Conditioned taste aversion (CTA) in mammals has several specific characteristics: (1) emergence of a negative symptom in subjects due to selective association with a taste-related stimulus, (2) robust long-term memory that is resistant to extinction induced by repeated presentation of the conditioned stimulus (CS), (3) a very-long-delay presentation of the unconditioned stimulus (US), and (4) single-trial learning. The pond snail, Lymnaea stagnalis, can also form a CTA. Although the negative symptoms, like nausea, in humans cannot be easily observed in invertebrate animal models of CTA, all the other characteristics of CTA seem to be present in snails. Selective associability was confirmed using a sweet sucrose solution and a bitter KCl solution. Once snails form a CTA, repeated presentation of the CS does not extinguish the CTA. A long interstimulus interval between the CS and US, like in trace conditioning, still results in the formation of a CTA in snails. Lastly, even single-trial learning has been demonstrated with a certain probability. In the present review, we compare, in detail, CTA in mammals and snails, and discuss the possible molecular events in CTA.

Steroid hormones play very important roles in gonadal differentiation in many vertebrate species. Previously, we have determined a threshold dosage of testosterone (T) to induce female-to-male sex reversal in Glandirana rugosa frogs. Genetic females formed a mixture of testis and ovary, the so-called ovotestis, when tadpoles of G. rugosa were reared in water containing the dosage of T, which enabled us to detect primary changes in the histology of the masculinizing gonads. In this study, we determined a threshold dosage of estradiol-17β (E2) to cause male-to-female sex reversal in this frog. We observed first signs of histological changes in the ovotestes, when tadpoles were reared in water containing the dosage of E2. Ovotestes were significantly larger than wild-type testes in size. By E2 treatment, male germ cells degenerated in the feminizing testis leading to their final disappearance. In parallel, oocytes appeared in the medulla of the ovotestis and later in the cortex as well. Quantitative polymerase chain reaction analysis revealed that the expression of sex-related genes involved in testis formation was significantly decreased in the ovotestis. In addition, immuno-positive signals of CYP17 that is involved in testis differentiation in this frog disappeared in the medulla first and then in the cortex. These results suggested that oocytes expanded in the feminizing gonad (ovary) contemporaneously with male germ cell disappearance. Primary changes in the histology of the gonads during male-to-female sex reversal occurred in the medulla and later in the cortex. This direction was opposite to that observed during female-to-male sex reversal in the G. rugosa frog.

Polymerase chain reaction (PCR)-based antigen tests are technically difficult, time-consuming, and expensive, and may produce false negative results requiring follow-up confirmation with computed tomography. The global coronavirus disease 2019 (COVID-19) pandemic has increased the demand for accurate, easy-to-use, rapid, and cost-effective antigen tests for clinical application. We propose a de novo antigen test for diagnosing COVID-19 using the combination of sandwich enzyme-linked immunosorbent assay and thio-nicotinamide adenine dinucleotide (thio-NAD) cycling. Our test takes advantage of the spike proteins specific to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The limit of detection of our test was 2.3 × 10−18 moles/assay. If the virus has ~25 spike proteins on its surface, our method should detect on the order of 10−20 moles of virus/assay, corresponding to ~104 copies of the virus RNA/assay. The detection sensitivity approaches that of PCR-based assays because the average virus RNA load used for PCR-based assays is ~105 copies per oro- or naso-pharyngeal swab specimen. To our knowledge, this is the first ultrasensitive antigen test for SARS-CoV-2 spike proteins that can be performed with an easy-to-use microplate reader. Sufficient sensitivity can be achieved within 10 min of thio-NAD cycling. Our antigen test allows for rapid, cost-effective, specific, ultrasensitive, and simultaneous multiple measurements of SARS-CoV-2, and has broad application for the diagnosis for COVID-19.

Despite the numerous risk factors for atherosclerotic cardiovascular diseases (ASCVD), cumulative evidence shows that electronegative low-density lipoprotein (L5 LDL) cholesterol is a promising biomarker. Its toxicity may contribute to atherothrombotic events. Notably, plasma L5 LDL levels positively correlate with the increasing severity of cardiovascular diseases. In contrast, traditional markers such as LDL-cholesterol and triglyceride are the therapeutic goals in secondary prevention for ASCVD, but that is controversial in primary prevention for patients with low risk. In this review, we point out the clinical significance and pathophysiological mechanisms of L5 LDL, and the clinical applications of L5 LDL levels in ASCVD can be confidently addressed. Based on the previously defined cut-off value by receiver operating characteristic curve, the acceptable physiological range of L5 concentration is proposed to be below 1.7 mg/dL. When L5 LDL level surpass this threshold, clinically relevant ASCVD might be present, and further exams such as carotid intima-media thickness, pulse wave velocity, exercise stress test, or multidetector computed tomography are required. Notably, the ultimate goal of L5 LDL concentration is lower than 1.7 mg/dL. Instead, with L5 LDL greater than 1.7 mg/dL, lipid-lowering treatment may be required, including statin, ezetimibe or PCSK9 inhibitor, regardless of the low-density lipoprotein cholesterol (LDL-C) level. Since L5 LDL could be a promising biomarker, we propose that a high throughput, clinically feasible methodology is urgently required not only for conducting a prospective, large population study but for developing therapeutics strategies to decrease L5 LDL in the blood.

Conditioned taste aversion (CTA) in the freshwater pulmonate Lymnaea stagnalis can be formed by presenting ten pairings of sucrose as the conditioned stimulus (CS) and KCl as the unconditioned stimulus (US). The CTA is consolidated to long-term memory (LTM) lasting longer than a month. In the present study, we examined the time course of protein synthesis-dependent period during the consolidation of Lymnaea CTA to LTM by pharmacological inhibition of transcription or translation. The robustness for CTA-LTM was then examined by extinction trials, i.e., repeated presentations of the CS alone. Furthermore, we evaluated the effects of the interstimulus interval (ISI) between the presentation of the CS and US. Our findings indicated that the protein synthesis-dependent period coincides with the CTA training. Repeated presentations of the CS alone after establishment of CTA did not extinguish the CTA, demonstrating the robustness of the CTA-LTM. The ISI ranged from 10 s to a few minutes, and there was no inverted U-shaped function between the ISI and the conditioned response (i.e., suppression of feeding). Thus, CTA still formed even when the presentation of the US was delayed. These features of Lymnaea CTA complement the knowledge for mammalian CTA.

The pleiotropic behavior of mesenchymal stem cells (MSCs) has gained global attention due to their immense potential for immunosuppression and their therapeutic role in immune disorders. MSCs migrate towards inflamed microenvironments, produce anti-inflammatory cytokines and conceal themselves from the innate immune system. These signatures are the reason for the uprising in the sciences of cellular therapy in the last decades. Irrespective of their therapeutic role in immune disorders, some factors limit beneficial effects such as inconsistency of cell characteristics, erratic protocols, deviating dosages, and diverse transfusion patterns. Conclusive protocols for cell culture, differentiation, expansion, and cryopreservation of MSCs are of the utmost importance for a better understanding of MSCs in therapeutic applications. In this review, we address the immunomodulatory properties and immunosuppressive actions of MSCs. Also, we sum up the results of the enhancement, utilization, and therapeutic responses of MSCs in treating inflammatory diseases, metabolic disorders and diabetes.

The Japanese wrinkled frog Glandirana rugosa is separated into five genetically different groups. One group in western Japan is further divided into three subgroups, found in Kyushu, Shikoku, and western Honshu. We collected G. rugosa frogs at 39 sites in Kyushu and determined nucleotide sequences of the mitochondrial 12S and 16S rRNA genes for phylogenetic analysis. Unexpectedly, we found a group of frogs in southeastern Kyushu that did not cluster with any of the pre-existing five groups of G. rugosa on the phylogenetic trees. The frogs in the new group and G. rugosa in Kyushu were externally similar, but there were a few significant differences in morphological features between the two populations. In addition, we observed significant differences in the frogs' calls . Thus, the group of the frogs in southeastern Kyushu may represent a new candidate species in the genus Glandirana. We discuss the possibility of a new species.

The role of serotonin in the immediate and delayed influence of physical exercise on brain functions has been intensively studied in mammals. Recently, immediate effects of intense locomotion on the decision-making under uncertainty were reported in the Great Pond snail, Lymnaea stagnalis (Korshunova et al., 2016). In this animal, serotonergic neurons control locomotion, and serotonin modulates many processes underlying behavior, including cognitive ones (memory and learning). Whether serotonin mediates the behavioral effects of intense locomotion in mollusks, as it does in vertebrates, remains unknown. Here, the delayed facilitating effects of intense locomotion on the decision-making in the novel environment are described in Lymnaea. Past exercise was found to alter the metabolism of serotonin, namely the content of serotonin precursor and its catabolites in the cerebral and pedal ganglia, as measured by high-performance liquid chromatography. The immediate and delayed effects of exercise on serotonin metabolism were different. Moreover, serotonin metabolism was regulated differently in different ganglia. Pharmacological manipulations of the serotonin content and receptor availability suggests that serotonin is likely to be responsible for the locomotor acceleration in the test of decision-making under uncertainty performed after exercise. However, the exercise-induced facilitation of decision-making (manifested in a reduced number of turns during the orienting behavior) cannot be attributed to the effects of serotonin.

As human-origin cells, human dental pulp stem cells (hDPSCs) are thought to be potentially useful for biological and medical experiments. They are easily obtained from lost primary teeth or extracted wisdom teeth, and they are mesenchymal stem cells that are known to differentiate into osteoblasts, chondrocytes, and adipocytes. Although hDPSCs originate from neural crest cells, it is difficult to induce hDPSCs to differentiate into neuron-like cells. To facilitate their differentiation into neuron-like cells, we evaluated various differentiation conditions. Activation of K+ channels is thought to regulate the intracellular Ca2+ concentration, allowing for manipulation of the cell cycle to induce the differentiation of hDPSCs. Therefore, in addition to a conventional neural cell differentiation protocol, we activated K+ channels in hDPSCs. Immunocyto-chemistry and real-time PCR revealed that applying a combination of 3 stimuli (high K+ solution, epigenetic reprogramming solution, and neural differentiation solution) to hDPSCs increased their expression of neuronal markers, such as β3-tubulin, postsynaptic density protein 95, and nestin within 5 days, which led to their rapid differentiation into neuron-like cells. Our findings indicate that epigenetic reprogramming along with cell cycle regulation by stimulation with high K+ accelerated the differentiation of hDPSCs into neuron-like cells. Therefore, hDPSCs can be used in various ways as neuron-like cells by manipulating their cell cycle.

Our goal was to develop an automated system to determine whether animals have learned and changed their behavior in real-time using a low calculation-power central processing unit (CPU). The bottleneck of real-time analysis is the speed of image recognition. For fast image recognition, 99.5% of the image was excluded from image recognition by distinguishing between the subject and the background. We achieved this by applying a binarization and connected-component labeling technique. This task is important for developing a fully automated learning apparatus. The use of such an automated system can improve the efficiency and accuracy of biological studies. The pond snail Lymnaea stagnalis can be classically conditioned to avoid food that naturally elicits feeding behavior, and to consolidate this aversion into long-term memory. Determining memory status in the snail requires real-time analysis of the number of bites the snail makes in response to food presentation. The main algorithm for counting bites comprises two parts: extracting the mouth images from the recorded video and measuring the bite rate corresponding to the memory status. Reinforcement-supervised learning and image recognition were used to extract the mouth images. A change in the size of the mouth area was used as the cue for counting the number of bites. The accuracy of the final judgment of whether or not the snail had learned was the same as that determined by human observation. This method to improve the processing speed of image recognition has the potential for broad application beyond biological fields.

For the diagnosis of disease, the ability to quantitatively detect trace amounts of the causal proteins from bacteria/viruses as biomarkers in patient specimens is highly desirable. Here we introduce a simple, rapid, and colorimetric assay as a de novo, ultrasensitive detection method. This ultrasensitive assay consists of a sandwich enzyme-linked immunosorbent assay (ELISA) and thionicotinamide-adenine dinucleotide (thio-NAD) cycling, forming an ultrasensitive ELISA, in which the signal substrate (i.e., thio-NADH) accumulates in a triangular manner, and the accumulated thio-NADH is measured at its maximum absorption wavelength of 405 nm. We have successfully achieved a limit of detection of ca. 10-18 moles/assay for a target protein. As an example of infectious disease detection, HIV-1 p24 could be measured at 0.0065 IU/assay (i.e., 10-18 moles/assay), and as a marker for a lifestyle-related disease, adiponectin could be detected at 2.3 × 10-19 moles/assay. In particular, despite the long-held belief that the trace amounts of adiponectin in urine can only be detected using a radioisotope, our ultrasensitive ELISA was able to detect urinary adiponectin. This method is highly versatile because simply changing the antibody enables the detection of various proteins. This assay system requires only the measurement of absorbance, thus it requires equipment that is easily obtained by medical facilities, which facilitates diagnosis in hospitals and clinics. Moreover, we describe an expansion of our ultrasensitive ELISA to a non-amplification nucleic acid detection method for nucleic acids using hybridization. These de novo methods will enable simple, rapid, and accurate diagnosis.

The ancestral insulin/relaxin peptide superfamily member relaxin-3 is an important regulator of food intake and behaviors related to anxiety and motivation. Relaxin family peptide receptor 1 (RXFP1) and RXFP3 are expressed in the rat hypothalamic paraventricular nucleus (PVN). Corticotropin-releasing factor (CRF) is produced in the PVN in response to stressors and promotes adrenocorticotropic hormone secretion from the anterior pituitary. We hypothesized that relaxin-3 directly regulates Crf expression in the hypothalamus and investigated its effect on Crf expression in cultured hypothalamic 4B cells. Relaxin-3 increased Crf mRNA levels and stimulated Crf promoter activity. Both protein kinase A and C pathways contributed to relaxin-3-induced Crf promoter activity. Rxfp1 and Rxfp3 mRNA and their proteins were expressed in cultured hypothalamic 4B cells. Relaxin-3 decreased Rxfp1 mRNA and protein levels and increased Rxfp3 mRNA and protein levels. These results suggested that the action of relaxin-3 in cultured hypothalamic 4B cells may be regulated through both RXFP1 and RXFP3.

Molluscan gastropods have long been used for studying the cellular and molecular mechanisms underlying learning and memory. One such gastropod, the pond snail Lymnaea stagnalis, exhibits long-term memory (LTM) following both classical and operant conditioning. Using Lymnaea, we have successfully elucidated cellular mechanisms of learning and memory utilizing an aversive classical conditioning procedure, conditioned taste aversion (CTA). Here, we present the behavioral changes following CTA training and show that the memory score depends on the duration of food deprivation. Then, we describe the relationship between the memory scores and the monoamine contents of the central nervous system (CNS). A comparison of learning capability in two different strains of Lymnaea, as well as the filial 1 (F1) cross from the two strains, presents how the memory scores are correlated in these populations with monoamine contents. Overall, when the memory scores are better, the monoamine contents of the CNS are lower. We also found that as the insulin content of the CNS decreases so does the monoamine contents which are correlated with higher memory scores. The present review deepens the relationship between monoamine and insulin contents with the memory score.

The pond snail Lymnaea stagnalis learns conditioned taste aversion (CTA) and consolidates it into long-term memory (LTM). How well they learn and form memory depends on the degree of food deprivation. Serotonin (5-HT) plays an important role in mediating feeding, and insulin enhances the memory consolidation process following CTA training. However, the relationship between these two signaling pathways has not been addressed. We measured the 5-HT content in the central nervous system (CNS) of snails subjected to different durations of food deprivation. One-day food-deprived snails, which exhibit the best learning and memory, had the lowest 5-HT content in the CNS, whereas 5-day food-deprived snails, which do not learn, had a high 5-HT content. Immersing 1-day food-deprived snails in 5-HT impaired learning and memory by causing an increase in 5-HT content, and that the injection of insulin into these snails reversed this impairment. We conclude that insulin rescues the CTA deficit and this may be due to a decrease in the 5-HT content in the CNS of Lymnaea.

To find a causal mechanism of learning and memory is a heuristically important topic in neuroscience. In the pond snail Lymnaea stagnalis, the following experimental facts have accrued regarding a classical conditioning procedure known as conditioned taste aversion (CTA): (1) one-day food-deprived Dutch snails have superior CTA memory formation; (2) the one-day food-deprived snails have a low monoamine content (e.g., serotonin, dopamine, octopamine) in their central nervous system (CNS); (3) fed or five-day food-deprived snails have poorer CTA memory and a higher monoamine content; (4) the Dutch snails form better CTA memory than the Canadian TC1 strain; and, (5) the F1 cross snails between the Dutch and Canadian TC1 strains also form poor CTA memory. Here, in one-day food-deprived snails, we measured the monoamine content in the CNSs of the 3 populations. In most instances, the monoamine content of the Dutch strain was lower than in the other two populations. The F1 cross snails had the highest monoamine content. A lower monoamine content is correlated with the better CTA memory formation.

Cannabinoids are hypothesized to play an important role in modulating learning and memory formation. Here, we identified mRNAs expressed in Lymnaea stagnalis central nervous system that encode two G-protein-coupled receptors (Lymnaea CBr-like 1 and 2) that structurally resemble mammalian cannabinoid receptors (CBrs). We found that injection of a mammalian CBr agonist WIN 55,212-2 (WIN 55) into the snail before operant conditioning obstructed learning and memory formation. This effect of WIN 55 injection persisted for at least 4 days following its injection. A similar obstruction of learning and memory occurred when a severe traumatic stimulus was delivered to L. stagnalis. In contrast, injection of a mammalian CBr antagonist AM 251 enhanced long-term memory formation in snails and reduced the duration of the effects of the severe traumatic stressor on learning and memory. Neither WIN 55 nor AM 251 altered normal homeostatic aerial respiratory behaviour elicited in hypoxic conditions. Our results suggest that putative cannabinoid receptors mediate stressful stimuli that alter learning and memory formation in Lymnaea. This is also the first demonstration that putative CBrs are present in Lymnaea and play a key role in learning and memory formation.

Each vertebrate species, as a general rule, has either the XX/XY or ZZ/ZW chromosomes by which sex is determined. However, the Japanese Rana (R.) rugosa frog is an exception, possessing both sex-determining combinations within one species, varying with region of origin. We collected R. rugosa frogs from 104 sites around Japan and South Korea and determined the nucleotide sequences of the mitochondrial 12S ribosomal RNA gene. Based on the sequences, R. rugosa frogs were divided into four groups from Japan and one from South Korea. The ZZ/ZW type is reportedly derived from the XX/XY type, although recently a new ZZ/ZW type of R. rugosa was reported. However, it still remains unclear from where the sex chromosomes in the five groups of this species were derived. In this study, we successfully isolated a sex-linked DNA maker and used it to classify R. rugosa frogs into several groupings. From the DNA marker as well as from nucleotide analysis of the promoter region of the androgen receptor (AR) gene, we identified another female heterogametic group, designated, West-Central. The sex chromosomes in the West-Central originated from the West and Central groups. The results indicate that a sex-linked DNA marker is a verifiable tool to determine the origin of the sex chromosomes in R. rugosa frogs in which the sex-determining system has changed, during two independent events, from the male to female heterogamety.

Each vertebrate species, as a general rule, has either the XX/XY or ZZ/ZW chromosomes by which sex is determined. However, the Japanese Rana (R.) rugosa frog is an exception, possessing both sex-determining combinations within one species, varying with region of origin. We collected R. rugosa frogs from 104 sites around Japan and South Korea and determined the nucleotide sequences of the mitochondrial 12S ribosomal RNA gene. Based on the sequences, R. rugosa frogs were divided into four groups from Japan and one from South Korea. The ZZ/ZW type is reportedly derived from the XX/XY type, although recently a new ZZ/ZW type of R. rugosa was reported. However, it still remains unclear from where the sex chromosomes in the five groups of this species were derived. In this study, we successfully isolated a sex-linked DNA maker and used it to classify R. rugosa frogs into several groupings. From the DNA marker as well as from nucleotide analysis of the promoter region of the androgen receptor (AR) gene, we identified another female heterogametic group, designated, West-Central. The sex chromosomes in the West-Central originated from the West and Central groups. The results indicate that a sex-linked DNA marker is a verifiable tool to determine the origin of the sex chromosomes in R. rugosa frogs in which the sex-determining system has changed, during two independent events, from the male to female heterogamety.

Introduction
In the Japanese frog Rana (R.) rugosa the androgen receptor (AR) gene on theWchromosome (W-AR) is barely expressed. Previously we showed that incomplete female-to-male sex-reversal occurred in Z-AR transgenic female frogs. To date, however, there is no report showing that AR with androgens can determine genetically programed male sex fate in any vertebrate species. Here, we examined whether AR together with androgens functions as a sex determinant in an amphibian species.
Methods
To examine whether complete female-to-male sex-reversal occurs in R. rugosa frogs, we produced AR-transgenic (Tg) and -knockdown (KD) female R. rugosa frogs by the I-SceI meganuclease-mediated gene trap and CRISPR/Cas9 system, respectively. AR-Tg and -KD tadpoles were reared in water containing testosterone (T) at 0 to 7.1 ng/ml. Frozen sections were prepared from the gonads of metamorphosed frogs and immunostained for laminin, Vasa, Pat1a, CYP17 and AR. We also employed PCR analysis to examine Dmrt1, Pat1a and CYP17 expression in the gonads of KD and placebo-KD female frogs.
Results
Complete female-to-male sex-reversal occurred in the AR-Tg ZW female frogs when a low dosage of T was supplied in the rearing water of tadpoles. However, no sex-reversal was observed in AR-KD ZW female frogs when the gonads were treated with dosages of T high enough to induce complete female-to-male sex-reversal even in wild type frogs.
Discussion
These results suggest that AR with its androgen ligand functions as a male sex-determinant in the ZW type R. rugosa frogs.

The pond snail Lymnaea stagnalis is capable of learning taste aversion by pairing presentations of a sucrose solution and an electric shock and consolidating it into long-term memory (LTM), which is referred to as conditioned taste aversion (CTA). We asked here if the neurotransmitter octopamine is involved in CTA. We first determined the levels of octopamine and its catabolites in the central nervous system (CNS) of snails with varying degrees of food deprivation, because CIA grades are correlated with degrees of food deprivation. We next manipulated the octopamine signaling using both an agonist and an antagonist of octopamine receptors and correlated their respective effects with CTA grades. We found that snails with the least amount of food-deprivation obtained the best CTA grade and had low levels of octopamine; whereas the most severely food-deprived snails did not form CTA and had the highest CNS octopamine levels. In modestly food-deprived snails, octopamine application increased the basal level of feeding response to a sucrose solution, and it did not obstruct CTA formation. Application of phentolamine, an octopamine receptor antagonist, to the most severely food-deprived snails decreased the basal level of feeding elicited by sucrose, but it did not enhance CTA formation. We conclude that octopamine involvement in OA formation in Lymnaea is at best weak, and that the changes in CNS octopamine content are an epiphenomenon. (C) 2017 Elsevier Inc. All rights reserved.

The pond snail Lymnaea stagnalis can acquire conditioned taste aversion (CTA) as a long-term memory. CTA is caused by the temporal pairing of a stimulus, such as sucrose (the conditioned stimulus; CS), with another stimulus, such as electric shock (the unconditioned stimulus; US). Previous studies have demonstrated changes in both cellular and molecular properties in a pair of neurons known as the cerebral giant cells (CGCs), suggesting that these neurons play a key role in CTA. Here we examined the necessity of the pair of CGC somata for the learning, memory formation and memory recall of CTA by using the soma ablation technique. There was no difference in the feeding response elicited by the CS before and after ablation of the CGC somata. Ablation of the CGC somata before taste-aversion training resulted in the learning acquisition, but the memory formation was not observed 24 h later. We next asked whether memory was present when the CGC somata were ablated 24 h after taste-aversion training. The memory was present before performing the somata ablation. However, when we tested snails five days after somata ablation, the memory recall was not present. Together the data show that: 1) the somata of the CGCs are not necessary for learning acquisition; 2) the somata are necessary for memory formation; and 3) the somata are necessary for memory recall. That is, these results demonstrate that the CGCs function in the long-term memory of CTA in Lymnaea.

The pond snail Lymnaea stagnalis learns and forms long-term memory (LTM) following both operant conditioning of aerial respiratory behavior and classical conditioning of taste aversive behavior. In the present study, we examined whether there are interstrain differences in the ability to form LTM following these two types of conditioning. A strain of Lymnaea (TC1) collected in Alberta, Canada exhibits superior memory-forming ability following aerial respiratory operant conditioning compared to a laboratory-reared strain of Lymnaea from Netherlands known as the Dutch strain. We asked whether the offspring of the Canadian TC1 and Dutch snails (i.e., filial 1 (F1) cross snails) would have the superior memory ability and found, rather, that their memory ability was average like the Dutch snails. That is, the Canadian TC1 snails have superior ability for LTM formation following aerial respiratory operant conditioning, but the Dutch and the generated F1 cross have average ability for memory forming. We next examined the Canadian TC1, Dutch and F1 cross snails for their ability to learn and form memory following conditioned taste aversion (CTA). All three populations showed similar associative CTA responses. However, both LTM formation and the ratio of good-to-poor performers in the memory retention test were much better in the Dutch snails than the Canadian TC1 and F1 cross snails. The memory abilities of the Canadian TC1 and F1 cross snails were average. Our present findings, therefore, suggest that snails of different strains have different memory abilities, and the F1 cross snails do not inherit the memory ability from the smart strain. To our knowledge, there have been a limited number of studies examining differences in memory ability among invertebrate strains, with the exception of studies using mutant flies.

The pond snail Lymnaea learns conditioned taste aversion (CTA) and remembers not to respond to food substances that initially cause a feeding response. The possible relationship between how well snails learn to follow taste-aversion training and brain dopamine contents is not known. We examined this relationship and found the following: first, snails in the act of eating just before the commencement of CTA training were poor learners and had the highest dopamine contents in the brain; second, snails which had an ad libitum access to food, but were not eating just before training, were average learners and had lower dopamine contents; third, snails food-deprived for one day before training were the best learners and had significantly lower contents of dopamine compared to the previous two cohorts. There was a negative correlation between the CTA grades and the brain dopamine contents in these three cohorts. Fourth, snails food-deprived for five days before training were poor learners and had higher dopamine contents. Thus, severe hunger increased the dopamine content in the brain. Because dopamine functions as a reward transmitter, CTA in the severely deprived snails (i.e. the fourth cohort) was thought to be mitigated by a high dopamine content.

Background: The pond snail Lymnaea stagnalis is capable of taste avoidance classical conditioning (TAC) with sucrose as the conditional stimulus (CS) and mechanical prodding as the unconditional stimulus (US). After successful training, feeding behavior is significantly suppressed in response to CS presentation.
New method: An automated apparatus is described for the training of multiple snails up to 10 snails at the same time. The new apparatus employs an electrical shock obtained from a 9-V dry cell to deliver a consistent and effective current amplitude of 0.4 mu A (i.e., the US).
Results: Using this apparatus, 10 snails can be conditioned simultaneously. We found that the optimal parameters to result in both short (STM) and long-term memory (LTM) were 15 paired presentations of the CS and US with a 5-min inter-trial interval (ITI) and 0.2-s current duration. However, both STM and LTM were observed with other ITIs tested. Successful TAC with only a single pairing of the CS US occurred with a CS of 100 mM sucrose solution for 60 s followed by a US of 9 V with 0.4 mu A for 5 s.
Comparison with existing method: The use of automated training apparatus for TAC will enable us to better examine the relationship between strength of CS and US. (C) 2015 Elsevier B.V. All rights reserved.

Each cell produces its own responses even if it appears identical to other cells. To analyze these individual cell characteristics, we need to measure trace amounts of molecules in a single cell. Nucleic acids in a single cell can be easily amplified by polymerase chain reaction, but single-cell measurement of proteins and sugars will require de novo techniques. In the present study, we outline the techniques we have developed toward this end. For proteins, our ultrasensitive enzymelinked immunosorbent assay (ELISA) coupled with thionicotinamide-adenine dinucleotide cycling can detect proteins at subattomoles per assay. For sugars, fluorescence correlation spectroscopy coupled with glucose oxidase-catalyzed reaction allows us to measure glucose at tens of nM. Our methods thus offer versatile techniques for single-cell-level analyses, and they are hoped to strongly promote single-cell biology as well as to develop noninvasive tests in clinical medicine.

To minimize patient suffering, the smallest possible volume of blood should be collected for diagnosis and disease monitoring. When estimating insulin secretion capacity and resistance to insulin in diabetes mellitus (DM), increasing insulin assay immunosensitivity would reduce the blood sample volume required for testing. Here we present an ultrasensitive ELISA coupled with thio-NAD cycling to measure immunoreactive insulin in blood serum. Only 5 mL of serum was required for testing, with a limit of detection (LOD) for the assay of 10-16 moles/assay. Additional recovery tests confirmed this method can detect insulin in sera. Comparisons between a commercially available immunoreactive insulin kit and our ultrasensitive ELISA using the same commercially available reference demonstrated good data correlation, providing further evidence of assay accuracy. Together, these results demonstrate our ultrasensitive ELISA could be a powerful tool in the diagnosis and treatment of not only DM but also many other diseases in the future.

Insulin is well known as a hormone regulating glucose homeostasis across phyla. Although there are insulin-independent mechanisms for glucose uptake in the mammalian brain, which had contributed to a perception of the brain as an insulin-insensitive organ for decades, the finding of insulin and its receptors in the brain revolutionized the concept of insulin signaling in the brain. However, insulin’s role in brain functions, such as cognition, attention, and memory, remains unknown. Studies using invertebrates with their open blood-vascular system have the promise of promoting a better understanding of the role played by insulin in mediating/modulating cognitive functions. In this review, the relationship between insulin and its impact on long-term memory (LTM) is discussed particularly in snails. The pond snail Lymnaea stagnalis has the ability to undergo conditioned taste aversion (CTA), that is, it associatively learns and forms LTM not to respond with a feeding response to a food that normally elicits a robust feeding response. We show that molluscan insulin-related peptides are up-regulated in snails exhibiting CTA–LTM and play a key role in the causal neural basis of CTA–LTM. We also survey the relevant literature of the roles played by insulin in learning and memory in other phyla.

Food deprivation for 1 day in the pond snail Lymnaea stagnalis before aversive classical conditioning results in optimal conditioned taste aversion (CTA) and long-term memory (LTM) formation, whereas 5-day food deprivation before training does not. We hypothesize that snails do in fact learn and form LTM when trained after prolonged food deprivation, but that severe food deprivation blocks their ability to express memory. We trained 5-day food-deprived snails under various conditions, and found that memory was indeed formed but is overpowered by severe food deprivation. Moreover, CTA-LTM was context dependent and was observed only when the snails were in a context similar to that in which the training occurred.

To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10(-18) moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10(-18) moles of the p24/assay corresponds to ca. 10(3) copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (10(2) copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.

Behavioural/motivational state is known to influence nearly all aspects of physiology and behaviour. The cellular basis of behavioural state control is only partially understood. Our investigation, performed on the pond snail Lymnaea stagnalis whose nervous system is useful for work on completely isolated neurons, provided several results related to this problem. First, we demonstrated that the behavioural state can produce long-term changes in individual neurons that persist even after neuron isolation from the nervous system. Specifically, we found that pedal serotonergic neurons that control locomotion show higher activity and lower membrane potential after being isolated from the nervous systems of hungry animals. Second, we showed that the modulatory state (the chemical neuroactive microenvironment of the central ganglia) changes in accordance with the nutritional state of an animal and produces predicted changes in single isolated locomotor neurons. Third, we report that observed hunger-induced effects can be explained by the increased synthesis of serotonin in pedal serotonergic neurons, which has an impact on the electrical activity of isolated serotonergic neurons and the intensity of extrasynaptic serotonin release from the pedal ganglia.

The pond snail Lymnaea stagnalis can learn conditioned taste aversion and then consolidate it into long-term memory (LTM). A high-voltage electric shock was used as the unconditioned stimulus, where we have previously used KCl. We varied the strength of both the conditioned and unconditioned stimuli to determine whether the so-called Yerkes-Dodson law prevailed. This is an empirical relationship between the state of arousal and LTM formation, showing that there is an optimal level of arousal leading to memory formation. However, too little or too much arousal results in poorer LTM. We found here that the most appropriate stimuli to use in taste aversion training in Lymnaea were a 10 mmol l(-1) sucrose solution as the conditioned stimulus and a 3 s electric shock as the unconditioned stimulus.

Serotonin (5-hydroxytryptamine: 5-HT) is a multimodal transmitter that controls both feeding response and heartbeat in snails. However, the effects of 5-HT on the hunger state are still unknown. We therefore examined the relation among the hunger state, the heartbeat rate and the 5-HT action in food-starved snails. We found that the hunger state was significantly distinguished by the heartbeat rate in snails. The heartbeat rate was high in the food-satiated snails, whereas it was low in the food-starved snails. An increase in 5-HT concentration in the body boosted the heartbeat rate in the food-starved snails, but did not affect the rate in the food-satiated snails. These results suggest that 5-HT application may mimic the change from a starvation to a satiation state normally achieved by direct ingestion of food.

The activity of thermo-transient receptor potential (TRP) channels is highly dependent on temperature, and thus thermo-TRP reactions have a high temperature coefficient Q10. In thermodynamics, a high value of Q10 indicates the existence of a large activation energy (i.e., a large enthalpy) over a short period during the transition process between the closed and open states of the channels. The Gibbs free energy equation shows that a large entropy is required to compensate for this large enthalpy and permit activation of the channels, suggesting a large conformational change of the channels. These large values of enthalpy and entropy seem to be a match for the values of the unfolding process of globular proteins. We outline these thermodynamic issues in thermo-TRPs.

The involvement of serotonin in mediating hunger-related changes in behavioral state has been described in many invertebrates. However, the mechanisms by which hunger signals to serotonergic cells remain unknown. We tested the hypothesis that serotonergic neurons can directly sense the concentration of glucose, a metabolic indicator of nutritional state. In the snail Lymnaea stagnalis, we demonstrate that completely isolated pedal serotonergic neurons that control locomotion changed their biophysical characteristics in response to glucose application by lowering membrane potential and decreasing the firing rate. Additionally, the excitatory response of the isolated serotonergic neurons to the neuroactive microenvironment of the pedal ganglia was significantly lowered by glucose application. Because hunger has been reported to increase the activity of select neurons and their responses to the pedal ganglia microenvironment, these responses to glucose are in accordance with the hypothesis that direct glucose signaling is involved in the mediation of the hunger-related behavioral state.

Conditioned taste aversion (CTA) in Lymnaea is brought about by pairing a sucrose solution (the conditioned stimulus, CS) with an electric shock (the unconditioned stimulus, US). Following repeated CS-US pairings, CTA occurs and it is consolidated into long-term memory (LTM). The best CTA is achieved, if snails are food-deprived for I day before training commences. With a longer period of food deprivation (5 days), learning and memory formation does not occur. It has been hypothesized that the levels of insulin in the central nervous system (CNS) are very important for CTA to occur. To test his hypothesis, we injected insulin directly into 5-day food-deprived snails. The injection of insulin, as expected, resulted in a decrease in hemolymph glucose concentration. Consistent with our hypothesis with insulin injection, learning and memory formation of CTA occurred. That is, the 'insulin spike' is more important than an increase in hemolymph glucose concentration for CTA-LTM. If we injected an insulin receptor antibody into the snails before the insulin injection, learning was formed but memory formation was not, which is consistent with our previous study. Therefore, a rise in the insulin concentration (i.e., insulin spike) in the CNS is considered to be a key determining factor in the process of CTA-LTM. (C) 2014 Elsevier Inc. All rights reserved.

Acetylcholine plays various important roles in the central nervous system of invertebrates as well as vertebrates. In the olfactory center of the terrestrial slug Limax, the local field potential (LFP) oscillates, and the change in its oscillatory frequency is thought to correlate with the detection of odor that potentially changes an ongoing behavior of the animal. Acetylcholine is known to upregulate the frequency of the LFP oscillation, and is one of the candidates for the neurotransmitters that are involved in such higher cognitive functions. However, there have been no histological data on the cholinergic system in gastropods, nor are there data on the receptors that are responsible for the upregulation of the oscillatory frequency of LFP due to the lack of analytical tools (such as antibodies or cDNA sequence information on cholinergic system-related genes). Here we cloned the cDNAs of choline acetyltransferase (ChAT), acetylcholinesterase, vesicular acetylcholine transporter, and several nicotinic acetylcholine receptors (nAChRs), and investigated their localization in the brain of Limax. We also generated a polyclonal antibody against ChAT to examine its localization, and investigated pharmacologically the involvement of nAChRs in the LFP oscillation. Our data showed: 1) dense distribution of the neurons expressing mRNAs of ChAT and vesicular acetylcholine transporter in the olfactory center
2) spatially unique expression patterns of different nAChRs in the olfactory center
3) involvement of nAChRs in the upregulation of the oscillation
4) localization of ChAT protein in nerve fibers and/or terminals
and 5) the presence of cholinergic nerves in the tentacles. © 2014 Wiley Periodicals, Inc.

The honeybee waggle dance communicates the location of profitable food sources, usually with a certain degree of error in the directional information ranging from 10-15 degrees at the lower margin. We simulated one-day colonial foraging to address the biological significance of information error in the waggle dance. When the error was 30 degrees or larger, the waggle dance was not beneficial. If the error was 15 degrees, the waggle dance was beneficial when the food sources were scarce. When the error was 10 degrees or smaller, the waggle dance was beneficial under all the conditions tested. Our simulation also showed that precise information (0-5 degrees error) yielded great success in finding feeders, but also caused failures at finding new feeders, i.e., a high-risk high-return strategy. The observation that actual bees perform the waggle dance with an error of 10-15 degrees might reflect, at least in part, the maintenance of a successful yet risky foraging trade-off.

The pond snail Lymnaea stagnalis is capable of being classically conditioned to avoid food and to consolidate this aversion into a long-term memory (LTM). Previous studies have shown that the length of food deprivation is important for both the acquisition of taste aversion and its consolidation into LTM, which is referred to as conditioned taste aversion (CTA). Here we tested the hypothesis that the hemolymph glucose concentration is an important factor in the learning and memory of CTA. One-day food deprivation resulted in the best learning and memory, whereas more prolonged food deprivation had diminishing effects. Five-day food deprivation resulted in snails incapable of learning or remembering. During this food deprivation period, the hemolymph glucose concentration decreased. If snails were fed for 2 days following the 5-day food deprivation, their glucose levels increased significantly and they exhibited both learning and memory, but neither learning nor memory was as good as with the 1-day food-deprived snails. Injection of the snails with insulin to reduce glucose levels resulted in better learning and memory. Insulin is also known to cause a long-term enhancement of synaptic transmission between the feeding-related neurons. On the other hand, injection of glucose into 5-day food-deprived snails did not alter their inability to learn and remember. However, if these snails were fed on sucrose for 3 min, they then exhibited learning and memory formation. Our data suggest that hemolymph glucose concentration is an important factor in motivating acquisition of CTA in Lymnaea and that the action of insulin in the brain and the feeding behavior are also important factors. (C) 2013 Elsevier Inc. All rights reserved.

An ultrasensitive method for the determination of proteins is described that combines an enzyme-linked immunosorbent assay (ELISA) and a thionicotinamide-adenine dinucleotide (thio-NAD) cycling method. A sandwich method using a primary and a secondary antibody for antigens is employed in an ELISA. An androsterone derivative, 3α-hydroxysteroid, is produced by the hydrolysis of 3α-hydroxysteroid 3-phosphate with alkaline phosphatase linked to the secondary antibody. This 3α-hydroxysteroid is oxidized to a 3-ketosteroid by 3α-hydroxysteroid dehydrogenase (3α-HSD) with a cofactor thio-NAD. By the opposite reaction, the 3-ketosteroid is reduced to a 3α-hydroxysteroid by 3α-HSD with a cofactor NADH. During this cycling reaction, thio-NADH accumulates in a quadratic function-like fashion. Accumulated thio-NADH can be measured directly at an absorbance of 400 nm without any interference from other cofactors. These features enable us to detect a target protein with ultrasensitivity (10–19 mol/assay) by measuring the cumulative quantity of thio-NADH. Our ultrasensitive determination of proteins thus allows for the detection of small amounts of proteins only by the application of thio-NAD cycling reagents to the usual ELISA system.

The brain of gastropod mollusks contains many giant neurons with polyploid genomic DNAs. Such DNAs are generated through repeated DNA endoreplication during body growth. However, it is not known what triggers DNA endoreplication in neurons. There are two possibilities: (1) DNAs are replicated in response to some unknown molecules in the hemolymph that reflect the nutritive status of the animal; or (2) DNAs are replicated in response to some unknown factors that are retrogradely transported through axons from the innervated target organs. We first tested whether hemolymph with rich nutrition could induce DNA endoreplication. We tested whether the transplanted brain exhibits enhanced DNA endoreplication like an endogenous brain does when transplanted into the homocoel of the body of a slug whose body growth is promoted by an increased food supply. However, no enhancement was observed in the frequency of DNA endoreplication when we compared the transplanted brains in the growth-promoted and growth-suppressed host slugs, suggesting that the humoral environment is irrelevant to triggering the body growth-dependent DNA endoreplication. Next, we tested the requirement of target innervation by surgically dissecting a unilateral posterior pedal nerve of an endogenous brain. Substantially lower number of neurons exhibited DNA endoreplication in the pedal ganglion ipsilateral to the dissected nerve. These results support the view that enhanced DNA endoreplication is mediated by target innervation and is not brought about through the direct effect of humoral factors in the hemolymph during body growth. (c) 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 609-620, 2013

The neural circuitry comprising the central pattern generator (CPG) that drives feeding behavior in the great pond snail (Lymnaea stagnalis (L., 1758)) has been worked out. Because the feeding behavior undergoes associative learning and long-term memory (LTM) formation, it provides an excellent opportunity to study the causal neuronal mechanisms of these two processes. In this review, we explore some of the possible causal neuronal mechanisms of associative learning of conditioned taste aversion (CTA) and its subsequent consolidation processes into LTM in L. stagnalis. In the CTA training procedure, a sucrose solution, which evokes a feeding response, is used as the conditioned stimulus (CS) and a potassium chloride solution, which causes a withdrawal response, is used as the unconditioned stimulus (US). The pairing of the CS-US alters both the feeding response of the snail and the function of a pair of higher order interneurons in the cerebral ganglia. Following the acquisition of CTA, the polysynaptic inhibitory synaptic input from the higher order interneurons onto the feeding CPG neurons is enhanced, resulting in suppression of the feeding response. These changes in synaptic efficacy are thought to constitute a "memory trace" for CTA in L. stagnalis.

The pond snail Lymnaea stagnalis learns taste aversion and consolidates it into long-term memory (LTM). This is referred to as conditioned taste aversion (CTA). The superfusion of molluscan insulin-related peptides (MIPs) over the isolated snail brain causes a long-term enhancement of synaptic input between the cerebral giant cell and the B1 buccal motor neuron. This enhancement is hypothesized to underlie CTA. The synaptic enhancement caused by the superfusion of MIPs can be blocked by the application of human insulin receptor antibody, which recognizes the extracellular domain of human insulin receptor and acts as an antagonist even for MIP receptors. An injection of the human insulin receptor antibody into the abdominal cavity of trained snails blocks the consolidation process leading to LTM, even though the snails acquire taste aversion. Here, we examined whether or not taste-aversion training changes the mRNA expression level of MIP receptor in the snail brain and found that it does not. This result, taken together with previous findings, suggest that the MIPs' effect on synaptic function in the snail brain is attributable to a change in the MIP concentration, and not to a change in the mRNA expression level of MIP receptor, which is thought to reflect the number of MIP receptors. © 2013 Landes Bioscience.

Insulin's action in the brain can directly alter cognitive functioning. We have recently shown that molluscan insulin-related peptides are upregulated following a conditioned taste aversion (CTA) training procedure. In addition, when mammalian insulin is superfused over the isolated Lymnaea stagnalis central nervous system, it elicits long-term synaptic enhancement at the monosynaptic connection between the cerebral giant cell and the buccal 1 (B1) motor neuron. This synaptic enhancement is thought to be a neural correlate of CTA. Here, we examined whether the observed changes in synaptic plasticity were the result of presynaptic and/or postsynaptic alterations using the paired pulse procedure. The paired pulse ratio was unaltered following insulin application, suggesting that insulin's effects on synaptic plasticity are mediated postsynaptically in the B1 motor neuron. Thus, it was suggested that postsynaptic changes need to be considered when insulin's actions on synaptic plasticity are examined.

The synaptology of the cell body layer of the olfactory center, procerebrum, was investigated in two prominent terrestrial pulmonate gastropod species, Helix pomatia and Limax valentianus. In addition, the analysis of the 5-HT-immunoreactive innervation, including ultrastructural level, was performed at high resolution in H. pomatia. A highly complex system of synaptic and non-synaptic connections was found in the procerebrum of both species connected to local neuropil areas of different size. The procerebral (globuli) cell perikarya were richly innervated by varicosities meanwhile the axon profiles also established contacts with each other. Synaptic configurations including convergence, divergence and presynaptic modulation were also revealed. The frequent occurrence of unspecialized but close axo-somatic and axo-axonic membrane contacts referring to the modulatory forms of transmitter release were also accompanied by membrane configurations indicative of active exocytosis. In H. pomatia, the cell mass layer was shown to receive a rich 5-HT-immunoreactive innervation, forming a dense network around the cell bodies. At ultrastructural level, 5-HT-immunoreactive varicosities contacted both cell bodies and different unlabeled axon profiles. Our results suggest that the local neuropil regions in the cell body layer are site of local circuits, which may play a decisive role in olfactory integrative processes bound to the procerebrum. The pattern and form of the 5-HT-immunoreactive innervation of extrinsic origin suggest an overall modulatory role in the cell body layer. The results may serve a basis for considering the role of local intercellular events, connected to microcircuits, within the procerebrum cell body layer involved in oscillation activities.

A simple spectrophotometric method for the assay of steroid 5α-reductase (5α-sr) was developed in which 5α-dihydrotestosterone (5α-dht) and 5α-androstane-3α,17β-diol (5α-diol), metabolites formed in the nadph-dependent reduction of testosterone with enzyme sources of 5α-sr, were measured by enzymatic cycling using 3α-hydroxysteroid dehydrogenase in the presence of excess thionicotinamide-adenine dinucleotide (thio-nad) and nadh. it was found that 5α-sr activity was proportional to the accumulated thio-nadh having an absorption maximum at 400 nm. because of the high cycling rate (&gt
600 cycle per min) and no interference from testosterone, enzymatic cycling can determine the sum of 5α-dht and 5α-diol at the picomole level without separation from excess testosterone. the present method was readily applicable to the assay of 5α-sr activity of rat liver and prostate microsomes as well as to the assay of inhibitory activity of finasteride, a synthetic inhibitor of 5α-sr. ©2013 The Japan Society for Analytical Chemistry.

Fluorescence correlation spectroscopy (FCS) is a technique in which measurement of fluorescence intensity fluctuations is used to clarify dynamic molecular interactions within a very small space in a solution containing a small number of fluorescent molecules. The FCS-based analysis gives the average number and average diffusion time of the fluorescent molecules during their passage through a very small space. One advantage of FCS is that physical separation between free and bound fluorescent probes is not required because the properties of fluorescence fluctuations are accounted for. Therefore, when fluorescent probes are bound with proteins by peroxidase and hydrogen peroxide (H2O2), FCS enables us to detect H2O2 with high sensitivity. In addition, because H2O2 is generated by oxidase-catalyzed reactions, a highly sensitive method for detecting H2O2 is applicable to the measurement of low levels of various oxidases and their substrates, such as glucose. We here describe the protocol of a de novo, highly sensitive method for the measurement of H2O2 and glucose using FCS.

The pond snail Lymnaea stagnalis is capable of learning taste aversion and consolidating this learning into long-term memory (LTM) that is called conditioned taste aversion (CTA). Previous studies showed that some molluscan insulin-related peptides (MIPs) were upregulated in snails exhibiting CTA. We thus hypothesized that MIPs play an important role in neurons underlying the CTA-LTM consolidation process. To examine this hypothesis, we first observed the distribution of MIP II, a major peptide of MIPs, and MIP receptor and determined the amounts of their mRNAs in the CNS. MIP II was only observed in the light green cells in the cerebral ganglia, but the MIP receptor was distributed throughout the entire CNS, including the buccal ganglia. Next, when we applied exogenous mammalian insulin, secretions from MIP-containing cells or partially purified MIPs, to the isolated CNS, we observed a long-term change in synaptic efficacy (i.e., enhancement) of the synaptic connection between the cerebral giant cell (a key interneuron for CTA) and the B1 motor neuron (a buccal motor neuron). This synaptic enhancement was blocked by application of an insulin receptor antibody to the isolated CNS. Finally, injection of the insulin receptor antibody into the snail before CTA training, while not blocking the acquisition of taste aversion learning, blocked the memory consolidation process; thus, LTM was not observed. These data suggest that MIPs trigger changes in synaptic connectivity that may be correlated with the consolidation of taste aversion learning into CTA-LTM in the Lymnaea CNS.

Conditioned taste aversion (CTA) can be classically conditioned in the pond snail Lymnaea stagnalis and subsequently be consolidated into long-term memory (LTM). The neural trace that subserves CTA-LTM can be summarized as follows: A polysynaptic inhibitory postsyn-aptic potential recorded in the neuron 1 medial (N1M) cell in the conditioned snails as a result of activation of the cerebral giant cell (CGC) is larger and lasts longer than that in control snails. The N1M cell is ultimately activated by the CGC via the neuron 3 tonic (N3t) cell. That is, the inhibitory monosynaptic inputs from the N3t cell to the N1M cell are facilitated. The N1M and N3t cells are the members of feeding central pattern generator, whereas the CGC is a multimodal interneuron thought to play a key role in feeding behavior. Here we examined the involvement of a second messenger, cAMP, in the establishment of the memory trace. We injected cAMP into the CGC and monitored the potentials of the B3 motor neuron activated by the CGC. B3 activity is used as an index for the synaptic inputs from the N3t cell to the N1M cell. We found that the B3 potentials were transiently enlarged. Thus, when the cAMP concentration is increased in the CGC by taste aversion training, cAMP-induced changes may play a key role in the establishment of a memory trace in the N3t cell. © 2013 THE BIOPHYSICAL SOCIETY OF JAPAN.

Background: The pond snail Lymnaea stagnalis can maintain a conditioned taste aversion (CTA) as a long-term memory. Previous studies have shown that the inhibitory postsynaptic potential (IPSP) evoked in the neuron 1 medial (N1M) cell by activation of the cerebral giant cell (CGC) in taste aversion-trained snails was larger and lasted longer than that in control snails. The N1M cell is one of the interneurons in the feeding central pattern generator (CPG), and the CGC is a key regulatory neuron for the feeding CPG.
Methodology/Principle Findings: Previous studies have suggested that the neural circuit between the CGC and the N1M cell consists of two synaptic connections: (1) the excitatory connection from the CGC to the neuron 3 tonic (N3t) cell and (2) the inhibitory connection from the N3t cell to the N1M cell. However, because the N3t cell is too small to access consistently by electrophysiological methods, in the present study the synaptic inputs from the CGC to the N3t cell and those from the N3t cell to the N1M cell were monitored as the monosynaptic excitatory postsynaptic potential (EPSP) recorded in the large B1 and B3 motor neurons, respectively. The evoked monosynaptic EPSPs of the B1 motor neurons in the brains isolated from the taste aversion-trained snails were identical to those in the control snails, whereas the spontaneous monosynaptic EPSPs of the B3 motor neurons were significantly enlarged.
Conclusion/Significance: These results suggest that, after taste aversion training, the monosynaptic inputs from the N3t cell to the following neurons including the N1M cell are specifically facilitated. That is, one of the memory traces for taste aversion remains as an increase in neurotransmitter released from the N3t cell. We thus conclude that the N3t cell suppresses the N1M cell in the feeding CPG, in response to the conditioned stimulus in Lymnaea CTA.

J. Neurochem. (2012) 122, 727737. Abstract The brain of gastropod mollusks contains giant neurons whose nuclei are enlarged with a large amount of genomic DNA. Such DNA is produced by repeated endoreplication. We have previously demonstrated that the frequency of the neuronal DNA endoreplication is correlative to the body growth of the adult land slug and to the increase in the amount of transcripts within the neuron. However, it has long been controversial whether the neuronal DNA endoreplication entails whole genome amplification (polyploidy), or whether only the necessary genomic loci are amplified (polyteny, polysomy, or cis-amplification by unequal recombination). In the present study, we adopted two modern techniques quantitative genomic PCR and 5'-bromodeoxyuridine labeling to distinguish between these two possibilities. Our results demonstrated that multiple genomic loci were amplified to the same extent irrespective of the transcriptional activities at these loci. Moreover, the visceral giant cell, the biggest neuron in the slugs brain, was estimated to contain approximately 10 000-times as much genomic DNA as the haploid amount. The 5'-bromodeoxyuridine-labeling experiments also revealed a uniform DNA synthesis within the nucleus. These results strongly support the idea that the giant neurons contain a polyploid genome rather than a locus-specific amplified genome.

Kobayashi S, Matsuo R, Sadamoto H, Watanabe S, Ito E. Excitatory effects of GABA on procerebrum neurons in a slug. J Neurophysiol 108: 989-998, 2012. First published May 9, 2012; doi:10.1152/jn.01137.2010.-Classical neurotransmitters, such as glutamate and gamma-aminobutyric acid (GABA), often have different actions on invertebrate neurons from those reported for vertebrate neurons. In the terrestrial mollusk Limax, glutamate was found to function as an inhibitory transmitter in the procerebrum (PC), but it has not yet been clarified how GABA Acts in the PC. We thus examined what effects GABA exerts on PC neurons in the present study. For this purpose, we first applied GABA to isolated PC preparations and recorded postsynaptic currents and potentials in PC neurons. The GABA Application reduced the amplitude of inhibitory postsynaptic currents and depolarization-induced outward currents recorded in nonbursting neurons and increased the number of spontaneous spikes of nonbursting neurons. However, direct GABA-induced currents were not observed in either bursting or nonbursting neurons. These results suggest a potential direct effect of GABA on outward currents resulting in enhanced excitability of PC neurons. Next, we measured the change in [Ca2+](i) in cultured PC neurons by application of GABA. The GABA Application increased spontaneous Ca2+ events in cultured neurons. These Ca2+ events were ascribable to the influx of extracellular Ca2+. We then confirmed the presence of GABA And GABA receptors in the PC. The GABA-like immunoreactivity was observed in the neuropil layers of the PC, and the mRNAs for both GABA(A) and GABA(B) receptors were expressed in the PC. In particular, GABA(B) receptor mRNA, rather than GABA(A), was found to be more abundantly expressed in the PC. These results suggest that GABA functions as an excitatory modulator for PC neurons via mainly GABA(B) receptors.

A honeybee informs her nestmates about the location of a profitable food source that she has visited by means of a waggle dance: a round dance and a figure-of-eight dance for a short- and long-distance food source, respectively. Consequently, the colony achieves an effective collection of food. However, it is still not fully understood how much effect the dance behavior has on the food collection, because most of the relevant experiments have been performed only in limited locations under limited experimental conditions. Here, we examined the efficacy of the waggle dances by physically preventing bees from dancing and then analyzing the changes in daily mass of the hive as an index of daily food collection. To eliminate place-and year-specific effects, the experiments were performed under fully natural conditions in three different cities in Japan from mid September to early October in three different years. Because the experiments were performed in autumn, all six of the tested colonies lost mass on most of the experimental days. When the dance was prevented, the daily reduction in mass change was greater than when the dance was allowed, i.e. the dance inhibited the reduction of the hive mass. This indicates that dance is effective for food collection. Furthermore, clear inhibition was observed on the first two days of the experiments; after that, inhibition was no longer evident. This result suggests that the bee colony adapted to the new environment.

The gating of ion channel of ionotropic glutamate receptors is controlled by the structural change of the ligand-binding domain of GluR2. We examined the roles of residues in the glutamate-binding and cleft-closing mechanisms by molecular dynamics (MD) simulations. A glutamate entered the cleft deeply within the order of nanoseconds and the cleft locked the glutamate completely at 15 ns in an MD run. TYR450 seemed to regulate the orientation of the glutamate upon binding by cation-pi interaction. A semi-open state was identified in the free energy profile evaluated with the structures on the spontaneously glutamate-bound and cleft-closed pathway by the unbiased MD simulations for the first time to our knowledge. In the semi-open state, the two sub-domains were bridged by two hydrogen bonds of GLU705 in the sub-domain 2 with TYR732 in the sub-domain 1 and with the glutamate bound to the sub-domain 1 until the transition to the closed state. (C) 2011 Elsevier RV. All rights reserved.

A honeybee informs her nestmates of the location of a flower by doing a waggle dance. The waggle dance encodes both the direction of and distance to the flower from the hive. To reveal how the waggle dance benefits the colony, we created a Markov model of bee foraging behavior and performed simulation experiments by incorporating the biological parameters that we obtained from our own observations of real bees as well as from the literature. When two feeders were each placed 400 m away from the hive in different directions, a virtual colony in which honeybees danced and correctly transferred information (a normal, real bee colony) made significantly greater numbers of successful visits to the feeders compared to a colony with inaccurate information transfer. Howerer, when five feeders were each located 400 m from the hive, the inaccurate information transfer colony performed better than the normal colony. These results suggest that dancing's ability to communicate accurate information depends on the number of feeders. Furthermore, because non-dancing colonies always made significantly fewer visits than those two colonies, we concluded that dancing behavior is beneficial for hives' ability to visit food sources.

The tripeptide Asn-d-Trp-Phe-NH2 (NdWFamide) is a D-amino acid-containing cardioexcitatory peptide initially isolated from Aplysia. Previously we detected NdWFamide immunoreactivity in the visceral giant cells, the largest neurons in the brain of the terrestrial slug Limax located at the dorsal surface of the visceral ganglia. In the present study, we further analyzed the morphological features of these neurons by an intracellular injection of Lucifer yellow, and found that these neurons extend neurites out of the brain through at least 5 nerve bundles. We then isolated a gene and a cDNA clone potentially encoding a NdWFamide precursor, and investigated expression at the levels of mRNA and protein in Limax. The NdWFamide gene consists of 5 exons spanning at least 17 kb of the genome, and its open reading frame extends over 3 exons. The spatial expression pattern of NdWFamide mRNA was almost identical to that of the NdWFamide peptide, with some minor discrepancies in between. Although the most remarkable expression was evident in the visceral giant cells, we also found the expression of NdWFamide mRNA and peptide in the cerebral and pedal ganglia. These results suggest the involvement of NdWFamide in the regulation of a broad area of the slug's body. (C) 2011 Elsevier Inc. All rights reserved.

Background: Because H(2)O(2) is generated by various oxidase-catalyzed reactions, a highly sensitive determination method of H(2)O(2) is applicable to measurements of low levels of various oxidases and their substrates such as glucose, lactate, glutamate, urate, xanthine, choline, cholesterol and NADPH. We propose herein a new, highly sensitive method for the measurement of H(2)O(2) and glucose using fluorescence correlation spectroscopy (FCS).
Methodology/Principal Findings: FCS has the advantage of allowing us to determine the number of fluorescent molecules. FCS measures the fluctuations in fluorescence intensity caused by fluorescent probe movement in a small light cavity with a defined volume generated by confocal illumination. We thus developed a highly sensitive determination system of H(2)O(2) by FCS, where horseradish peroxidase (HRP) catalyzes the formation of a covalent bond between fluorescent molecules and proteins in the presence of H(2)O(2). Our developed system gave a linear calibration curve for H(2)O(2) in the range of 28 to 300 nM with the detection limit of 8 nM. In addition, by coupling with glucose oxidase (GOD)-catalyzed reaction, the method allows to measure glucose in the range of 80 nM to 1.5 mu M with detection limit of 24 nM. The method was applicable to the assay of glucose in blood plasma. The mean concentration of glucose in normal human blood plasma was determined to be 4.9 mM.
Conclusions/Significance: In comparison with commercial available methods, the detection limit and the minimum value of determination for glucose are at least 2 orders of magnitude more sensitive in our system. Such a highly sensitive method leads the fact that only a very small amount of plasma (20 nL) is needed for the determination of glucose concentration in blood plasma.

In the central nervous system of the pond snail Lymnaea stagnalis, serotonergic transmission plays an important role in controlling feeding behavior. Recent electrophysiological studies have claimed that only metabotropic serotonin (5-HT(2)) receptors, and not ionotropic (5-HT(3)) receptors, are used in synapses between serotonergic neurons (the cerebral giant cells, CGCs) and the follower buccal motoneurons (the B1 cells). However, these data are inconsistent with previous results. In the present study, we therefore reexamined the serotonin receptors to identify the receptor subtypes functioning in the synapses between the CGCs and the B1 cells by recording the compound excitatory postsynaptic potential (EPSP) of the B1 cells evoked by a train of stimulation to the CGC in the presence of antagonists: cinanserin for 5-HT(2) and/or MDL72222 for 5-HT(3). The compound EPSP amplitude was partially suppressed by the application of these antagonists. The rise time of the compound EPSP was longer in the presence of MDL72222 than in that of cinanserin. These results suggest that these two subtypes of serotonin receptors are involved in the CGC-B1 synapses, and that these receptors contribute to compound EPSP. That is, the fast component of compound EPSP is mediated by 5-HT(3)-like receptors, and the slow component is generated via 5-HT(2)-like receptors.

Cyclic AMP-responsive element binding protein 1 (CREB1) plays multiple functions as a transcription factor in gene regulation. CREB1 proteins are also known to be expressed in several spliced isoforms that act as transcriptional activators or repressors. The activator isoforms, possessing the functional domains for kinase induction and for interaction with other transcriptional regulators, act as transcriptional activators. On the other hand, some isoforms, lacking those functional domains, are reported to be repressors that make heterodimers with activator isoforms. The complex and ingenious function for CREB1 arises in part from the variation in their spliced isoforms, which allows them to interact with each other. To date, however, the dimerization between the activator and repressor isoforms has not yet been proved directly in living cells. In this study, we applied fluorescence cross-correlation spectroscopy (FCCS) to demonstrate direct observation of dimerization between CREB1 activator and repressor. The FCCS is a well established spectroscopic method to determine the interaction between the different fluorescent molecules in the aqueous condition. Using differently labeled CREB1 isoforms, we successfully observed the interaction of CREB1 activator and repressor via dimerization in the nuclei of cultured cells. As a result, we confirmed the formation of heterodimer between CREB1 activator and repressor isoforms in living cells.

Endoreplication is DNA synthesis without cell division. Giant neurons observed in the brains of mollusks are thought to be generated as a result of DNA endoreplication. It has been hypothesized that neuronal size becomes larger in parallel with an increase in body size and that DNA endoreplication is involved in this process to meet the increasing demand for macromolecules in neurons. Thereis, however, no experimental evidence for this hypothesis to date. In the present study, we investigated the following quantitatively: (1) the size of the brain and each ganglion, (2) the size of identified neurons,(3) the total number of neurons undergoing DNA endoreplication, (4) the total number of the neurons containing a cardioexcitatory peptide, and (5) the gene expression level per neuron, using terrestrial slugs whose body growth was regulated through the amount of food supplied in the laboratory. The body growth was accompanied by increases in the sizes of both neurons and ganglia and triggered more frequent DNA endoreplication events in each ganglion of the growth-promoted slugs, without increasing the total number of neurons. Increase in the neuronal size also involved the increase in the amount of transcripts expressed in a single neuron. This is the first quantitative evidence showing that the DNA endoreplication, neuronal size, and gene expression are increased concomitantly with body growth in adult mollusks. © 2011 the authors.

Immunohistochemical techniques were used to study the distribution of cholinergic neurons containing choline acetyltransferase of the common type (cChAT), the synthetic enzyme of acetylcholine, in the central nervous system of the slug Limax maximus and Limax valentianus. Because the antiserum applied here was raised against a recombinant protein encoded by exons 7 and 8 of the rat gene for ChAT, three methods were used in order to validate antibody specificity for the Limax counterpart enzyme. Western blot combined with ChAT activity assay following native gel electrophoresis and immunoprecipitation analysis both indicated that immunoreactive Limax brain molecules were capable of synthesizing acetylcholine. Western blot after denatured gel electrophoresis of Limax brain extracts revealed a single band of about 67 kDa. All findings obtained with these three methods clearly indicated that the antiserum effectively recognized Limax cChAT. 1400 neuronal cell bodies positive for cChAT, mainly small to medium-sized, were found in various brain regions in the buccal, cerebral, pleural, parietal, visceral and pedal ganglia. cChAT immunoreactive nerve fibers were distributed extensively in the neuropil, connectives and commissures of these central ganglia. The map of cChAT-positive cells provided here are valuable for understanding the cholinergic mechanism in the slug brain, as well as giving an important hint to clarifying the mechanisms of learning and memory in higher vertebrates including humans. © 2011 Elsevier Ltd. All rights reserved.

Terrestrial pulmonates can learn olfactory-aversion tasks and retain them in their long-term memory. To elucidate the cellular mechanisms underlying learning and memory, researchers have focused on both the peripheral and central components of olfaction: two pairs of tentacles (the superior and inferior tentacles) and a pair of procerebra, respectively. Data from tentacleamputation experiments showed that either pair of tentacles is sufficient for olfactory learning. Results of procerebrum lesion experiments showed that the procerebra are necessary for olfactory learning but that either one of the two procerebra, rather than both, is used for each olfactory learning event. Together, these data suggest that there is a redundancy in the structures of terrestrial pulmonates necessary for olfactory learning. In our commentary we exemplify and discuss functional optimization and structural redundancy in the sensory and central organs involved in olfactory learning and memory in terrestrial pulmonates. © 2011 Published by The Company of Biologists Ltd.

In conditioned taste aversion (CTA) training performed on the pond snail Lymnaea stagnalis, a stimulus (the conditional stimulus, CS; e.g., sucrose) that elicits a feeding response is paired with an aversive stimulus (the unconditional stimulus, US) that elicits the whole-body withdrawal response and inhibits feeding. After CTA training and memory formation, the CS no longer elicits feeding. We hypothesize that one reason for this result is that after CTA training the CS now elicits a fear response. Consistent with this hypothesis, we predict the CS will cause (1) the heart to skip a beat and (2) a significant change in the heart rate. Such changes are seen in mammalian preparations exposed to fearful stimuli. We found that in snails exhibiting long-term memory for one-trial CTA (i.e., good learners) the CS significantly increased the probability of a skipped heartbeat, but did not significantly change the heart rate. The probability of a skipped heartbeat was unaltered in control snails given backward conditioning (US followed by CS) or in snails that did not acquire associative learning (i.e., poor learners) after the one-trial CTA training. These results suggest that as a consequence of acquiring CTA, the CS evokes conditioned fear in the conditioned snails, as evidenced by a change in the nervous system control of cardiac activity.

Of all organs in mammals including humans, the brain has the most limited regenerative capacity after injury or damage. In spite of extensive efforts to treat ischemic/stroke injury of the brain, thus far no reliable therapeutic method has been developed. However, some molluscan species show remarkable brain regenerative ability and can achieve full functional recovery following injury. The terrestrial pulmonates are equipped with a highly developed olfactory center, called the procerebrum, which is involved in olfactory discrimination and odor-aversion learning. Recent studies revealed that the procerebrum of the land slug can spontaneously recover structurally and functionally relatively soon after injury. Surprisingly, no exogenous interventions are required for this reconstitutive repair. The neurogenesis continues in the procerebrum in adult slugs as in the hippocampus and the olfactory bulb of mammals, and the reconstitutive regeneration seems to be mediated by enhanced neurogenesis. In this review, we discuss the relationship between neurogenesis and the regenerative ability of the brain, and also the evolutionary origin of the brain structures in which adult neurogenesis has been observed. © 2011 Marine Biological Laboratory.

It is well established that honeybees inform their nestmates of the location of flowers they have visited by a unique behavior called a "waggle dance". To evaluate the efficacy of this dance in terms of food collection, we constructed a Markov model for foraging behavior. The computer simulation showed that our model faithfully expressed actual bee behavior; revealed that a colony in which honeybees danced and correctly transferred information made 2.15 times more successful visits to food sources compared to a colony with non-accurate information transfer; and suggested that the waggle dance is effective only under particular conditions.

In many in vitro cultures, cells may change their morphology, probably caused by adherence to the surface of the culture dish. Since a fractal alkyl ketene dimer (AKD) surface provides super water-repellency with a contact angle of 174, we considered that it might provide an improved surface environment for the growth and differentiation of cells by preventing intimate adhesion. C6 glioma cells which were selected to test the effects of the fractal surface, were cultured on a conventional surface, a smooth AKD surface or a fractal AKD surface. On the conventional and smooth AKD surfaces, cells developed bipolar or multipolar shapes with enlarged cell bodies and neurite-like processes. In contrast, cells cultured on the fractal AKD surface presented fine filopodium-like processes like protoplasmic astrocytes in vivo, and higher morphological complexity was revealed by fractal analysis. Reconstruction of three-dimensional shape indicated that cells on the fractal surface were globular, whereas those on the conventional surface were rather flat. Our results suggest that C6 glioma cells on a fractal AKD surface show features of natural astrocytes with their elaborate morphology. The fractal surface thus may provide a new and natural culture environment for experimental assessment of glial structure and function.

To create precise neural circuits in the nervous system, neuritogenesis and synaptogenesis are the critical cellular processes during neuronal differentiation. We examined the cyclic AMP (cAMP)-responsible signaling pathways for regulating neuritogenesis and synaptogenesis in NG108-15 cells. A rise in intracellular cAMP concentration by a membrane-permeable cAMP analog, dibutyryl cAMP (DBcAMP), led to an increase in the number of neurites and varicosities. Inhibition of cAMP-dependent protein kinase (PKA) activity by a PKA inhibitor (H89) accelerated this neuritogenesis and neurite outgrowth rate. Treatment with H89, however, decreased the number of varicosities and the frequency of postsynaptic miniature current recorded in the cultured cells, resulting in suppression of synaptogenesis. Immunoblot analyses revealed that PKA activity mediates phosphorylation of a gene transcription factor, cAMP-response element binding protein (CREB). On the other hand, inhibition of a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway by a MAPK/ERK kinase (MEK) inhibitor (PD98059) suppressed both neuritogenesis and neurite outgrowth without CREB phosphorylation. These results suggest strongly that PKA simultaneously plays two different roles in neuronal differentiation: inhibition of neuritogenesis and stimulation of synaptogenesis, via CREB-mediated gene expression. © 2003 Wiley-Liss, Inc.

A- and D-type K+ channels (KA and KD channels) have unique physiological properties that play important roles in the integration of excitatory post synaptic potentials (EPSPs) in neuronal dendrites. These functions were analyzed using a computer program, NEURON, to simulate high-frequency sequential synaptic inputs, that can induce long-term potentiation (LTP). We paid close attention to the stability of the reduction of sequential EPSPs. When either KA or KD channels were included in models, the EPSP reduction ratios were less stable than containing both KA and KD channels. When both KA and KD channels were present in the model, the variance of EPSP reduction ratios was significantly smaller in comparison with that in the presence of either KA or KD channels alone. We thus concluded that the co-existence of KA and KD channels is necessary to produce stable EPSPs during the high-frequency synaptic stimulation necessary for induction of LTP. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

We observed the time-dependent morphological alteration of astrocytes during their adhesion by atomic force microscopy (AFM) and investigated the relationships between this morphological alteration and the localization of actin filaments and connexin 43 by immunocytochemistry. The fine processes observed as fine ridge-like structures by AFM were closely concerned with ac tin filaments by immunocytochemistry, During the adhesion of astrocytes, actin filaments appeared to be aligned regularly beyond the borders among different cells. Detectable connexin immunoreactivity was changed in the following regions: 1) the tips of fine cell processes and the cell margin when astrocytes started to adhere; 2) the border of cells when astrocytes tightly adhered; and 3) non-specific sites when astrocytes became a cluster. In the former two cases, the immunopositive spots for connexin were observed to colocalize with the tips of cell processes with actin filaments. These results strongly suggest that connexin associated with actin filaments at the tip of cell processes plays an important role in the early stage of the adhesion of astrocytes. These observations afford valuable clues for understanding the glial communication.

We have investigated viscoelastic properties of living mouse fibroblasts (NIH3T3) in a physiological condition using both force mapping and force modulation modes in atomic force microscopy. Spatial distribution of the elastic moduli has been measured using the force mapping mode. The nucleus area of the cell is about 10 times softer than the cytoplasm. Temporal changes in shape and viscoelasticity of developing lamellipodia have been captured using the force modulation mode. The dynamic changes were succeeded in capturing with time resolution of 10 minutes.

The topographic and viscoelastic images of the nerve cell (NG108-15) living in the culture medium were successfully obtained in the force modulation mode using atomic force microscopy(AFM), We found that there exists variation of elasticity in the cell, To compare the elastic results with F-actin network of cytoskeleton which is considered as a possible origin for cell stiffness, the fluorescence observation was made on the fu;ed cells just after the AFM measurements. The results show no clear correspondence between density of F-actin and stiffness of the cells.

Abstract: Abstract: Previous observations have implicated GABA as a neurotransmitter released by the vestibular sensory neurons („hair cells”) of the snail Hermissenda onto visual sensory neurons, the type B cells, whose cell bodies are the sites of biophysical and biochemical changes during and following Pavlovian conditioning. Still other observations demonstrated that light‐GABA pairings that simulate stimuli presented during Pavlovian conditioning cause prolonged elevatiori of intracellular Ca2+ and transformation of GABA‐induced synaptic inhibition into excitation. Intracellular Ca2+ signals in response to GABA perfused onto the postsynaptic: type B terminal branches are shown here to be prolonged on days after conditioning, but not after control paradigms. These and past results demonstrate two separate sites, i.e., the cell body and the terminal branches, for learning‐induced changes after Pavlovian conditioning. Copyright © 1994, Wiley Blackwell. All rights reserved

The bond lengths, bond orders, electron densities and optical absorption of the ground state of retinal forming a protonated Schiff-base linkage with a lysine residue (PRSB-Lys) are calculated using the extended INDO-CI molecular orbital method, and the result is compared with the previous one obtained by the π-electron approximation. It is, thus, shown that the π-electron system of PRSB-Lys is appreciably polarized, and that the σ – π interaction in PRSB-Lys is too large to be neglected when a C-C bond in its polyene chain is twisted. It is also shown that the negative counter point-charge exerts influence on the π-electron distribution in a similar way to that shown by the π-electron approximation. © 1990, THE PHYSICAL SOCIETY OF JAPAN. All rights reserved.

We consider the ground state (GS), the excited state on absorption (ESA), the virtual ground (VGS) and the deformed excited state (DES) of π-electron system in hexaene, retinal, retinal Schiff-base (RSB), protonated retinal Schiff-base (PRSB) or our model for rhodopsin chromophore, and we semiempirically calculate the following two kinds of force constants for the pairs of bonded atoms in each of the four π-electronic states: (1) the force constants of torsional vibrations
(2) those of the interactions between torsional and stretching vibrations. The calculations are carried out by our LCAO-ASMO-SCF-CI method for π-electrons, selecting such a part of the total energy of π-electron system that gives a significant contribution to the force constants. © 1988, THE PHYSICAL SOCIETY OF JAPAN. All rights reserved.

We consider retinal and its related compounds in planar configuration provided they have no ionic structure, and we extend the ordinary INDO-CI method to calculate their bond lengths in consistence with their electronic states. One of the important features of our extended INDO-CI method is that each bond length is calculated as a function of bond order which is similar to the Coulson type of relation derived by the π-electron approximation. The extended INDO-CI calculations of the bond lengths, electronic structure and optical absorptions of the molecules indicate that the previous results, which were obtained by Suzuki et al. using the π-electron approximation, are reasonable. © 1988, THE PHYSICAL SOCIETY OF JAPAN. All rights reserved.

In the olfactory center of terrestrial animals, changes in the oscillatory frequency of the local field potential (LFP) are thought to be involved in olfaction-based behavior and olfactory memory. The terrestrial slug Limax has a highly developed olfactory center, the procerebrum, in which the LFP spontaneously oscillates. Although changes in the oscillatory frequency are thought to correspond to the preference for specific odors, our knowledge about the mechanism of this frequency regulation is limited. To clarify the mechanism of the bidirectional frequency changes in the procerebrum, we focused on the neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide), which is known to have neuromodulatory functions in invertebrate nervous systems. Application of FMRFamide decreased the oscillatory frequency via G-protein-mediated cascades. Immunohistochemistry showed that FMRFamide-like-immunoreactive neuronal cell bodies are located in the cell mass layer of the procerebrum, projecting their neurites to the neuropile layers. The procerebrum was shown to also receive innervation from other regions of the cerebral ganglion. Furthermore, according to their morphological and projection characteristics, FMRFamide-containing neurons belong to the subpopulations of both bursting and nonbursting neurons in the procerebrum. The mRNA splice variant encoding multiple copies of canonical FMRFamide was specifically expressed in the procerebrum. Taking into account previous results showing that serotonin increases the oscillatory frequency, our results indicate that FMRFamide and serotonin both regulate the LFP frequency but in exactly the opposite direction in the olfactory center of the terrestrial slug.

The tentacles of pulmonates regenerate spontaneously following amputation. The regenerated tentacle is equipped with all the elements necessary for normal olfactory functioning, and the slugs can behave as well as they did before the tentacle amputation. However, it is not known what changes occur to the olfactory center procerebrum in the brain at the morphological and physiological levels. Here, we investigated the innervation of tentacular nerves into the procerebrum by examining the size of the terminal mass (input layer from tentacular nerves) of the procerebrum and also by staining afferent nerves immunohistochemically at 15, 58 and 75 days following unilateral amputation of the superior and inferior tentacles. The size of the terminal mass was significantly decreased, and the Phe-Met-Arg-Phe-NH(2)ergic (FMRFamidergic) afferent nerves disappeared by 15 days following the tentacle amputation. However, the size of the terminal mass had recovered substantially by 58 days, as the tentacle regenerated. The FMRFamidergic innervation into the cerebral ganglion was also restored by this time. An extended recovery (75 days), however, did not result in any further increase in the size of the terminal mass. We also recorded the local field potential (LFP) oscillation in the procerebrum. We found that the oscillatory frequency of the LFP had decreased at 15 days following the tentacle amputation but had recovered at 58 and 75 days. These results suggest that the amputation and regrowth of the tentacle are accompanied by the respective degeneration and re-innervation of olfactory nerves, and these changes in the innervation status affect the basal state of LFP oscillation.

Previous studies on glutamate (GLU) and its receptors in the pond snail Lymnaea stagnalis have suggested that GLU functions as a neurotransmitter in various behaviors, particularly for generation of feeding rhythm. The uptake mechanism of GLU is not yet known in Lymnaea. In the present study, we characterized the GLU transporters and examined their functions in the feeding circuits of the central nervous system (CNS) in Lymnaea. First, measurement of the accumulation of (3)H-labeled GLU revealed the presence of GLU transport systems in the Lymnaea CNS. The highest accumulation rate was observed in the buccal ganglia, supporting the involvement of GLU transport systems in feeding behavior. Second, we cloned two types of GLU transporters from the Lymnaea CNS, the excitatory amino acid transporter (LymEAAT) and the vesicular GLU transporter (LymVGLUT). When we compared their amino acid sequences with those of mammalian EAATs and VGLUTs, we found that the functional domains of both types are well conserved. Third, in situ hybridization revealed that the mRNAs of LymEAAT and LymVGLUT are localized in large populations of nerve cells, including the major feeding motoneurons in the buccal ganglia. Finally, we inhibited LymEAAT and found that changes in the firing patterns of the feeding motoneurons that have GLUergic input were similar to those obtained following stimulation with GLU. Our results confirmed the presence of GLU uptake systems in the Lymnaea CNS and showed that LymEAAT is required for proper rhythm generation, particularly for generation of the feeding rhythm. (C) 2009 Wiley-Liss, Inc.

In most sensory modalities, neuronal inputs are bilaterally processed in a higher center. In some animal species, however, functional lateralization is sometimes observed in the sensory processing at the higher level. For the terrestrial slug Limax, olfaction is the most important sensory modality and this slug can acquire odor-aversion memories. Previously, it has been demonstrated in bilateral PC ablation experiments that the procerebrum (PC) is necessary for odor-aversion memory, and that the PC is the memory storage site. On the other hand, it has been hypothesized that only the unilateral PC is used for odor-aversion learning. Here we demonstrated that the number of the slugs with intact memory performance was reduced by approximately 50% when the PC was surgically ablated only unilaterally before or after conditioning. There was no difference in the memory performance of the right vs. the left PC-ablated slugs. However, memory deficit from unilateral PC ablation was not observed when the ipsilateral tentacles were also amputated at the same time. We also showed that there was no lateral memory transfer from one PC to the other, after up to 7 days post-conditioning. Our results demonstrated clearly that either the left or right PC is randomly used for olfactory learning, and that the side of use is determined at the level of the olfactory ascending pathway to the PC. (c) 2009 Elsevier Inc. All rights reserved.

The pond snail Lymnaea stagnalis moves along the sides and bottom of an aquarium, but it can also glide upside down on its back below the water's surface. We have termed these two forms of locomotion "standard locomotion" and "upside-down gliding," respectively. Previous studies showed that standard locomotion is produced by both cilia activity on the foot and peristaltic contraction of the foot muscles. whereas upside-down gliding is mainly caused by cilia activity. The pedal A neurons are thought to receive excitatory octopaminergic input, Which ultimately results in increased cilia beating. However, the relationship between locomotory speed and the responses of these neurons to octopamine is not known. We thus examined the effects of both an agonist and an antagonist of octopamine receptors on locomotory speed and the firing rate of the pedal A neurons. We also examined. at the electron and light-microscopic levels, whether structural changes occur in cilia following the application of either an agonist or an antagonist of octopamine receptors to the central nervous system (CNS). We found that the application of an octopamine antagonist to the CNS increased the speed of both forms of locomotion, whereas application of octopamine increased only the firing rate of the pedal A neurons. Microscopic examination of the cilia proved that there were no chan-e in their morphology after application of octopamine me ligands. These data suggest that there is an unidentified octopaminergic neuronal network in the CNS whose activation reduces cilia movement and thus locomotory speed.

Background: Of all organs and tissues in adult mammals, the brain shows the most limited regeneration and recovery after injury. This is one reason why treating neurological damage such as ischemic injury after stroke presents such a challenge. Here we report a novel mode of regeneration which the slug's cognitive center, the procerebrum, shows after surgical lesioning in the adult. It is well known that the land slug Limax possesses the capacity to demonstrate conditioned food aversion. This learning ability critically depends on the procerebrum, which is the higher olfactory center in the brain of the terrestrial mollusk.
Principal Findings: In the present study, after a 1-month recovery period post-surgical lesioning of the procerebrum we investigated whether the brain of the slug shows recovery from damage. We found that learning ability, local field potential oscillation, and the number of cells in the procerebrum (PC) all recovered spontaneously within 1 month of bilateral lesioning of the PC. Moreover, neurogenesis was enhanced in the lesioned PC. However, memory acquired before the surgery could not be retrieved 1 month after surgery although the procerebrum had recovered from injury by this time, consistent with the notion that the procerebrum is the storage site of odor-aversion memory, or deeply involved in the memory recall process.
Significance: Our findings are the first to demonstrate that a brain region responsible for the associative memory of an adult organism can spontaneously reconstitute itself, and can recover its function following injury.

Cyclic AMP-responsive element binding protein1 (CREB1) has multiple functions in gene regulation. Various studies have reported that CREB1-dependent gene induction is necessary for memory formation and long-lasting behavioral changes in both vertebrates and invertebrates. In the present study, we characterized Lymnaea CREB1 (LymCREB1) mRNA isoforms of spliced variants in the central nervous system (CNS) of the pond snail Lymnaea stagnalis. Among these spliced variants, the three isoforms that code a whole LymCREB1 protein are considered to be the activators for gene regulation. The other four isoforms, which code truncated LymCREB1 proteins with no kinase inducible domain, are the repressors. For a better understanding of the possible roles of different LymCREB1 isoforms, the expression level of these isoform mRNAs was investigated by a real-time quantitative RT-PCR method. Further, we examined the changes in gene expression for all the isoforms in the CNS after conditioned taste aversion (CTA) learning or backward conditioning as a control. The results showed that CTA learning increased LymCREB1 gene expression, but it did not change the activator/repressor ratio. Our findings showed that the repressor isoforms, as well as the activator ones, are expressed in large amounts in the CNS, and the gene expression of CREB1 isoforms appeared to be specific for the given stimulus. This was the first quantitative analysis of the expression patterns of CREB1 isoforms at the mRNA level and their association with learning behavior.

The terrestrial slug Limax has the ability to learn odor associations. This ability depends on the function of the procerebrum, the secondary olfactory center in the brain. Among the various neurotransmitters that are thought to be involved in the function of the procerebrum, glutamate is one of the most important molecules. However, the existence and function of glutamate in this system have been proposed solely on the basis of a few lines of indirect evidence from pharmacological experiments. In the present study, we demonstrated the existence and release of glutamate as a neurotransmitter in the procerebrum of Limax, by using three different techniques: 1) immunohistochemistry of glutamate, 2) in situ hybridization to mRNA of the vesicular glutamate transporter, and 3) real-time imaging of glutamate release within the procerebrum using the glutamate optical sensor EOS2. The release of glutamate within the cell mass layer of the procerebrum was synchronized with oscillation of the local field potential and had the same physiological properties as this oscillation; both were blocked by a serotonin antagonist and were propagated in an apical to basal direction in the procerebrum. Our observations suggest strongly that the oscillation of the local field potential is driven by the glutamate released by bursting neurons in the procerebrum. (C) 2009 wiley-Liss, Inc.

Nitric oxide (NO) plays important roles in the olfactory center of various animals. In the terrestrial slug, NO is indispensable for field potential oscillation in the higher olfactory center, the procerebrum (PC), and also for odor learning. Here we identify a novel NO synthase (NOS) gene, limNOS2, in the terrestrial slug. The mRNA (similar to 10 kb) of limNOS2 encodes a protein consisting of 1616 amino acids, including a PDZ domain. The protein has 70.0% sequence identity with the previously identified limNOS1 gene. In contrast to limNOS1, however, limNOS2 is expressed specifically in the PC. Moreover, most of the cells in the PC contain limNOS2 mRNA, indicating that the nonbursting neurons, the major constituent of the PC, have this mRNA. The expression pattern of limNOS2 conforms well to the pattern of NOS enzymatic histochemical staining. Our present findings indicate that limNOS2 is responsible for most of the NO generation in the PC. (C) 2009 Elsevier Inc. All rights reserved.

The pond snail Lymnaea stagnalis can often be observed moving upside down on its back just below the surface of the water. We have termed this form of movement "upside-down gliding." To elucidate the mechanism of this locomotion, we performed a series of experiments involving behavioral analyses and microscopic observations. These experiments were designed (1) to measure the speed of this locomotion; (2) to determine whether the mucus secreted from the foot of Lymnaea repels water, thereby allowing the snail to exploit the surface tension of the water for upside-down gliding; and (3) to observe the beating of foot cilia in this behavior. The beating of these cilia is thought to be the primary driving force for upside-down gliding. Our results demonstrate that upside-down gliding is an efficient active process involving the secretion of mucus that floats up to the water surface to serve as a substrate upon which cilia beat to cause locomotion at the underside of the water surface.

Atomic force microscopy (AFM) creates a topographic map of a sample surface and has enabled the imaging of biological surfaces. As an initial step in elucidating gene expression mechanisms from the perspective of structural biology, we have started to characterize the structure of nucleosomes that consist of core histones and DNA promoter regions, including specific transcription factor-related binding sites. We formed nucleosomes using core histones and tyrosine hydroxylase promoter regions that include cAMP- and TPA-responsive elements, and then we observed these nucleosomes using the dynamic-force (tapping) mode in AFM. Good correspondence was seen between the lengths of the DNA molecules observed by AFM and the theoretical values estimated from the base pairs of DNA molecules, thus confirming that the present AFM study acquired images of nucleosomes, including promoter regions.

Elevation of the intracellular cAMP level induces morphological changes of astrocyte-like differentiation in C6 glioma cells. Such changes may be accompanied with expression of cytoskeletal protein genes. We therefore analyzed morphological changes after a treatment with dibutyryl cAMP (dbcAMP) and then assessed the expression of cytoskeletal protein genes by a quantitative real-time polymerase chain reaction. The cell number remained unaltered upon incubation with 1 mM dbcAMP in medium supplemented with 0.1% fetal bovine serum (FBS), whereas the number and lengths of processes increased, when compared with those of cells incubated in medium supplemented with 0.1% or 10% FBS only. The amounts of beta-actin, gamma-actin, and beta-tubulin mRNAs in C6 cells, but not alpha-tubulin mRNA, increased during the early proliferation in DMEM containing 10% FBS. The expression of cytoskeletal protein genes decreased when incubated with 0.1% FBS or 1 mM dbcAMP in 0.1% FBS, compared with those of cells cultured in 10% FBS. These results indicated that, during the early proliferation in normal culture condition, the expression of cytoskeletal protein genes in C6 cells, except alpha-tubulin, increased, while in differentiating or differentiated C6 glioma cells, cAMP-induced morphological changes were not accompanied with elevation of gene expression for cytoskeletal proteins, such as actin and tubulin.

Terrestrial slugs have the ability to learn and remember a food odor paired with an aversive stimulus. Olfaction in slugs involves the tips of two pairs of tentacles, the superior and the inferior tentacles. Sensory nerves in both pairs of the tentacles transmit olfactory information to the structure in the CNS, the procerebrum where learning and memory formation occur. We investigated the role of each pair of tentacles in odor-aversion learning, and examined the ability of slugs to recall memory after selective surgical amputation. Our results show that memory formation was not altered by the amputation of either one of the pairs before or after odor-aversion learning, while the odor sensibility of the slugs was maintained. These data suggest that either pair of tentacles is sufficient for the acquisition and retrieval of aversive olfactory memory.

The analysis of biological function produced by both of individual and assemblage activity of animals is a challenging topic of behavioral ecology. Social insects are possible tool for the analysis because numerous studies have been already done to clarify the behavioral factors of individuals. Especially, honeybee is an ideal animal for the experiments because of the development of breeding techniques, existence of clear caste according to their age, efficient forage behavior adaptive for variable feeding places and high ability of honey storage in their colony. Seeley et al. conducted many behavioral experiments in order to scrutinize honeybee behavior inside and outside of the nests. However, these experiments normally have to put numbers on each body of thousands of honeybees to identify individuals and follow their behavior by hand, which require huge time and labors. In this study, we developed a system to identify honeybee in a nest image and to follow plural honeybees simultaneously. The system is composed by three parts, determination of the region of honeybee body from the other parts with vector quantization, extraction of the region of single honeybee bodies, and reorganization of single honeybees. Our system succeeded to identify nearly 70% of honeybees in a hive automatically.

A honeybee informs her nestmates of flower locations by a unique behavior called a 'waggle dance'. We regard this behavior as a good model of the 'propagation and sharing of knowledge' to maintain a society. We have attempted to reveal how this dance benefits the colony using mathematical models and computer simulation based on parameters obtained from observations of bee behavior. Our simulation indicated that the most successful forages were made by a putative bee colony that used the dance to communicate. Video analysis of worker honeybee behavior in the field showed that a bee does not dance in a single, random place in the hive, but waggles several times in one place and several times in another. The orientation and duration of waggle runs varied from run to run, within ranges of +/- 15 degrees and +/- 15%, respectively. We also found that most of the bees that listened to the waggle dance turned away from the dancer after listening to one or two runs. These data suggest that honeybees use the waggle dance as a method of communication, but that they must base their forages oil ambiguous information about the location of a food source. (C) Koninklijke Brill NV, Leiden and The Robotics Society of Japan, 2008

A honeybee informs her nestmates of the location of a flower she has visited by a unique behavior called a "waggle dance." On a vertical comb, the direction of the waggle run relative to gravity indicates the direction to the food source relative to the sun in the field, and the duration of the waggle run indicates the distance to the food source. To determine the detailed biological features of the waggle dance, we observed worker honeybee behavior in the field. Video analysis showed that the bee does not dance in a single or random place in the hive but waggled several times in one place and then several times in another. It also showed that the information of the waggle dance contains a substantial margin of error. Angle and duration of waggle runs varied from run to run, with the range of +/- 15 degrees and +/- 15%, respectively, even in a series of waggle dances of a single individual. We also found that most dance followers that listen to the waggle (lance left the dancer after one or two sessions of listening.

The pond snail, Lymnaea stagnalis, can locomote on its back utilizing the surface tension of the water. We have called this form of movement 'back-swimming'. In order to perform this behavior, the snail must flip itself over on its back so that its foot is visible from above. Little is known about the mechanism of this back-swimming. As a first step for the elucidation of this mechanism, we measured the speed of back-swimming of Lymnaea at the different times of the day. They back-swam significantly faster in the morning than just before dark. These data are consistent with Our earlier findings on circadian-timed activity pattern in Lymnaea.. Lymnaea appear to secrete a thin membrane-like substance from their foot that may allow them to back-swim. To confirm the existence of this substance and to examine whether this substance is hydrophobic or hydrophilic, we applied a detergent onto the foot during back-swimming. A single drop of 1% Tween 20 drifted Lymnaea away that were still kept at the water surface. These results suggest that Lymnaea secrete a hydrophobic substance from their foot that floats to the water surface allowing Lymnaea to back-swim.

Gamma-aminobutyric acid (GABA)-containing neurons are capable of controlling network oscillations and their patterns in the widely divergent species. In the terrestrial slug, Limax valentianus, oscillatory activity of the procerebrum neurons is considered to encode the odor information. Previous studies showed that GABA is present in the central nervous system and may be involved in the control of neuronal activity of Limax. In the present study, we examined the effects of GABA on the generation of oscillatory activity in the procerebrum. The results suggest that the GABAergic synaptic transmission may be involved in the oscillatory neural network of the procerebrum.

The procerebrum (PC) is indispensable for odor-aversion learning in Limax. On the other hand, the central nervous system (CNS) of some Pulmonata shows robustness against injury, recovering from nerve injury both at the histological and functional levels. To investigate whether the PC of Limax also shows robustness against nerve injury, we tested whether or not the slugs can acquire and retrieve odor-aversion memory after a long recovery period from PC ablation. When the recovery period is short (7 days), the PC-ablated slugs failed to avoid the conditioned odor. But when the recovery period is long (I month), the PC-ablated slugs successfully avoided the conditioned odor. These results indicate that the CNS including the PC can recover from injury at least at the functional level.

The serotonin transporter, SERT, is reported as a key molecule that regulates serotonergic neurotransmission. In the present study, we analyzed the localization of Lymnaea SERT (LymSERT) mRNA-containing neurons by in situ hybridization using frozen sections of the central nervous system (CNS) of Lymnaea. To precisely demonstrate the distribution of LymSERT mRNA-containing neurons, colocalization with serotonin immunoreactivity was also examined. The results showed that LymSERT mRNA was constitutively expressed and localized in the serotonin-containing neurons in the CNS.

We investigated the distribution and projection patterns of central and peripheral glutamate-like immunoreactive (GLU-LIR) neurons in the adult and developing nervous system of Lymnaea. Altogether, 50-60 GLU-LIR neurons are present in the adult central nervous system. GLU-LIR labeling is shown in the interganglionic bundle system and at the varicosities in neuropil of the central ganglia. In the periphery, the foot, lip, and tentacle contain numerous GLU-LIR bipolar sensory neurons. In the juvenile Lymnaea, GLU-LIR elements at the periphery display a pattern of distribution similar to that seen in adults, whereas labeled neurons increase in number in the different ganglia of the central nervous system from juvenile stage P1 up to adulthood. During embryogenesis, GLU-LIR innervation can be detected first at the 50% stage of embryonic development (the E50% stage) in the neuropil of the cerebral and pedal ganglia, followed by the emergence of labeled pedal nerve roots at the E75% stage. Before hatching, at the E90% stage, a few GLU-LIR sensory cells can be found in the caudal foot region. Our findings indicate a wide range of occurrence and a broad role for glutamate in the gastropod nervous system; hence they provide a basis for future studies on glutamatergic events in networks underlying different behaviors.

Repeated spaced training sessions of contingent tactile stimulation to the pneumostome as it opens are required to cause long-term memory (LTM) formation of aerial respiratory behaviour making if difficult to determine exactly when memory forms. We have devised a single-trial aversive operant conditioning training procedure in Lymnaea to be better able to elucidate the causal mechanisms of LTM formation. Observations of baseline breathing behaviour in hypoxia were first made. Twenty-four hours later the snails were trained using the single trial procedure, by placing them in a small Petri dish containing 4 ml of 25 mM KCl for 30-35 s as soon as the first pneumostome opening in hypoxia was attempted. LTM was present if (1) breathing behaviour following training was significantly less than before; and (2) breathing behaviour post-training was significantly less in experimental groups than in yoked control groups. LTM persisted for 24 It but not 48 h. Yoked controls that received an aversive stimulus not contingent with pneumostome opening had no evidence of memory. Cooling directly after, but not at any other time, blocks LTM formation. LTM formation was also prevented by removal of the cell body of the neuron RPeD1 before training. (C) 2007 Elsevier Inc. All rights reserved.

In the majority of studies designed to elucidate the causal mechanisms of memory formation, certain members of the experimental cohort, even though subjected to exactly the same conditioning procedures, remember significantly better than others, whereas others show little or no long-term memory (LTM) formation. To begin to address the question of why this phenomenon occurs and thereby help clarify the causal mechanism of LTM formation, we used a conditioned taste aversion (CTA) procedure on individuals of the pond snail Lymnaea stagnalis and analyzed their subsequent behavior. Using sucrose as an appetitive stimulus and KCI as an aversive stimulus, we obtained a constant ratio of 'poor' to 'good' performers for CTA-LTM. We found that approximately 40% of trained snails possessed LTM following a one-trial conditioning procedure. When we examined the time-window necessary for the memory consolidation, we found that if we cooled snails to 4 degrees C for 30 min within 10 min after the one-trial conditioning, LTM was blocked. However, with delayed cooling (i.e. longer than 10 min), LTM was present. We could further interfere with LTM formation by inducing inhibitory learning (i.e. backward conditioning) after the one-trial conditioning. Finally, we examined whether we could motivate snails to acquire LTM by depriving them of food for 5 days before the one-trial conditioning. Food-deprived snails, however, failed to exhibit LTM following the one-trial conditioning. These results will help us begin to clarify why some individuals are better at learning and forming memory for specific tasks at the neuronal level.

Nitric oxide (NO), hydrogen sulfide (H2S), and carbon monoxide (CO) are thought to act as gaseous neuromodulators in the brain across species. For example, in the brain of honeybee Apis mellifera, NO plays important roles in olfactory learning and discrimination, but the existence of H2S- and CO-mediated signaling pathways remains unknown. In the present study, we identified the genes of nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC), cystathionine beta-synthase (CBS), and heme oxygenase (HO) from the honeybee brain. The honeybee brain contains at least one gene for each of NOS, CBS, and HO. The deduced proteins for NOS, CBS, and HO are thought to contain domains to generate NO, H2S, and CO, respectively, and to contain putative Ca2+/calmodulinbinding domains. On the other hand, the honeybee brain contains three subunits of sGC: sGC alpha 1, sGC beta 1, and sGC beta 3. Phylogenetic analysis of sGC revealed that Apis sGC alpha 1 and sGC beta 1 are closely related to NO- and CO-sensitive sGC subunits, whereas Apis sGC beta 3 is closely related to insect O-2-sensitive sGC subunits. In addition, we performed in situ hybridization for Apis NOS mRNA and NADPH-diaphorase histochemistry in the honeybee brain. The NOS gene was strongly expressed in the optic lobes and in the Kenyon cells of the mushroom bodies. NOS activity was detected in the optic lobes, the mushroom bodies, the central body complex, the lateral protocerebral lobes, and the antennal lobes. These findings suggest that NO is involved in various brain functions and that H2S and CO can be endogenously produced in the honeybee brain. (c) 2007 Wiley Periodicals, Inc.

Gonadotropin-releasing hormone (GnRH) is a hypophysiotropic decapeptide that stimulates the release of gonadotropins from the pituitary. In addition, there are extra-hypothalamic GnRH neurons that project to all regions of the brain and whose function remains unknown. Here, we investigated the effects of GnRH on retinotectal synaptic transmission, the synapses of which are formed between retinal fibers and tectal periventricular neurons that express GnRH receptor mRNA. We used rainbow trout as our study model. The excitatory postsynaptic currents (EPSCs), which were evoked by electrical stimulation of the retinal fibers and recorded in periventricular neurons, were suppressed by antagonists of ionotropic glutamate receptors. EPSCs were increased by application of each of two types of GnRH (GnRH2 and GnRH3) in the trout tectum. Such facilitation lasted for at least 10 min after application of the GnRH. To our knowledge, this is the first report of GnRH modulating conventional synaptic transmission in the brain, suggesting that tectal GnRH enhances tectal sensitivity for retinal inputs. Furthermore, such long-lasting facilitation might occur across all the brain regions innervated by GnRH neurons, and GnRH might simultaneously switch neuronal activities in the brain regions relevant to reproductive behaviors.

Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.

Hybrid quantum mechanics/molecular mechanics simulations, coupled to the recently introduced metadynamics method, performed on the adenosine triphosphate (ATP) of the bovine Hsc70 ATPase protein, show which specific water molecule of the solvation shell of the Mg2+ metal cation acts as a trigger in the initial phase of the ATP hydrolysis reaction in ATP synthase. Furthermore, we provide a detailed picture of the reaction mechanism, not accessible to experimental probes, that allows us to address two important issues not yet unraveled: (i) the pathway followed by a proton and a hydroxyl anion, produced upon dissociation of a putative catalytic H2O molecule, that is crucial in the selection of the reaction channel leading to the hydrolysis; (ii) the unique and cooperative role of K+ and Mg2+ metal ions in the reaction, acting as cocatalysts and promoting the release of the inorganic phosphate via an exchange of the OH- hydroxyl anion between their respective solvation shells. This is deeply different from the proton wire mechanism evidenced, for instance, in actin and lowers significantly the free energy barrier of the reaction.

The efferent connections and axonal and dendritic morphologies of periventricular neurons were examined in the optic tectum of rainbow trout to classify periventricular efferent neurons in salmonids. Among the target nuclei of tectal efferents, tracer injections to the following four structures labeled periventricular neurons: the area pretectalis pars dorsalis (APd), nucleus pretectalis superficialis pars magnocellularis (PSm), nucleus ventrolateralis of torus semicircularis (TS), and nucleus isthmi (NI). Two types of periventricular neurons were labeled by injections to the APd. One of them had an apical dendrite ramifying at the stratum fibrosum. et griseum. superficiale (SFGS), with an axon that bifurcated into two branches at the stratum griseum. centrale (SGC), and the other had an apical dendrite ramifying at the SGC. Two types of periventricular neurons were labeled after injections to the TS. One of them had an apical dendrite ramifying at the boundary between the stratum opticum (SO) and the SFGS, and the other had dendritic branches restricted to the stratum album centrale or stratum periventriculare. Injections to the PSm and NI labeled periventricular neurons of the same type with an apical dendrite ramifying at the SO and a characteristic axon that split into superficial and deep branches projecting to the PSm and NI, respectively. This cell type also possessed axonal branches that terminated within the tectum. These results indicate that periventricular efferent neurons can be classified into at least five types that possess type-specific axonal and dendritic morphologies. We also describe other tectal neurons labeled by the present injections.

The pond snail Lymnaea stagnalis is capable of learning conditioned taste aversion (CTA) and then consolidating that learning into long-term memory (LTM) that persists for at least 1 month. LTM requires cle novo protein synthesis and altered gene activity. Changes in gene activity in Lymnaea that are correlated with, much less causative, memory formation have not yet been identified. As a first step toward rectifying this situation, we constructed a cDNA microarray with mRNAs extracted from the central nervous system (CNS) of Lymnaea. We then, using this microarray assay, identified genes whose activity either increased or decreased following CTA memory consolidation. We also identified genes whose expression levels were altered after inhibition of the cyclic AMP response element-binding protein (CREB) that is hypothesized to be a key transcription factor for CTA memory. We found that the molluscan insulin-related peptide II (MIP II) was up-regulated during CTA-LTM, whereas the gene encoding pedal peptide preprohormone (Pep) was down-regulated by CRE132 RNA interference. We next examined mRNAs, of MIP II and Pep using real-time RT-PCR with SYBR Green. The MIP II mRNA level in the CNS of snails exhibiting "good" memory for CTA was confirmed to be significantly higher than that from the CNS of snails exhibiting.,poor" memory. In contrast, there was no significant difference in expression levels of the Pep mRNA between "good" and "poor" performers. These data suggest that in Lymnaea MIP II may play a role in the consolidation process that forms LTM following CTA training. (c) 2006 Wiley-Liss, Inc.

Interaction between the activator type of cyclic AMP response element binding protein (CREB1) and the repressor type (CREB2) results in determining the emergence of long-lasting synaptic enhancement involved in memory consolidation. However, we still do not know whether the constitutively expressed forms of CREB are enough or the newly synthesized forms are required for the synaptic enhancement. In addition, if the newly synthesized forms are needed, we must determine the time for translation of CREB from its mRNA. We applied the methods of RNA interference and real-time polymerase chain reaction (PCR) to CREB in the cerebral giant cells of Lymnaea. The cerebral giant cells play an important role in associative learning and employ a CREB cascade for the synaptic enhancement to neurons such as the B1 moto-neurons. We injected the small interfering RNA (siRNA) of CREB1 or CREB2 into the cerebral giant cells and examined the changes in amplitude of excitatory postsynaptic potential (EPSP) recorded in the B1 motoneurons. The changes in the amounts of CREB1 and CREB2 mRNAs were also examined in the cerebral giant cells. The EPSP amplitude was suppressed 15 min after injection of CREB1 siRNA, whereas that was augmented 60 min after injection of CREB2 siRNA. In the latter case, the decrease in the amount of CREB2 mRNA was confirmed by real-time PCR. Our results showed that the de novo synthesized forms of CREB are required within tens of minutes for the synaptic enhancement in memory consolidation. (c) 2006 Wiley-Liss, Inc.

Recent studies have shown that astrocytes release various transmitters including glutamate and thus directly affect synaptic neurotransmission. The mechanisms involved in the release of glutamate from astrocytes remain unclear, however. In the present study, we examined differences in 1) the amount of glutamate released, 2) the appearance of exocytosis, and 3) the expression of SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor) proteins between cyclic AMP-treated and non-treated astrocytes in culture. Extracellular glutamate was detected in the recording solution of cyclic AMP-treated astrocytes after stimulation with ATP by high-performance liquid chromatography and NADH imaging. Exocytosis, which was observed by FM1-43 imaging, appeared in cyclic AMP-treated astrocytes in a punctiform fashion, but not in non-treated cells, after stimulation with ATP and glutamate. Immunocytochemistry and Western blotting showed that the amount of SNARE proteins increased during cAMP-induced morphologic changes, and in particular, a v-SNARE, synaptobrevin, appeared as punctiform staining in the cytosol of cyclic AMP-treated astrocytes. These findings show that astrocytes acquire SNARE proteins during cyclic AMP-induced differentiation, and suggest that glutamate is released by exocytosis in cyclic AMP-treated astrocytes in response to ATP released from neighboring neurons and astrocytes. (c) 2006 Wiley-Liss, Inc.

Super water repellency of solid surfaces can be realized by formation of fractal structures, which were verified and explained by various materials, such as alkylketene dimer (AKD), platinum-palladium alloy-covered AKD, and poly(alkylpyrrole), and also by comparison of fractal surfaces with smooth ones. Novel potential applications of the fractal solid surfaces to unique biological studies are suggested based on our previous and the present biological studies on fractal AKD surfaces. (c) 2005 Elsevier B.V. All rights reserved.

The midbrain roof is a retinorecipient region referred to as the optic tectum in lower vertebrates, and the superior colliculus in mammals. The retinal fibers projecting to the tectum transmit visual information to tectal retinorecipient neurons. Periventricular neurons are a subtype of these neurons that have their somata in the deepest layer of the teleostean tectum and apical dendrites ramifying at more superficial layers consisting of retinal fibers. The retinotectal synapses between the retinal fibers and periventricular neurons are glutamatergic, and ionotropic glutamate receptors mediate the transmission in these synapses. This transmission involves long-term potentiation, and is modulated by hormone action. Visual information processed in the periventricular neurons is transmitted to adjacent tectal cells and target nuclei of periventricular neuron axonal branches, some of which relay the visual information to other brain areas controlling behavior. We demonstrated that periventricular neurons play a principal role in visual information processing in the teleostean optic tectum; the effects of tectal output on behavior is discussed also in the present review. (c) 2006 Elsevier Ltd. All rights reserved.

Conditioned taste aversion (CTA) in the pond snail Lymnaea stagnalis has been widely used as a model for gaining an understanding of the molecular and behavioral mechanisms underlying learning and memory. At the behavioral level, however, it is still unclear how taste discrimination and CTA interact. We thus examined how CTA to one taste affected the feeding response induced by another appetitive food stimulus. We first demonstrated that snails have the capacity to recognize sucrose and carrot juice as distinct appetitive stimuli. We then found that snails can become conditioned (i.e. CTA) to avoid one of the stimuli and not the other. These results show that snails can distinguish between appetitive stimuli during CTA, suggesting that taste discrimination is processed upstream of the site where memory consolidation in the snail brain occurs. Moreover, we examined second-order conditioning with two appetitive stimuli and one aversive stimulus. Snails acquired second-order conditioning and were still able to distinguish between the different stimuli. Finally, we repeatedly presented the conditional stimulus alone to the conditioned snails, but this procedure did not extinguish the long-term memory of CTA in the snails. Taken together, our data suggest that CTA causes specific, irreversible and rigid changes from appetitive stimuli to aversive ones in the conditioning procedure.

Some specific transcription factors are essential for memory consolidation across species. However, it is still unclear whether only the activation of constitutively expressed forms of these conserved transcription factors is involved in memory consolidation or their de novo synthesis also occurs after learning. This question has remained unanswered partly because of the lack of an efficient method for the determination of copy numbers of particular mRNAs in single neurons, which allows the detection of new transcription at the cellular level. Here we applied a newly developed protocol of single-cell quantitative real-time polymerase chain reaction (qRT-PCR) to single neurons playing an important role in associative learning. Specifically, we examined the changes in the mRNA and protein expression levels of a highly conserved transcription factor, CCAAT/enhancer binding protein (C/EBP), in the paired B2 motoneurons of the pond snail Lymnaea stagnalis. These buccal neurons are involved in the motor control of feeding behavior, with a potentially important role in conditioned taste aversion (CTA). Single-cell qRT-PCR revealed a significant decrease in LymC/EBP mRNA copy numbers in the B2 motoneurons during memory consolidation after CTA training. By contrast, isoelectric focusing and immunoblotting of extracts of the buccal ganglia showed that translation and phosphorylation levels of LymC/EBP significantly increased during memory consolidation. The C/EBP-like immunoreactivity in the B2 motoneurons, which are the major immunopositive component in the buccal ganglia, also significantly increased during memory consolidation, suggesting that the main source of increase in the level of protein in the buccal ganglia are the B2 motoneurons. Thus, early memory consolidation after CTA learning in L. stagnalis involves both the rapid synthesis and phosphorylation of LymC/EBP as well as the rapid breakdown of LymC/EBP mRNA in the neural network controlling feeding, suggesting that all of these processes play a role in the function of C/EBP in memory consolidation. (c) 2005 Elsevier Ltd. All rights reserved.

The fractal alkylketene dimer (AKD) surface is an artificial super water-repellent one with a high contact angle of 174 degrees, therefore, may provide special surface circumstances for studies of biological cells such as cell cultures. The experimental results indicated that the distribution of F-actin in the astrocytes cultured on the fractal AKD-coated dishes showed the stellate shape, while that in the astrocytes cultured on the poly-L-lysine-coated coverslips showed the formation of long alignment. The morphological change of astrocytes is induced by the fractal AKD surface, and the result suggests that astrocyte differentiation is stimulated by the fractal AKD surface.

A transcription factor, cyclic AMP-response element binding protein (CREB), which is phosphorylated by protein kinases (PKA and PKC), is known to be involved in the regulation of oligodendrocyte differentiation. However, it is still unclear whether protein kinase A (PKA) and protein kinase C (PKC) are used simultaneously or at different time points to phosphorylate CREB in oligodendrocytes and whether CREB phosphorylation advances oligodendrocyte differentiation or vise versa. Our previous experiments have shown that in the differentiation process from immature to mature cells, CREB phosphorylation depends on PKC activity and leads to the progression of differentiation. In order to gain a better understanding of the process of differentiation from progenitor to immature cells, we identified which protein kinase, i.e., PKA or PKC, regulates CREB phosphorylation and we determined whether CREB phosphorylation advances differentiation or the reverse. Our results showed that CREB phosphorylation is principally regulated by PKA activity in progenitor cells but not by PKC activity, and that this phosphorylation advances the differentiation of progenitor cells to immature cells in oligodendrocytes. (c) 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Gene expression is differently regulated in every cell even though the cells are included in the same tissue. For this reason, we need to measure the amount of mRNAs in a single cell to understand transcription mechanism better. However, there are no accurate, rapid and appropriate methods to determine the exact copy numbers of particular mRNAs in a single cell. We therefore developed a procedure for isolating a single, identifiable cell and determining the exact copy numbers of mRNAs within it. We first isolated the cerebral giant cell of the pond snail Lymnaea stagnalis as this neuron plays a key role in the process of memory consolidation of a learned behavior brought about by associative learning of feeding behavior. We then determined the copy numbers of mRNAs for the cyclic AMP-responsive element binding proteins (CREBs). These transcription factors play an important role in memory formation across animal species. The protocol uses two techniques in concert with each other: a technique for isolating a single neuron with newly developed micromanipulators coupled to an assay of mRNAs by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The molecular assay determined the mRNA copy numbers, each of which was compared with a standard curve prepared from cDNA solutions corresponding to the serially diluted solutions of Lymnaea CREB mRNA. The standard curves were linear within a range of 10 to 10(5) copies, and the intra-assay variation was within 15%. Each neuron removed from the ganglia was punctured to extract the total RNA directly and was used for the assay without further purification. Using this two-step procedure, we found that the mRNA copy number of CREB repressor (CREB2) was 30-240 in a single cerebral giant cell, whereas that of CREB activator (CREB1) was below the detection limits of the assay (< 25). These results suggest that the CREB cascade is regulated by an excess amount of CREB2 in the cerebral giant cells. Our procedure is the only quantitative analysis for elucidation of the dynamics of gene transcription in a single cell.

Previous experiments showed that the expression and phosphorylation levels of cyclic AMP-response element binding protein (CREB) are important factors that regulate oligodendrocyte differentiation. The present study was designed to determine whether CREB phosphorylation advances oligodendrocyte differentiation or vice versa and to identify the protein kinase that primarily regulates CREB phosphorylation. We examined the expression and phosphorylation levels of CREB in developing oligodendrocytes at a specific differentiation stage by double-immunocytochemical staining with specific differentiation markers and antibody for phosphorylated CREB. We found that the CREB expression level increased along oligodendrocyte differentiation, and that its phosphorylated level was highest in immature oligodendrocytes. We also showed that CREB phosphorylation was regulated principally by protein kinase C (PKC) activity in immature oligodendrocytes. Our findings suggest that CREB phosphorylation is dependent on a PKC signaling cascade, and this phosphorylation activates CREB-mediated transcription and advances the differentiation of immature to mature oligodendrocytes. (c) 2005 Wiley-Liss, Inc.

Retinotectal transmission has not yet been well characterized at the cellular level in the optic tectum. To address this issue, we used a teleost, the rainbow trout, and characterized periventricular neurons as postsynaptic cells expected to receive the retinotectal inputs to the optic tectum. The somata of periventricular neurons are localized in the upper zone of the stratum periventriculare (SPV), whereas the lower zone of the SPV comprises the cell body layer of radial glial cells. Ca2+ imaging identified functional ionotropic glutamate receptors in periventricular neurons. We also cloned cDNAs encoding the NR1 subunit of N-methyl-D-aspartic acid (NMDA) receptors and the GluR2 subunit of (+/-)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors, and detected their mRNAs in periventricular neurons by in situ hybridization. The presence of the receptor subunit proteins was also confirmed in the dendrites of periventricular neurons by immunoblotting and immunohistochemistry. On the other hand, radial glial cells in the lower zone of the SPV did not respond to glutamate applications, and mRNA and immunoreactivities of ionotropic glutamate receptors were not detected in glial cells. The present findings suggest that glutamatergic transmission at synapses between retinotectal afferents and periventricular neurons is mediated by the functional NMDA and AMPA receptors. (C) 2005 Wiley-Liss, Inc.

We have isolated and characterized the cDNAs for nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) from the terrestrial slug Limax marginatus, and examined the presence and distribution of their mRNAs in the central nervous system using histological techniques and a reverse transcription-polymerase chain reaction method. Our results showed that both bursting and nonbursting neurons in the procerebral lobes contain the mRNAs for both NOS and sGC. We further found that the oscillation frequency of electrical activity in the procerebral lobes increases with increasing intracellular concentrations of cyclic GMP (cGMP). Taken together with previous data on the NO-induced cGMP-like immunoreactivity and on the anatomical distribution of neurites and the localization of synapses of bursting and nonbursting neurons, our present results suggest that NO-induced changes in cGMP concentration modulate the oscillation frequency in the procerebral lobes by acting on the olfactory input pathways, but possibly not on the output pathways, in Slugs. (C) 2004 Wiley Periodicals, Inc.

The transcription factor, CCAAT/enhancer-binding protein (C/EBP), is involved in important physiological processes, such as cellular proliferation and differentiation, homeostasis, and higher-order functions of the brain. In the present study, we investigated the distribution of mRNA and protein of C/EBP in the central nervous system of the pond snail, Lymnaea stagnalis, by in situ hybridization and immunohistochemistry. Specificity of the anti-mammalian C/EBP antibody against Lymnaea C/EBP (LymC/EBP) was confirmed by combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis or isoelectric focusing and immunoblotting. Cells positive for in situ hybridization were immunoreactive for LymC/EBP in all 11 ganglia. The motoneurons (B1, B2, B4, and B4 clusters) in the buccal ganglia and interneurons ( cerebral giant cell, CGC) in the cerebral ganglia were positive for in situ hybridization and were immunopositive. In the pedal ganglion, the right pedal dorsal 1 (RPeD1), pedal A, and pedal C clusters exhibited positive signals of in situ hybridization and immunohistochemistry for LymC/EBP. CGC and RPeD1 are key neurons for associative learning. In addition, the neuropeptidergic cells in the cerebral, pleural, parietal, and visceral ganglia were positive for in situ hybridization and immunoreactive. Interestingly, although the cytoplasm of almost all immunopositive cells was stained, some neuropeptidergic cells located in the light parietal and visceral ganglia exhibited immunoreactivity in nuclei. Our results suggest that LymC/EBP is involved in learning and memory and in the expression and/or secretion of neuropeptides in Lymnaea.

We examined synaptic plasticity in the optic tectum of rainbow trout by extracellular recordings. We found that the field-excitatory postsynaptic potential in the retinotectal synapses was potentiated by repetitive stimuli of 1.0 Hz for 20 s to the retinotectal afferents. The long-term potentiation (LTP) developed slowly, and was maintained for at least 2 h. Applications of an antagonist for N-methyl-D-aspartic acid (NMDA) receptors or Mg2+-free saline showed that activation of NMDA receptors was required to form the UP beyond the induction period. The present findings indicate that presynaptic stimulation in the retinotectal synapses causes LTP mediated by NMDA receptors in the optic tectum of rainbow trout. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Ionotropic glutamate receptors (iGluRs) are postsynaptic ion channels involved in excitatory neurotransmission. iGluRs play important roles in development and in forms of synaptic plasticity that underlie higher order processes such as learning and memory. Neurobiological and biochemical studies have long characterized iGluRs in detail. However, the structural basis for the function of iGluRs has not yet been investigated, because there is insufficient information about their three-dimensional structures. In 1998, a crystal structure called S1S2 lobes was first solved for the extracellular bilobed ligand-binding domain of the GluR2 subunit. Since then, the crystal structures for the S1S2 lobes both in the apo and in various liganded states have been reported, and recent biophysical studies have further elucidated the dynamic aspects of the structure of the S1S2 lobes. In this review, the dynamic structures of the S1S2 lobes and their ligands are summarized, and the importance of their structural flexibility and fluctuation is discussed in light of the mechanisms of ligand recognition, activation, and desensitization of the receptor. (C) 2004 Wiley-Liss, Inc.

Calexcitin (CE) is a low molecular weight Ca2+- and guanosine triphosphate- binding protein, which is phosphorylated during associative learning in both vertebrates and invertebrates. The purpose of this study was to determine the presence of CE in the central nervous system (CNS) of the pond snail Lymnaea stagnalis, which can acquire classical and operant conditioning. Immunoblotting of CE showed that the anti-CE antibody prepared from squid can detect Lymnaea CE. In the cerebral ganglia, CE-like immunoreactivity was exhibited in two pairs of cell clusters that receive taste signals from the superior or median lip nerves. In both pedal ganglia, CE-like immunoreactivity was detected in 1-4 cell of the PeA clusters, which are involved in the withdrawal response. Our results therefore showed that CE is involved in the feeding and withdrawal neural networks, suggesting that CE may function in associative learning of feeding and withdrawal behavior in L. stagnalis.

In the present study, we identified the key protein that modulates the initiation of functional glutamatergic synapses in developing cortical neurons. First, we found a day in vitro that marked a critical increase in the number of functional synapses by the application of cyclic AMP, as demonstrated with Ca2+ imaging. We then examined the changes in the expression levels of proteins, which were expected to play a role in glutamatergic synapses, by the application of cyclic AMP. Our findings suggest that the expression of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors modulates the initiation of functional synapses in developing neurons, and that immature neurons already contain N-methyl D-aspartate (NMDA) receptors and presynaptic proteins such as synaptophysin.

The digit-like extensions (the digits) of the tentacular ganglion of the terrestrial slug Limax marginatus are the cell body rich region in the primary olfactory system, and they contain primary olfactory neurons and projection neurons that send their axons to the olfactory center via the tentacular nerves. Two cell clusters (the cell masses) at the bases of the digits form the other cell body rich regions. Although the spontaneous slow oscillations and odor responses in the tentacular nerve have been studied, the origin of the oscillatory activity is unknown. In the present study, we examined the contribution of the neurons in the digits and cell masses to generation of the tentacular nerve oscillations by surgical removal from the whole tentacle preparations. Both structures contributed to the tentacular oscillations, and surgical isolation of the digits from the whole tentacle preparations still showed spontaneous oscillations. To analyze the dynamics of odor-processing circuits in the digits and tentacular ganglia, we studied the effects of gamma-aminobutyric acid, glutamate, and acetylcholine on the circuit dynamics of the oscillatory network(s) in the peripheral olfactory system. Bath or local puff application of gamma-aminobutyric acid to the cell masses decreased the tentacular nerve oscillations, whereas the bath or local puff application of glutamate and acetylcholine to the digits increased the digits' oscillations. Our results suggest the existence of two intrinsic oscillatory circuits that respond differentially to endogenous neurotransmitters in the primary olfactory system of slugs. (C) 2004 Wiley Periodicals, Inc.

The pond snail Lymnaea stagnalis is an excellent model system in which to study the neuronal and molecular substrates of associative learning and its consolidation into long-term memory. Until now, the presence of cyclic AMP (cAMP)-responsive element binding protein (CREB), which is believed to be a necessary component in the process of a learned behavior that is consolidated into long-term memory, has only been assumed in Lymnaea neurons. We therefore cloned and analyzed the cDNA sequences of homologues of CREB1 and CREB2 and determined the presence of these mRNAs in identifiable neurons of the central nervous system (CNS) of L stagnalis. The deduced amino acid sequence of Lymnaea CREB1 is homologous to transcriptional activators, mammalian CREB1 and Aplysia CREB1a, in the C-terminal DNA binding (bZIP) and phosphorylation domains, whereas the deduced amino acid sequence of Lymnaea CREB2 is homologous to transcriptional repressors, human CREB2, mouse activating transcription factor-4, and Aplysia CREB2 in the bZIP domain. In situ hybridization revealed that only a relatively few neurons showed strongly positive signals for Lymnaea CREB1 mRNA, whereas all the neurons in the CNS contained Lymnaea CREB2 mRNA. Using one of the neurons (the cerebral giant cell) containing Lymnaea CREB1 mRNA, we showed that the injection of a CRE oligonucleotide inhibited a cAMP-induced, long-lasting synaptic plasticity. We therefore conclude that CREBs are present in Lymnaea neurons and may function as necessary players in behavioral plasticity. (C) 2003 Wiley Periodicals, Inc.

Differentiating neurons must acquire many unique morphological and functional characteristics in creating the precise neural circuits of the mature nervous system. The phenomenon of 'neuronal differentiation' includes a special set of simple, separate processes, that is, neuritogenesis, neurite outgrowth, pathfinding, targeting and synaptogenesis. All of these processes are critically dependent on the reorganization of actin cytoskeleton by many actin-binding proteins that function downstream of Rho-family GTPases. Furthermore, de novo synthesis of key proteins are critically involved in the reorganization of actin cytoskeleton during neuronal differentiation. In this article, we review recent progresses in the general mechanisms that control actin dynamics by various actin-binding proteins in differentiating neurons, including a series of recent studies from our laboratory on de novo synthesis of several key proteins that are essential for actin reorganization induced by second messengers. We demonstrated that dual regulation of cyclic AMP and Ca2+ determines cofilin (an actin-binding protein) phosphorylation states and LIM kinase 1 (a colifin kinase) expression level during neuritogenesis. (C) 2004 Elsevier Ltd. All rights reserved.

Conformational changes of proteins are dominated by the excitation and relaxation processes of their vibrational states. To elucidate the mechanism of receptor activation, the conformation dynamics of receptors must be analyzed in response to agonist-induced vibrational excitation. In this study, we chose the bending vibrational mode,of the guanidinium group of Arg485 of the glutamate receptor subunit GluR2 based on our previous studies, and we investigated picosecond dynamics of the glutamate receptor caused by the vibrational excitation of Arg485 via molecular dynamics simulations. The vibrational excitation energy in Arg485 in the ligand-binding site initially flowed into Lys730, and then into the J-helix at the subunit interface of the ligand-binding domain. Consequently, the atomic displacement in the subunit interface around an intersubunit hydrogen bond was evoked in about 3 ps. This atomic displacement may perturb the subunit packing of the receptor, triggering receptor activation.

To analyze the expression pattern of genes of cAMP responsive element binding protein (CREB), we performed in situ hybridization for the whole central nervous system (CNS) of the pond snail Lymnaea stagnalis. The CREB1 (activator) and CREB2 (repressor) homologues have already been cloned in L. stagnalis, and they are referred to as LymCREB1 and LymCREB2. Using the frozen sections and the whole mount preparations of the CNS, we mapped the distribution of LymCREB1 and LymCREB2 mRNA containing neurons. The present findings showed that the LymCREB1 mRNA containing neurons are a relatively few, whereas LymCREB2 mRNA is contained ubiquitously in the whole CNS of L. stagnalis.

The fluorescence-based real-time reverse transcription polymerase chain reaction (RT-PCR) is becoming widely used to quantity mRNA level in cells and tissues and is now a crucial tool for basic biological researches and biotechnology. In the present study, on the basis of the real-time quantitative RT-RCR, we detected and quantified mRNA copies of the transcription factor, CCAAT/enhancer binding protein (C/EBP; an immediate-early gene that is involved in synaptic plasticity and teaming and memory) in the central nervous system of the pond snail Lymnaea stagnalis. We designed the primer set and the probe in the specific insert for the detection of Lymnaea C/EBP (LymC/EBP) clone 1. This insert is not contained in LymC/EBP clone 2 by alternative splicing. The copy number of LymC/EBP clone I was linearly decreased relative to the dilution of cDNA, and it was estimated 30 copies/mu1 in test sample. The availability of the present study showed that the real-time quantitative RT-PCR technique is more accurate and more specific for the detection and quantification of the mRNA level of genes in L. stagnalis than the other PCR methods.

The pond snail Lymnaea stagnalis acquires conditioned taste aversion (CTA) and maintains its memory for more than a month. Snails in our laboratory were cultured at 20 degreesC on a 12: 12 light-dark cycle (light from 7 am to 7 pm). To examine the hours during which snails acquire CTA effectively, we trained some snails in the morning and others in the afternoon, and then compared their scores. CTA developed in both cases, but scores were significantly better in the morning than in the afternoon. To elucidate the cause of this difference in scores, we observed the voluntary activity of snails and found the circadian rhythm reflected in the snails' free-movement distances; distances at the circadian time 0-12 (daytime) were significantly longer than those at the circadian time 12-24 (nighttime). This rhythm was kept up for at least 3 days, even in constant darkness. In conclusion, L. stagnalis should be trained in the morning to acquire associative learning, possibly because of its greater propensity to roam about at that time as opposed to the afternoon.

The tentacular ganglion, the primary olfactory system of terrestrial slugs, exhibits spontaneous oscillations with a spatial coherence. The digit-like extensions (digits) of the tentacular ganglion presumably house the cell bodies of the neurons underlying the oscillations. The present study was designed to identify the anatomical and physiological determinants of these oscillations with a special focus on whether the neurons located in the digits contribute to the coherent oscillations. We recorded field potentials from the spatially separated sites in the digits in the terrestrial slug Limax marginatus. We also simultaneously recorded tentacular nerve to monitor the coherent oscillations. The spatially separated regions in the digits oscillated at the same frequency as the tentacular nerve, indicating a single coherent activity. To study the neural networks underlying the coherent oscillations, we examined the distributions of acetylcholinesterase (AChE)-containing and gamma-aminobutyric acid immunoreactive (GABA-ir) neurons. AChE-containing and GABA-ir fibers were found to connect the neurons in a branch of the digits with those in other branches. We also used a vital staining technique with 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate to examine the projections of neurons in the digits. Large stained cells were detected in many branches of the digits after placing the dye on one of the cell masses located in right and left sides of the tentacular ganglion. They were detected in the cell masses and in many branches of the digits after placing the dye on a branch of the digits. Our results showed that the slug primary olfactory system has highly interconnected neural networks.

Although primary olfactory systems in various animals display spontaneous oscillatory activity, its functional significance in olfactory processing has not been elucidated. The tentacular ganglion, the primary olfactory system of the terrestrial slug Limax marginatus, also displays spontaneous oscillatory activity at 1-2 Hz. In the present study, we examined the relationship between odor-evoked spike activity and spontaneous field potential oscillations in the tentacular nerve, representing the pathway from the primary olfactory system to the olfactory center. Neural activity was recorded from the tentacular nerve before, during and after application of various odors (garlic, carrot, and rat chow) to the sensory epithelium and the changes in firing rate and spontaneous oscillations were analyzed. We detected the baseline amplitude of the oscillations and baseline spike activity before stimulation. Odor stimulations for 20 s or 60 s evoked a transient increase in the firing rate followed by a decrease in the amplitude of spontaneous oscillations. The decrease in the amplitude was larger in the first 8 s of stimulation and subsequently showed recovery during stimulation. The amplitude of the recovered oscillations often fluctuated. Odor-evoked spikes appeared when the amplitude of the recovered oscillations was transiently small. These results suggest that the large oscillations could inhibit spike activity whereas the first transient increase in spike activity was followed by the decrease in the oscillation amplitude. Our results indicate that there is a significant negative correlation between spontaneous oscillations and odor-evoked spike activity, suggesting that the spontaneous oscillations contribute to the olfactory processing in slugs.

Conformational changes of receptors are dominated by the excitation of their collective motions. The most likely energy source of this excitation is considered to be a collision of an agonist with the binding site of a receptor and a consequential excitation of their vibrational modes. In the present study, as an approach to elucidating the mechanism for receptor activation, we chose both the symmetric stretching vibration of the 1C-carboxyl group of glutamate and the bending vibration of the guanidinium group of Arg485 of the glutamate receptor subunit GluR2 based on our previous study, and we quantum-mechanically calculated the vibrational excitation probability of these two vibrations by glutamate collision. The excitation probability of these two vibrations was found to exceed 0.5 about 260 fs after the onset of collision within the first-order perturbation approximation. Taking into account that the period of these vibrations is about 20 fs, we can expect that the vibrational excitation occurs in Arg485 after tens of vibrations during the collision. This vibrational energy may redistribute to the collective motions of the receptor, resulting in global conformational changes in the receptor. We also confirmed that the charge transfer was small between an agonist and the binding site of GluR2. The glutamate receptor may be oscillatory systems that require the energy injection into the specific vibrational modes of the specific amino acid residues to trigger their activation. Such an injection of energy is provided by agonist collision. (C) 2003 Elsevier B.V. All rights reserved.

The present study was designed to elucidate the roles of dendritic voltage-gated K+ channels in Ca2+ influx mechanism of a rat Purkinje cell using a computer simulation program. First, we improved the channel descriptions and the maximum conductance in the Purkinje cell model to mimic both the kinetics of ion channels and the Ca2+ spikes, which had failed in previous studies. Our cell model is, therefore, much more authentic than those in previous studies. Second, synaptic inputs that mimic stimulation of parallel fibers and induce sub-threshold excitability were simultaneously applied to the spiny dendrites. As a result, transient Ca2+ responses were observed in the stimulation points and they decreased with the faster decay rate in the cell model including high-threshold Ca2+-dependent K+ channels than in those excluding these channels. Third, when a single synaptic input was applied into a spiny dendrite, Ca2+-dependent K+ channels suppressed Ca2+ increases at stimulation and recording points. Finally, Ca2+-dependent K+ channels were also found to suppress the time to peak Ca2+ values in the recording points. These results suggest that the opening of Ca2+-dependent K+ channels by Ca2+ influx through voltage-gated Ca2+ channels hyperpolarizes the membrane potentials and deactivates these Ca2+ channels in a negative feedback manner, resulting in local, weak Ca2+ responses in spiny dendrites of Purkinje cells. (C) 2003 Elsevier B.V. All rights reserved.

The present study was designed to elucidate the cellular and molecular mechanisms of neuritogenesis in differentiating neurons. For this purpose. we used pharmacological and immunochemical techniques to determine the intracellular signal transduction pathways that regulate actin dynamics during neuritogenesis. We confirmed that a rise in intracellular cyclic AMP (cAMP) concentration stimulated cells to increase their neurite numbers, and that this increase of neurites was suppressed by activation of calcineurin induced by a Ca2+ influx through voltage-dependent Ca2+ channels. Expression of a specific cofilin kinase (LIM kinase 1) was increased and decreased by cAMP and Ca2+ cascades. respectively. The phosphorylation state, but not the level of expression, of a potent regulator of actin dynamics (cofilin) was strongly correlated with the expression level of LIM kinase 1. Our results suggest that polymerization and depolymerization of actin by cofilin phosphorylation is necessary for neuritogenesis in differentiating neurons. (C) 2003 Elsevier B.V. All rights reserved.

To understand the mechanism of molecular recognition of glutamate receptors, we calculated the dipole moments, net charges, and electrostatic potentials of the agonists and antagonists of the glutamate receptors under aqueous conditions by the ab initio molecular orbital method at the HF/6-311++G(3df,2pd) level. All of the ligands had negative net charges at both termini and, consequently, formed negative electrostatic potentials at these termini. We further calculated the net charges and electrostatic potential of the S1S2 lobes of the glutamate receptor subunit GluR2 under aqueous conditions by the semempirical PM3 molecular orbital method, and found that the ligand-binding cleft of the S1S2 lobes formed a primarily positive electrostatic potential. A strongly positive electrostatic potential was formed particularly around Arg485 in the S1 lobe. A negative electrostatic potential was observed only in a small region around Glu657 in the S2 lobe in the ligand-binding cleft. When a ligand approaches the ligand-binding cleft, it may proceed to Arg485 in the S1 lobe, due to both repulsion by the negative electrostatic potential of Glu657 as well as the attraction of the strongly positive electrostatic potential of Arg485 itself. (C) 2003 Elsevier B.V. All rights reserved.

Even though glutamic acid contains only one more carboxyl group than gamma-aminobutyric acid (GABA), these neurotransmitters are recognized by their own specific receptors. To understand the ligand-recognition mechanism of the receptors, we must determine the geometric and electronic structures of GABA and glutamic acid in aqueous conditions using the ab initio calculation. The results of the present study showed that the stable structure of GABA was the extended form, and it attracted both cations and anions. Glutamic acid only attracted cations and was stabilized in four forms in aqueous conditions: Type 1 (an extended form), Type 2 (a rounded form), and Types 3 and 4 (twisted forms of Type 1). The former two types had low energy and the energy barrier between them was estimated to be small. These results showed that most free glutamic acid is present as Type a, Type 2, and transient forms. The present results therefore suggest that the flexibility of the geometric structures of ligands should be taken into account when we attempt to elucidate the mechanism of recognition between ligands and receptors, in addition to the physicochemical characteristics of ligands and receptors.

To map the functional synaptic organization over a wide area in the optic tectum, we directly monitored two-dimensional propagation of postsynaptic depolarization evoked by firing of retinotectal afferents in optic tectum slices prepared from rainbow trout (Oncorhynchus mykiss), using a voltage-sensitive dye and a photodiode array system. The postsynaptic responses to afferent stimulation first propagated in the stratum opticum and stratum fibrosum et griseum superficiale in an anterograde fashion in the afferents and then expanded vertically into the deep layers. This vertical propagation appeared to occur along a bundle-like structure that corresponded well with a cluster of neurons whose somata are located in the stratum periventriculare. Pharmacological studies showed that these postsynaptic responses were mediated by ionotropic glutamate receptors. On the other hand, the optical signals appeared to consist of at least two components (a transient signal and a slow signal). The second transient signal summated with the first slow signal by paired stimulation, suggesting that the transient and slow signals originated from different cell types. Taken together, these results showed that the functional synaptic organization of the teleost optic tectum comprises of two depolarization-signal propagating paths along a horizontal layer structure and a vertical bundle-like structure and that these synaptic responses occur via glutamatergic transmission.

Based on the results of previous behavioral experiments, researchers believe that sensitivity to light stimuli is not restricted to the eyes in the pond snail Lymnaea stagnalis. To determine the presence of a non-ocular dermal photoreception system and to examine the synaptic connections between this peripheral system and the central nervous system, we electrophysiologically examined the activities of the pedal nerves in L. stagnalis by light stimulation. The results demonstrated that light stimulation evokes non-ocular dermal photosensitive responses in the foot, that these responses exert inhibitory, afferent influences through the inferior pedal nerves to the pedal ganglia, and that these responses were independent of the ocular photoreception system in L. stagnalis. (C) 2002 Elsevier Science B.V. All rights reserved.

Nitric oxide (NO), synthesized by the enzyme nitric oxide synthase (NOS), acts as an intercellular messenger associated with various physiological and pathological events. In this study, we investigated whether there exits a difference in the vulnerability to NO-induced cytotoxicity between undifferentiated and differentiated NG108-15 cells, and if so, the mechanisms responsible for the difference. Following a 7- to 8-day exposure to dibutyryl cAMP (dbcAMP), NG108-15 cells exhibited a neuron-like morphology associated with the expression of the neuronal protein, synaptophysin, and with increased NADPH-d activity. Neuron-like differentiated NG108-15 cells acquired resistance to exogenously applied NO. This increased resistance to NO toxicity in differentiated cells was almost completely cancelled out by inhibiting the activity of superoxide dismutase (SOD), but not by inhibiting the activity of NOS. The present study suggested that the activity of SOD increased in parallel with the activity of NOS associated with differentiation and was crucial for the acquired resistance to NO toxicity in differentiated cells.

Astrocytes are connected by gap junctions, which provide intercellular pathways that allow a direct exchange of ions and small metabolites including second messengers and the propagation of electric currents. The roles of gap junctional communication on whole-cell morphology, cytoskeletal organization, and intercellular communication in astrocytes are not yet clear even in vitro, though there are many studies that have examined the active relation between gap junctions and actin filaments in astrocytes. Here we examined the effects of gap junction inhibitors, which do not interrupt the formation but rather the function of gap junctions, on whole-cell morphology, cytoskeletal organization. and intercellular communication in rat cultured astrocytes. Functional blockade of gap junctions during the formation of an astrocytic monolayer resulted in discordance of actin stress fibers between neighboring cells. even though whole-cell morphology of these cells did not change by such treatment. Mechanical stimulation-induced calcium wave propagation was significantly reduced in these actin-discordance cells even after thorough wash out. Differentiation of astrocytes in the presence of gap junction inhibitors was associated with morphological disarrangement among neighboring cells due to disordered alignment of actin stress fibers between cells.
Our results indicate that gap junctional communication enables cell-to-cell coordination of actin stress fibers in astrocytes, thus enhancing intercellular communication through calcium spread. (C) 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved.

To examine the distribution of nitric oxide (NO)-generative cells and NO-responsive cells in the tentacles and procerebral lobes (olfactory processing center) of terrestrial slugs, we applied NADPH diaphorase (NADPH-d) histochemistry and NO-induced cyclic GMP (cGMP)-like immunohistochemistry. We found that NADPH-d reactive cells/fibers and cGMP-like immunoreactive cells/fibers were different, but they were localized adjacent to each other, in both the tentacles and the procerebral lobes. Then, we measured the concentration of NO that was generated around the procerebral lobes using an NO sensitive electrode, when the olfactory nerve was electrically stimulated as a replacement for an odorant stimulus. Stimulation of the olfactory nerve evoked an increase in NO concentration at nanomolar levels, suggesting that binding of nanomolar concentrations of NO to the prosthetic heme group activates soluble guanylyl cyclase. Taken together with previously reported physiological data, our results, therefore, showed that the NO/cGMP pathways are involved in slug olfactory processing.

Multiple site optical recording was used to analyze the neural activity changes caused by conditioned taste aversion (CTA) training in the pond snail Lymnaea stagnalis. In response to electrical stimulation of the median lip nerve, which transmits chemosensory signals of appetitive taste to the central nervous system, we optically detected large numbers of spikes in several parts of the buccal ganglion. The effects of CTA training on the spike responses were examined in two areas of the ganglion where the most active neural responses occurred. In one area (termed Area I) that included the N1 medial (NUM) cells, a class of central pattern generator interneurons involved in feeding behavior, the number of spikes in a period 1500-2000 ms after median lip nerve stimulation was significantly reduced in conditioned animals compared to control animals. In another area (termed Area II) positioned between buccal motoneurons, the B3 and B4CL (cluster) cells, the evoked spike responses were unaffected by CTA training. These results, taken together with our previous results indicating an enhancement of an inhibitory input to the NIM cells during CTA, suggest that an appetitive taste signal transmitted to the NIM cells through the median lip nerves is suppressed during CTA, resulting in a decrease of the feeding response. (C) 2001 John Wiley & Sons, Inc.

Although the mechanisms of Ca2+, wave propagation in astrocytes induced,by mechanical stimulation have been well studied, it is still not known how the [Ca2+](i) increases in the stimulated cells. Here, we have analyzed the mechanisms of [Ca2+](i) increase in single, isolated astrocytes. Our results showed that there was an autocrine mechanism of Ca2+ regulation mediated by ATP in mechanically stimulated astrocytes. This autocrine mechanism induced the activation of phospholipase C via a G-protein, resulting in Ca2+ release from intracellular Ca2+ stores. A second pathway mediating a [Ca2+](i) increase was via a Ca2+ influx from the extracellular space, which, interestingly, suppressed an intracellular Ca2+ oscillation. These two different Ca2+ cascades are involved in signal transduction and may function separately during intercellular communication. NeuroReport 12:2619-2622 (C) 2001 Lippincott Williams & Wilkins.

As a first approach to understanding the mechanism for the recognition of a ligand by its receptor, we first calculated the electronic and structural states of ionized gamma -aminobutyric acid (GABA) and ionized glutamic acid using the ab initio method with the 6311++G (3df, 2pd) basis set. We paid special attention to the physicochemical characteristics of these molecules, such as the electric dipole moment, electrostatic potential, and electrostatic force, Even though GABA and glutamic acid are known to exert completely opposite influences in the mammalian brain by binding their specific receptors, the only difference in their chemical structures is that glutamic acid contains one more carboxyl group than GABA. As a result, we succeeded in showing that a difference of only one carboxyl group induces significant differences in the electronic and structural states between these molecules. These differences have a crucial influence on the electric dipole moments, the electrostatic potentials, and the electrostatic forces. The most remarkable finding of the present research is that the electrostatic potential formed by glutamic acid is composed of only negative parts, while that formed by GABA is separated into positive and negative parts. These results strongly suggest that GABA can approach either positively or negatively charged amino acids by adjusting its own orientation, while glutamic acid can approach only a positively charged binding site.

To understand the mechanism of activation of a receptor by its agonist, the excitation and relaxation processes of the vibrational states of the receptor should be examined. As a first approach to this problem, we calculated the normal vibrational modes of agonists (glutamate and kainate) and an antagonist (6-cyano-7-nitroquinoxaline-2,3-dione: CNQX) of the glutamate receptor, and then investigated the vibrational interactions between kainate and the binding site of glutamate receptor subunit GluR2 by use of a semiempirical molecular orbital method (MOPAC2000-PM3). We found that two local vibrational modes of kainate, which were also observed in glutamate but not in CNQX, interacted through hydrogen bonds with the vibrational modes of GluR2: (i) the bending vibration of the amine group of kainate, interacting with the stretching vibration of the carboxyl group of Glu705 of GluR2, and (ii) the symmetric stretching vibration of the carboxyl group of kainate, interacting with the bending vibration of the guanidinium group of Arg485, We also found collective modes with low frequency at the binding site of GluR2 in the kainate-bound state. The vibrational energy supplied by an agonist may flow from the high-frequency local modes to the low-frequency collective modes in a receptor, resulting in receptor activation.

We studied the neural oscillatory activity in the peripheral olfactory system of the tentacles in the terrestrial slug, Limax marginatus, by extracellular recording, Recordings from the cut-ends of the inferior tentacular nerves connected to the inferior tentacular ganglia and sensory epithelia showed spontaneous oscillatory activity at frequencies of 0.1-30 Hz. This spontaneous activity was dominated by the 0.6-6 Hz band. Ethanol odor stimulation decreased the amplitude in the 0.6-6 Hz band and increased those in the 6-15 and 15-30 Hz bands. Antagonists of the gamma -aminobutyric acid (GABA) receptor, bicuculline and picrotoxin, resulted in suppression of spontaneous activity and modulated the odor response in the 0.6-6 Hz band. Our results indicate the involvement of GABA-mediated oscillatory activity in the tentacular nerves in the olfactory processing in molluscs. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Complement receptor 3 (CR3), one of cell adhesion molecules, plays a crucial role in secretion of mammalian neutrophils. To study whether CR3 is also involved in neurosecretion of gastropod molluscs, we examined the CR3-like immunoreactivity in the central nervous system (CNS) and the superior and inferior tentacles of the terrestrial slug, Limax marginatus. In the CNS of L. marginatus, we did not detect the specific immunoreactivity at all. In contrast, the CR3-like immunoreactivity was observed in the cell bodies and processes of collar cells of the superior and inferior tentacles. In particular, granules contained in the cell bodies of collar cells exhibited the CR3-like immunoreactivity. Retrograde labeling of horseradish peroxidase applied on the sensory epithelia (SE) of superior and inferior tentacles showed that the collar cells of these tentacles project their processes to the SE. in the previous study, homogenate of the superior tentacles injected into the body cavity of slugs stimulated spermatogenesis and simultaneously inhibited egg-laying behavior, suggesting that the tentacular hormone acts as gonadotropic hormone. Taken together, CR3 expressed in the collar cells is considered to be involved in the secretion of tentacular hormone in L. marginatus.

Although synapsin has been localized to presynaptic structures, its function remains poorly understood. In the present study, we investigated the presynaptic function of synapsin II using a synaptic vesicle recycling process using synapsin-II-overexpressing NG108-15 cells. Western blot analysis with antibodies for synaptic-vesicle-associated protein indicated that the number of synaptic vesicles was approximately doubled in synapsin II transfectants as reported previously. In differentiated synapsin-II-overexpressing and control cells, the application of high potassium induced strong intracellular calcium elevation along neurites and varicosities after differentiation and a weak calcium rise in the cell bodies. The uptake and release of the fluorescent dye FM1-43 revealed that synaptic vesicle recycling in synapsin-LI-transfected cells occurred with the same kinetics in the cell body and neuritic varicosities. Furthermore, the area labeled with FM1-43 fluorescence in the synapsin-II-transfected cells was approximately twice as much as in control cells after stimulation, and ATP released after synaptic vesicle fusion with the plasma membrane in synapsin-II-expressing cells was significantly elevated relative to controls. The number of synaptic vesicles paralleled the amount of transmitter released from the cells leading to the conclusion that the number of releasable synaptic vesicles were increased by synapsin II transfection into NG108-15 cells, suggesting that synapsin II may have a role in the regulation of synaptic vesicle number in presynapse-like structures in NG108-15 cells. (C) 2000 Elsevier Science BN. All rights reserved.

Although there are many lines of evidence for both the presence of nitric oxide synthase (NOS) in the central nervous system (CNS) and the effects of NO on activating and modulating the feeding circuit in Lymnaea stagnalis, there has been no direct evidence that NO generation in the CNS accompanies feeding behavior. In the present study, we used a NO specific electrode to measure the increase in NO concentration around the buccal ganglia when the lips of semi-intact preparations of L. stagnalis were stimulated by sucrose. The NO concentration of the buccal ganglia was significantly increased by an application of sucrose to the lips. A NO scavenger and a NOS inhibitor suppressed this increase in NO concentration. A pair of putative NO-generative neurons in the buccal ganglia, the B2 cells, are active during the inter-feeding phase, and the bursting of the B2 cell elicited by sucrose application starts simultaneously with the feeding response. The rhythmic pulses of NO generation corresponded well with the rhythmic bursting of the B2 cells, which itself corresponds to the 'fictive feeding response'. The present data provide the first direct evidence that NO is generated in the buccal ganglia of L, staganalis and is involved in a specific behavior such as Feeding. (C) 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

A- and D-type K+ channels (KA and KD channels) have unique physiological properties that play important roles in the integration of excitatory post synaptic potentials (EPSPs) in neuronal dendrites. These functions were analyzed using a computer program, NEURON, to simulate high- frequency sequential synaptic inputs, that can induce long-term potentiation (LTP). We paid close attention to the stability of the reduction of sequential EPSPs. When either KA or KD channels were included in models, the EPSP reduction ratios were less stable than containing both KA and KD channels. When both KA and KD channels were present in the model, the variance of EPSP reduction ratios was significantly smaller in comparison with that in the presence of either KA or KD channels alone. We thus concluded that the co-existence of KA and KD channels is necessary to produce stable EPSPs during the high-frequency synaptic stimulation necessary for induction of LTP. (C) 2000 Elsevier Science Ireland Ltd.

In the present study, the 'clockwork' hypothesis proposed by Schrodinger was examined from the viewpoint of thermodynamics. Firstly, noticing a unidirectional transfer of entropy in a heat engine, the logic was briefly explained about a close relation between this entropy transfer and an irreversible cycle performed by a working body. Next, paying attention to two fundamental differences between a heat engine and a biological system, we considered an isolated system A(Sigma) consisting of three one-component systems (A(i), A, A(o)) and noted a case that the same molecules as the component ones flowed quasistatically into A(i) from the outside. Then, the unidirectional flows of the molecules, energy and entropy, which were induced by the above inflow in A(Sigma), were formulated on the basis of the equilibrium thermodynamics for an open system. Furthermore, it was clarified that the fundamental equation for these flows is the Schrodinger inequality and that the necessary-sufficient condition for this inequality is the existence of an irreversible cycle performed by A. Here A corresponds to a working body in a heat engine. It was, thus, concluded that the 'clockwork' hypothesis by Schrodinger is considered to be reasonable for a biological system composed of various irreversible subsystems. (C) 2000 Elsevier Science B.V. All rights reserved.

To identify the types of neurons and to infer the patterns of connectivity in slug tentacles, we stained the neurons in the superior and inferior tentacles in the terrestrial slug, Limax marginatus, by backfilling of the tentacular nerves with Lucifer yellow. Four types of stained neurons, '(1) sensory neurons', '(2) gamma cells', '(3) ganglion cells', '(4) lateral cells', were identified both in the superior and inferior tentacles. Three subtypes of the sensory neurons, '(la) round sensory neurons', '(lb) spindle-shaped sensory neurons', and '(lc) small sensory neurons', were found in the digits. The gamma cells and the ganglion cells were interneurons. Three subtypes of gamma cells, '(2a) round monopolar gamma cells', '(2b) round bipolar gamma cells', and '(2c) large gamma cells', were present in the digits. The ganglion cells were composed of '(3a) monopolar ganglion cells','(3b) bipolar ganglion cells', and '(3c) elongated ganglion cells'. The monopolar and bipolar types were located both in the tentacular ganglia and digits, whereas the elongated type was present only in the tentacular ganglia. The lateral cells, whose function is unknown, were found in the dermo-muscular sheaths of the tentacles. Our study provides the first description of the neuronal map of inferior tentacles in gastropods. The results showed no differences in the morphological features of stained neurons between the superior and inferior tentacles in L. marginatus. (C) 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

The differentiated type of neuroblastoma x glioma hybrid cell line, NG108-15, has widely been used in in vitro studies instead of primary-cultured neurons. Here we examined whether NG108-15 cells can be used as a model for studying the neuronal differentiation process. We compared the expression of neuronal proteins (neurofilament 200 (NF200), phosphorylated-NF200 (p-NF200), microtubule associated protein 2, synaptophysin, syntaxin 1, choline acetyltransferase, and acetylcholinesterase (AChE)) and a glial protein (vimentin) between undifferentiated and differentiated NG108-15 cells by immunocytochemistry and immunoblot analysis. The expression of all neuronal proteins, with the exception of NF200 and p-NF200, was positive in differentiated cells, but almost negative in undifferentiated cells. On the other hand, cytoskeletal intermediate filaments (NF200 and p-NF200) for neurons and that (vimentin) for glia were present in both undifferentiated and differentiated cells. Furthermore, a high expression of AChE mRNA was confirmed in differentiated cells by reverse transcription-PCR analysis. Our results showed that even though the expression of cytoskeletal filaments does not change during differentiation of NG108-15 cells, these cells during differentiation can serve as an appropriate tool for investigating and understanding the mechanisms involved in neuronal development and differentiation. (C) 2000 Elsevier Science Ireland Ltd and the Japanese Neuroscience Society. All rights reserved.

In order to examine the mechanical properties of the membrane surface of astrocytes, we observed living astrocytes by atomic force microscopy (AFM) both in contact mode and force-mapping mode. Ridge-like structures reflecting actin filaments were observed in the topographic images in contact mode, but not in force-mapping mode, using a zero-loading force. When we measured the elasticity of astrocytes, we observed that the cell membrane above the nucleus was soft and the cell membrane above the cytosol was stiff. In particular, the parts reflecting actin filaments were very stiff. This effect of actin filaments on the elasticity of astrocytes was confirmed by the loss of actin filaments after application of actin-polymerization inhibitor.

Fictive feeding activity was monitored in the buccal ganglia of semi-intact preparations of the pond snail, Lymnaea stagnalis, to examine the effects of nitric oxide (NO) released from motoneurons innervating the esophagus on the feeding response. The present results suggest that first
even the low concentration of constitutive NO precisely regulates the feeding rhythm by suppressing high frequency feeding responses
second, that the high concentration of NO released after activation of the feeding central pattern generator following appetitive stimulation of the lips suppresses the feeding rate, resulting in recurrent inhibition. This is the first direct evidence that NO can function to suppress rhythmic activity in the brain. Copyright (C) 2000 Elsevier Science Ireland Ltd.

We observed CR3-like immunoreactivity in the central nervous system (CNS) and its surrounding peripheral nerves of the pond snail, Lymnaea stagnalis. Ln the CNS of L. stagnalis, the immunoreactivity presenting meshwork-like structure was detected in some neurosecretory cells, which an the light green cells (LGCs) and the canopy cells (CCs), both controlling the body growth. The immunoreactivity was also observed along the edges of median lip nerves. The immunoreactive regions in the median lip nerves appeared to form the axonal plates, from which the LGCs and the CCs release molluscan insulin-related peptides (MIPs) into the blood. By contrast, no immunoreactivity was detected in other neurosecretory cells or their release sites, fur example the caudodorsal cells and the cerebral commissure, which release ovulation hormones. The present findings, therefore, suggested that CR3 expresses only in the neurosecretory cells releasing MIPs in L. stagnalis. (C) 2000 Elsevier Science B.V. All rights reserved.

We examined three-dimensionally the arrangement of gamma-aminobutyric acid; (GABA)-like immunoreactive neurons in the central nervous system (CNS) of the pond snail, Lymnaea stagnalis, by a combination of immunohistochemistry and confocal laser scanning microscopy on whole-mount preparations. GABA-like immunoreactivity was detected in all ganglia of the adult CNS. The following distribution of immunoreactive cell bodies was noted in the adult snail. Buccal ganglia: one cell body and five pairs of cell bodies, cerebral ganglia: one pair of cell bodies, pedal ganglia: two single cell bodies, two pairs of cell bodies, and three pairs of cell clusters, and pleural ganglia: one pair of cell bodies. In the asymmetrical parietal ganglia, three cell bodies were located in the left parietal ganglion; three cell bodies and three cell clusters were located in the right parietal ganglion. In the single visceral ganglion, a few scattered individual cell bodies and a cell cluster were GABA-like immunoreactive. Our results showed that the occurrence of GABA is widely spread in the CNS of adult L. stagnalis. GABA-like immunoreactivity in the CNS was not detected in the embryo but; was observed after hatching, although the number of stained cells was less than in the adult, with the exception of those in the cerebral ganglia where their number decreased with maturation. Our results provide detailed maps of the central GABA-like immunoreactive neurons in juveniles, immatures, and adults oft. stagnalis. J. Comp. Neurol. 418:310-322, 2000. (C) 2000 Wiley-Liss, Inc.

The pond snail, Lymnaea stagnalis, is a useful preparation for analyzing the commonality between development and learning. To promote this analysis, the anatomical substrate should be provided upon which learning is superposed during development. Because we previously demonstrated that L. stagnalis change their ability of conditioned taste aversion (CTA) as a long-term memory from veliconcha embryos to immatures, we examined in the present study the numbers of cells and the volume of the buccal and cerebral ganglia in the snails at the critical developmental stages. The buccal and cerebral ganglia include the majority of neurons involved in the CTA. We found that the numbers of cells in these ganglia are almost saturated in the immatures, but the volumes of these ganglia still increase from the immatures to the adults. These results suggested that most of the cells indispensable to the CTA emerge at the immature stage, but that individual cells in the ganglia continue to enlarge even in adulthood. Furthermore, the key neuron for the CTA was found to mature at the immature stage. The present study provided the anatomical substrate upon the long-term CTA, by which snails can eat safe food in a wide territory.

Using the force mapping mode of atomic force microscopy (AFM), we measured spatial distribution of elastic moduli of living mouse fibroblasts (NIH3T3) in a physiological condition. The nuclear portion of the cellular surface is about 10 times softer than the surroundings. Stiffer fibers are confirmed in the elastic images. In order to investigate origin of the softer nuclear portion and the stiffer fibers, we fixed the identical cells imaged by the AFM, and carried out immunofluorescence observation for three types of cytoskeletal filaments - actin filaments, microtubules, and intermediate filaments, using confocal laser scanning microscopy (CLSM). A comparison between the AFM and the CLSM images revealed that the elasticity of the cells was concerned not only with the distribution of actin network, but also with intermediate filaments, whereas microtubules had no large effect on the measured elasticity. (C) 2000 Elsevier Science B.V. All rights reserved.

We examined the interior structure of exocytotic apertures in synaptic vesicles of neuroblastoma x glioma hybrid cells using atomic force microscopy. The atomic force microscopy detected apertures of 50-100 nm in diameter at various depths within the varicosities of these cells. We were also able to image a regular radial pattern on the wall and lump-like structures at the bottom of these apertures. In contrast, scanning electron microscopy could only detect the apertures but not the fine details of their interior. The cells examined here exhibited the same electrophysiological properties and expression of synaptophysin and syntaxin 1 as presynaptic terminals, as studied by various electrophysiological and imaging techniques.
Our results indicate that atomic force microscopy allows three-dimensional viewing of the fine structures located inside exocytotic apertures in nerve cells. (C) 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved.

To directly monitor the glial activity in the CNS of the pond snail, Lymnaea stagnalis, we optically measured the electrical responses in the cerebral ganglion and median lip nerve to electrical stimulation of the distal end of the median lip nerve. Using a voltage-sensitive dye, RH155, we detected a composite depolarizing response in the cerebral ganglion, which consisted of a fast transient depolarizing response corresponding to a compound action potential and a slow depolarizing response. The slow depolarizing response was observed more clearly in an isolated median lip nerve and also detected by extracellular recording. In the median lip nerve preparation, the slow depolarizing response was suppressed by an L-type Ca2+ channel blocker, nifedipine, and was resistant to tetrodotoxin and Na+-free conditions. Together with the fact that a delay from the compound action potential to the slow depolarizing response was not constant, these results suggested that the slow depolarizing response was not a postsynaptic response. Because the signals of the action potentials appeared on the saturated slow depolarizing responses during repetitive stimulation, the slow depolarizing response was suggested to originate from glial cells. The contribution of the L-type Ca2+ current to the slow depolarizing response was confirmed by optical recording in the presence of Ba2+ and also supported by intracellular Ca2+ measurement.
Our results suggested that electrical stimulation directly triggers glial Ca2+ entry through L-type Ca2+ channels, providing evidence for the generation of glial depolarization independent of neuronal activity in invertebrates. (C) 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved.

We have focused on effects of local mechanical properties of the cell on cell motion. By using atomic force microscopy, we measured spatial distribution of local elastic modulus on mouse fibroblasts (NIH3T3), which is living in a physiological condition. In order to examine validity of AFM elastic measurements, we measured local elastic modulus of gels as elastic reference materials. The results obtained with AFM were compared with values obtained by tensile creep method. It is veryfied that these values are proportional each other. The AFM experiments on living cells revealed that center area of cell surface is about 10 times softer than the surroundings and looks like a big softer hole in the elasticity image. We fixed the cell just after the AFM measurements and carried out immunofluorescence observation for cytoskeletal filaments of actin filaments, microtubules and intermediate filaments. A comparison between distribution of local elasticity and cytoskeletones indicates that harder area on the cell results mainly from concentration of actin filaments. However, We found that some areas like the big softer hole do not correspond to distribution of actin filaments.

Using the force modulation mode in atomic force microscopy, we have succeeded in capturing time-lapse viscoelastic images of living mouse fibroblasts (NIH3T3) for several hours in a physiological condition without damaging the fibroblasts. Elongation of the lamellipodia and swelling of blabs were observed in time-lapse topographic images, which were taken every 10 min. The corresponding viscoelastic responses at a frequency of 600 Hz were visualized as consecutive images. The stiffer part of the cell body was fairly stable and did not show morphological changes for over 1 h. This is probably due to excess condensation of the actin network, hardening the cell cortex, and lowering the cytoskeletal activity. The nuclear portion of the cell body seems to be slightly less viscous than the peripheral region.

The topography and elasticity of living and fixed astrocytes cultured from the rat cerebra were studied quantitatively by atomic force microscopy (AFM). Ridge-like structures reflecting F-actin beneath the cell membrane were prominent in the contact-mode images of living astrocytes. Many of these ridges became unclear after fixation (2%, glutaraldehyde). In addition, the ridge-like structures were invisible in the topography of living cells observed at zero-loading force in the force mapping mode, which is considered to show the real cell surface not pressed down by an AFM tip. The topography of fixed cells observed both in the contact mode and at zero-loading force in the force mapping mode was similar to that of living cells observed at zero-loading force in the force mapping mode, although some deformed areas were detected in the fixed cells. The elasticity map images of living astrocytes showed that the cell membrane above the nucleus was softer (2-3 kPa) than the surroundings, and that the cell membrane above F-actin was stiffer (10-20 kPa) than the surroundings. In the elasticity map images of fixed astrocytes, on the other hand, the elasticity of the cells was found to be relatively uniform (200-700 kPa) irrespective of the inner structures of cells. These results show that images observed by AFM should be carefully examined in consideration of the force introduced to specimens and the elasticity of specimens to find out the real surface topography.

The pond snails, Lymnaea stagnalis, change their ability of conditioned taste aversion (CTA) during their development, for example, stage 29 embryos can acquire the CTA, whereas immature snails come to use a long-term memory to maintain the conditioned response. We thus examined the relationships between the learning ability and the development of key neurons (cerebral giant cells: CGCs) for this CTA. The immunoreactivity of serotonin, which is a main neurotransmitter employed in the feeding circuitry, was first observed in the CGCs at the stage 29. After hatching, the CGCs developed their neuropile faster than other cells in the buccal and cerebral ganglia, resulting in their early innervation at the immature stage. The present results, therefore, indicate that the development of key neurons for learning stimulates the developmental changes in learning ability. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

As the first step in the study df morphological changes in neurons associated with their functional changes, we applied atomic force microscopy (AFM) for the observation of fine three-dimensional morphological changes in rat cerebellar granule cells stimulated by an agonist of glutamate receptors, N-methyl-D-aspartate (NMDA). The AFM revealed that NMDA changed the cross-sections of cell bodies from a trapezoid-like form to a triangle-like form within a minute. The fine hill-like structures on the top surfaces of the cell bodies became wider during the same period. These results were suggested to be induced by the depolymerization; of filamentous actin triggered by the entry of Ca2+ via cation channels complexed with the activated NMDA receptors.

The cerebral giant cell (CGC) is known to play a crucial role in the regulation of feeding response in the pond snail, Lymnaea stagnalis. However, the mechanisms of signal transduction from the CGC to the following buccal motor neurons are not clear. In the present study, we examined whether cyclic AMP (cAMP)-dependent protein kinase (PKA) contributes to enhancement of a monosynaptic connection between the presynaptic CGC and the postsynaptic buccal motor neuron 1 (B1 cell). Injection of cAMP into the CGC or inhibition of phosphodiesterase by isobutylmethylxanthine in the CGC increased the amplitude of excitatory postsynaptic potential (EPSP) in the B1 cell, whereas no changes were detected in the electrical properties of the CGC. The synaptic enhancement in the B1 cell was completely blocked by inhibition of PKA in the CGC but did not require a de novo protein synthesis due to a PKA phosphorylation. The increase in the EPSP amplitude of B1 cell was associated with the increase in the amount of serotonin release from the CGC. These results hence provided the physiological evidence of the direct regulation of a synaptic enhancement by PKA in the CNS of L. stagnalis, indicating the completely different mechanism from that in the well-studied siphon- and gill-withdrawal reflex in Aplysia.

The lip and tentacle nerves of the pond snail, Lymnaea stagnalis, were characterized using electrophysiological techniques. When the activity of those nerves was induced in lip-tentacle preparations, aversive taste signals were transmitted through all the lip and tentacle nerves, but appetitive signals could be recorded only through the superior lip nerve. In the CNS immersed in high Mg2+-high Ca2+ saline, electrical stimuli applied to any of the nerves failed to induce action potentials in one of the regulatory neurons (cerebral giant cell: CCC) involved in feeding responses, implying that the signals are polysynaptically transmitted to the CGC. Intracellular recordings revealed that the CGCs in semi-intact half-body preparations received both appetitive and aversive taste signals not only through the superior lip nerve but also through the median lip nerve. In addition, an osphradium was ruled out as a candidate for appetitive reception. The present results, together with our preceding data arrived at by the histochemical analyses, indicate that the appetitive taste transduction responsible for generating feeding responses is performed through the superior lip nerve with some contribution of the median lip nerve. The data showing that the CGC can receive various taste signals suggests that it may play a crucial role in feeding behavior as demonstrated in the study of conditioned taste-aversion. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

Although it has long been believed that glial cells play a major role in transmitter uptake at synapses in the CNS, the relative contribution of glial and neuronal cells to reuptake of synaptically released glutamate has been unclear. Recent identification of the diverse glutamate transporter subtypes provides an opportunity to examine this issue. To monitor glutamate transporter activity, we optically detected synaptically induced changes of membrane potential from hippocampal CA1 field in slice preparations using a voltage-sensitive dye, RH155. In the presence of ionotropic glutamate-receptor blockers, synaptic inputs gave rise to a slow depolarizing response (SDR) in the dendritic field. The amplitude of SDR correlated well with presynaptic activities, suggesting that it was related to transmitter release. The SDR was found to be caused by the activities of glutamate transporters because it was not affected by blockers for GABA(A), nACh, 5-HT3, P-2x, or metabotropic glutamate receptors but was greatly reduced by dihydrokainate (DHK), a specific blocker for GLT-1 transporter, and by D,L-threo-beta-hydroxyaspartate (THA), a blocker for EAAC, GLAST, and GLT-1 transporters. When SDR was detected with RH482 dye, which stains both glial and neuronal cells, 1 mM DHK and 1 mM THA were equally effective in suppressing SDR. The SDR was very small in GLT-1 knockout mice but was maintained in gerbil hippocampi in which postsynaptic neurons were absent because of ischemia. Because GLT-1 transporters are exclusively expressed in astrocytes, our results provide direct evidence that astrocytes play the dominant role in sequestering synaptically released glutamate.

The lip and tentacle nerves of the pond snail, Lymnaea stagnalis, were characterized using histological techniques. Anatomical drawings showed the detailed distributions of the superior lip, median lip, and tentacle nerves in the lip and tentacle; in particular it was found that the mouth is mainly innervated by the superior lip nerve. The tentacle nerve was clarified to form a zigzag structure along the extension direction in a shrinking tentacle. By backfilling of the superior lip nerve and/or the median lip nerve with fluorescent dyes, the neurons in the CNS made some clusters, whereas those stained from the tentacle nerve made other clusters. These stained neurons were not part of the central pattern generator or its regulatory neurons for feeding. The present results, therefore, suggest that the superior lip nerve may be employed as a principal factor in the chemosensory transduction from the mouth, and that no direct inputs occur through the lip and tentacle nerves to the central pattern generator or its regulatory neurons for feeding. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

As the first step to study relationships between development and learning in the molluscan central nervous system,we examined developmental changes in acquisition and retention of a conditioned taste aversion (CTA) in the pond snail, Lymnaea stagnalis. We found that snails developed ability of a CTA as a long-term memory through three critical stages. Embryos in veliconcha started to respond to appetitive sucrose at the first critical stage. This response was in good agreement with morphological observations that embryos at this developmental stage seemed to be physically ready to eat. However, they could not associate this appetitive stimulus (conditioned stimulus: CS) with an aversive stimulus of KCI (unconditioned stimulus: UCS). At the second critical stage, embryos just before hatching acquired the CTA, but the conditioned response did not persist. Through this stage, they may acquire learning ability to safely seek out food in an external environment. At the third critical stage, immature snails with a 10 mm shell could use a long-term memory to maintain the conditioned response. This memory persisted for at least a month, showing that now they are able to maintain a long-term memory so that they can safely eat a variety of food when they cover wide territory to search for a mate. The present findings indicate that the development of learning ability in snails, which secures acquisition of better survival ability, is coincident with the major changes in their life cycle.

The incomplete morphological characterization of type 2 astrocytes is in pari responsible for the slow progress of studies on these cells. To examine and characterize type 2 astrocytes morphologically, three-dimensional fine structures of the surfaces of type 2 astrocytes cultured from rat cerebella were studied by a combination of atomic force microscopic and immunocytochemical techniques. Atomic force microscopy (AFM) revealed irregular ridge-like structures that form a meshwork distributed throughout the cell body surfaces and the thick processes. These ridges were found to be of two heights (31 nm and 82 nm). This finding indicates two possible configurations responsible for shaping the meshwork: (1) two structures of different thickness are beneath the cell membrane; and (2) two structures are located at two different depths from the cell membrane. On the other hand, immunocytochemical studies for tubulin and glial fibrillary acidic protein (GFAP) revealed that these cytoskeletal filaments are similarly distributed within the resolution power of a light microscope. However, no detectable structures were obtained by actin staining. The immunocytochemical findings suggest that the AFM-revealed ridges forming the irregular meshwork on the cell surfaces may reflect very fine bundles of tubulin and/or GFAP. Therefore, AFM study, with the help of immunocytochemical study, is a powerful tool for characterizing cell morphology. The results of the present study reveal the first morphological characterization of type 2 astrocytes.

We observed the time-dependent morphological alteration of astrocytes during their adhesion by atomic force microscopy (AFM) and investigated the relationships between this morphological alteration and the localization of actin filaments and connexin 43 by immunocytochemistry. The fine processes observed as fine ridge-like structures by AFM were closely concerned with actin filaments by immunocytochemistry. During the adhesion of astrocytes, actin filaments appeared to be aligned regularly beyond the borders among different cells. Detectable connexin immunoreactivity was changed in the following regions: 1) the tips of fine cell processes and the cell margin when astrocytes started to adhere
2) the border of cells when astrocytes tightly adhered
and 3) non-specific sites when astrocytes became a cluster. In the former two cases, the immunopositive spots for connexin were observed to colocalize with the tips of cell processes with actin filaments. These results strongly suggest that connexin associated with actin filaments at the tip of cell processes plays an important role in the early stage of the adhesion of astrocytes. These observations afford valuable clues for understanding the glial communication.

To find the primary mechanism for the frequency changes of electrical oscillations in the procerebral (PC) lobe of a slug, we electrically stimulated the tip, middle and basal regions of the digits of the superior and inferior tentacles and recorded the local field potentials from the PC lobe. Stimuli to the middle and basal regions of the digits of the inferior tentacle significantly decreased the frequency of electrical oscillations in the PC lobe, whereas those to the tip region of the digits of the inferior tentacle and all regions of the digits of the superior tentacle increased it. These findings suggest that the change in the frequency of electrical oscillations in the PC lobe depends on the excited region in the digits, providing the first presentation of the physiological difference in the olfactory function between the superior and inferior tentacles. (C) 1999 Elsevier Science B.V. All rights reserved.

We examined the roles of A- and D-type K channels in the integration of EPSPs, particularly EPSP reduction, in a model dendrite using a computer simulation program (NEURON), instead of conventional electrophysiological methods which present technical difficulties. We obtained two important results: (1) KD channels play a crucial role in every synaptic input event, and (2) KA channels affect reduction of the second EPSP only when the second synaptic input is applied with a short interval (less than or equal to 100 ms). These results suggest that KD channels, which have not yet been analyzed experimentally, play a major role in synaptic integration in dendrites by altering their cable properties. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

We demonstrated a sensory preconditioning in the pond snail, Lymnaea stagnalis. An appetitive sucrose solution (a conditioned stimulus: CS1) and weak vibration (another conditioned stimulus: CS2) were first associated, and then the CS2 and an aversive KCl solution (an unconditioned stimulus: UCS) were done. To build the conditioning, two different training procedures, spaced and massed, were examined. After the both training, the sensory preconditioning was built: significantly fewer feeding response to the CS1 became elicited; slower latency to the first bite to the CS1 was induced. No significant differences on the memory retention between these training procedures were found in the sensory preconditioning. (C) 1998 Elsevier Science B.V. All rights reserved.

Operant conditioning that the pond snail, Lymnaea stagnalis, suppressed its naturally occurring behavior of escape from a water tank was examined by using a negative reinforcement (i.e. an aversive stimulus) prepared outside the tank. During the training period, the number of escapes from a tank was strongly suppressed. One of behavioral factors for this suppression was confirmed as the elongation of latency to the first escape after training. The effects on the memory retention were examined in the massed and spaced training procedures. The latter procedure interposes a rest interval between three sets of 20-min training sessions, whereas the former has the same number of training sessions with no rest interval within 60 min. The memory retention by the massed training was observed within 20 min after training. By the spaced training, the learning acquisition was found to be stronger, which was observed as the slower latency to the first escape, than by the massed training, but the longer-lasting memory retention, which had been expected first, was not formed. These results suggest that once Lymnaea recognize the external environment is safe after training, they may extinguish their memory of the past situation quickly, resulting in no or very little difference in the memory retention by two different training procedures in this operant conditioning. Together with the facts that classical conditioning and its neuronal mechanisms in Lymnaea were previously clarified, the present findings may help to address not only the neuronal basis of operant conditioning but also the relation between classical and operant conditioning.

To examine whether nitric oxide (NO)-generative neurons are included in the central circuitry for generation of feeding pattern in the pond snail, Lymnaea stagnalis, two staining techniques for NADPH diaphorase and serotonin (5-HT) were applied for its central nervous system (CNS). The former technique is known to show localization of NO synthase; the latter is well employed as a marker for the feeding circuitry because 5-HT is a main transmitter in it. In the buccal ganglion, B2 motoneuron was found to be a putative NO-generative neuron. This motoneuron is not involved directly in the coordination of feeding pattern but is activated simultaneously with the feeding to control the oesophageal and gut tissues for the digestion. Taking account of the diffusion effects of NO, the NO released from B2 motoneuron, when the feeding is started, is thought to sufficiently modulate the feeding circuitry. In the cerebral ganglion, the superior lip nerve, the median lip nerve and the tentacle nerve included both putative NO-generative fibers and serotonergic fibers. These fibers are not identical, but the NO released in the nerves may activate the serotonergic fibers, resulting in the influence upon the initiation of the feeding. Therefore, our present findings clearly showed that NO is not involved in transmission within the central circuitry for the feeding, but suggested that NO can crucially affect the feeding behavior, such as initiation and modulation of the feeding pattern. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

We first showed a general theory that reception of information from the outside in a receptor system is accompanied both by an inflow of entropy and by a generation of entropy depending on the reliability of the actual reception mechanism. Then, considering a case for the absorption of light by the visual-pigment system of visual cell, we calculated the time(t)-dependent change in the number N-2(t) of excited visual-pigments to obtain the entropy increase. We thus arrived at the following four conclusions: (1) One or two photons can be detected with a reliability of at least 54%; (2) In compensation for this detection, entropy > 1.12 x log2 is generated; (3) An incident photon of frequency nu from a light source of temperature T-s yields an entropy of h nu/T-s; and (4) Depending on the characteristics of the visual-pigment system, another entropy being different from (2) is generated in proportion to N-2(t). (C) 1998 Elsevier Science B.V. All rights reserved.

Using the force modulation mode in atomic force microscopy (AFM), we have succeeded in imaging elastic properties of agar gels immersed in water. The elastic images of ap ar have been captured simultaneously with the topographic images. Stiffer grains of agar whose size is about 200 nm can be clearly seen in the elastic image of 3.0% agar, while they are not so visible in the case of 1.5% agar. These grains probably correspond to aggregation of agar which cannot be observed in the topographic images. We also measured force-versus-distance curves using AFM to confirm that the absolute values of elastic modulus (Young's modulus) of agar coincide with the bulk values measured using the conventional stress-strain method. The estimated values of the elastic moduli with the AFM were 40 and 90 kPa for 1.5% and 3.0% agar gels, respectively. These are in good agreement with the respective bulk values of 30 and 80 kPa obtained using the conventional method.

Although we can routinely obtain fine structural images of cells by atomic force microscopy (AFM), the adequacy and reliability of morphological information acquired from these AFM images remain to be examined. In this report, we compared images of the line structures of nerve cells as observed by both AFM and scanning electron microscopy (SEM). Although AFM revealed the structure of the top views of cells in greater detail than SEM, their side structures were better observed by SEM. The linear structures in the neural processes detected only by AFM were confirmed, by immunofluorescence staining, to be reflections of the cytoskeletal structures located beneath the cell membrane. These differences between the AFM and the SEM images reflected the characteristics of the detection systems and methods used for sample preparation. Therefore, these results revealed that more detailed information on cell morphology can be obtained by using both AFM and SEM to advantage.

The morphology of cultured glial cells was examined using a combination of atomic force microscopy (AFM) and immunofluorescence staining for cytoskeletons. The meshwork of type-1 astrocytes consisted of thick longitudinal and thin lateral lines on the cell surfaces observed by AFM, the former lines were confirmed to be reflections of actin filaments. The astrocytic processes of type-2 astrocytes were observed to be rugged on AFM. These structures were mainly affected by microtubules. Immunofluorescence imaging of microglia revealed that actin filaments and microtubules were arranged radially and wavily along the cell edge, respectively. AFM could detect these radial and wavy structures clearly. These results show that AFM can provide information on the cytoskeletons of glial cells, indicating that AFM is a useful tool for the morphological characterization of cells.

Using the force modulation mode in atomic force microscopy, we measured elastic properties of living mouse fibroblasts (NIH3T3) in a culture medium, The topographic images of the cellular surface and the corresponding elastic images of the cellular surface were able to be captured simultaneously with high spatial resolution, The consecutive images were useful for examining time-dependent changes in the cellular surface, We observed that some cells continued to shrink and change their softness for 2 hours, Then the force modulation mode in atomic force microscopy shows potential use in analyzing time-dependent regional elastic properties of living cells with high spatial resolution.

To study the neuronal mechanism of a conditioned taste-aversion (CTA) learning in the pond snail Lymnaea stagnalis, we examined the synaptic connection between the neuron 1 medial (N1M) cell and the cerebral giant cell (CGC), the former is an interneuron in central pattern generator for the feeding response and the latter is a regulatory neuron to the central pattern generator. Inhibitory postsynaptic potential (IPSP) which was evoked in the N1M cell by activation of the CGC was larger and lasted longer in the conditioned animal than that in the control animal. The electrical properties of the cell body of CGC and the responses of the CGC to the chemosensory inputs were not changed during the CTA learning. These results, together with the previous report indicating the existence of excitatory projection from the N1M cell to the feeding motoneuron, suggest that enhanced IPSP in the N1M cell may underlie the suppression of feeding responses in the Lymnaea CTA learning. (C) 1997 Elsevier Science Ireland Ltd.

We examined the practical scan speed for the observation of live neurons in a physiological solution using atomic force microscopy (AFM) with a desired vertical resolution of the order of 10(-8) mi which was reasonable when taking into account that a flicker of extracellular protein and saccharide on the neurons in the solution occurred during an observation period of a couple of minutes. The practical scan speed was found to be under 40 mu m/s, therefore, if we applied AFM using 100 lines and 100 pixels per line to an observation area of 20 mu m x 20 mu m, the minimum period for acquiring one image was estimated to be about 2 min. This procedure gave us good images that represented the slow three-dimensional dynamics in live neurons, such as the retrograde movement of surface protuberances, but suggested that another approach was required to detect fast structural changes induced by stimulation.

It is necessary to determine whether, in the same species and for the same behavior, aversive and appetitive conditioning yield different strengths and periods of either acquisition or retention. To this end, we first examined the effects of various chemo-sensory and physical stimuli on feeding and avoidance behavioral responses in the pond snail, Lymnaea stagnalis. Then, using these findings, we constructed classical-conditioning paradigms with aversive and appetitive stimuli. In the aversive conditioning paradigm, an appetitive stimulus (sucrose), which increased the feeding response, was paired with an aversive stimulus (KCI, quinidine sulfate or electric shock), which inhibited the feeding behavior. Upon presentation of KCI, the first type of aversive conditioning, which is generally called ''taste-aversion learning with cessation of feeding response'', was acquired quickly and persisted for up to a month. When using a noxious stimulus (quinidine sulfate or electric shock) inducing pain we additionally found the second type of aversive conditioning, in which the previously appetitive stimulus (sucrose) not only failed to increase the feeding response, but came to elicit an avoidance response. This second type conditioning took longer to acquire and persisted for a shorter period of time than the first type. On the other hand, the appetitive conditioning paradigm paired a neutral stimulus (vibratory) with an appetitive stimulus (sucrose). The strength and period for acquisition and retention of this appetitive leamed response were very similar to those of the second type aversive conditioning but not to the first one. On the basis of these behavioral analyses, the neuronal mechanisms of the two types of aversive and appetitive conditioning were discussed.

We reported that malignant cells are inactivated by photo-excited TiO2 particles (Cai, R.-X., et al. (1992) Cancer Res. 52, 2346-2348). In the present study, the process of cell death of a human bladder cell line T24 with TiO2 irradiated TiO2 was investigated by monitoring the time course change in intracellular calcium concentration ([Ca2+](i)) under UV irradiation using calcium fluorescence indicator, fura-2. In the presence of photo-excited TiO2 particles (100 mu g/ml), [Ca2+](i) showed a sapid two-step increase; while in the absence of TiO2, it exhibited only a slight and monotonous increase or maintained a constant level. The rapid elevation of [Ca2+](i) in the presence of photo-excited TiO2 particles was caused by the influx of extracellular Ca2+ through the plasma membrane and cell death occurred only after the second rapid elevation of [Ca2+](i). These results suggested that the cell membrane permeability to Ca2+ was promoted prior to cell death.

Primary cultures of rabbit and skate lens epithelia were used to investigate the effect of calcium release from intracellular stores upon the actin cytoskeleton. Primary cultures were loaded with fura-2 AM and intracellular calcium, i.e. (Ca2+)i, quantitated using a Hamamatsu Photonics digital imaging system. Agonists used were bombesin, inositol-1,4,5-trisphosphate (IP3), thapsigargin (Tg), neuropeptide Y (NPY) and calcium chloride. Recordings were typically made on seven cells in each case. We found that IP3 caused a 6-8-fold immediate release of (Ca2+)i in rabbit cells, but skate cells showed no response unless permeabilized with saponin, whereupon an increase of about 50% occurred. Tg induced release from internal stores in rabbit cells, but had no effect on skate cells. Bombesin caused a large increase in (Ca2+)i release in both, while NPY had no effect in either. Skate cells incubated in calcium-free EGTA-Ringer's solution responded rapidly to addition of 5 mM CaCl2, whereas only three of 35 rabbit cells responded, and in gradual fashion. After calcium imaging, the cells were fixed and stained with rhodamine phalloidin or with an antibody against IP3 receptor (IP3R) conjugated to FITC. Fluorescence microscopy revealed that the actin cytoskeleton had reorganized from the normal stress fiber pattern into polygonal networks. Tg caused the same structures to form in rabbit cells, but bombesin had no effect. IP3 receptor was located intracellularly, presumably on endoplasmic reticulum, and was not associated with plasma membranes. The rapid response of rabbit cells may have been caused by the DMSO in which fura-2 was dissolved.
We have found an interesting difference in agonist-induced calcium release between rabbit and skate cells. The latter may utilize either a Ca-Na exchanger or capacitative calcium entry, which could reflect a difference in lens accommodative mechanisms. This seems relevant in view of the fact that the rabbit lens accommodates through change in shape, whereas the skate lens does so through translation of position.

Although beta-amyloid is the main constituent of neurite plaques and may play a role in the pathophysiology of Alzheimer's disease, mechanisms by which soluble beta-amyloid might produce early symptoms such as memory loss before diffuse plaque deposition have not been implicated. Treatment of fibroblasts with beta-amyloid (10 nM) induced the same potassium channel dysfunction previously shown to occur specifically in fibroblasts from patients with Alzheimer's disease-namely, the absence of a 113-picosiemen potassium channel. A tetraethylammonium-induced increase of intracellular concentrations of calcium, [Ca2+]i, a response that depends on functional 113-picosiemen potassium channels. was also eliminated or markedly reduced by 10 nM beta-amyloid. Increased [Ca2+]i induced by high concentrations of extracellular potassium and 166-picosiemen potassium channels were unaffected by 10 nM beta-amyloid. In Alzheimer's disease, then, beta-amyloid might alter potassium channels and thus impair neuronal function to produce symptoms such as memory loss by a means other than plaque formation.

Previous observations have implicated GABA as a neurotransmitter released by the vestibular sensory neurons (''hair cells'') of the snail Hermissenda onto visual sensory neurons, the type B cells, whose cell bodies are the sites of biophysical and biochemical changes during and following Pavlovian conditioning. Still other observations demonstrated that light-GABA pairings that simulate stimuli presented during Pavlovian conditioning cause prolonged elevation of intracellular Ca2+ and transformation of GABA-induced synaptic inhibition into excitation. Intracellular Ca2+ signals in response to GABA perfused onto the postsynaptic type B terminal branches are shown here to be prolonged on days after conditioning, but not after control paradigms. These and past results demonstrate two separate sites, i.e., the cell body and the terminal branches, for learning-induced changes after Pavlovian conditioning.

Microspectrofluorometry and video imaging techniques were used to study and to compare the changes in intracellular calcium concentrations ([Ca2+](i)) of individual Fura-2 loaded Spisula oocytes treated with serotonin (5-hydroxytryptamine, 5-HT) or tricyclic antidepressants. In the present study, we showed that 5-HT increased [Ca2+](i) in freshly isolated Spisula oocytes suspended in artificial sea water. In the absence of extracellular Ca2+, 5-HT did not influence [Ca2+](i). Stimulation of [Ca2+](i) by 5-HT was blocked by calcium channel blocker, e.g. verapamil, and by tricyclic antidepressants. These observations combined with our previous results on the effects of 5-HT, tricyclic antidepressants and verapamil on calcium uptake suggest that the increase in [Ca2+](i) induced by 5-HT results from an influx of extracellular calcium through calcium channels, which can be blocked by tricyclic antidepressants.
The use of the Fura-P imaging technique allowed single-cell measurements and defined changes induced by 5-HT In [Ca2+](i) which is the net result of calcium uptake and release of intracellular calcium from storage sites in individual Spisula oocytes.

The recent demonstration of Kf channel dysfunction in fibroblasts from Alzheimer disease (AD) patients and past observations of Ca2+-mediated K+ channel modulation during memory storage suggested that AD, which is characterized by memory loss and other cognitive deficits, might also involve dysfunction of intracellular Ca2+ mobilization. Bombesin-induced Ca2+ release, which is inositol trisphosphate-mediated, is shown here to be greatly enhanced in AD fibroblasts compared with fibroblasts from control groups. Bradykinin, another activator of phospholipase C, elicits similar enhancement of Ca2+ signaling in AD fibroblasts. By contrast, thapsigargin, an agent that releases Ca2+ by direct action on the endoplasmic reticulum, produced no differences in Ca2+ increase between AD and control fibroblasts. Depolarization-induced Ca2+ influx data previously demonstrated the absence of between-group differences of Ca2+ pumping and/or buffering. There was no correlation between the number of passages in tissue culture and the observed Ca2+ responses. Furthermore, cells of all groups were seeded and analyzed at the same densities. Radioligand binding experiments indicated that the number and affinity of bombesin receptors cannot explain the observed differences. These and previous observations suggest that the differences in bombesin and bradykinin responses in fibroblasts and perhaps other cell types are likely to be due to alteration of inositol trisphosphate-mediated release of intracellular Ca2+.

Since memory loss is characteristic of Alzheimer disease (AD), and since K+ channels change during acquisition of memory in both molluscs and mammals, we investigated K+ channel function as a possible site of AD pathology and, therefore, as a possible diagnostk index as well. A 113-pS tetraethylammonium (TEA)-sensftive K+ channel was consistendy absent from AD ribroblasts, while it was often present in young and aged control fibroblasts. A second (166-pS) K+ channel was present in all three groups. Elevated external potassium raised intracellular Ca2+ in all cases. TEA depolarized and caused intracellular Ca2+ elevation in young and aged control fibroblasts but not AD fibroblasts. The invariable absence of a 113-pS TEA-sensitive K+ channel and TEA-induced Ca2+ signal indicate K+ channel dysfunction in AD fibroblasts. These results suggest the possibility of a laboratory method that would diagnostically distinguish AD patients, with or without a family history of AD, from normal age-matched controls and also from patients with non-AD neurological and psychiatric disorders.

Previous studies of molluscan and mammalian neural networks have implicated potassium channels, calcium-dependent kinases and a small G-protein (Cp20) in associative memory storage. Since Alzheimer's disease is characterized by memory deficits and possible changes in PKC activation, we studied potassium channel function in AD. Patch-clamp experiments revealed the absence of a 113 pS TEA-sensitive K+ channel in fibroblasts from Alzheimer's but not control patients. These results suggest a role for potassium channel dysfunction in Alzheimer's disease.

For a constant membrane potential, a predominantly inhibitory GABAergic synaptic response is shown to undergo long-term transformation into an excitatory response after pairing of exogenous gamma-aminobutyric acid (GABA) with postsynaptic depolarization or pairing of pre- and postsynaptic stimulation. Current- and voltage-clamp experiments suggest that this synaptic transformation is due to a shift from a net increase of conductance to a net decrease of conductance in response to GABA. GABA-induced elevation of intracellular calcium is prolonged after the same stimulus pairing and may, therefore, contribute to this synaptic transformation via Ca2+-activated phosphorylation pathways. This synaptic transformation, which does not follow unpaired stimulus presentations, occurs in a neuronal compartment spatially separated from the soma, which also changes during stimulus pairing.

To investigate the relation between the function of Ca2+-activated K+ channels and phosphoinositide turnover, we have examined the physiological and pharmacological characteristics of ionotropic and metabotropic quisqualate (QA) receptors in rat cerebellar Purkinje cells during development using the slice-patch method combined with Ca2+ imaging. The typical response to QA obtained from a rat on postnatal day (PND) 21 consisted of three components: (1) a fast inactivating inward current, (2) a slow inward current, and (3) a slow outward current. The slow inward current was abolished in Ca2+-free medium. while the fast inactivating inward current and the slow outward current remained unaffected. The slow outward current which appeared to be activated via a metabotropic receptor was not observed in the Purkinje cell of PND 7 rat, in which dendrites were poorly developed but its amplitude increased linearly with PND. QA caused significant increases in [Ca2+]i in the fully developed dendritic region of the Purkinje cells even in Ca2+-free medium, suggesting a dendritic localization of the metabotropic receptors.

Caudal hair cell impulses cause postsynaptic inhibition of ipsilateral type B photoreceptors in the snail Hermissenda. This inhibition is shown to be GABAergic according to a number of criteria. HPLC, mass spectrophotometric, and immunocytochemical techniques demonstrated the presence of GABA in the hair cells and their axons. GABA agonists and antagonists mimic and block the synaptic effect in a manner consistent with endogenous GABAergic transmission. Other properties, including I-V relations, conductance changes and reversal potentials, are comparable for exogenous GABA responses and endogenous effects of the hair cell impulses. This inhibitory synapse has been found to undergo a long-lasting transformation into an excitatory synapse if GABA release is paired with post-synaptic depolarization. GABA, via GABA(A) and GABA(B) receptors in the B cell, causes the opening of calcium sensitive chloride and potassium channels that leads to the post-synaptic hyperpolarization. GABA also induces a long-lasting intracellular calcium elevation at the terminal branches of the B cell that greatly outlasts the voltage responses. Synaptic transformation induced by pairings is caused by a decrease in both GABA induced chloride and potassium conductances in the post-synaptic B cell, as well as a significant prolongation of the intracellular calcium accumulation in the B cell's terminal axonal branches.

The distribution of functional glutamate receptor subtypes was visualized as regional changes in intracellular Ca2+ concentration induced by administration of a specific agonist for each subtype. An image of the fura-2 fluorescence from a whole hippocampal slice was directly excited at 340 or 380 nm through an optical fibre and observed via a low-magnification objective lens (X 4). A heterogeneous distribution of N-methyl-D-aspartate (NMDA) and non-NMDA subtypes was demonstrated by the ratio of fluorescence intensities before and after administration of each agonist. This 'macro' image analysis can reveal distributions of multiple functional receptors in a living brain slice.

The postnatal development of calcium-mobilizing systems was studied by both microfluorometric imaging analysis of Ca2+ on living rat cerebellar slices and immunohistochemical labeling of phosphatidylinositol 4,5-bisphosphate (PIP2) and inositol 1,4,5-trisphosphate binding protein (IP3BP) in fixed rat cerebellum. Stimulation with quisqualate (QA) or N-methyl-D-aspartate (NMDA) enhanced the Ca2+ level only diffusely on postnatal day (PND) 3, but more discretely on PNDs 7 and 15. On PND 21, QA-induced responses were localized in the molecular layer especially, but not in the granular layer. By contrast, NMDA mobilized Ca2+ prominently in the granular layer, but only weakly in the molecular layer. Localized expression of PIP2 in the molecular layer paralleled QA-induced Ca2+ mobilization, but IP3BP was expressed more diffusely. The present study offers the first direct evidence that PIP2, but not IP3BP, is essential for QA-induced Ca2+ mobilization in the cerebellar cortex.

In order to examine whether a transient appearance of glutamate receptor in developing rat Purkinje cell in coupled to PI turnover, we have studied Ca2+ mobilization induced by quisqualate or NMDA using fura-2 analyzing system in combination with immunohistochemical staining analysis with anti-PIP2 antibody and/or anti-IP3 binding protein antibody.

我々は超高感度なタンパク測定ELISA法を開発した実績があり、今回は、さらにこの測定系を拡張するために、今までにない核酸検出に取り組んだ。それは「核酸の増幅を必要としない」超高感度核酸測定の実現である。本研究では、結核菌群に特異的に存在するタンパク質MPB64の１本鎖を用い、かつコントロールとして非結核菌群を用いた。結果として、MPB64の１本鎖に対して、3.7 × 10^5 copies/assay（すなわち4.2 × 10^3 copies/μL）の測定に成功し、また非結核菌群は測定に掛らなかった。すなわち予定通りに、 非増幅・迅速・簡便核酸測定法の開発に成功した。

  脊椎動物でも無脊椎動物でも、その脳内でインスリンがはたらくことが、近年わかってきた。ただし、このインスリンが学習記憶機構に対して「増強」としてはたらくのか、または「減弱」としてはたらくのかは、不明な点が多い。本研究では、学習記憶研究で永年使用されてきている軟体動物モノアラガイを用いて、①インスリンのシグナルカスケードとセロトニンカスケードが長期記憶形成において拮抗的にはたらくことが明らかとなった。また、②転写因子CREBのシグナルカスケード、ならびにそれに続くCRTCのシグナルカスケードの研究を開始することができた。なお本研究は、同一課題にて特定課題Bでも研究が遂行された。

  過度のストレスから解放されると、動物の学習成績が改善されるのか？この問題を神経生物学で永く利用されている軟体動物モノアラガイで調べた。ストレスからの解放はカンナビノイドの投与によって得た。ほ乳類の脳内には内在性カンナビノイドならびにその受容体が存在する。一方、無脊椎動物はストレスや学習の研究に十分利用可能であるが、それらの存在は不明であった。そこで軟体動物モノアラガイを用いて受容体の存在を調べたところ、ほ乳類と相同な受容体が見つかった、さらには学習行動やストレスが及ぼす影響に対して、カンナビノイド受容体のアゴニスト・アンタゴニストがどのように関与するのかを調べる実験も開始することができた。

脊椎動物でも無脊椎動物でも、その脳内でインスリンがはたらくことが、近年わかってきた。ただし、このインスリンが学習記憶機構に対して「増強」としてはたらくのか、または「減弱」としてはたらくのかは、不明な点が多い。本研究では、学習記憶研究で永年使用されてきている軟体動物モノアラガイを用いて、①インスリンのシグナルカスケードとセロトニンカスケードが長期記憶形成において拮抗的にはたらくことが明らかとなった。また、②転写因子CREBのシグナルカスケード、ならびにそれに続くCRTCのシグナルカスケードの研究を開始することができた。なお本研究は、同一課題にて特定課題（新任の教員等）でも研究が遂行された。