To determine sex and regional differences in the properties of serotonin (5-HT) neurons of the raphe nuclei, the responsiveness to parachlorophenylalanine (PCPA) of 5-HT neurons in the dorsal and median raphe nuclei (DR and MR) and the nucleus raphe magnus (RMg) was analyzed by counting 5-HT-immunoreactive (5-HT-ir) cells. Gonadectomized male (OCX) and female (OVX) rats were treated with 100 mg/kg b.wt PCPA or saline daily for 4 days. The brains were removed and fixed one day after the last injection. Frozen sections were stained with serotonin antibody and the numbers of 5-HT-ir cells in the raphe nuclei were counted. As a result, in female rats, the densities of 5-HT-ir cells in these 3 raphe nuclei were almost the same when compared the PCPA-treated and saline-treated groups. On the other hand, in male rats, the densities of 5-HT-ir cells in the DR and MR of PCPA-treated rats were lower than in saline-treated rats. In the male RMg, no difference was seen. These results suggest that responsiveness of 5-HT neurons to PCPA in the DR and MR, but not in the RMg, were sexually dimorphic. (C) 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

To determine sex and regional differences in the properties of serotonin (5-HT) neurons of the raphe nuclei, the responsiveness to parachlorophenylalanine (PCPA) of 5-HT neurons in the dorsal and median raphe nuclei (DR and MR) and the nucleus raphe magnus (RMg) was analyzed by counting 5-HT-immunoreactive (5-HT-ir) cells. Gonadectomized male (OCX) and female (OVX) rats were treated with 100. mg/kg b.wt PCPA or saline daily for 4 days. The brains were removed and fixed one day after the last injection. Frozen sections were stained with serotonin antibody and the numbers of 5-HT-ir cells in the raphe nuclei were counted. As a result, in female rats, the densities of 5-HT-ir cells in these 3 raphe nuclei were almost the same when compared the PCPA-treated and saline-treated groups. On the other hand, in male rats, the densities of 5-HT-ir cells in the DR and MR of PCPA-treated rats were lower than in saline-treated rats. In the male RMg, no difference was seen. These results suggest that responsiveness of 5-HT neurons to PCPA in the DR and MR, but not in the RMg, were sexually dimorphic. © 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society.

Sensitivity of neurons to estrogen in down-regulation of estrogen receptor alpha (ER alpha) can be thought to make a sex difference in regulatory system of reproductive activities. In this study, to investigate the sex difference of expression of ER alpha in the hypothalamus and midbrain, the number of ER alpha immunoreactive (-ir) cells was counted in orchidectomized (OCX) and ovariectomized (OVX) rats with or without treatment with estrogen. A week after the gonadectomy, 5 rats in each female and male were injected with 1 mg estradiol benzoate (EB). The remaining 5 rats in both sexes did not receive EB. The brain was fixed 24 h after EB-injection and 50 mu m-serial frozen sections were made. After immunohistochemical staining for ER alpha, the number of ER alpha-ir cells was counted in a 0.2-mm(2) frame in the anteroventral periventricular nucleus (AVPvN), the ventrolateral part of the ventromedial hypothalamic nucleus (vIVMN), the arcuate nucleus (ARCN), and the lateral mesencephalic central gray (IMCG) in 2 or 3 sections. The total number of ER alpha-ir cells was changed to a density value (number per 1 mm(3)). As the results, in EB-treated rats, the density of ER alpha-ir cells in all regions, except the male AVPvN and male IMCG, were lower than those in untreated rats of both sexes. In the vIVMN, the density of ER alpha-ir cells in OVX rats was higher than in OCX rats. These results suggest that there are sex and regional differences in the mechanisms of down-regulation of ER alpha by estrogen in the rat brain. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

In order to investigate the relationship between GABA(A) and GABA(B) receptors in the induction of lordosis behavior, agonists of these receptor subtypes were injected simultaneously to estrogen-treated ovariectomized rats and lordosis behavior was observed before and after the injections. The GABAA receptor agonist, muscimol (MUS). at a dose in the range from 1.0 to 1.4 mg/kg body weight (bw) or the GAEA(B) receptor agonist, baclofen (BAC) at a dose in the range from I to 10 mg/kg bw, was injected intraperitoneally. The lordosis quotient (LQ) decreased after treatments with MUS or BAC and a dose-dependent decrease of LQ was observed in MUS or BAC-treated rats. When 1.2 mg/kg bw MUS and 5 mg/kg bw BAC were injected simultaneously, the mean LQ decreased strongly and was significantly lower than the values obtained after single injections of the agonists at these doses (P < 0.05). In addition, to ascertain the time-course of changes, a behavioral test was carried out 7 times from 15 to 180 min after the injection of agonists. The low LQ in the rats injected with both MUS and BAC continued longer than in rats given single injections. These results indicate that both GABAA and GABAB receptors are involved in lordosis-inhibiting mechanisms by the GABA neuron and operate independently. (c) 2008 Elsevier Inc. All rights reserved.

The objective of this study was to clarify the 5-HT projections from the right and left sides of the dorsal (DRD), ventral (DRV) and lateral (DRL) subdivisions of the middle level of the dorsal (DR) raphe nucleus and median (MR) raphe nucleus to the lateral septum (LS), preoptic area (POA) or ventromedial hypothalamus (VMH), which are important neural substrates for neuroendocrine regulation of reproduction. A retrograde neural tracer, Fluoro-Gold (FG), was infused into the right side of these regions in ovariectomized rats and the numbers of FG and/or 5-HT immunopositive cells in the right and left sides of the raphe nuclei were counted. It was found that the POA and VMH received more 5-HT projections than the LS from the DR and MR. In the subdivisions of the DR, 70% of all 5-HT projections from the DR to these 3 areas originated from the DRL. Furthermore, ipsilateral projections from the DR to the POA and VMH but not to the LS were dominant, compared to the contralateral projections. A right-left difference was not seen among the MR 5-HT projections. Thus, laterality of the projections is thought to be strong in the 5-HT clusters located far from the midline of the midbrain raphe nuclei. (c) 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

In order to examine the effects of estrogen, androgen, and phytoestrogen on maternal behavior induced by exposure to fresh pups in ovariectomized nulliparous rats, 1 mg estradiol benzoate (EB), 1 mg testosterone propionate (TP), 1 mg coumestrol (CM), or oil (female control) was injected subcutaneously daily for 10 days. To elucidate the sex difference, 1 mg EB or oil (male control) was injected in orchidectomized rats by the same method as that used in nulliparous rats. Exposure to fresh pups was started 6 days after the first injection. Behavioral tests were carried out daily for 5 days from the first exposure to the last on the 10th day. In the behavioral test, the onset of retrieving and licking behaviors was recorded. In female control rats, the median onset day of retrieving behavior was day 5. Onset in the EB female group was day 1.5, which was shorter than that in the female control (P<0.05). TP female and CM female rats started to show retrieving at day 5 and day 4.5, respectively, comparable to the female controls. In males, the median day of retrieving onset in the control and EB groups was over day 5 and day 4.5, respectively. No statistical difference was seen between the female and male controls. In contrast, there was a difference between the EB-treated female and EB male groups. Licking activity was less frequent than seen in the retrieving behavior among all groups, but there was no statistical difference among the groups. These results suggest that estrogen facilitates retrieving behavior in female, but not in male rats. TP and CM have no effect on retrieving behavior in female rats.

The neural, projection of the lateral septum (LS) to the rostral mesencephalic central gray (MCG) is sexually dimorphic and plays an important role in inhibiting female reproductive behavior. In this experiment, development of the LS-MCG connection from birth to 15 days after birth was examined in female rats by a tract-tracing method with DiI. On the birth day (D1 rat), and 5, 10 or 15 days after birth (D5, D10 or D15 rat, respectively) or 8 weeks after birth (adult), the brain was fixed by perfusion of a mixture of 4% PFA and 0.1% glutaraldehyde. DiI was pasted on the coronally cut-surface of the LS and the sample was incubated in PEA at 40 degrees C for up to 4 months. After incubation, 200-mu m frozen parasagital sections were prepared and observed by fluorescence microscopy. As a result, numerous DiI labeled fibers were found in the preoptic area, the anterior and posterior hypothalamus, and the MCG in adult rats. In D1 rats, several labeled axons extended caudal to the anterior hypothalamic area. In D5 rats, a few labeled fibers reached the MCG. Some labeled fibers were observed in the rostral MCG of D10 rats. In D15 rats, a considerable number of labeled fibers were seen to reach the rostral MCG and relative density of the fibers was comparable to that of adult. These results suggest that the neural pathway from the LS to the rostral MCG develops acutely during the period from 5-10 days up to more than 15 days after birth. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

The inhibitory effects of 8-OH-DPAT, a 5-HT1A-receptor agonist, and baclofen, a GABA(B)-receptor agonist, on lordosis were examined in estrogen and progesterone-treated ovariectomized rats with lesions in either the dorsal raphe nucleus (DRN) or septum and in rats with either sham lesions or no lesions. The first behavior test series was carried out 6 days after implantation of the rats with silicon tubes containing estradiol. Four hours after injection with 0.5 mg progesterone, behavioral tests were performed before and 30 min after an injection with 1 mg/kg body weight 8-OH-DPAT. As a result, the mean lordosis quotient (LQ)s were changed from 100 to less than 20 before and after the injection in all groups. These results suggest that 8-OH-DPAT acts on areas other than the DRN and the septum, leading to a decrease in lordosis. Two weeks after implantation with estradiol, the next behavioral test series was carried out after injection with progesterone. Behavioral tests were performed before and after an injection with 10 mg baclofen. The results showed that the mean LQs decreased after the injection in all groups, but the mean LQ in the DRN lesion group was higher than that in the sham groups. These results indicate that baclofen may act partially on the DRN in inhibiting lordosis in female rats.

The neural control systems for the ovulatory cycle and lordosis behavior are sexually differentiated by estrogen during the perinatal period in rats. In the present study, the effects of a single neonatal injection with the phytoestrogen, coumestrol, on female reproductive functions were investigated. Female rats were injected subcutaneously with 1 or 3 mg coumestrol (CM1, CM3), 1 mg genistein (GS1), 1 mg estradiol (E-2) or oil at day 5 after birth (birth day = day 1) and an estrous cycle check and lordosis behavior test were performed. As a result, vaginal opening was advanced in CM1-, CM3- or E-2-treated females. A vaginal smear check indicated that oil- or GS1-treated females showed a constant 4- or 5-day estrous cycle, whereas CM1-, CM3- or E2-treated rats showed a persistent or prolonged estrus. Ovariectomy was performed in all females at 60 days of age. The ovary weights in the CM1-, CM3- or E-2-treated groups were lower than those in the oil- and GS1-treated groups and no corpora lutea were found in any rats of these three groups, except for two E-2-treated rats. Behavioral tests were carried out after implantation of E-2-tubes. All rats in the CM1-, GS1-treated groups showed a high lordosis quotient (LQ), being comparable to that in the oil-treated females. On the other hand, LQs in the CM3, E2 or male groups were lower than that in the control female group. These results suggest that a single neonatal injection of 3 mg coumestrol was effective in suppressing the functions of ovulation-inducing mechanisms and the induction of lordosis, but 1 mg coumestrol was effective in only the estrous cycle of female rats. (C) 2004 Elsevier Inc. All rights reserved.

Effects of the serotonin (5-HT) receptor 1A antagonist, WAY-100635, on lordosis in female rats were examined. Ovariectomized rats were implanted with a silicon tube containing estradiol and behavioural tests were performed. Next, 5, 10 or 20 mg/kg bw WAY-100635 or saline was injected subcutaneously in female rats with a lordosis quotient (LQ) from 10 to 30 and the behavioural test was performed again. As a result, the mean LQs in 10 or 20 mg WAY-100635-treated groups were higher than in the saline-treated group (P < 0.01 and P < 0.05, 10 or 20 mg groups versus saline, respectively). In the experiment on the time-course of change in LQ after injection with 10 mg WAY-100635, the mean LQ was increased (P < 0.01, versus saline) 15 min after the injection and high levels persisted for 1 h. This finding shows that WAY-100635 has the potency to enhance lordosis behaviour acutely in female rats with a low estrous state. In order to investigate relationships between the 5-HT1A receptor and the GABA(B) receptor in regulating lordosis, 10 mg baclofen, a GABA(B) receptor agonist, was injected and this was followed 1 h later by the injection of 10 mg/kg WAY-100635. Mean LQ decreased after the injection of baclofen (P < 0.0001, versus placebo-treated control), but the decrease in LQs was not reversed by injection with WAY-100635. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

To determine whether apoptosis is involved in the formation of the structure and morphological sex difference of the lateral septum (LS), the postnatal developmental changes in the number of apoptotic cells were examined in the LS on postnatal day 1 (PD1 = birth day), 4, 6, 8, 11, 16, and 31 in male and female rats. Apoptotic cells were imimmohistochemically detected by antibody against single-stranded DNA (ssDNA) or active caspase-3. The volume of the LS was also measured and was found to increase with age. The number of apoptotic cells detected by anti-ssDNA in the LS increased from PD1 to PD8 but decreased after PD11. Also, the LS was divided into dorsal, intermediate, and ventral parts (LSd, LSi, and LSv), and the volume and number of ssDNA-immunoreactive cells in each part were measured on PD6, 8, 11, 16, and 31. In both sexes, a large number of ssDNA-immunoreactive cells was found in the LSd and LSi on PD8 (but not on PD6) and in the LSv on PD6 and PD8. On PD6, the number of active caspase-3-immunoreactive cells was significantly greater in the LSv than in the LSd or LSi, in both sexes. Only the LSi of males had a high number of ssDNA-immunoreacitve cells on PD16; the number was significantly greater than that of females of the same age. However, there was no significant sex difference in the number of active caspase-3-immunoreacitve cells in the LSi on PD16. On PD31, the volume of the LSi was significantly greater in females than in males. There was no sex difference in volume or number of apoptotic cells in the LSd or LSv. These findings indicate that loss of cells due to apoptosis, which is partially caused by activation of caspase-3, occurs in the LS during postnatal development, with regional differences. They also indicate that sex difference in caspase-3-independent apoptosis contributes to morphological sexual differentiation of the LSi. (C) 2004 Wiley-Liss, Tue.

Neurons in the lateral septum (LS) with projecting axons to the midbrain central gray (MCG) exert an inhibitory influence on lordosis. The number of such neurons is greater in female than in male rats. In this experiment, effects of neonatal estrogen on the density of the LS-MCG connections and on lordosis behavior were examined in female rats. On postnatal day 4 (day 0 = day of birth), females were injected subcutaneously with 50 or 100 mug estradiol benzoate (EB) or oil. On postnatal day 60, females and control males were gonadectomized. Behavioral tests were carried out after the implantation of silicone tubes containing estradiol. Lordotic activities in both males and EB-treated females were lower than in oil-treated females. After completing the behavioral tests, the animals were injected with Fluoro-Gold (FG), a retrograde tracer, into the right-side MCG and the number of FG-labeled neurons in the LS was measured. In all groups, the right-side LS ipsilateral to the FG injection had more FG-labeled neurons than the left-side LS. The number of FG-labeled neurons in the LS of oil-treated females was larger than that of males on both right and left sides. In the females treated with 100 mug EB (EB100), the number of FG-labeled neurons was comparable with that of males and lower than that of oil-treated females. The number of FG-labeled neurons in the EB50 females was also lower than that in oil-treated females, but tended to be larger than that observed in the EB100 group. These results indicate that neonatal estrogen decreases both lordotic activity and the density of the LS-MCG neural connections in female rats. Copyright (C) 2003 S. Karger AG, Basel.

To investigate the role of serotonin (5-HT) receptor 1 A or 7 in regulating lordosis behavior in female rats, ovariectomized rats were treated with 3 kinds of receptor agonists and lordosis behavior was observed. The injected agents were the selective 5-HT1A receptor agonist, buspirone (BUS), the highly selective 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin ((+/-)8-OH-DPAT), and the 5-HT1A and 5-HT7 receptor agonist, (R)-8-hydroxy-2-(di-n-propylamino)tetralin ((+)8-OH-DPAT). A behavioral test was performed after ovariectomy and subcutaneous implantation of a silicon tube containing estradiol. Female rats in which the lordosis quotient (LQ) was over 70 were intraperitoneally injected with several doses of these agents. As a result, in the BUS group, the dose of 3 mg/kg bw, but not 1 mg/kg was effective for suppressing lordosis. On the other hand, an inhibitory effect was observed from 0.25 mg/kg and 0.5 mg/kg in the (+)8-OH-DPAT and ()8-OH-DPAT groups, respectively. In the time-course experiment, in all drug-treated groups, LQ decreased to lower than 20 after 15 min and low LQ continued for 1 hr at least. Measurement of locomotor activity using an infrared sensor system showed no relation between the decrease in lordosis by these agents and spontaneous locomotion. These results indicate that 5-HT1A is strongly involved in the lordosis-inhibiting circuit of the serotonin neurons.

It is well known that neonatal exposure to estrogen induces masculinization or defeminization of the brain. In this study, the effects of neonatal treatment with two kinds of soybean isoflavone aglycone, genistein (GS) and daidzein (DZ), on the estrous cycle and lordosis behavior were investigated. Female rats were injected subcutaneously with 1 mg GS, 1 mg DZ, 100 mug estradiol (E-2), or oil daily for 5 days from birth. As a result, vaginal opening was advanced in GS- or E-2-treated females. A vaginal smear check indicated that oil- or DZ-treated females showed a constant 4- or 5-day estrous cycle, whereas GS- or E-2-treated rats showed a persistent or prolonged estrus. Ovariectomy was performed in all females at 60 days of age. The ovaries in the GS- or E-2-treated groups were smaller than those in the oil- and DZ-treated groups and contained no corpora lutea. In the DZ group, although corpora lutea were seen, ovaries were smaller than that of control females. Behavioral tests were carried out after implantation of E-2-tubes. All of the oil- or DZ-treated females showed lordosis with a high lordosis quotient (LQ). On the other hand, as male rats, LQs were extremely low in the E-2-treated group, when compared to the oil-treated group. In the GS-treated group, the mean LQ was lower than that in the oil-treated group, but higher than those in the E-2-treated female or male groups. These results suggest that genistein acts as an estrogen in the sexual differentiation of the brain and causes defeminization of the brain in regulating lordosis and the estrous cycle in rats. In addition, neonatal daidzein also has some influence on ovarian function. (C) 2003 Elsevier Inc. All rights reserved.

Sex difference in the number of neurons projecting axons from the lateral septum (LS) to the midbrain central gray (MCG) that are concerned with the lordosis-inhibiting system was investigated by injection of Fluoro-Gold (FG), a retrograde tracer, into the rostral MCG on the right side in male and female rats. Immunohistochemistry for ER-alpha and -beta was also performed with or without combination with FG immunostaining. All animals were gonadectomized. Lordosis was observed after treatment with E2 in some animals. In the results, lordosis was rare in males, compared with females. FG-immunoreactive (ir) cells were concentrated in the intermediate LS on the right side, and its number in the females was significantly higher than that in the males. There was no sex difference in the distribution and number of ERalpha-ir and ERbeta-ir cells in the LS. Furthermore, the number of ERs-ir cells was not influenced by E2 in either males or females. Double FG-ERbeta-ir cells were less than 20% of total FG-ir cells in the LS in both males and females. These data suggest that the LS-MCG connection is sexually dimorphic but that there is no sex difference in the expression of ERs in the LS.

To verify the anatomical and functional connection of the lateral septum (LS) and periaqueductal gray (PAG) in inhibiting female sexual behavior, lordosis, in male rats, retrograde (Fluoro-Gold, FG) or anterograde (Phaseolus vulgaris-leucoagglutinin, PHA-L) tracer was injected into the PAG or LS on the right side, respectively, and FG-labeled cells or PHA-L-labeled axons in the forebrain and mesencephalon were determined in estrogen-treated castrated male rats. A ventral cut (VC) of the septum and a behavioral test were also conducted in some FG-injected rats. Furthermore, lordosis behavior was observed after chemical destruction of the septum by ibotenate. As a result, the lordosis quotient (LQ) in VC males was higher than that in control males without VC. FG-labeled neuronal cell bodies were found in the ipsilateral intermediate part of the LS in the control males but not in this area of the VC males. When neuronal cells in the intermediate part of the bilateral LS were completely destroyed by ibotenate, the LQ was higher than that in sham-lesioned male rats. These results suggest that a direct neural connection of the intermediate LS to the PAG has an inhibitory role in regulating lordosis in male rats. In addition, neuronal cell bodies in the intermediate LS exert an inhibitory influence. In the PHA-L experiment, labeled axons were seen in the ventral part of the LS, the medial forebrain bundle at the chiasmatic level, the lateral hypothalamus, the median region of the mesencephalon, and the rostral PAG in the side ipsilateral to the tracer injection site of the LS. Thus, these areas are thought to be involved in the pathway for lordosis-inhibition from the intermediate part of the LS to the PAG in male rats. © 2001 Wiley-Liss, Inc.

The inhibitory role of progesterone (P) in regulating lordosis was investigated in male and female rats with septal lesions (SL). Male rats with SL showed lordosis quotients (LO) as high as female rats with SL and female control rats without brain surgery after injection of 50 mug/kg estradiol benzoate (EB) followed by 0.5 mg P 44 h later. Even when primed with 5 mg P 1 h prior to the 50 mug EB-injection, the mean LQs were still high in all groups. When the dose of EB was decreased to 5 mug/kg, all rats showed high-score LQs. In contrast, all animals in both male and female in which 5 mg P was injected 1 h before 5 mug EB, showed low LQs. These results suggest that P is effective in suppressing lordosis enhanced by estrogen in either male rats or females. Furthermore, the high dose of estrogen overcomes the inhibitory action of P on lordosis in both sexes. (C) 2000 Elsevier Science Inc.

To clarify the role of the ventromedial (VMH) or dorsomedial (DMH) hypothalamic nucleus in regulating male sexual behavior in female rats, radiofrequency or ibotenic acid lesions were made in ovariectomized rats, and three behavioral tests in total were carried out after implantation of Silastic tubes containing testosterone. As a result, females with radiofrequency or ibotenic acid lesions in the VMH showed higher levels of mounting behavior than those in females with no brain surgery or with sham-operation. The incidence of intromissive pattern in females with ibotenic acid lesions but not in females with radiofrequency lesions was higher than those in other female control groups. In the group of radiofrequency lesions in the DMH, both frequencies of mounting and intromissive pattern were lower than those in control and sham groups. No ejaculatory pattern was seen in any of the female groups. These results indicate that neuronal cell bodies in the VMH, but not in the DMH, exert an inhibitory influence on mounting activity in female rats. © 2000 Elsevier Science Ireland Ltd.

To clarify the role of the ventromedial (VMH) or dorsomedial (DMH) hypothalamic nucleus in regulating male sexual behavior in female rats, radiofrequency or ibotenic acid lesions were made in ovariectomized rats, and three behavioral tests in total were carried out after implantation of Silastic tubes containing testosterone. As a result, females with radiofrequency or ibotenic acid lesions in the VMH showed higher levels of mounting behavior than those in females with no brain surgery or with sham-operation. The incidence of intromissive pattern in females with ibotenic acid lesions but not in females with radiofrequency lesions was higher than those in other female control groups. In the group of radiofrequency lesions in the DMH, both frequencies of mounting and intromissive pattern were lower than those in control and sham groups. No ejaculatory pattern was seen in any of the female groups. These results indicate that neuronal cell bodies in the VMH, but not in the DMH, exert an inhibitory influence on mounting activity in female rats. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

To clarify the role of the extrahypothalamic input to the preoptic area (POA)-medial basal hypothalamus (MBH) on spontaneous ovulation, the dorsal cut of the POA-MBH was performed on various days and ova were counted in female rats. An anterior half-circle cut (anterior dorsal cut
ADC) was performed at the dorsal of the POA on the day of proestrus. As a result, ovulation was seen on the day of estrus when ADC was performed in the evening (18:00-19:30 h) but did not occur when performed in the morning (10:00-11:30 h). Furthermore, the suppressive effect of ADC was observed, even when cut was performed in the evening 2-5 days before estrus. These results suggest that the dorsal input to the POA-MBH plays an important role in ovulation-triggering mechanisms. In addition, about 7 days after the ADC, regular estrous cycle and normal ovulation were seen. Furthermore, destruction of the medial or bilateral lateral septum was done in the morning of proestrus, to investigate the role of gonadotropin-releasing hormone (GnRH) neurons in these areas. Lesions in the septal area had no effect, suggesting that the inhibitory effect on ovulation of the ADC is not due to interruption of the fibers of the septum including GnRH neurons.

The role of estrogen in lordosis-inhibiting systems in the lateral septum or the dorsal raphe nucleus was investigated in female and male rats. Ovariectomized rats received implantation of 22-gauge guide cannulae to the bilateral or right side of the lateral septum (LS and rLS, respectively), the dorsal raphe nucleus (DRN) or bilateral cortex (CX). In castrated male rats, bilateral implantations of the cannulae to the LS were carried out (mLS). Three behavioral tests in total were performed at 2-week intervals. In the first test, all animals were subcutaneously injected with 1.5 mu g/kg estradiol benzoate (EB). Forty-four hours after EB, 0.5 mg progesterone (P) was injected and a behavioral test was started 4 h after P. These hormonal regimes were used in all tests. In the second test, 2 h before EB injection, 27-gauge cannulae filled with estradiol (E-2) were inserted into the DRN, LS or CX through the guide cannulae and were kept there for 4 h. In the third test, cholesterol was implanted instead of Eg into these areas. In the first test, most females showed low levels of lordosis quotient (LQ) and most males showed no lordosis. In the second test, mean LQs in the LS or rLS groups of females increased but not in the DRN and CX groups. In the mLS group no increase of LQ was observed. When cholesterol was implanted in the third test, mean LQs in all groups were as low as in the first test. These results suggest the possibility that estrogen releases the inhibition when it acts on the LS, but not on the DRN female rats. On the other hand, inhibition in the male LS may not be released by the direct action of estrogen.

The role of estrogen in lordosis-inhibiting systems in the lateral septum or the dorsal raphe nucleus was investigated in female and male rats. Ovariectomized rats received implantation of 22-gauge guide cannulae to the bilateral or right side of the lateral septum (LS and rLS, respectively), the dorsal raphe nucleus (DRN) or bilateral cortex (CX). In castrated male rats, bilateral implantations of the cannulae to the LS were carried out (mLS). Three behavioral tests in total were performed at 2-week intervals. In the first test, all animals were subcutaneously injected with 1.5 μg/kg estradiol benzoate (EB). Forty-four hours after EB, 0.5 mg progesterone (P) was injected and a behavioral test was started 4 h after P. These hormonal regimes were used in all tests. In the second test, 2 h before EB injection, 27-gauge cannulae filled with estradiol (E2) were inserted into the DRN, LS or CX through the guide cannulae and were kept there for 4 h. In the third test, cholesterol was implanted instead of E2 into these areas. In the first test, most females showed low levels of lordosis quotient (LQ) and most males showed no lordosis. In the second test, mean LQs in the LS or rLS groups of females increased but not in the DRN and CX groups. In the mLS group no increase of LQ was observed. When cholesterol was implanted in the third test, mean LQs in all groups were as low as in the first test. These results suggest the possibility that estrogen releases the inhibition when it acts on the LS, but not on the DRN female rats. On the other hand, inhibition in the male LS may not be released by the direct action of estrogen.

The effect of complete (CC), anterior (AC) or posterior (PC) cut of the dorsal raphe nucleus (DRn) on induction of the nocturnal prolactin (PRL) surge by electrical vaginal stimulation (VS) was investigated in ovariectomized rats. Plasma level of PRL was measured by radioimmunoassay before and after VS. The data revealed that PRL levels increased in early morning on the day following VS in the rats without brain surgery or with sham-operation. In contrast, the nocturnal PRL surge did not occur in the CC, AC, or PC rats. These results suggest that both the anterior and the posterior fibers of the DRn plays an important role in induction of nocturnal PRL surge by VS in ovariectomized rats. (C) 1998 Elsevier Science B.V. All rights reserved.

Androgen has a potency to induce female sexual behavior in ovariectomized rats. This study investigated the inhibitory effect of progesterone (P) on lordosis behavior facilitated by androgen. Ovariectomized female rats were given 100 to 800 mu g/kg testosterone propionate (TP) or 5 mu g/kg estradiol benzoate (EB). Forty-four hours after administration of the TP or the EB, all females received 0.5 mg P, and the first behavioral test was carried out four hours later. TP-treated animals showed lordosis responses in a dose dependent manner. Two weeks after the first test, the animals were retested by using the same hormonal regime as in the first test, except that an additional 5 mg P was administered 1 h prior to the TP or the EB. As a result, significant reductions in sexual receptivity were observed in the TP-treated as well as in the EB-treated females. These results indicate that progesterone can inhibit androgen-induced lordosis behavior in female rats.

The role of the dorsal raphe nucleus in the maintenance of pseudopregnancy induced by reserpine and/or vaginal stimulation was investigated in female rats. In order to induce pseudopregnancy, either the vagina was stimulated electrically (VS) on the day of proestrus, or 1 mg/kg b.w. reserpine (R) was injected on the day of diestrus I. In some females, both VS and R treatments (VS+R) were applied. Radiofrequency lesions were made in the dorsal raphe nucleus (DRL) 5 days after VS and 3 days after R. At the same time, the left uterine horn was traumatized in order to induce deciduoma. As a result, positive decidual responses were seen in most control and sham-control rats with VS or R-treatments. In contrast, the incidence of deciduoma in DRL females with R injection was lower than that in control and sham groups, but the incidence in DRL females with VS or VS+R was comparable to that in control and sham females. These results suggest that the dorsal raphe nucleus plays an important role in maintaining the mechanism of pseudopregnancy induced by reserpine, but not in that induced by vaginal stimulation.

The effect of radiofrequency lesions in the median or dorsal raphe nucleus (MRL or DRL) on copulatory behavior was examined in sexually inexperienced male rats. Three weeks after castration and the brain surgery, all males were subcutaneously implanted with Silastic capsules containing testosterone. In the first behavioral test, the frequency of ejaculation in the MRL group was significantly higher than that in sham and DRL males, but mount and intromission were not. Seven days after the first test, the second test was carried out after treatments with 100 mg/kg p-chlorophenylalanine (PCPA), a serotonin synthesis inhibitor, or saline daily for 4 days in MRL and DRL males. The frequencies of male sexual behavior in PCPA treated DRL males were higher than those in saline treated DRL males. In contrast, even after treatments with PCPA, male sexual activity in MRL males was comparable to those in saline treated MRL males. These results suggest that serotonergic neurons in the median raphe nucleus play an inhibitory role in the regulation of male sexual activity, especially ejaculation. Furthermore, it can be thought that PCPA acts on the median raphe neurons and facilitates ejaculatory behavior.

The efferents and/or afferents of the dorsal raphe nucleus (DRN) were transected by several types of cut in castrated male rats, and lordosis behavior was observed after implantation with Silastic tubes containing estradiol. Throughout the behavioral tests, low incidences of lordosis were observed in control male rats without brain surgery or with a sham operation. in contrast, all male rats with a horizontal circle cut at the ventral area of the DRN displayed lordosis, and the mean lordosis quotient (LQ) was higher than that in control rats, while rats with a horizontal cut at the dorsal area of the DRN did not. Furthermore, mean LQs in male rats with an anterior half-circle horizontal cut at the ventral area of the DRN were higher than those in control groups. A posterior half-circle cut at the ventral area had no effect. In addition, male rats with a half-dome cut located anterior to the DRN showed a high LQ score, but rats with a posterior half-dome cut did not. These results suggest that anterior and anteroventral neural fibers of the DRN are involved in the lordosis inhibiting mechanism in male rats. (C) 1997 Academic Press.

The role of serotonergic neurons in the mesencephalic raphe nuclei in regulating male sexual behavior in female rats was examined. The median or dorsal raphe nucleus lesions (MRL or DRL) were made in ovariectomized rats and behavioral tests were performed after implantation of Silastic tubes containing testosterone and treatment with serotonin synthesis inhibitor p-chlorophenylalanine (pCPA). Half of the animals in each group received 4 100 mg/kg pCPA injections before the behavioral test. As a result, the incidences and frequencies of mounts and intromissive patterns in the MRL and DRL groups were comparable to those in control females without brain surgery. Mount latency in the MRL females was shorter than that in the control females. When pCPA was given, most females with or without brain surgery showed mounts and intromissive patterns, and frequencies were higher than those in females without pCPA. These results suggest that the median raphe nucleus plays an inhibitory role in the onset mechanism for mounting. On the other hand, a stronger inhibitory influence in regulating male sexual behavior exists in other serotonergic neurons than those in the median and dorsal raphe nuclei in female rats.

To clarify the role of serotonin in penile erection, testosterone-primed castrated male rats were treated with the serotonin-synthesis inhibitor, p-chlorophenylalanine (pCPA), and reflexive erection (RE; male supine, penile sheath retracted) and noncontact erection (NCE; penile erection evoked by remote sexual stimuli) tests were performed. Half the males were injected with 100 mg/kg pCPA 4 times before each test; control males were treated with saline instead of pCPA. In the RE test, compared to the control group, pCPA-treated males had a shorter erection latency, but they also displayed fewer erections. NCE tests were conducted as a 2 x 2 factorial experiment: pCPA or saline, and estrous female present or absent. Only the pCPA-female Group had a high proportion of responders (68%), compared to 14-27% in the other Groups (p < 0.02). These results suggest that the serotonergic system exerts facilitative and inhibitory influences on different systems in regulating reflexive erection. On the other hand, serotonin appears to play an inhibitory role in the induction of noncontact erection, because pCPA did not directly induce erection, but rather facilitated the response to females. Copyright (C) 1997 Elsevier Science Inc.

The efferents and/or afferents of the dorsal raphe nucleus (DRN) were transected by several types of cut in castrated male rats, and lordosis behavior was observed after implantation with Silastic tubes containing estradiol. Throughout the behavioral tests, low incidences of lordosis were observed in control male rate without brain surgery or with a sham operation. In contrast, all male rats with a horizontal circle cut at the ventral area of the DRN displayed lordosis, and the mean lordosis quotient (LQ) was higher than that in control rats, while rats with a horizontal cut at the dorsal area of the DRN did not. Furthermore, mean LQs in male rats with an anterior half-circle horizontal cut at the ventral area of the DRN were higher than those in control groups. A posterior half-circle cut at the ventral area had no effect. In addition, male rats with a half-dome cut located anterior to the DRN showed a high LQ score, but rats with a posterior half-dome cut did not. These results suggest that anterior and anteroventral neural fibers of the DRN are involved in the lordosis inhibiting mechanism in male rats.

The inhibitory effect of progesterone (P) injected at various times on female sexual behavior was investigated in estradiol benzoate (EB) treated ovariectomized rats. Four behavioral tests were carried out at two-week intervals. All females received 5 mu g/kg b.w. EB and 0.5 mg P 44 hr after the EB. In the P-control group, an additional 5 mg P was administered at the same time as the injection of EB in four tests. Instead of P, oil was given concurrently with EB in the Oil control group. In the experimental groups, female rats were treated with 5 mg P from 1 to 40 hr before (PB group) or after (PA group) the EB-injection. A sexual behavioral test was started 4 hr after 0.5 mg P. The results show that low levels of lordosis and soliciting behavior were observed in the P group, compared to the Oil-control group. In the PB groups, lordosis quotient (LQ) was low when P was given from 1 to 24 hr before EB. Moreover, animals in which P was given 27-40 hr before EB showed lower LQ than Oil-control animals, but higher LQ than rats in the P-control group. In the PA groups, when P was administered from 1 to 24 hr after EB, low levels of lordosis response were observed, whereas animals which received P 27-40 hr after EB showed a high score of LQ, being comparable to that in the Oil control. These results suggest that the period of 24 hr before and after EB injection is a critical period for inhibitory action of P on female sexual behavior in female rats.

The role of mesencephalic raphe nuclei in the induction of pseudopregnancy was investigated in female rats. The dorsal or median raphe nucleus lesions (DRL or MRL, respectively) were made by means of a radiofrequency lesion generator. Two or 3 weeks after the operation, in order to induce pseudopregnancy, the vagina was stimulated electrically on the day of proestrus or 1 mg/kg b.w. reserpine was injected on the day of diestrus I. Traumatization by passing thread to one uterine horn was performed to induce deciduoma 5 days after vaginal stimulation or 3 days after reserpine injection. As the results, decidual response was seen in most control and sham females in both vaginal stimulation and reserpine-treated groups. In contrast, incidences of deciduoma in DRL females with vaginal stimulation or reserpine-injection were significantly lower than those in control and sham groups. In the MRL females with either vaginal stimulation or reserpine-treatment, incidences of deciduoma were comparable to those of the control and sham operated groups. These results suggest that the dorsal raphe nucleus plays an important role in pseudopregnancy-inducing mechanisms in female rats.

The inhibitory effect of baclofen, a GABA(B) receptor agonist, on lordosis was examined in female and male rats with dorsal raphe nucleus lesions (DRL) or cut of the septal fibers (ARD). Both female and male DRL and ARD rats showed higher lordosis quotients (LQ) than corresponding controls without brain surgery. This indicates that the dorsal raphe nucleus and the septum exert lordosis-inhibiting influences in female and male rats. After treatment with 10 mg/kg baclofen, the mean LQs in female control and female ARD groups were significantly lower than those of vehicle-treated control and ARD females. In DRL females, however, LQs did not decrease, even after the injection of baclofen. In males, baclofen also diminished lordosis in ARD rats, but not in DRL rats. These results suggest that the GABA(B) receptor system plays an inhibitory role in regulating lordosis behavior not only in female but also in male rats. Furthermore, the function of the GABA neurons depends on the inhibitory mechanism of the dorsal raphe nucleus in the regulation of female sexual behavior.

Radiofrequency lesions in the septum (SL), the preoptic area (POAL), or the dorsal raphe nucleus (DRL) were made in ovariectomized rats. In a control group of 16 females, ovariectomy, but no brain surgery, was performed. All animals except half of the control rats received injections of 5 mg progesterone (P) 1 h prior to the injection of 5 μg/kg b.w. of estradiol benzoate (EB). Instead of 5 mg P, oil was administered to half of the controls. Forty-four hours after EB, all females received 0.5 mg P. A sexual behavior test was performed 4 h after the last injection of P. The result was that oil-treated control rats showed high lordosis quotient (LQ) and soliciting behavior. in contrast, low scores of LQ and no soliciting behavior were observed in all of the 5 mg P-treated rats, even if the SL, POAL, or DRL was made. These results suggest that the septum, the preoptic area, and the dorsal raphe nucleus are not essential for the female sexual behavior-inhibiting mechanisms of progesterone.

The effect of serotonin synthesis inhibitor, p-chlorophenylalanine (PCPA), on induction and maintenance of pseudopregnancy as indicated by deciduoma formation was examined in female rats. Animals were injected with 1 mg/kg b.wt. of reserpine on the day of metestrus, and silk thread was passed through and placed in the left uterine horn 3 days after reserpine to induce deciduoma. PCPA (100 mg/kg b.wt.) was injected daily for 4 days before or after reserpine in 15 and 13 rats, respectively. A single injection of PCPA was administered before reserpine in nine females. In another group of rats (N = 16), instead of PCPA, saline was injected four times before reserpine. Nineteen female rats were treated with reserpine only as a control group. Results showed 89% of the control and 81.3% of the saline-treated females had massive deciduoma in traumatized uterine hem. In contrast, only 33.3% or 46.2% females with daily treatments of PCPA for 4 days before or after reserpine showed positive decidual reaction. In addition, 88.9% of females with single injection of PCPA possessed uterine hems with deciduoma. These results suggest that 4 days of treatment with PCPA eliminate induction and/or maintenance of pseudopregnancy. Thus, some levels of serotonin are required to induce and maintain pseudopregnancy.

The inhibitory effect of baclofen, a GABAβ receptor agonist, on lordosis was examined in female and male rats with dorsal raphe nucleus lesions (DRL) or cut of the septal fibers (ARD). Both female and male DRL and ARD rats showed higher lordosis quotients (LQ) than corresponding controls without brain surgery. This indicates that the dorsal raphe nucleus and the septum exert lordosis-inhibiting influences in female and male rats. After treatment with 10 mg/kg baclofen, the mean LQs in female control and female ARD groups were significantly lower than those of vehicle-treated control and ARD females. In DRL females, however, LQs did not decrease, even after the injection of baclofen. In males, baclofen also diminished lordosis in ARD rats, but not in DRL rats. These results suggest that the GABAB receptor system plays an inhibitory role in regulating lordosis behavior not only in female but also in male rats. Furthermore, the function of the GABA neurons depends on the inhibitory mechanism of the dorsal raphe nucleus in the regulation of female sexual behavior. © 1996 S. Karger AG, Basel.

The effect of medial amygdala lesions on male sexual behavior in male rats with stria terminalis cut was examined, First, castrated male rats received bilateral transections of the stria terminalis (STC) or sham cut (SC). Most STC males showed no ejaculation, but displayed mount and intromission, although the frequencies were not high compared to those of males with SC. Next, bilateral lesions of the medial amygdala (MAL) or sham lesion (SL) were performed in males with STC or SC. The MAL caused severe loss of all aspects of copulatory behavior in males with STC as well as in males with SC. The suppressive effect of the MAL on copulatory activity was stronger than that of the STC. These results indicate that a neural pathway other than the stria terminalis is involved in the regulation of male sexual behavior by the amygdala in male rats.

In order to clarify the functional relationships between the lateral septum (LS) and the mesencephalic central gray (MCG) in regulating lordosis behavior, ovariectomized female rats received dual lesions in these two areas. In the first experiment, females with unilateral (right or left, R-MCGL or L-MCGL) or bilateral MCG (B-MCG) lesions were subjected to behavioral tests after the implantation of a Silastic tube containing estradiol. Lordosis was observed in only one B-MCGL female. In the R-MCGL and L-MCGL groups, most females displayed lordosis, but lordosis quotients (LQ) were significantly lower than that of the control group. These results suggest the importance of the MCG in lordosis regulation, and that there is no functional laterality in the MCG. In the second experiment, B-MCGL or R-MCGL females received bilateral LS lesions (LSL). The lordotic activity in the LSL + B-MCGL group was extremely low, being comparable to that of B-MCGL alone. On the other hand, in the LSL + R-MCGL females, the LQ was significantly higher than that of females with R-MCGL alone and was comparable to that of controls. Thus. the lateral septum plays an inhibitory role in regulating lordosis, but the influence of the lateral septum is not stronger than the facilitatory influence of the mesencephalic central gray, because the LSL could not recover the suppressive effect of the MCGL.

雄ラットを去勢し、大量のエストロゲンを投与しても雌型性行動であるロードーシスはほとんど発現しない。雌ラットにも雄ラットにも、中隔と背側縫線核のセロトニン神経によるロードーシス抑制回路が発達しており、その抑制力の違いにより、雄と雌のロードーシスの発現の違いが生じているものと考えられる。本実験ではその抑制機構の性質の違いを明確にするめに雌と雄ラットの脳に直接エストロゲンを投与しロードーシスの発現を調べた。　麻酔下で雌ラットの卵巣を除去し、中隔または背側縫線核にステンレスガイドチューブを設置する。２週間後、閾値下のエストロゲンを注射し、インナーチューブにエストロゲンかコレステロールをいれ挿入し、ロードーシスの強さをみた。その結果、中隔にエストロゲンをいれるとロードーシスの発現が見られたが、背側縫線核にエストロゲンをいれても生じなかった。コレステロールではどちらの部位ににいれても全く影響がなかった。従って、中隔の抑制力はエストロゲンが直接作用して解除されるが、背側縫線核の抑制機構は直接作用をうけないものと考えられる。次に、雄ラットを去勢し同様の実験を行なった結果、雌ラットとは異なり、中隔にエストロゲンをいれてもロードーシスは生じなかった。　これらの結果より、雄ラットが雌の性行動であるロードーシスをしないのは、中隔にエストロゲンにより解除できないほど強い抑制回路が形成されるためであることが明かとなった。研究成果の発表Lordosis in male rats： Effect of dorsal raphe nucleus cuts. Horm. Behav. 32 (1), 60-67,1997Kakeyama, M. and Yamanouchi, K. Facilitatory effect of ventral cut of dorsal raphe nuclues on lordosis in female rats. Endocrine J. 44 (4) 589-593,1997Kakeyama, M., Negishi, M. and Yamanouchi, K.Inhibitory effect of progesterone on lordosis in male and female rats with septal lesions.In 'Advances in Comparative Endocrinology 2Kawashima, S. and Kikuyama, S.(eds) Monduzzi/Bologna, pp. 1759-1763, 1997.Satou, M. and Yamanouchi, K.

　ラットの妊娠は下垂体前葉から分泌されるプロラクチンの周期的分泌により、卵巣の黄体からプロゲステロンが分泌されて維持される。妊娠状態は腟の刺激や脳のモノアミン枯渇剤投与により人工的に誘起でき、子宮の脱落膜腫形成により形態的に検証できる。　本実験では人工的な妊娠（偽妊娠）誘起に対して、セロトニンニュ－ロンがどのような影響をもつかセロトニン合成阻害剤であるp-chlorophenylalanine (PCPA)を投与し影響をみ、さらに、セロトニンニュ－ロンの最も豊富な中脳の背側縫線核の働きを破壊により調べた。　正常な4日周期の雌ラットの発情間期Iに1 mg/kg reserpine(R)を投与すると19匹中17のラットに脱落膜腫形成が生じたが、R投与3日前から4回、5 mg/kg PCPAを投与された群では15匹中5匹のみ脱落膜腫がみられた。また、R投与後4回PCPAを投与されても13中6匹で脱落膜腫が形成されたにすぎなかった。この結果より偽妊娠誘起と維持にはセロトニンニュ－ロンの働きが必要であることが示された。　次に雌ラットに背側縫線核か正中縫線核を破壊しておき、R投与または腟刺激によって偽妊娠状態が生じるかどうか検証した。背側縫線核を破壊しておくと、腟刺激をしても15匹中7匹のみで脱落膜腫がみられ、Rを投与しても18匹中10匹のみ脱落膜腫が生じた。一方、正中縫線核を破壊したラットでは、腟刺激でも、Rを投与した場合も、9匹中すべての動物が脱落膜腫を生じた。このように中脳の背側縫線核のセロトニンニュ－ロンが偽妊娠誘起に必須のものであることが示された。　排卵前日の午前中に背側縫線核を破壊すると翌日の排卵が生じなくなる事から、背側縫線核は偽妊娠と同様，排卵に関しても、重要な働きをしていることが示されている。

雌哺乳類の中枢神経系には性行動発現に対する促進神経機構と抑制神経機構が発達している。性行動抑制力は前脳の中隔－視索前野と中脳背側縫線核に存在している。雌哺乳類の性行動はエストロゲンとプロゲステロンが中枢神経系に直接作用し制御を行っている。エストロゲンは中枢神経系内の促進機構と抑制機構に働いて性行動を促進する。一方，プロゲステロンはエストロゲンと同様に雌性行動を促進する働きがあるが，投与するタイミングによってはエストロゲンによって引きおこされる雌性行動が抑制される。すなわちプロゲステロンは発情の停止作用をもつ。本実験ではプロゲステロンによる発情抑制作用が脳がどのような機構と関係しているか，その解明の第一ステップとして，雌性行動抑制機構である中隔，視索前野，背側縫線核との関係を調べた。 雌ラットの卵巣を除去すると同時に，中隔，視索前野，背側縫線核を高周波破壊装置で破壊した。プロゲステロンの抑制作用を見るために5mgのプロゲステロンを5μg/kgのエストラディオールベンゾエート（EB）投与1時間前に注射し，EB投与44時間後に0.5mgのプロゲステロンを投与して性行動を調べた。その結果，中隔，視索前野，背側縫線核を破壊された雌ラットも性行動発現の低下が見られた。この結果から，プロゲステロンは中枢神経系の抑制機構に働いて性行動の抑制をしているのではないことが明らかとなった。さらに，それらの部位を破壊し，雌の性行動を示すようになった雄ラットに，EB投与前にプロゲステロンを注射しても雌性行動の低下が見られた。このように，雄ラットにおいても，プロゲステロンの雌性行動抑制作用がみられることが示された。