Neonatal septal lesions prevent behavioral defeminization caused by neonatal treatment with estradiol in female rats.

Male rats rarely show lordosis, a female sexual behavior, because of strong inhibition of the behavior in the lateral septum. Because neonatal treatment with estradiol (E2) in female rats decreases lordosis, it is believed that the lateral septum is a target of E2 action to defeminize or masculinize the lordosis-inhibiting system. Here, we tested the hypothesis that disruption of the lateral septum before E2 treatment prevents the effect of neonatal E2 on lordosis. Female rats that underwent radiofrequency-induced septal lesions or sham operation on postnatal day 4 (PD4, day of birth = PD1) were subcutaneously injected with E2 or sesame oil vehicle alone on PD5. Vaginal opening and smears were checked. After sexual maturation, lordosis tests were performed. The effects of neonatal septal lesions on lordosis in male rats were also observed. Sham-operated and E2-treated female rats showed a reduction in lordosis and irregular estrous cycles. Conversely, septal lesioned and E2-treated females exhibited higher levels of lordosis, although their estrous cycles were irregular. These results suggest that neonatal septal lesions prevent females from being behaviorally defeminized by neonatal E2. Additionally, neonatally septal lesioned males displayed higher levels of lordosis than sham-operated males. These results suggest that E2, which is produced by the aromatization of testicular testosterone in the neonatal period, acts on the lateral septum to organize the lordosis-inhibiting system.

Efferent and afferent connections of the ventromedial hypothalamic nucleus determined by neural tracer analysis: implications for lordosis regulation in female rats.

Neural connections of the ventromedial hypothalamic nucleus (VMN) to and from forebrain and midbrain structures, which are involved in the neuroendocrine regulation of reproduction, were investigated. A retrograde (fluoro-gold [FG]) or an anterograde neural tracer (phaseolus vulgaris-leucoagglutinin [PHA-L]) was injected into the left side of the VMN in ovariectomized rats. Six days after injection with FG or 11 days after injection with PHA-L, brains were fixed and sectioned. After immunohistochemistry, digital images of FG-labeled neural cell bodies (FG-cells) or PHA-L-labeled fibers (PHA-L-fibers) were analyzed. Injection sites of FG and PHA-L were mainly in the ventrolateral VMN. Considerable numbers of FG-cells and PHA-L-fibers were present in the left side of the medial amygdala, ventral lateral septum, preoptic area, bed nucleus of stria terminalis, dorsomedial hypothalamic nucleus, arcuate nucleus, periventricular nucleus of thalamus, and midbrain central gray. The lateral dorsal raphe nuclei contained many PHA-L-fibers but few FG-cells. By contrast, both sides of the median raphe nucleus contained many FG-cells but few PHA-L-fibers. Reciprocal direct neural connection between the right and left side of the VMN were observed. The present results provide an anatomical basis for functional relationships between the VMN and these nuclei.

Neuroanatomy and sex differences of the lordosis-inhibiting system in the lateral septum.

Female sexual behavior in rodents, termed lordosis, is controlled by facilitatory and inhibitory systems in the brain. It has been well demonstrated that a neural pathway from the ventromedial hypothalamic nucleus (VMN) to the midbrain central gray (MCG) is essential for facilitatory regulation of lordosis. The neural pathway from the arcuate nucleus to the VMN, via the medial preoptic nucleus, in female rats mediates transient suppression of lordosis, until female sexual receptivity is induced. In addition to this pathway, other regions are involved in inhibitory regulation of lordosis in female rats. The lordosis-inhibiting systems exist not only in the female brain but also in the male brain. The systems contribute to suppression of heterotypical sexual behavior in male rats, although they have the potential ability to display lordosis. The lateral septum (LS) exerts an inhibitory influence on lordosis in both female and male rats. This review focuses on the neuroanatomy and sex differences of the lordosis-inhibiting system in the LS. The LS functionally and anatomically links to the MCG to exert suppression of lordosis. Neurons of the intermediate part of the LS (LSi) serve as lordosis-inhibiting neurons and project axons to the MCG. The LSi-MCG neural connection is sexually dimorphic, and formation of the male-like LSi-MCG neural connection is affected by aromatized testosterone originating from the testes in the postnatal period. The sexually dimorphic LSi-MCG neural connection may reflect the morphological basis of sex differences in the inhibitory regulation of lordosis in rats.

Inhibitory role of the serotonergic system on estrogen receptor α expression in the female rat hypothalamus.

The role of the serotonergic system in regulating the expression of estrogen receptor (ER) α in the hypothalamus was investigated in ovariectomized rats by injecting a serotonin synthesis inhibitor, parachlorophenylalanine (PCPA), or by destroying the dorsal raphe nucleus (DR). The number of ERα-immunoreactive (ir) cells was counted in the anteroventral periventricular nucleus in the preoptic area (AVPV), ventrolateral ventromedial hypothalamic nucleus (vlVMN), and arcuate nucleus (ARCN). Seven days after ovariectomy, 100mg/kg PCPA or saline was injected daily for 4 days. Alternatively, radiofrequency lesioning of the DR (DRL) or sham lesions were made on the same time of ovariectomy. One-day after the last injection of PCPA or 7 days after brain surgery, the brain was fixed for immunostaining of ERα and the number of ERα-ir cell were counted in the nuclei of interest. The mean number of ERα-ir cells/mm(3) (density) in the AVPV of the PCPA or DRL groups was statistically higher than that in the saline or sham group. In the vlVMN and ARCN of the PCPA or DRL groups, the mean density of ERα-ir cells was comparable to the saline or sham groups. These results suggest that the serotonergic system of the DR plays an inhibitory role on the expression of ERα in the AVPV, but not in the vlVMN and ARCN.

Postnatal changes in the number of serotonin-immunoreactive cells in midbrain raphe nuclei of male rats.

To clarify the developmental changes in serotonergic neurons in the subdivisions of the dorsal (DR) and median raphe (MR) nuclei before puberty, the extent of the nuclei and the number of serotonin (5-HT) immunoreactive (ir) cells were measured in 5-, 15-, and 30-day-old rats and 8-week-old (adult) castrated male rats. The brains were fixed and 50 µm frozen sections prepared. After immunostaining for 5-HT, the number of 5-HT-ir cells in a 0.2 × 0.2 mm frame in the dorsal, ventral and lateral subdivisions of the DR (dDR, vDR and lDR, respectively) and MR were counted. Total numbers of 5-HT-ir cells counted in the frame of three sections in each rat were expressed as the number of cells per cubic millimeter (density). The results indicated that the densities of 5-HT-ir cells in the MR were almost the same in all age groups. On the other hand, among the subdivisions of the DR, the mean density of 5-HT-ir cells in 15-day-old rats was higher than that in the 5-day-old group in the lDR only. The area of the three sections of the DR and of the MR was also measured. The area of the DR in 15-day-old rats was found to be twice that in the 5-day-old rats, and differed from the area in 30-day-old rats and adults. There were no differences among the age groups in the areas of the MR. The results indicate that the expression of 5-HT in the lDR and extent of the DR increased to adult levels from days 5 to 15 after birth. In the dDR, vDR and MR, expression of 5-HT at postnatal day 5 was at adult levels already.

Synthesis and bioassay of a boron-dipyrromethene derivative of estradiol for fluorescence imaging in vivo.

C7α-substituted estradiols bind to estrogen receptors in cell nuclei, yet these derivatives remain little used in bioimaging. Here, we describe a fluorescent derivative of estradiol (E2) with a boron-dipyrromethene (BODIPY) moiety attached to C7α, synthesized by olefin metathesis reaction of 7α-allylestradiol and 9-decenyl-BODIPY. In ovariectomized rats and non-ovariectomized mice, E2-BODIPY promoted the growth of uterine tissue similar to the effect of estradiol. Twenty-four hours after subcutaneous injection of E2-BODIPY in non-ovariectomized mice, we observed fluorescence of E2-BODIPY in the nuclei of uterine epithelial cells. Our findings suggest that fluorescence microscopy can localize this derivative in E2-responsive cells during normal development and tumorigenesis in vivo.

Defeminization of brain functions by a single injection of estrogen receptor α or ß agonist in neonatal female rats.

Sexual differentiation of brain function is regulated by estrogen in the perinatal period of rodents. However, the role of the estrogen receptor subtypes ERα and ERß is still in question. Accordingly, the effects of neonatal treatment with the ERα agonist propyl pyrazole triol (PPT) or the ERß agonist diarylpropionitrile (DPN) on female reproductive functions were investigated in rats. Female rats were injected subcutaneously with 100-500 µg/10 g body weight (b.w.) PPT or DPN, 100 µg/10 g b.w. estradiol (E(2)), or saline at day 5 (birth day = day 1), and then vaginal opening and vaginal smears were examined. On day 60, their ovaries were removed and lordosis behavior was observed after subcutaneous implantation of a silicon tube containing E(2). As a result, in most PPT and all E(2) rats, vaginal opening was advanced and an irregular estrous cycle was observed. In contrast, in most rats of the DPN groups, vaginal opening was comparable to that of the control and there was a regular estrous cycle. Lordosis tests revealed that the mean lordosis quotients (LQs) in the 250- and 500-µg PPT groups was lower than in the saline group, but higher than in the E(2) group. Mean LQs in all DPN groups were comparable to those in the saline group. These results suggest that ERα plays a major role in masculinization of the system regulating the estrous cycle in the rat brain. In behavioral defeminization of the lordosis-regulation system, ERα was also found to be the main target of estrogen.

Acceleration of irregular estrous cycle in forced running by midbrain raphe lesions in female rats.

It is known that over-exercise or forced running interrupts the regular ovulatory (estrous) cycle in female mammals, including women. The serotonin content of the brain changes under stress conditions. In this experiment, radiofrequency lesions were made in the dorsal (DRL) or median (MRL) raphe nuclei of the midbrain, in which serotonergic neurons are abundant, and changes in the estrous cycle with forced running using an electric-motor running wheel were examined in female rats. Through the tests, the estrous cycle was checked by taking vaginal smears. Female rats with a regular 4-day estrous cycle were forced to run in the wheel for 30 min daily over 15 days. As a result, 27.3% of the control and 30.0% of the sham-operated rats showed an irregular estrous cycle. In contrast, 100% of the DRL and 87.5% of the MRL rats showed an irregular cycle (P<0.05 vs. control and sham). Statistical analysis revealed that the median onset day of an irregular cycle was in excess of 15 days in both the control and sham groups. In the DRL and MRL groups, the median onset days of the irregular cycle were day 5 and 3, respectively, being shorter than those in control and sham groups (P<0.01). These results indicate that the dorsal and median raphe nuclei play an important role in preventing the effect of stress conditions in the ovulatory system in female rats.

Sex and regional differences in decrease of serotonin-immunoreactive cells by parachlorophenylalanine in rat raphe nuclei.

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 100mg/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.

Sex and regional differences in decrease of estrogen receptor alpha-immunoreactive cells by estrogen in rat hypothalamus and midbrain.

Sensitivity of neurons to estrogen in down-regulation of estrogen receptor alpha (ERalpha) 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 ERalpha in the hypothalamus and midbrain, the number of ERalpha 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 1mg estradiol benzoate (EB). The remaining 5 rats in both sexes did not receive EB. The brain was fixed 24h after EB-injection and 50 microm-serial frozen sections were made. After immunohistochemical staining for ERalpha, the number of ERalpha-ir cells was counted in a 0.2-mm2 frame in the anteroventral periventricular nucleus (AVPvN), the ventrolateral part of the ventromedial hypothalamic nucleus (vlVMN), the arcuate nucleus (ARCN), and the lateral mesencephalic central gray (lMCG) in 2 or 3 sections. The total number of ERalpha-ir cells was changed to a density value (number per 1mm3). As the results, in EB-treated rats, the density of ERalpha-ir cells in all regions, except the male AVPvN and male lMCG, were lower than those in untreated rats of both sexes. In the vlVMN, the density of ERalpha-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 ERalpha by estrogen in the rat brain.

Additive inhibition of lordosis by simultaneous treatments with GABA(A) and GABA(B) receptor agonists, muscimol and baclofen, in female rats.

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 GABA(A) receptor agonist, muscimol (MUS), at a dose in the range from 1.0 to 1.4 mg/kg body weight (bw) or the GABA(B) receptor agonist, baclofen (BAC) at a dose in the range from 1 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 GABA(A) and GABA(B) receptors are involved in lordosis-inhibiting mechanisms by the GABA neuron and operate independently.

Ipsilateral and contralateral serotonergic projections from dorsal and median raphe nuclei to the forebrain in rats: immunofluorescence quantitative analysis.

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.

Effects of estrogen, androgen, and phytoestrogen on retrieving and licking behaviors in nulliparous and male rats.

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.

Postnatal development of septal projections to the midbrain central gray in female rats: tract-tracing analysis with DiI.

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 PFA at 40 degrees C for up to 4 months. After incubation, 200-microm frozen parasagittal 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.

Effects of 5-HT1A-receptor agonist, 8-OH-DPAT, and GABAB-receptor agonist, baclofen, on lordosis in female rats with lesions in either the dorsal raphe nucleus or septum.

The inhibitory effects of 8-OH-DPAT, a 5-HT1A-receptor agonist, and baclofen, a GABAB-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.

Sex difference and postnatal change of maternal behavioral patterns in juvenile male and female rats.

Juvenile rats are known to show certain elements of maternal behavior. In this experiment, to investigate sex difference and postnatal change of retrieving and pup-cleaning (licking) behaviors in juvenile rats, these behaviors were recorded using new observation method at 20, 30 and 45 days of age in female and male Wistar rats. At 20 days of age, maternal behavior was observed in a common plastic observation cage (test A) and then test B was performed. In the test B, observation was carried out using a cage with a wooden box that was open on one side, helping the juveniles to establish a nest. As the results of day 20, most rats in all groups showed licking behavior in both the test A and B. The incidence of retrieving behavior increased from the test A to the test B with the box in both sexes, especially in males (p<0.01). The box is thought to play a facilitative role in induction of retrieving. Moreover, the incidence in males was higher than that in females in the test B (p<0.001). At 30 and 45 days of age, only a test B with box was performed. The incidences of licking and retrieving behaviors at 30 days of age were decreased significantly compared to those at 20 days of age in both sexes(p<0.001). Further decrease from 30 days to 45 days was observed. These results suggest that in juvenile rat, incidence of retrieving behavior in males is higher than that in females but there is no sex difference in incidence of licking behavior. Potency to show these behaviors decreases acutely before puberty in rats.

Suppressive effect of neonatal treatment with a phytoestrogen, coumestrol, on lordosis and estrous cycle 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 3mg coumestrol (CM1, CM3), 1mg genistein (GS1), 1mg estradiol (E2), 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 E2-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 E2-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 E2-treated rats. Behavioral tests were carried out after implantation of E2-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 1mg coumestrol was effective in only the estrous cycle of female rats.

Facilitatory effects of WAY-100635, a 5-HT1A receptor antagonist, on lordosis in female rats.

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 behavioral 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 behavioral 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 behavior acutely in female rats with a low estrous state. In order to investigate relationships between the 5-HT(1A) 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.

Postnatal apoptosis, development, and sex difference in the lateral septum of rats.

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 immunohistochemically 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.

Effects of highly or relatively selective 5-HT1A receptor agonists on lordosis in female rats.

To investigate the role of serotonin (5-HT) receptor 1A 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.