required to modulate the FP amplitude and the NMDA receptor is involved in the long-lasting FP enhancement induced by Me-B12. The present results suggest that Me-B12 modifies the photic entrainment of the circadian clock in the suprachiasmatic nucleus via an activation of NMDA receptors.

We examined the role of serotonin IA (5-HT1A) receptors in the inhibitory effects of methamphetamine (MA) on photic entrainment to the circadian pacemaker in the suprachiasmatic nucleus (SCN) of rodents. MA inhibited optic nerve stimulation-evoked field potential in the SCN, light-induced Fos expression in the SCN and light-induced phase shift of hamster wheel-running rhythm. NAN-190, a 5-HT1A receptor antagonist, eliminated the inhibitory effects of MA. NAN-190 has also been reported to antagonize alpha 1 adrenergic receptors. However, prazosin, which selectively antagonizes alpha(1) adrenergic receptors, did not affect the inhibitory action of RIA on light-induced Fos expression. In addition, parachloroamphetamine, which is known to be a 5-HT releaser, dose-dependently inhibited light-induced phase shift of wheel-running rhythm. These findings suggest that elevation of endogenous 5-HT levels by RIA inhibits the photic entraining responses of the circadian pacemaker in the SCN via 5-HT1A receptor stimulation of the 5-HT released by MA.

Adenosine is widely accepted to act as an inhibitory neuromodulator in the mammalian central nervous system. In the present study, we examined whether adenosine receptor agonist modifies the photic entraining responses in the rat suprachiasmatic nucleus both in vivo and in vitro. Light (200 lux, 15 min)-induced phase shifts of hamster wheel-running rhythms was attenuated by a systemic administration of AI-adenosine receptor agonist N-6-cyclohexyladenosine (N-CHA) in a dose-dependent manner; 0.5 mg/kg N-CHA caused 60% inhibition of light-induced phase shifts. On the other hand, A2-adenosine receptor agonist N-6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA) failed to inhibit light-induced phase shifts. Systemic administration of N-CHA but not of DPMA inhibited light (300 lux, 1 h)-induced Fos expression in the suprachiasmatic nucleus in a dose-dependent manner; 1 mg/kg N-CHA caused 73% inhibition of light-induced Fos expression. Bath application of N-CHA but not of DPMA inhibited optic nerve stimulation-evoked field potentials in rat suprachiasmatic nucleus slices. The present results suggest that activation of adenosine Al-receptor attenuates the photic input through the inhibition of retinohypotalamic pathway to the SCN.

An intracerebral microdialysis technique was applied to study the effect of metabotropic glutamate receptor (mGluR) agonist on dopamine release in the striatum of methamphetamine (MAP)-sensitized rats. Rats were treated with MAP (1 mg/kg, i.p.) once daily for 6 consecutive days, followed by a 6-day withdrawal. Perfusion of 0.1 mM (1S:3R)-1-aminocyclopentane-trans-1,3-dicarboxylic acid through a microdialysis probe placed in the striatum enhanced the extracellular dopamine level, and induced stereotyped behavior in MAP-sensitized rats. The enhancement of dopamine release and the stereotyped behavior were attenuated by co-perfusion of 0.4 mM RS-alpha-methyl-4-carboxyphenyl-glycine, a mGluR antagonist. The present results sus est that mGluRs may be involved in the expression of MAP-induced sensitization.

We examined the roles of nitric oxide (NO) in methamphetamine (MAP)-induced behavioral sensitization and enhancement of striatal dopamine (DA) release using both in vivo and in vitro methods. Repeated administration of MAP produced augmentation of MAP-induced locomotor activity after 3-day withdrawal of MAP and an enhancement of MAP-evoked DA release from striatal slices after 6-day withdrawal. When the NO synthase (NOS) inhibitor N-G-nitro-L-arginine methyl ester (L-NAME) was administered only during the period of MAP withdrawal, the behavioral sensitization and enhancement of DA release were attenuated significantly. In contrast, N-G-nitro-D-arginine methyl ester, an inactive isomer of L-NAME, exhibited no such effect. When L-NAME was administered acutely before the challenge injection of MAP, behavioral sensitization was also attenuated only when the dose of L-NAME was high. Coadministration of L-NAME with MAP did not block the development of sensitization to MAP. We also examined whether MAP-induced behavioral sensitization and enhancement of DA release could be observed in vivo in a microdialysis experiment. Challenge injection of MAP caused marked enhancement of DA release in MAP-sensitized rats compared with saline-treated controls corresponding to robust augmentation of locomotor activity. When L-NAME was injected during the MAP withdrawal period, the enhancement of DA release and locomotor activity induced by challenge injection of MAP were attenuated. These results suggest that NO production plays a role in the maintenance (expression) of MAP-induced behavioral sensitization and enhancement of DA release but not in the development of these effects.

The in vivo uptake and distribution of 6-deoxy-6-[F-18]fluoro-L-ascorbic acid (F-18-DFA) were investigated in rat brains following postischemic reperfusion. Global cerebral ischemia was in induced in male Wistar rats for 20 min by occlusion of four major arteries. Two time points were chosen for F-18-DFA injection to rats subjected to cerebral ischemia, at the start of recirculation and 5 days following recirculation. The rats were then killed at 2 h after tail vein administration of F-18-DFA and tissue radioactivity concentration was determined. Increased uptake of radioactivity in particular brain regions, including the cerebral cortex, hypothalamus, and amygdala following injection of F-18-DFA, compared to the sham operated control, was observed 5 days after reperfusion. Similar results were also obtained in in vitro experiments using brain slices. Abnormal in vivo accumulation of Ca-45, a marker of regional postischemic injury, was observed in these brain regions in tissue dissection experiments. Furthermore, metabolite analysis of nonradioactive DFA using F-19-NMR showed that DFA remained intact in the postischemic reperfusion brain. The present results indicate that F-18-DFA increasingly accumulates in damaged regions of postischemic reperfusion brain.

We examined whether methamphetamine modifies the photic entraining responses in the rat suprachiasmatic nucleus. Optic nerve stimulation increased vasoactive intestinal polypeptide release from rat suprachiasmatic nucleus slices, and methamphetamine inhibited this increase in a concentration-dependent manner. Optic nerve stimulation has been reported to evoke field potentials in rat suprachiasmatic nucleus slices. Methamphetamine attenuated this held potential, and maximal inhibition (75.5%) was achieved at a concentration of 100 mu M. Systemic administration of methamphetamine (1-5 mg/kg) inhibited light (300 lux, 1 h)-induced Fos expression in the suprachiasmatic nucleus; methamphetamine at a dose of 5 mg/kg, i.p. caused 40% inhibition of light-induced Fos expression. We examined whether the inhibitory effect of methamphetamine on photic entraining responses mediates serotonin release from the suprachiasmatic nucleus. High-performance liquid chromatographic analysis revealed that methamphetamine application increased serotonin release from rat suprachiasmatic nucleus slices in a concentration-dependent manner, but did not affect noradrenaline release. In addition, reduction of serotonin content attenuated the effect of methamphetamine on field potential induced by optic nerve stimulation in vitro and also light-induced phase advances of wheel running activity rhythm in vivo.
The present results support the idea that methamphetamine produces an inhibitory effect on photic entrainment in the suprachiasmatic nucleus via serotonin release.

The effects of chronic administration of methamphetamine (MAP) on rat locomotor activity rhythm under light-dark (LD) housing and on neuronal activity rhythms from the suprachiasmatic nucleus (SCN) in vitro were investigated. Control rats exhibited an LD-entrained nocturnal locomotor rhythm. The firing rate of SCN neuronal activity was high during the light period and low during the dark period in control normal rats, and peak of firing activity occurred around zeitgeber time (ZT) 6. On the other hand, chronic MAP administration caused various disorganization of locomotor activity rhythms with a long free-running period (25-35 h). Neuronal activity rhythms of the SCN were unaffected by chronic MAP administration, that is, high during the light period and low during the dark period. The present findings suggested that the SCN maintained as a circadian pacemaker even under chronic MAP administration which affected overt rhythms.

When feeding was restricted to a single meal scheduled at a fixed time (1300-1700 h) in the day, rats exhibited increased prefeeding locomotor activity 1-3 h before the feeding time, which increased over 6 days. The activity increase was seen for about 5 h(1100-1600 h) on the seventh day when no food was provided (fasting day), as the rat anticipated mealtime. Thus, mealtime-associated activity rhythm on the fasting day was considered to be another good index of food-anticipatory activity. In this study, we investigated the effects of the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, which has been reported to cause impairment of spatial learning, on food-anticipatory activity in rats. Daily injection of MK-801 (0.2 mg/kg) at 1700 h for 6 successive days impaired the development of increased prefeeding activity. The appearance of mealtime-associated activity was impaired by daily injection of MK-801 (0.2 mg/kg) at 1700 h (immediately after food restriction, FR) or at 1900 h (2 h after FR) but not at 0900 (16 h after FR). In addition, mealtime-associated activity was impaired by MK-801 injected at 1700 h in a dose-dependent manner(0.1-1 mg/kg). The present results demonstrate that NMDA receptor mechanisms were involved in learning processes of food anticipation and suggest that an impairment of food-anticipatory activity may be considered an animal model of a deficit of ''temporal learning.''

The in vivo uptake and distribution of 6-deoxy-6-[18F]fluoro-L-ascorbic acid (18F-DFA) were investigated in rat brains following postischemic reperfusion. Global cerebral ischemia was induced in male Wistar rats for 20 min by occlusion of four major arteries. Two time paints were chosen for 18F-DFA injection to rats subjected to cerebral ischemia, at the start of recirculation and 5 days following recirculation. The rats were then killed at 2 h after tail-vein administration of 18F-DFA and tissue radioactivity concentration was determined. Increased uptake of radioactivity in particular brain regions, including the cerebral cortex, hypothalamus, and amygdala following injection of 18F-DFA, compared to the sham operated control, was observed 5 days after reperfusion. Similar results were also obtained in in vitro experiments using brain slices. Abnormal in vivo accumulation of 45Ca, a marker of regional postischemic injury, was observed in these brain regions in tissue dissection experiments. Furthermore, metabolite analysis of nonradioactive DFA using 19F-NMR showed that DFA remained intact in the postischemic reperfusion brain. The present results indicate that 18F-DFA increasingly accumulates in damaged regions of postischemic reperfusion brain.

The aim of the present study was to determine whether calcium channel antagonists attenuated hypoxia/hypoglycemia- or glutamate-induced reduction in 2-deoxyglucose (2-DG) uptake of hippocampal slices obtained from ethanol withdrawal rats, Ethanol withdrawal significantly potentiated the hypoxia/hypoglycemia- and glutamate-induced reductions in 2-DG uptake of hippocampal slices, Both nifedipine and flunarizine exhibited attenuating effects on ethanol withdrawal-induced potentiation of impairment of 2-DG uptake caused by hypoxia/hypoglycemia or glutamate, Hypoxia/hypoglycemia-induced deficit of 2-DG uptake was prevented by ethanol, but chronic consumption of ethanol resulted in the development of tolerance to neuroprotective effect, These findings suggested that the increased sensitivity of neurons to ischemic damage by ischemia may involve in the increased activity of calcium channels in the hippocampus.

Optic nerve stimulation caused a postsynaptic field potential in the rat suprachiasmatic nucleus (SCN) of hypothalamic slices. In the present experiment, we demonstrated whether tetanic stimulation of optic nerve can produce a long-term potentiation (LTP) in the SCN postsynaptic field potential. The amplitude of SCN field potential was higher in the subjective day animals than that in the subjective night animals. Tetanic stimulation of optic nerve (100 Hz, 1 s) at subjective daytime (projected zeitgeber time: ZT 0-8) produced a LTP in this field potential, although the onset of LTP was slow. When tetanic stimulation was applied at ZT4, the percent increase of amplitude was 116.6% immediately after, 159.8% 30 min after and 215.4% 120 min after tetanic stimulation, whereas tetanic stimulation of optic nerve at subjective night-time caused a weak LTP in the SCN. Although tetanic stimulation of Schaffer collaterals induced a LTP formation in the CA1 region of rat hippocampal slices, there were no obvious circadian changes in this LTP formation. The present results demonstrated that excitatory influence on the SCN caused a synaptic plasticity such as LTP. Although the physiological meaning of this LTP is uncertain at present, LTP may be related to adaptation mechanism to photic stimulation.

The effects of aging on neuronal activity in the suprachiasmatic nucleus (SCN) were examined in hamsters kept under light-dark (LD) or constant light (LL) conditions. The free-running period in wheel-running rhythm of 24-month-old hamsters (24.2 +/- 0.04) was shorter than that of the 2-month-old hamsters (24.4 +/- 0.057). There was a significant difference in the mean firing rates of SCN neuron activity between old and young hamsters during subjective day (6.58 +/- 0.36 spikes/s in young and 5.63 +/- 0.24 in old hamsters), but not during subjective night (4.33 +/- 0.47 in young and 4.05 +/- 0.39 in old). Similar to LL condition, the firing activity during zeitgeber time 3-11 (4.33 +/- 0.27) in old hamsters kept under LD condition, was significantly lower than that of young hamsters (6.22 +/- 0.32). These results suggest that deterioration of SCN neuronal activity in old hamsters assessed as reduction of daytime activity may reflect changes in the interaction between SCN clocks and the overt behaviors and/or pacemaking properties of SCN cells.

The effects of methylcobalamin, a vitamin B-12 analogue, on the hypoxia/hypoglycemia- or glutamate-induced reduction in hippocampal CA1 presynaptic fiber spikes elicited by Schaffer collateral stimulation in rat brain slices were evaluated. Hippocampal slices were exposed to 15 min of hypoxia/hypoglycemia, and then these slices were returned to oxygenated and glucose-containing buffer for 3 h. Hypoxia/hypoglycemia reduced CA1 presynaptic potentials in vitro. Treatment with 10 mu M methylcobalamin attenuated the impairment of CA1 presynaptic potentials induced by hypoxia/hypoglycemia or glutamate application (10 mM). Daily injection of methylcobalamin (0.5 mg/kg i.p./day) for 3 days in vivo also attenuated the hypoxia/hypoglycemia- or glutamate-induced reduction in presynaptic potentials in hippocampal slices. Pretreatment with cyanocobalamin at 10 mu M failed to attenuate the impairment of CA1 presynaptic potentials. However, daily injection of cyanocobalamin (0.5 mg/kg i.p./day) for 3 days caused a protective action against the hypoxia/hypoglycemia- or glutamate-induced functional deficit. Furthermore, co-treatment of L-arginine (100 mu M), a substrate for nitric oxide synthase, with methylcobalamin in vitro reversed the methylcobalamin-induced functional recovery. The present results demonstrate that methylcobalamin application in vivo or in vitro leads to functional recovery from hypoxia/hypoglycemia- or glutamate-induced impairment of CA1 presynaptic potentials. Neuroprotection was obtained by in vivo application of cyanocobalamin, but not by its in vitro application. It is reported that in vivo injected cyanocobalamin converted to methylcobalamin in the hepatic cells. Therefore, the results suggest that a transmethylation reaction in the hippocampal regions may be involved in the methylcobalamin-induced functional recovery from ischemic impairment.

Prolyl endopeptidase (PEP; EC 3.4.21.26) cleaves the Pro-Arg bond of arginine-vasopressin (AVP), This study investigated the effects of PEP inhibitors, 1-[1-(benzyloxycarbonyl)-L-prolyl]prolinal (Z-Pro-prolinal) and 1-[3-(2-indanylacetyl)-L-thioprolyl]pyrrolidine (Z-321), on the AVP content in the septum of rats. Oral administration of Z-Pro-prolinal (100 mg/kg) and Z-321 (100 mg/kg) significantly increased the septal AVP content. At 10 mg/kg, Z-321 slightly increased the content. In contrast, the D-thioprolyl form of Z-321 (100 mg/kg), which lacks an inhibitory effect on the enzyme, failed to affect the AVP content. These results indicate that PEP inhibitors increase the content of AVP through inhibition of the enzyme activity in vivo. Therefore, it is suggested that PEP may contribute to the degradation of endogenous AVP in the brain.

Daily injection of methamphetamine (MAP) increased the locomotor activity and produced the development of MAP-induced anticipatory activity on the next withdrawal day. Daily restricted feeding also causes a feeding-associated anticipatory activity rhythm. The importance of catecholaminergic neurons has been suggested in the manifestation of a feeding-associated corticosterone rhythm. In our study, we examined the role of dopaminergic neurons in the development of MAP-induced anticipatory activity. The development of MAP-induced anticipatory activity was blocked by coadministration of dopamine D2 receptor antagonists such as YM-09151-2 and sulpiride, the D1 receptor antagonist SCH23390 acid haloperidol weak D1/D2 receptor antagonist, but not by clozapine. The anticipatory activity increase was reproduced by daily injection of the D2 receptor agonist quinpirole as well as moderately by the D1 receptor agonist SKF38393. Moreover, MAP-induced anticipation was blocked by coadministration of the N-methyl-D-aspartate receptor antagonist MK-801 or sigma ligands rimcazol and NE-100. Our results suggest that activation of the dopaminergic system, especially via D2 receptors may be required for the development of MAP-associated anticipatory activity increases and that N-methyl-D-aspartate receptors and sigma receptor mechanisms are also involved in the development of this behavior.

We examined the effects of μ-opioid receptor agonist and antagonists, and κ-opioid receptor agonist on the hypoxia/hypoglycemia-induced reduction in 2-deoxyglucose uptake of rat hippocampal slices. Naloxone, a μ-opioid receptor antagonist and (5,7,8)-(+)-3,4-dichloro-N-methyl-N-(7,8,1-pyrrolidinyl)-1-oxaspirol 4,5 dec-8-yl)-benzeneacetamide methanesulfonate, U-62,066E, a κ-opioid receptor agonist, showed neuroprotective actions against the hypoxia/hypoglycemia-induced deficit in glucose uptake. In contrast, morphine exhibited an exacerbating action. These results suggest that blockade of μ-opioid receptor-and stimulation of κ-opioid receptor-mediated functions has a protective role against the hypoxia/hypoglycemia-induced decreases in glucose metabolism in hippocampal slices. Chronic administration of morphine (10 mg/kg) for 9 days affected neither the basal nor the hypoxia/hypoglycemia-induced reduction in 2-deoxyglucose uptake. Rats treated with morphine chronically exhibited not only tolerance to the analgesic effect but also tolerance to the exacerbating action. However, chronic morphine did not modify U-62,066E-induced neuroprotection. These findings indicate that the receptor mechanisms of neuroprotection produced by the activation of κ-opioid receptors may not be involved in μ-opioid receptor function. © 1995.

Under constant darkness hamsters demonstrate free-running activity rhythms and light exposure during the early subjective night results in permanent phase delays of the activity rhythm. Recently, we reported that application of glutamate receptor agonists such as N-methyl-D-aspartate could reset the phase of the circadian rhythm of suprachiasmatic nucleus firing activity in vitro via nitric oxide production. In order to confirm this result by in vivo experiment, we examined the effect of nitric oxide synthesis inhibitor on the light-induced phase delay of circadian rhythms of wheel-running activity in hamsters. In vehicle-treated animals, light stimulation at circadian time 13.5 resulted in stable phase delays (1.3 +/- 0.63 h), whereas pre-treatment with 150 mg/kg of N-nitro-L-arginine methylester (L-NAME) significantly attenuated light-induced phase delays (0.72 +/- 0.18 h). L-NAME administration alone without light exposure, did not cause phase changes. The L-NAME-induced attenuating effect was reversed by co-administration of L-arginine (300 mg/kg). The present results suggest that nitric oxide production is involved in the light-induced phase delay of the hamster's circadian system.

In the present study, we examined attenuating effect of serotonin (5-HT) receptor antagonists on the impairment of the time perception presented by daily scheduled feeding in old rats. When feeding was restricted to a single meal at a fixed time of day (1300-1700 h) for six consecutive days, young rats exhibited intense locomotor activity from 1-3 h before feeding time. Intense locomotor activity was observed between 1200 and 1700 h in young animals even on the fasting day (day 7) (mealtime-associated activity). However, this mealtime-associated activity was impaired in 24-mo-old rats. Daily injections of 5-HT2 receptor antagonists, mianserin or ritanserin, or a 5-HT3 receptor antagonist, Y25130, at 1700 h for 6 consecutive days significantly and dose-dependently attenuated the impairment of mealtime-associated activity on the fasting day in old rats without affecting the food intake. Our results suggest that the blockade of 5-HT2 and/or 5-HT3 receptors attenuates impairment of the manifestation of mealtime-associated anticipatory activity related to temporal learning in old rats.

We studied the effects of high frequency tetanic stimulation of the striatum on the KCl (20 mM)-evoked dopamine release in rat striatal slices. The KCl-evoked dopamine release was potentiated by high frequency tetanic stimulation (10-20 Hz) of the striatum including the corticostriatal fibers, and this potentiation was observed until 3 h after high frequency tetanic stimulation. Potentiation of dopamine release after high frequency tetanic stimulation was induced not only by KCl but also by glutamate in Mg2+-free medium, N-methyl-D-aspartate in Mg2+-free medium, and by DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid. 2-amino-5-phosphovalerate, 3-[(+/-)-2-carboxypiperazine-4-yl]-propy-1-phosphonate or dibenzocycloheptaneimine, N-methyl-D-aspartate receptor inhibitors, abolished enhancement by tetanus, whereas, 6,7-dinitroquinoxaline-2,3-dione, an antagonist of DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ionotropic receptors, or L-2-amino-4-phosphonobutyrate, an antagonist of glutamate metabotropic receptors, showed no effect. Moreover, pretreatment with glutamate or N-methyl-D-aspartate in the absence of Mg2+ also facilitated dopamine release evoked by KCl concentrations. When extracellular Ca2+ was removed from the medium during pretreatment, potentiation by glutamate disappeared.
We conclude that activation of N-methyl-D-aspartate receptors on dopaminergic nerve terminals in the striatum produces the long-term changes in efficacy of the response to KCl or glutamatergic agents. That is, plastical phenomena could exist at presynaptic levels between glutamatergic neurons and dopaminergic neurons in striatum.

The aim of the present study was to determine whether calcium channel antagonists attenuated hypoxia/hypoglycemia- or glutamate-induced reduction in 2-deoxyglucose (2-DG) uptake of hippocampal slices obtained from ethanol withdrawal rats. Ethanol withdrawal significantly potentiated the hypoxia/hypoglycemia- and glutamate-induced reductions in 2-DG uptake of hippocampal slices. Both nifedipine and flunarizine exhibited attenuating effects on ethanol withdrawal-induced potentiation of impairment of 2-DG uptake caused by hypoxia/hypoglycemia or glutamate. Hypoxia/hypoglycemia-induced deficit of 2-DG uptake was prevented by ethanol, but chronic consumption of ethanol resulted in the development of tolerance to neuroprotective effect. These findings suggest that the increased sensitivity of neurons to ischemic damage by ischemia may involve in the increased activity of calcium channels in the hippocampus. © 1995 Elsevier Science B.V. All rights reserved.

We studied the effects of transient forebrain ischemia in vivo on long-term enhancement of dopamine (DA) release from rat striatal slices. One hour after the high-frequency tetanic stimulation (HFTS) or L-glutamate (10(-6) M) application in Mg2+-free medium to striatal slices, the high concentration of KCl (high K+)-evoked DA release was measured. Tetanic stimulation or L-glutamate application significantly potentiated the high-K+-evoked DA release. When striatal slices were prepared from rats exposed to 3 min of ischemia followed by 24-h survival, the enhancement of DA release by HFTS was unaffected by ischemia. In contrast, the enhancement of DA release by HFTS was impaired in rats exposed to 5 min or 10 min of ischemia. In addition, high K+-evoked DA release per se was significantly impaired by 10 min of ischemia. The enhancement of DA release elicited by pretreatment with L-glutamate was also impaired in the rats exposed to 5 min of ischemia. When striatal slices were prepared from rats exposed to 5 min of ischemia with 7-day survival, the enhancement of DA release by HFTS was still impaired. The present results indicate that the neuronal mechanisms of the enhancement of DA release may be more sensitive to impairement from short periods of ischemia. Furthermore, the results suggest that an impairment of long-term enhancement of DA release by ischemia may be related the dysfunction of motor performance in rats exposed to ischemia.

The effects of nitric oxide (NO) synthase inhibitors on the hypoxia/hypoglycemia-induced decrease in CA1 presynaptic fiber spikes elicited by stimulation of the Schaffer collaterals were investigated using rat hippocampal slices. Drugs were added to normal medium for 10 min before incubation under hypoxic/hypoglycemic conditions (15 min), and after a 3-h washout, the CA1 presynaptic potential was measured. Treatment with N-G-nitro-L-arginine methyl ester but not with N-G-nitro-D-arginine methyl ester produced a concentration-dependent attenuation of the hypoxia/hypoglycemia-induced decrease in presynaptic fiber spikes. In contrast, treatment with precursors of NO in the arginine-to-NO pathway, such as sodium nitroprusside, S-nitro-N-acetylpenicillamine and N-morpholino sydnonimine exacerbated the 15-min hypoxia/hypoglycemia-induced decrease in the CA1 presynaptic potential. The neuroprotective effect of N-G-nitro-L-arginine methyl aster was significantly attenuated by co-treatment with L-arginine. The present results suggest a facilitatory role of NO production in hypoxia/hypoglycemia-induced presynaptic dysfunction in CA1 regions of hippocampal slices.

In the present experiment, we examined the attenuating effect of bifemelane hydrochloride (BF), 4-(o-benzyl phenoxy)-N-methylbutylamine hydrochloride, on the impairment of time perception caused by daily scheduled feeding using aged and MK-801-treated rats. When feeding was restricted to a single meal at a fixed time of day (1300-1700 h) for six successive days, young rats exhibited intense locomotor activity 1-3 h before feeding time. Intense locomotor activity was observed for 1200-1700 h even on the fasting day (day 7; mealtime-associated activity). Mealtime-associated activity was impaired in 24-mo-old rats and also in N-methyl-D-aspartate receptor antagonist, MK-801-treated rats. Daily injections of bifemelane at 1700 h for six successive days significantly attenuated the impairment of mealtime-associated activity on the seventh day in a dose-dependent manner in aged rats. In addition, cotreatment of MK-801 with bifemelane blocked the MK-801-induced impairment of mealtime-associated activity. The present study suggests that bifemelane has an enhancing effect on learning and memory performance, such as spatial and temporal perception.

We studied the effects of high frequency tetanic stimulation of the striatum on the KCl (20 mM)-evoked dopamine release in rat striatal slices. The KCl-evoked dopamine release was potentiated by high frequency tetanic stimulation (10-20 Hz) of the striatum including the corticostriatal fibers, and this potentiation was observed until 3 h after high frequency tetanic stimulation. Potentiation of dopamine release after high frequency tetanic stimulation was induced not only by KCl but also by glutamate in Mg2+-free medium,N-methyl-d-aspartate in Mg2+-free medium, and bydl-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid. 2-amino-5-phosphovalerate, 3-[(±)-2-carboxypiperazine-4-yl]-propyl-1-phosphonate or dibenzocycloheptaneimine,N-methyl-d-aspartate receptor inhibitors, abolished enhancement by tetanus, whereas, 6,7-dinitroquinoxaline-2,3-dione, an antagonist ofdl-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ionotropic receptors, orl-2-amino-4-phosphonobutyrate, an antagonist of glutamate metabotropic receptors, showed no effect. Moreover, pretreatment with glutamate orN-methyl-d-aspartate in the absence of Mg2+ also facilitated dopamine release evoked by KCl concentrations. When extracellular Ca2+ was removed from the medium during pretreatment, potentiation by glutamate disappeared. We conclude that activation ofN-methyl-d-aspartate receptors on dopaminergic nerve terminals in the striatum produces the long-term changes in efficacy of the response to KCl or glutamatergic agents. That is, plastical phenomena could exist at presynaptic levels between glutamatergic neurons and dopaminergic neurons in striatum. © 1995 IBRO.

In the present study, we examined whether cholinergic drugs such as arecoline and physostigmine attenuated an impairment of time perception presented by daily scheduled feeding in aged rats. When feeding was restricted to a single meal at a fixed time of day (13:00-17:00) for 6 successive days, young rats exhibited intense locomotor activity from 1-3 h before feeding time. Intense locomotor activity was observed between 12:00-17:00 in young animals even on the fasting day (on day 7) (mealtime-associated activity). However, this mealtime-associated activity was impaired in old rats. Daily injection of arecoline (10 mg/kg) or physostigmine (0.1 and 0.2 mg/ kg) at 17:00 for 6 successive days attenuated the impairment of mealtime associated activity on the fasting day in a dose-dependent manner in old rats, whereas daily treatment with D-glucose (100 or 2000 mg/kg) did not. The results of the present study suggest that cholinergic drugs attenuate the impairment of the manifestation of mealtime-associated anticipatory activity related to 'temporal learning' in old rats.

The effects of glutathione, its analogue: YM737 (N-(N-gamma-L-glutamyl-L-cysteinyl) glycine-l-isopropyl ester sulfate monohydrate), a monoester of glutathione, and N-acetyl-L-cysteine on hypoxia/hypoglycemia-induced decreases in CA1 presynaptic fiber spikes and 2-deoxyglucose uptake were investigated using rat hippocampal slices. The drugs were added to normal medium for 30 min before the incubation under hypoxic/hypoglycemic conditions (20 min), and, after a 3-h washout, presynaptic potential or 2-deoxyglucose uptake in hippocampal slices was measured. Treatment with glutathione, YM737 and N-acetyl-L-cysteine produced an attenuation of the hypoxia/hypoglycemia-induced decrease in presynaptic fiber spikes and 2-deoxyglucose uptake. The order of potency for neuroprotective action was YM737 greater than or equal to N-acetyl-L-cysteine &gt; glutathione. The present results suggest a role for glutathione in improving hypoxia/hypoglycemia-induced dysfunction of hippocampal regions.

Effects of sigma receptor agonists or antagonists on hypoxia/hypoglycemia-induced decrease in CA1 presynaptic fiber spikes elicited by the stimulation of Schaffer collaterals were investigated using rat hippocampal slices. Treatment with sigma receptor antagonists such as haloperidol, NE-100 and rimcazole produced a concentration-dependent attenuation of the hypoxia/hypoglycemia-induced decrease in CA1 presynaptic fiber spikes. The order of potency of protection against hypoxia/ hypoglycemia-induced reduction in CA1 presynaptic potential was: NE-100 = haloperidol &gt; rimcazole. Treatment with sigma receptor agonist DTG potentiated the hypoxia/hypoglycemia-induced decrease in the CA1 presynaptic potential, whereas SKF10047 which possesses an affinity for phencyclidine site attenuated the decrease of potential. NE-100 antagonized a functional deficit induced by DTG, but unaffected the improving effect induced by SKF10047. The present results suggest a facilitatory role of sigma receptor stimulation in hypoxia/hypoglycemia-induced an impairment of neurophysiological functions in CA1 presynaptic regions of hippocampal slices.

The aim of the present study was to determine whether U-50,488H and U-62,066E, κ-opioid receptor agonists cause a neuroprotective action against hypoxia/hypoglycemia-induced reduction in 2-deoxyglucose (2-DG) uptake of hippocampal slices from U-50,488H-tolerant rats. Both U-50,488H and U-62,066E exhibited an attenuating effect on hypoxia/hypoglycemia-induced reduction in 2-DG uptake of hippocampal slices. Hypoxia/hypoglycemia-induced deficit of 2-DG uptake was prevented by cotreatment with naloxone, an opioid receptor antagonist, but potentiated by cotreatment with morphine, a μ-opioid receptor agonist. Chronic administration of U-50,488H resulted in the development of tolerance to the analgesic effect as well as the neuroprotective effect whereas this treatment affected neither basal- nor hypoxia/hypoglycemia-induced decreases in 2-DG uptake. Chronic administration of U-50,488H did not modify naloxone-induced attenuation of 2-DG uptake deficit but slightly potentiated the morphine-induced exacerbation. These findings suggest that the tolerance to κ-opioid receptors does not affect the μ-opioid receptor-mediated neuroprotective or neurotoxic action. © 1994.

Somatostatin is synthesized in the suprachiasmatic nucleus (SCN), a circadian pacemaker in mammals. To explore the functional significance of somatostatin in the circadian system, we examined rhythms of rat locomotor activity and electrical firing rate of SCN neurons in the brain slice after temporal depletion of somatostatin levels in the SCN. Intraperitoneal administration of cysteamine (200 mg/kg), a somatostatin depletor, significantly reduced somatostatin level in the in vivo SCN 5 min after injection and kept low level as long as 3 to 4 days. This administration, on the other hand, induced significant phase advances of about 51 min in the subsequent free-running rhythm of locomotor activity of the rat. A marked phase advance in the circadian rhythm of firing rate in the SCN was also observed after administration of cysteamine in coronal hypothalamic slices. These persistent phase shifts after administration of a somatostatin depletor may suggest that the change of somatostatin level in the SCN have a feedback influence on the circadian pacemaker.

The aim of the present study was to determine whether the 2-deoxyglucose uptake of amygdala slices was affected by olfactory bulbectomy. At 7, 14 and 21 days post-lesion, bilateral olfactory bulbectomized rats exhibited a significant increase of 2-deoxyglucose uptake in amygdala slices, but not in the cerebral cortex. In addition, unilateral olfactory bulbectomized rats showed a high uptake of 2-deoxyglucose uptake in the ipsilateral amygdala, but not in the contralateral amygdala. These results suggest that the enhancement of 2-deoxyglucose uptake is related to the hyperexcitability of amygdala neurons following bulbectomy, and that this enhancement may be responsible for the behavioral changes in olfactory bulbectomized rats.

The effect of dopamine (DA) receptor agonists and antagonists on hypoxia/hypoglycemia (ischemia)-induced decrease in CA1 presynaptic fiber spikes elicited by the stimulation of Schaffer collateral were investigated using hippocampal slices. Treatment with D-1 dopamine receptor antagonist, SCH23390 produced a concentration-dependent attenuation of the ischemia-induced decrease of presynaptic potentials. The magnitude of recovery of the CA1 presynaptic potential in SCH233390-treated slices at 10 and 100 mu M was 28 and 54%, respectively. Whereas, treatment with D1 dopamine receptor agonist, SKF38393 exacerbated the ischemia-induced decrease in the CA1 presynaptic potential. The decrease of CA1 presynaptic potential by ischemia was affected by neither D-2 dopamine receptor agonist, bromocriptin and quinpirole nor D-2 dopamine receptor antagonist, sulpiride. The neuroprotective effect of SCH23390 was completely blocked by cotreatment with SKF38393. The present results demonstrated that the blockade of D-1 dopamine receptor function played a neuroprotective role in ischemic damage, suggesting a facilitatory role of D-1 dopamine receptor-operated function in ischemia-induced neuronal deficits.

The aim of the present study was to determine whether or not the reduction of 2-deoxyglucose uptake by the cerebral cortical slices in aged rats (22-23 months old) was attenuated by somatostatin or carbachol. In 8-week-old rats, somatostatin and carbachol produced concentration-dependent increases in 2-deoxyglucose uptake. 2-Deoxyglucose uptake of the cortical slices in 22-23-month-old rats was significantly facilitated by treatment with 0.1-1 mu M somatostatin or 1-100 mu M carbachol. Metabolic responses to somatostatin or carbachol were quite similar in young and aged rats. The present results demonstrated that 2-deoxyglucose uptake by the cerebral cortex was facilitated by somatostatin and carbachol in both young and old rats.

The circadian rhythm in mammals is under control of the pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus. This tiny nucleus contains a number of neurochemicals, including peptides, amines and amino acids. Heterogeneous distribution of these neurochemicals defines the substructures of the SCN. In the present review, functional significance of such neurochemical heterogeneity in the SCN is discussed in the light of circadian patterns of the concentrations of these neurochemicals in the SCN and their effects on SCN neurons in in vitro slice preparation. In particular, the hypothesis that the dorsomedial SCN is involved in maintaining the circadian rhythm, while the ventrolateral SCN is involved in adjusting the phase of the rhythm, is critically discussed. These considerations suggest that distinct sub-components of the SCN as marked by neurochemicals, interact with each other and this organizational architecture could be the basis of the proper operation of the circadian time keeping system in this nucleus.

In rats of various ages (11 weeks to 24 months), restricted feeding was carried for 6 successive days and food was withheld completely on the 7th day to assess the effect of aging on food anticipatory activity. When feeding was restricted to a single meal at a fixed time of day (1330-1730), rats exhibited intense locomotor activity from 1-3 h before feeding time (prefeeding activity). This prefeeding activity was still detected on the fasting day. In addition, intense locomotor activity was observed for 1330-1730 even on the fasting day (mealtime-associated activity). Thus, mealtime-associated activity of the fasting day became an another good index for food anticipatory activity. These results indicate that the rats had come to anticipate the mealtime. Both prefeeding and mealtime-associated activities were similar at 11 weeks and 9 months of age, but they were impaired at 12 and 24 months of age. In 11-week-old rats, there was a negative correlation between the level of mealtime-associated activity and the daily duration of access to food, but this relationship was absent in 12-month-old rats. The present study suggests that aging strongly impairs the manifestation of food anticipatory activity in rats.

We investigated the effects of vasopressin-related neuropeptides on the hypoxia/hypoglycemia (ischemia)-induced decrease of the CAI presynaptic potential elicited by stimulation of Schaffer collaterals in rat hippocampal dices. Treatment with arginine-vasopressin (AVP) potentiated the ischemic decrease of the CA1 presynaptic potential. In contrast, a V, receptor antagonist produced a dose-dependent neuroprotective effect, whereas a V, receptor antagonist had no effect. The AVP-induced decrease of the CAI presynaptic potential was completely blocked by simultaneous application of the V, receptor antagonist. Because AVP(4-9) is regarded as the major proteolytic product of AVP in the rat brain, we examined its effect on the ischemic decrease of the CA1 presynaptic potential. Treatment with AVP(4-9) produced a more marked reduction of the potential than treatment with AVP itself. The present study demonstrates that stimulation of the V-1, receptor has a detrimental effect on the development of ischemic damage whereas V-1, receptor blockade has a neuroprotective effect, suggesting that AVP may potentiate ischemic neuronal deficits via V-1 receptor stimulation.

In the present experiment, under drinking of 0.01% methamphetamine (MA), young rats exhibited free-running locomotor rhythm ranging from 24.6 to 30.9 h period under light-dark cycle. In contrast, 24-month-old rats showed a low activity and did not exhibit MA-induced free-running rhythm. When MA was injected at a fixed time of day (14:00), young rats exhibited intense locomotor activity from 1-2 h before drug-injection time. This preinjection activity was still detected on the withdrawal day. In addition, intense locomotor activity was observed for 12:00-17:00, even on the withdrawal day (MA-associated anticipatory activity). Thus, MA-associated activity of the withdrawal day became an another good index for MA anticipatory activity rhythms whereas old rats did not exhibit this anticipatory activity. The present study suggests that aging strongly impairs the manifestation of MA-induced free-running and anticipatory activity rhythms in rats.

Excitatory amino acid (EAA) receptors such as N-methyl-D-aspartate (NMDA) and non-NMDA receptors have been suggested to play an important role in the regulation of photic information from the retina to the suprachiasmatic nucleus (SCN). Therefore, we investigated the role of glutamate as a retinohypothalamic transmitter by analyzing the phase-resetting effects of NMDA and a non-NMDA agonist, (R,S)-alpha-amino-3-hydroxy-5-methylisoxazo-4-propionic acid (AMPA), on the circadian rhythm of SCN firing activity. Nitric oxide (NO) production is believed to be an essential intermediate in NMDA-induced cGMP production in the CNS. Thus, we examined the effects of blockers of NO production on NMDA- or AMPA-induced phase delay of SCN activity rhythm. N-nitro-L-arginine methylester (L-NAME) blocked NMDA- but not AMPA-induced phase shift, indicating the involvement of NO synthesis in NMDA-induced phase changes. L-arginine but not D-arginine caused a phase delay, and L-NAME blocked L-arginine-induced phase delay. In addition, cotreatment with NMDA and L-arginine did not have an additive effect. These results suggest that NO production itself is involved in the phase change of SCN neuron activity, and NMDA-induced phase changes are also mediated via activation of NO synthesis in this nucleus.

The effects of cholecystokinin (CCK) receptor agonists and antagonists on hypoxia/hypoglycemia (ischemia)-induced decrease in CA1 presynaptic fiber spikes elicited by the stimulation of Schaffer collaterals were investigated using rat hippocampal slices. Treatment with the CCKB receptor agonist CCK tetrapeptide (CCK4, 0.01-10 mu M) exacerbated the ischemia-induced decrease in the CA1 presynaptic potential in a concentration-dependent manner. Whereas, treatment with the CCKB receptor antagonist [(3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl)-N1-(3-methylphenyl)-urea] (L365260), and not with CCKA receptor antagonist [(3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5phenyl-1H-1,4-benzodiazepin-3-yl)-1H-indole-2-carboxamide] (L364718), produced a concentration-dependent attenuation of the ischemia-induced decrease. The magnitude of recovery of the CAI field potentials in L365260-treated groups at 10 and 100 nM was 34% and 45%, respectively. The neuroprotective effect of L365260 (0.01 and 0.1 mu M) was completely blocked by co-treatment with CCK4 (0.1 mu M), a concentration that did not affect the decreased presynaptic potential induced by ischemia. These results demonstrated that the stimulation of the CCKB receptor played a detrimental role in the development of ischemic damage, whereas the blockade of CCKB receptors played a neuroprotective role in ischemic damage, suggesting a facilitatory role of CCK receptor-operated function in ischemia-induced neuronal deficits.

The mammalian suprachiasmatic nucleus (SCN) has been identified as a circadian pacemaker. N-methyl-D-aspartate (NMDA), non-NMDA and substance P receptors have been suggested to be involved in handling: of photic information in the SCN. In the Aplysia eyes, in which the circadian clocks are involved, serotonin- or cAMP-induced phase changes of the circadian rhythm were reported to be blocked by protein-synthesis inhibitors. Therefore, we investigated whether protein-synthesis inhibitor can block the non- NMDA receptor agonist (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA)- or substance P (SP)-induced phase changes of SCN activity rhythm. Although application of 10 mu M cycloheximide alone during the early part of the subjective night did not cause phase change, it blocked both 10 mu M AMPA- and 1 mu M SP-induced phase delay. The present result suggests that protein synthesis may be required in the manifestation of AMPA- and SP-induced phase change of circadian clock.

Living organisms including humans drive circadian rhythm, and this rhythm influences the social structure and daily life of human beings. The suprachiasmatic nucleus (SCN) has been established as a pacemaker for mammalian circadian rhythm. There are many kinds of neurotransmitters and neuromodulators in this nucleus. The circadian systems are involved in oscillation, input (entrainment) and output (overt rhythm). Therefore in this review, pharmacological characteristics of circadian systems in relation to the SCN are discussed by focussing especially upon the roles of various neurotransmitters and modulators.

The excitatory amino acid, glutamate plays a crucial role in the pathogenesis of brain damage caused by anoxia and/or hypoglycemia. Although vasopressin (VP) also acts as an excitatory transmitter in the CNS, little is known about its effect on hypoxic and/or ischemic brain damage. In this study, we investigated the effect of arginine vasopressin (AVP) on hypoxia/ hypoglycemia-induced impairment of dopamine release from striatal slices. Striatal slices were incubated in hypoxia-/ hypoglycemia-inducing medium with or without AVP (0.01-1.0 mu M) for 20 min. After 1-3 h of washout in normal medium, high K+-evoked dopamine release from the slices were examined. Hypoxia/hypoglycemia-induced decrease of striatal dopamine release was reversed by the removal of Ca2+ in the medium, but not by VP1- or VP2-receptor antagonist. In contrast, AVP potentiated the hypoxia/hypoglycemia-induced decrease of dopamine release in the striatum. This AVP-induced deterioration of the striatal response was antagonized by VP2 receptor antagonist, but not by VP1 receptor antagonist. The present results suggest that AVP may play a facilitatory role in hypoxia/hypoglycemia-induced dopamine release deficit mediated through the activation of VP2 receptor.

The effect of calmodulin inibitors on the circadian rhythm of locomotor activity and on the rhythm of suprachiasmatic nuclear (SCN) neuron firing rate recorded in vitro from hypothalamic slices was examined. Trifluoperazine produces changes in a dose-dependent manner in the phase of the activity rhythm, with phase advances throughout most of the subjective day extending into the subjec tive night. These phase changes in the activity rhythm occur rapidly and without induction of locomotor activity at the time of treatment. Similarly, trifluoperazine and the naphthalenesulfonamide W-7 produce changes in phase delays in the subjective night extending into early subjective day. The effects are greater with respect to amplitude when measured acutely after treatment than in the next cycle, and both the acute and next-day effects are greater than those observed in vivo, indicating that data from in vitro studies need to be interpreted with caution. These observations indicate that calmodulin inhibitors affect rhythms directly in vivo by altering SCN neuron pacemaker function, as this reflects involvement of calcium-calmodulin binding with activation of a calmodulin-dependent kinase, either to alter intracellular cAMP levels or to alter gene expression directly to modulate the phase of the SCN clock. © 1994, Sage Publications. All rights reserved.

Glutamate has been reported to be involved in the transmission of photic information from the retina to the suprachiasmatic nucleus (SCN). Therefore, we investigated whether the application of N-methyl-d-aspartate (NMDA), a glutamate receptor agonist could, reset the circadian rhythm of SCN firing activity in vitro. Treatment with NMDA for 1 h between projected zeitgeber time (ZT) 13-14 produced a phase delay in a concentration-dependent manner. The NMDA-induced phase delay was antagonized by an NMDA-receptor antagonist, MK-801 (100 μM). The retinohypothalamic tract has been reported to make terminals on neurons possessing vasoactive intestinal polypeptide (VIP). Therefore, we investigated the effects of NMDA on VIP release from the SCN and on VIP immunoreactivity in the SCN. Application of NMDA for 15 min between ZT 13-15 increased release of VIP from the SCN. In contrast to release, the content of VIP in the SCN tissue was reduced by application of NMDA. Immunohistochemical analysis revealed that application of NMDA for 4 h or 1 h reduced VIP immunoreactivity in the SCN. To investigate the possibility that VIP released by NMDA could reset SCN neuronal activity, we examined the effects of VIP on the SCN neuronal activity rhythm. Cotreatment with VIP (1 μM) and gastrin-releasing peptide (1 μM) for 1 h between ZT 13-14 caused a phase-delay of SCN activity rhythm. These findings suggest that activation of NMDA receptors during early subjective night causes a phase delay of the SCN neuronal activity via facilitation of VIP release in this nucleus.

We investigated the phase-resetting effect of muscimol, gamma-amino butyric acid (GABA)(A) receptor agonist, on the circadian neural activity rhythm of the rat suprachiasmatic nucleus (SCN), which contains a circadian pacemaker. Acute application of muscimol inhibited the neural activity of the SCN in a dose-dependent manner. Under the tissue culture condition, the treatment with 10 mu M muscimol during the early- to mid-subjective day on the first day (day 1) in vitro produced the largest phase advance in neural activity rhythm of the SCN on day 2. By contrast, the administration of muscimol during the subjective night produced no change. These phase changes were similar to those reported for dark pulses in constant light. These findings indicate that muscimol can directly affect SCN neurons and reset the circadian pacemaker in the SCN. The GABA neural function through the activation of GABA(A) receptors may play a role in modulating the phase of the SCN clock, especially during the subjective day.

The suprachiasmatic nucleus (SCN) acts as a pacemaker for mammalian circadian rhythms. Receptors for excitatory amino acids like N-methyl-D- aspartate (NMDA) and non-NMDA receptors have both been found to play an important role in the transmission of photic information from the retina to the SCN. Therefore, we investigated whether the application of glutamate receptor agonists could reset the phase of the circadian rhythm of SCN firing activity in vitro. Treatment with NMDA (0.1-10 μM) for 15 min or 1 h during the early part of the subjective night produced phase delay, whereas treatment during the late subjective night caused an advance in phase. The phase-response curve for NMDA was similar to that previously obtained in response to light pulses in vivo. Application of DL-α-amino-3-hydroxy-5- methylisoxazole-4-propionic acid hydrobromide (AMPA) (1 or 10 μM), a non- NMDA-receptor agonist, also produced a dose-dependent phase delay of SCN activity. The NMDA-induced phase delay was antagonized by an NMDA-receptor antagonist MK-801. These findings suggest that both NMDA and non-NMDA receptors may be involved in the transmission of information to the SCN via the retinohypothalamic tract. In addition, both the advances and delays in phase caused by NMDA were potentiated by cotreatment with neuropeptide Y, whereas AMPA-induced phase delay was not potentiated by neuropeptide Y. This points to a functional link between NMDA and neuropeptide Y receptor-mediated mechanisms in the SCN.

In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus appears to act as a circadian clock. The SCN vasoactive intestinal polypeptide-like immunoreactive neurons, which may act to mediate photic information in the SCN, receive input from neurons immunoreactive for somatostatin (SST). Therefore we investigated the role of SST as a transmitter for entrainment by analyzing the phase-resetting effect of SST on the circadian rhythm of SCN firing activity. Perfusion of SST increased 2-deoxyglucose uptake at circadian time (CT) 18, but not at CT6. A 1-h or 15-min treatment with SST produced phase delays when it was administered at CT13-14 and phase advances at CT22-23. Thus SST-induced phase changes are similar to those for light pulses to animals under constant darkness. The present findings suggest that SST is a transmitter for mediating information of entrainment to circadian clocks within the SCN.

The retinohypothalamic tract (RHT) is a direct pathway from the retina to the suprachiasmatic nucleus (SCN). Electrical stimulation of the optic nerve or optic chiasm activates the RHT and produces shifts in phase of a circadian rhythm in SCN neuron activity in rat hypothalamic slices in vitro. The phase response curve (PRC) for this effect is very similar to that obtained from administration of light pulses to intact animals maintained in constant darkness. The effect of optic chiasm stimulation is blocked by tetrodotoxin. In addition to the RHT, there is a second entraining pathway, the geniculohypothalamic tract, which arises from neuropeptide Y (NPY)-containing neurons of the intergeniculate leaflet of the lateral geniculate complex. In contrast to optic chiasm stimulation, NPY produces phase shifts in the rhythms of SCN neuron firing rate in vitro with a PRC that similar to that for NPY infusion into the SCN in intact animals as well as that produced by a series of treatments that induce locomotor activity. These results indicate that phase shifts of the circadian rhythm of SCN neuron activity may be produced by activation of two different entraining pathways and that the physiological actions of these pathways on pacemaker function are markedly different. © 1993.

It is well known that synaptic potentiation in the hippocampus can be produced by phorbol ester, a protein kinase C activator. The 2-deoxyglucose uptake is an index of regional glucose utilization which predominantly reflects activity in the axonal terminal of neuronal pathways. In the present experiment, therefore, we examined whether application of phorbol ester produces a facilitatory effect on 2-deoxyglucose uptake by the rat hippocampus in vitro. The application of phorbol-12,13-dibutyrate (PdBU) produced an elevation of 2-deoxyglucose uptake, while pretreatment with PdBU for 60 min eliminated the pdBU-induced elevation. Pretreatment with protein kinase C inhibitors, K252a (0.1 and 1 muM) or staurosporine (0.1 and 1 muM), was found to block significantly the PdBU-induced elevation of 2-deoxyglucose uptake. In addition, the facilitatory effect of glutamate, quisqualate and carbachol on 2-deoxyglucose uptake was reduced by pretreatment with PdBU. In the present experiment, we demonstrated that application of phorbol ester caused an elevation of 2-deoxyglucose uptake, which is linked in turn to neuronal activity, suggesting a positive relationship between protein kinase C activation and energy consumption.

In a working memory task with three-panel runway paradigm, cysteamine, a depletor of somatostatin, at 100 or 200 mg/kg i.p. given 24 h before testing, had no effect on the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points). Cysteamine at 100 mg/kg caused a significant reduction in somatostatin-like immunoreactivity in the rat brain, including the hippocampus and cerebral cortex. Working memory errors were significantly increased by scopolamine, a muscarinic receptor antagonist, at 0.32 mg/kg i.p. given 20 min before testing, whereas errors were not affected by the 0.1 mg/kg dose. Combined administration of 100 mg/kg cysteamine and 0.1 mg/kg scopolamine significantly increased the number of working memory errors. However, cysteamine at 100 mg/kg and scopolamine at 0.1 mg/kg had no effect on reference memory errors, whether they were administered alone or in combination. These results suggest that depletion of brain somatostatin aggravates working memory deficits induced by blockade of muscarinic receptors.

Single unit activity of suprachiasmatic nucleus (SCN) neurons recorded from hamster and rat hypothalamic slices in vitro exhibits a circadian rhythm in firing rate. Tetrodotoxin (TTX) produces electrical silence during the period of administration but has no effect on the oscillatory activity of the circadian pacemaker in both hamster and rat SCN. TTX does not affect either the phase of the rhythm in firing rate at any time of the circadian day, or that of the rhythm in glucose metabolism as demonstrated by the 2-deoxyglucose method. These data are in accord with the view that SCN neuron firing rate and glucose utilization is an expression of pacemaker activity but that pacemaker function is maintained in the SCN independent of neuronal function manifested by sodium dependent action potentials.

The effects of histamine (HA) receptor antagonists on hypoxia + hypoglycemia (ischemia)-induced impairment of 2-deoxyglucose (2-DG) uptake by rat hippocampal slices was evaluated. Hippocampal slices were exposed to 20-min ischemia and then returned to oxygenated and glucose-containing Krebs-Ringer solution for 6 h. Ischemia reduced 2-DG uptake in the hippocampal slices. The ischemia-induced reduction in 2-DG uptake was attenuated by pretreatment with H-1 receptor antagonist but not with H-2 receptor antagonist. Treatment with HA exacerbated the ischemia-induced decrease. The H-1 receptor antagonist-induced neuroprotective effect was blocked by co-treatment with HA. The present study suggests that the blockade of H-1 receptor-mediated function has a protective role in ischemia-induced decreases in glucose metabolism in hippocampal slices.

2-Deoxyglucose (2-DG) uptake is an index of regional glucose utilization which reflects predominantly activity in the axonal terminal of neuronal pathways. The present experiments showed that somatostatin elevated 2-DG uptake in rat cortex and hippocampus slices. Treatment with somatostatin-14 and somatostatin-28 markedly enhanced 2-DG uptake, whereas the amino-terminal fragment of somatostatin-28 did so only slightly. This effect appeared to be mediated by an interaction with somatostatin receptors because cyclo-somatostatin, a somatostatin antagonist, abolished the effect of somatostatin-14. The increase in 2-DG uptake caused by somatostatin-14 was blocked by the calcium channel antagonist, nifedipine, but not by tetrodotoxin, suggesting that the action of somatostatin does not require the initiation of impulse activity. somatostatin enhanced the KCl-induced release of acetylcholine, suggesting that a cholinergic mechanism is involved in the somatostatin-induced cellular responses. We therefore examined whether acetylcholine receptor antagonists block the somatostatin-induced increase in 2-DG uptake. Neither muscarinic nor nicotinic receptor antagonists affected the somatostatin-14-induced response. The present results suggest that somatostatin has a stimulatory effect on energy metabolism and that this effect is independent of acetylcholine receptor mechanism.

The aim of the present study was to determine whether the facilitation of 2-deoxyglucose (2-DG) uptake in the cerebral and hippocampal slices by nicotinic and muscarinic receptor agonists is compromised in the aged rat brain. For this, the effects of the nicotinic receptor agonist nicotine, the muscarinic receptor agonists oxotremorine and McN-A-343, and the ACh esterase inhibitors physostigmine and NK247 on 2-DG uptake in the brain slices of young (2-month-old) and aged (24-26-month-old) rats were tested. The decrements of 2-DG uptake in the cortical slices of aged rats were significantly attenuated by treatment with oxotremorine, nicotine and amiridine. In contrast, the metabolic responsivity of hippocampal slices to these drugs was reduced. To assess whether age-related changes in 2-DG uptake may be due to deficits in cholinergic function, we tested these drugs on the decrements of 2-DG uptake in ethylcholine aziridinium (a neurotoxic analog of choline) injected rats. The reductions of 2-DG uptake by injection of ethylcholine aziridinium was attenuated by oxotremorine but not by physostigmine. The present results reveal that metabolic decrements in the cerebral cortex from aged or ethylcholine aziridinium-injected rats were attenuated by muscarinic and nicotinic receptor agonists, suggesting that the muscarinic and nicotinic receptor mechanism in the cerebral cortex may be involved in cholinergic drug-induced functional recovery in aged rats.

A series of pentanthrene type heterocyclic compounds were synthesized and evaluated for anxiolytic activities by three kinds of behavioral pharmacological tests. Several compounds showed anxiolytic activities. In particular, s-triazolo[3,4-a]phthalazine (Tri-P) and 3-propyl derivatives of Tri-P(PTP) showed remarkable activities, although the activities were slightly lower than those of diazepam. The results suggested that Tri-P or PTP is a useful lead compound for the development of the antianxietic agents. The relationship between the structure and anxiolytic activity, and the inducing mechanism of the activity was discussed.

Various in vitro models have been developed to study ischemia and/or hypoxia. In the present experiment, we examined whether hypoxia/hypoglycemia (ischemia) in rat hippocampal slices reduced the 2-deoxyglucose (2-DG) uptake and CA1 field potentials evoked by stimulation of Schaffer collaterals. Autoradiograms revealed that ischemia for 15 or 20 min reduced 2-DG uptake in the stratum radiatum of the CA1 and the dentate gyrus. Similarly, the CA1 field potentials of slices exposed to ischemia for 15 and 20 min decreased by about 70 and 90% after a 6-h washout. In the second experiment, we evaluated the neuroprotective effect of the 5-HT1A receptor agonists 8-OH-DPAT and buspirone, and the 5-HT2 receptor antagonists cyproheptadine, mianserin and ketanserin on deficits of 2-DG uptake and Schaffer-CA1 field potentials induced by ischemia. The 5-HT1A receptor agonists and 5-HT2 receptor antagonists exhibited significant neuroprotective actions against ischemia-induced deficits. Therefore, impairments of 2-DG uptake and CA1 field potentials induced by ischemia may be good markers of ischemia-induced functional deficits. The attenuating action of 5-HT1A receptor agonists and 5-HT2 receptor antagonists were assessed using this model of ischemia.

The suprachiasmatic nuclei (SCN) have been identified as a pacemaker for many circadian rhythms in mammals. Although substance P (SP) fibers from retina are found to terminate the SCN, the physiological role of this peptide is uncertain. The 2-deoxyglucose (2-DG) uptake and firing activity in the SCN show a robust circadian change. SP causes an increase in 2-DG uptake by SCN during the subjective night but not during subjective day. SP-induced increase in 2-DG uptake is blocked by co-treatment with the SP receptor antagonist, spantide. Treatment with SP produces phase shifts of circadian rhythm in spontaneous neural activity in SCN neurons with a phase-response curve that is similar to the effect of light pulses to animals under constant darkness. SP-induced phase change is also blocked by pretreatment with spantide. SP-induced increase in 2-DG uptake and phase changes in firing activity occur only during subjective night, at circadian times when photic phase shifting of activity occurs. The present results suggest that SP may be an important transmitter for conveying environmental light-dark information from retina to the SCN.

The effect of 5-HT3 receptor agonists and antagonists on the hypoxia/hypoglycemia (ischemia)-induced decrease in CA1 field potential elicited by stimulation of Schaffer collaterals was investigated using rat hippocampal slices. Treatment with the 5-HT3 receptor agonist, 2-methyl-5-HT (1-10 muM), exacerbated the ischemia-induced decreased in CA1 field potential, whereas treatment with the 5-HT3 receptor antagonist, Y-25130 (0.1-100 muM), or the 5-HT2 receptor antagonist, ketanserin (10, 100 muM), produced dose-dependent neuroprotection against the ischemia-induced decrease. However, in normal non-ischemic solution these treatments did not significantly change the CA1 field potential. The protective action of Y-25130 was blocked by co-treatment with 2-methyl-5-HT. The magnitude of protection in the Y-25130-treated group (EC50, 1.8 muM) was about 20 times greater than that in the ketanserin-treated group (EC50, 33 muM). The present study demonstrated that stimulation of 5-HT3 receptors plays a detrimental role in the development of ischemic damage, whereas blockade of the 5-HT3 receptor plays a neuroprotective role in ischemic damage, suggesting a facilitatory role of 5-HT neurons in ischemia-induced neuronal deficits.

To clarify the role of muscarinic acetylcholine receptors in the hypoxia/ hypoglycemia (ischemia)-induced functional deficit in hippocampal neurons, we examined the effect of cholinergic drugs on ischemia-induced impairments of glucose uptake and CA1 field potentials in hippocampus slices. Muscarinic receptors were subdivided into M1 (high affinity for pirenzepine) and M2 (low affinity for pirenzepine) subtypes. The M1 receptor subtype is coupled to an increase in phosphoinositide hydrolysis and the M2 receptor subtype is associated with inhibition of adenylate cyclase. The greater potency of carbachol in stimulating phosphoinositide hydrolysis resulted in exacerbated ischemia-induced deficits. Treatment with the muscarinic receptor antagonists scopolamine and pirenzepine (M1 receptor-selective antagonist) had a strong dose-dependent protective effect against ischemia-induced deficits. Oxotremorine and McN-A-343, weak stimulators of phosphoinositide hydrolysis and strong inhibitors of adenylate cyclase, had a weak neuroprotective action against ischemia-induced deficits. These results suggest that stimulation of M1 musarinic receptors coupled with an increase in phosphoinositide hydrolysis may play a facilatory role in ischemia-induced deficits. Stimulation of M2 muscarinic receptors may play an inhibitory role in ischemia-induced neuronal deficits.