Neuronal activity in the sagittalis nucleus of the hypothalamus after ovarian steroid hormone manipulation and sexual behavior in female rat.

During extended observation of estrogen receptor (ER) α-immunoreactive neurons in the hypothalamus, we previously identified a novel nucleus, the Sagittalis Nucleus of the Hypothalamus (SGN), in the interstitial area between the arcuate nucleus and the ventromedial hypothalamic nucleus. The SGN exhibits sexual dimorphism in its volume and cell count, and estrous cycle related variations in ERα-immunoreactivity. These characteristics of the SGN implicate the nucleus in sex-biased brain functions and behaviors. In this study, we examined involvement of the SGN in sexual arousal in female rats. Immunohistochemical staining of c-Fos, a marker of neuronal activity was performed after administration of an estrus-inducing dose of estrogen and progesterone in ovariectomized female rats. Analysis of microscopic images showed a significant increase in the number of c-Fos-expressing neurons in the SGN following hormonal manipulation. Moreover, neuronal activity in the region exhibited a further increase after each animal was coupled with a male and allowed to mate. These results suggest that the SGN plays an important role in sexual activity in female rat.

Strain differences in intermale aggression and possible factors regulating increased aggression in Japanese quail.

The National Institute for Environmental Studies (NIES) of Japan established a strain of Japanese quail (Coturnix japonica) known as NIES-L by rotation breeding in a closed colony for over 35years; accordingly, the strain has highly inbred-like characteristics. Another strain called NIES-Brn has been maintained by randomized breeding in a closed colony to produce outbred-like characteristics. The current study aimed to characterize intermale aggressive behaviors in both strains and to identify possible factors regulating higher aggression in the hypothalamus, such as sex hormone and neuropeptide expression. Both strains displayed a common set of intermale aggressive behaviors that included pecking, grabbing, mounting, and cloacal contact behavior, although NIES-Brn quail showed significantly more grabbing, mounting, and cloacal contact behavior than did NIES-L quail. We examined sex hormone levels in the blood and diencephalon in both strains. Testosterone concentrations were significantly higher in the blood and diencephalon of NIES-Brn quail compared to NIES-L quail. We next examined gene expression in the hypothalamus of both strains using an Agilent gene expression microarray and real-time RT-PCR and found that gene expression of mesotocin (an oxytocin homologue) was significantly higher in the hypothalamus of NIES-Brn quail compared to NIES-L quail. Immunohistochemistry of the hypothalamus revealed that numbers of large cells (cell area>500µm2) expressing mesotocin were significantly higher in the NIES-Brn strain compared to the NIES-L strain. Taken together, our findings suggest that higher testosterone and mesotocin levels in the hypothalamus may be responsible for higher aggression in the NIES-Brn quail strain.

Sexual behavior-associated c-Fos induction in the sagittalis nucleus of the hypothalamus in male rat.

The sagittalis nucleus of the hypothalamus (SGN) is a small nucleus located in the interstitial area between the arcuate and ventromedial nuclei of the hypothalamus in rats. The SGN exhibits male-biased sexual dimorphism and expresses estrogen receptor α and calbindin-D28K. This suggests a contribution of the SGN to sexually differentiated brain function, but its functional role is unknown. In this study, neuronal activation in the SGN during sexual behavior in male rats was examined by c-Fos immunohistochemistry. The number of c-Fos-immunoreactive (c-Fos-ir) cells was elevated with only exposure to chemosensory cues of estrous females and significantly increased after the first mount. The first intromission and ejaculation did not induce further increases in the number of c-Fos-ir cells in the SGN. These findings suggest that the SGN is involved in regulation of the early phase of male sexual behavior, including motivation.

A Sexually Dimorphic Area of the Dorsal Hypothalamus in Mice and Common Marmosets.

We found a novel sexually dimorphic area (SDA) in the dorsal hypothalamus (DH) of mice. The SDA-DH was sandwiched between 2 known male-biased sexually dimorphic nuclei, the principal nucleus of the bed nucleus of the stria terminalis and the calbindin-sexually dimorphic nucleus, and exhibited a female-biased sex difference in neuronal cell density. The density of neurons in the SDA-DH was increased in male mice by orchidectomy on the day of birth and decreased in female mice by treatment with testosterone, dihydrotestosterone, or estradiol within 5 days after birth. These findings indicate that the SDA-DH is defeminized under the influence of testicular testosterone, which acts via both directly by binding to the androgen receptor, and indirectly by binding to the estrogen receptor after aromatization. We measured the activity of SDA-DH neurons with c-Fos, a neuronal activity marker, in female mice during maternal and sexual behaviors. The number of c-Fos-expressing neurons in the SDA-DH of female mice was negatively correlated with maternal behavior performance. However, the number of c-Fos-expressing neurons did not change during female sexual behavior. These findings suggest that the SDA-DH contains a neuronal cell population, the activity of which decreases in females exhibiting higher performance of maternal behavior, but it may contribute less to female sexual behavior. Additionally, we examined the brain of common marmosets and found an area that appears to be homologous with the mouse SDA-DH. The sexually dimorphic structure identified in this study is not specific to mice and may be found in other species.

Sex differences in cells expressing green fluorescent protein under the control of the estrogen receptor-α promoter in the hypothalamus of mice.

Estradiol that originates from testicular testosterone and binds to estrogen receptor-α (ERα) during developing period acts to organize the male-type brain in mice. Here, we examined transgenic mice expressing green fluorescent protein (GFP) under the control of the ERα promoter, in which ERα-expressing cells in the brain can be visualized by GFP. Fluorescence microscopy revealed the existence of many GFP-expressing cells in the medial preoptic area, medial preoptic nucleus (MPN), bed nucleus of the stria terminalis (BNST), and striohypothalamic nucleus (StHy) of adult transgenic mice. Neuronal nuclear antigen, a neuron marker, but not glial fibrillary acidic protein, an astrocyte marker, was mostly expressed by GFP-expressing cells. Analysis of GFP expression area showed that adult females had higher GFP expression in a region including the ventral part of the BNST, StHy, and dorsal part of the MPN than in adult males. Such female-biased sex difference was also found in transgenic pups on postnatal day 5 and 8. The GFP expression area of adult females was decreased by postnatal treatment with testosterone or estradiol. These results indicate that GFP visualizes a sex difference of ERα-expressing neurons. The transgenic mice may be useful for the analysis of the sexual differentiation of the brain.

Athymic nude mice are insensitive to low-level toluene-induced up-regulation of memory-related gene expressions in the hippocampus.

The function of the N-methyl-d-aspartate (NMDA) subtype of glutamatergic receptors is known to be antagonized by toluene, a well-characterized neurotoxic chemical known to impair memory functions. Recently, peripheral T cells have been clearly shown to play an important role in cognitive and behavioral functions. In the present study, we investigated the role of peripheral T cells in the hippocampal mRNA expression of memory-related genes induced by low levels of toluene exposure in mice. BALB/c wild-type (WT) and nude mice were exposed to 9ppm of toluene or filtered air (0ppm toluene; control groups) in a nose-only exposure chamber for 30min on 3 consecutive days followed by weekly sessions for 4 weeks. Twenty-four hours after the last exposure, the hippocampi were collected and the inducibility of memory-related genes was examined using a real-time quantitative PCR method. NMDA NR2A, calcium/calmodulin-dependent protein kinase IV (CaMKIV), cyclic AMP-responsive element binding protein 1 (CREB1), and BDNF were significantly up-regulated in the hippocampi of WT mice exposed to 9ppm of toluene, compared to the expressions observed in WT mice exposed to filtered air, but similar results were not observed in nude mice. To investigate the possible involvement of peripheral T cells in the toluene-induced up-regulation of memory-related genes in WT mice, we examined the mRNA expression of Thy-1 (a pan T cell-specific marker) and quantified the number of cells that were immunoreactive to a T cell antigen receptor, CD3 (CD3-ir). Both the expression of Thy-1 mRNA and the number of CD3-ir cells were significantly higher in the hippocampi of the WT mice exposed to 9ppm of toluene, compared with that in WT mice exposed to filtered air; similar results were not observed in nude mice. We also examined the expression of chemokine genes like CCL2 and CCL3. The expression of CCL3 mRNA was significantly up-regulated only in the toluene-exposed WT mice. Although other differences unrelated to immune function may exist between WT and nude mice from the same background, the findings of the present study strongly suggest that the recruitment of peripheral T cells in the hippocampi of BALB/c WT mice exposed to low levels of toluene may be involved in the toluene-induced up-regulation of memory-related genes at the mRNA level.

Increased hippocampal mRNA expression of neuronal synaptic plasticity related genes in mice chronically exposed to toluene at a low-level human occupational-exposure.

Although neurological symptoms in individuals exposed to toluene both inside and outside the homes have been reported well, the chronic effects of low-level toluene-exposure on the hippocampal expression of neuronal synaptic plasticity related genes have not been studied in vivo. In the present study, to understand the possible adult hippocampal neurobiological responses of mice chronic exposure to toluene at a low-level human occupational-exposure, we exposed 10-week-old C3H/HeN female mice to 50 ppm toluene or filtered air for 6 h a day, on 5-consecutive days of a week for 6 and 12 weeks, in a whole-body exposure chamber. Then, by a quantitative real-time PCR method, we investigated the hippocampal mRNA-expression of several genes, functions of which are necessary to maintain the homeostasis of neuronal synaptic plasticity. We observed that chronic exposure of mice to 50 ppm toluene for a longer period (12 weeks) caused a significant up-regulation of NMDA receptor subunit 2B (NMDA NR2B) expression associated with a simultaneous induction of CaMKIV, CREB-1, and FosB/DeltaFosB in the same hippocampal tissues. Our data indicate that the in vivo transcriptional up-regulation of these genes in the adult hippocampus of our experimental mouse model following the chronic exposure to toluene may be an NMDA-receptor related neuroprotective mechanism of gene expression.

Distribution of glutamic acid decarboxylase, neurotensin, enkephalin, neuropeptide Y, and cholecystokinin neurons in the septo-preoptic region of male rats.

Neurons in the lateral septum (LS) and preoptic area (POA) are known to play an inhibitory role in feminine sexual behavior regulation in male rats. In this study, the distribution of neurons containing glutamic acid decarboxylase (GAD) and of the peptidergic neurotransmitters neurotensin (NT), enkephalin (ENK), neuropeptide Y (NPY), and cholecystokinin (CCK), was examined immunohistochemically in the LS and POA of castrated male rats subcutaneously implanted with estrogen-containing Silastic tubes. Colchicine was injected into the lateral ventricle of the animals. The forebrain sections were immunostained for each substance. A large number of GAD-immunoreactive (ir) cells were found in the LS. Many NT-ir cells were seen in the intermediate and ventral parts of the LS at the rostral and middle levels. A considerable number of ENK-ir cells were scattered over the LS at the rostral and middle levels and were observed in the ventral part of the caudal LS. There were only a few NPY-ir cells in the LS. No CCK-ir cells were observed in the LS. In the POA, GAD-ir cells were observed in abundance. Many NT-ir cells were seen, especially in the medial preoptic nucleus. Some ENK-ir cells and a few NPY-ir cells were found in the medial POA. CCK-ir cells of the POA were restricted to the periventricular and paraventricular hypothalamic nuclei.