Shinji Tsukahara

Estrogen modulates Bcl-2 family protein expression in the sexually dimorphic nucleus of the preoptic area of postnatal rats.

In the sexually dimorphic nucleus of the preoptic area (SDN-POA) of postnatal rats, apoptotic cells are detected more frequently in females than males. This sex difference is under the influence of aromatized androgen. We have reported that there are sex differences in the levels of Bcl-2 (femalemale) in the central division of the medial preoptic nucleus (MPNc), a significant component of the SDN-POA, followed by a sex difference in induction of apoptosis via caspase-3 activation (female>male). In the present study, we examined effects of estradiol benzoate (EB) on expression of Bcl-2 and Bax in the MPNc. Female rats were subcutaneously injected with EB (25 or 50 microg per head) on postnatal day 5. MPNc and caudate putamen (CP) tissues were obtained from EB-treated female and male rats on postnatal day 6. Protein levels of Bcl-2 and Bax were determined by Western blotting. In the MPNc of female rats, EB at a dose of 50 microg/head but not 25 microg/head significantly increased Bcl-2 protein level and decreased Bax protein level. The levels of Bcl-2 and Bax of female rats treated with 50 microg of EB were comparable to those of male rats. However, the protein levels of Bcl-2 and Bax in the CP did not change with EB treatment. These results suggest that estrogen up-regulates Bcl-2 expression and down-regulates Bax expression in the MPNc of postnatal rats. Effects of estrogen on the Bcl-2 family are presumably responsible for sex difference in postnatal apoptosis of the SDN-POA.

Inhalation of Low-Level Formaldehyde Increases the Bcl-2/Bax Expression Ratio in the Hippocampus of Immunologically Sensitized Mice

Objective: A recent study from our research group showed that repeated exposure to low-level formaldehyde (FA) increases the production of nerve growth factor, involving the survival and maintenance of neurons, in the hippocampus of immunized mice. In the present study, we examined the effects of FA on apoptotic mechanisms regulating survival and death of cells and on N-methyl-D-aspartate (NMDA) receptors related to hippocampal functions in the mouse hippocampus. Methods: Western blot analyses were performed for Bcl-2, Bax and NMDA receptor subtypes 2A and 2B of the hippocampus taken from C3H mice exposed to 0 or 400 ppb of FA with or without ovalbumin (OVA) immunization. Immunohistochemical analysis for active caspase-3 was also carried out for these mice. Results: The ratio of Bcl-2 to Bax expression levels significantly increased with 400-ppb FA exposure in OVA-immunized mice but not in mice without OVA immunization, although differences in each protein level were not significant among groups. Active caspase- 3-immunoreactive cells were found in the hippocampus. However, the number was only a few and not significantly affected by FA exposure and OVA immunization. NMDA receptor type 2A and 2B expression levels of FA-exposed mice were sustained at comparative levels with those for the control mice with or without OVA immunization. Conclusions: These results indicate that changes in the Bcl-2/Bax expression ratio, which occurs with low-level FA exposure and immunization and may follow enhancement of nerve growth factor production, exerts a protective effect against cell death by apoptosis.

Age-related change and its sex differences in histoarchitecture of the hypothalamic suprachiasmatic nucleus of F344/N rats

The present study examined the effects of aging and sex differences on the suprachiasmatic nucleus (SCN) of F344/N rats. In juveniles (1.6-1.9 months of age), adults (11.7-16.3 months of age), and old (29.2-34 months of age) rats, the volume, size of neuronal nucleus and neuronal cell number of the SCN were determined with cresyl fast violet-stained sections. In addition, immunohistochemical analysis was performed for glial fibrillary acidic protein (GFAP). There was no significant effect of aging and sex differences on the SCN volume. The number of neurons in the SCN gradually decreased from juvenile to old age in females. However, in males, the number was significantly decreased in adult and old age rats. The size of neuronal nuclei in the SCN was significantly decreased by increasing age in both sexes, except for the ventrolateral part of the SCN of males. In the dorsomedial part of the SCN of females, the density of GFAP-immunoreactive components was significantly higher in adult age rats than in rats of other ages. However, there was no significant increase in the density of the SCN in adult males. These results suggest that morphological changes in neuronal and astroglial cells occur in the SCN with aging in a sex-specific manner.

Effects of low-level formaldehyde exposure on synaptic plasticity-related gene expression in the hippocampus of immunized mice.

We examined the effects of inhalative exposure to formaldehyde (FA, 400 ppb) on N-methyl-D-aspartate (NMDA) receptor subunits (NR2A and NR2B), dopamine receptor subtypes (D1 and D2), cyclic AMP responsive element-binding protein (CREB)-1, CREB-2, FosB/DeltaFosB, and transient receptor potential vanilloid receptor (TRPV1) in the hippocampus of ovalbumin-immunized mice using quantitative real-time PCR. Western blot analyses for pCREB were performed. The mRNA levels of NR2A, D1 and D2 receptors, and CREB-1 were significantly increased by FA, but NR2B, CREB-2, FosB/DeltaFosB, and TRPV1 mRNA levels remained unchanged. Treatment with MK-801 normalized the mRNA levels induced by FA. There was no significant effect of FA exposure and MK-801 treatment on the protein level of pCREB. These results indicate that FA exposure selectively up-regulates hippocampal gene expression in immunologically sensitized mice. The FA effects are presumably mediated by glutamatergic neurotransmission through NMDA receptors.

Up-regulation of neurotrophin-related gene expression in mouse hippocampus following low-level toluene exposure.

To investigate the role of strain differences in sensitivity to low-level toluene exposure on neurotrophins and their receptor levels in the mouse hippocampus, 8-week-old male C3H/HeN, BALB/c and C57BL/10 mice were exposed to 0, 5, 50, or 500 ppm toluene for 6h per day, 5 days per week for 6 weeks in an inhalation chamber. We examined the expressions of neurotrophin-related genes and receptors in the mouse hippocampus using real-time reverse transcription polymerase chain reaction (RT-PCR). The expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), tyrosine kinase (Trk) A, and TrkB mRNAs in the C3H/HeN mice hippocampus was significantly higher in the mice exposed to 500 ppm toluene. Among the three strains of mice, the C3H/HeN mice seemed to be sensitive to toluene exposure. To examine the combined effect of toluene exposure and allergic challenge, the C3H/HeN mice stimulated with ovalbumin were exposed to toluene. The allergy group of C3H/HeN mice showed significantly elevated level of NGF mRNA in the hippocampus following exposure to 50 ppm toluene. Then, we also examined the expression of transcription factor, dopamine markers and oxidative stress marker in the hippocampus of sensitive strain C3H/HeN mice and found that the expression of CREB1 mRNA was significantly increased at 50 ppm toluene. In immunohistochemical analysis, the density of the NGF-immunoreactive signal was significantly stronger in the hippocampal CA3 region of the C3H/HeN mice exposed to 500 ppm toluene in non-allergy group and 50 ppm in allergy group. Our results indicate that low-level toluene exposure may induce up-regulation of neurotrophin-related gene expression in the mouse hippocampus depending on the mouse strain and an allergic stimulation in sensitive strain may decrease the threshold for sensitivity at lower exposure level.

Increased Fos Immunoreactivity in Suprachiasmatic Nucleus before Luteinizing Hormone Surge in Estrogen-Treated Ovariectomized Female Rats

Background/Aims: The suprachiasmatic nucleus (SCN) is thought to control the timing of luteinizing hormone (LH) surges. The present study was designed to examine temporal patterns of Fos expression in the dorsomedial and ventrolateral parts of the SCN (SCNdm and SCNvl) of female rats during an LH surge. It also included examination of temporal changes in plasma LH levels and temporal changes in Fos levels in the anteroventral periventricular nucleus (AVPV) and gonadotropin-releasing hormone (GnRH) neurons. Methods: Ovariectomized rats injected with 20 µg estradiol benzoate (EB) or vehicle were sacrificed at various times from Zeitgeber time (ZT) 8:00 to 16:30 h (ZT8–16.5; ZT0 = lights on; ZT12 = lights off) on the 2nd day after the injection. Immunohistochemical analyses for Fos and GnRH and enzyme-linked immunosorbent assays for LH were then performed. Results: In both the SCNdm and SCNvl of EB rats, the number of Fos-immunoreactive cells significantly increased between ZT9.5–10.5 and ZT11–12. On the other hand, in EB rats there were significant peaks of LH levels and Fos levels in GnRH neurons and the AVPV between ZT11–12 and ZT13–14. There was no significant difference in the number of Fos-immunoreactive cells between EB and control rats in either the SCNdm or SCNvl at ZT9.5–10.5, or in the SCNdm at ZT11–12, whereas the SCNvl of EB rats contained more Fos-immunoreactive cells than that of control rats at ZT11–12. Conclusion: These results suggest that in female rats during an LH surge, a peak in the Fos level in the SCN precedes peaks in Fos levels in the AVPV and GnRH neurons.

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. J. Comp. Neurol. 475:177–187, 2004.

Activation of cholecystokinin neurons in the dorsal pallium of the telencephalon is indispensable for the acquisition of chick imprinting behavior.

Chick imprinting behavior is a good model for the study of learning and memory. Imprinting object is recognized and processed in the visual wulst, and the memory is stored in the intermediate medial mesopallium in the dorsal pallium of the telencephalon. We identified chicken cholecystokinin (CCK)‐expressing cells localized in these area. The number of CCK mRNA‐positive cells increased in chicks underwent imprinting training, and these cells expressed nuclear Fos immunoreactivity at high frequency in these regions. Most of these CCK‐positive cells were glutamatergic and negative for parvalbumin immunoreactivity. Semi‐quantitative PCR analysis revealed that the CCK mRNA levels were significantly increased in the trained chicks compared with untrained chicks. In contrast, the increase in CCK‐ and c‐Fos‐double‐positive cells associated with the training was not observed after closure of the critical period. These results indicate that CCK cells in the dorsal pallium are activated acutely by visual training that can elicit imprinting. In addition, the CCK receptor antagonist significantly suppressed the acquisition of memory. These results suggest that the activation of CCK cells in the visual wulst as well as in the intermediate medial mesopallium by visual stimuli is indispensable for the acquisition of visual imprinting.

Effects of maternal toluene exposure on testosterone levels in fetal rats

The goal of our study was to determine if toluene affected the synthesis and secretion of testosterone in fetal rats. Dams were exposed to atmospheres that contained 0.09 ppm, 0.9 ppm or 9 ppm of toluene for 90 min/day from gestational days (GDs) 14.5 to 18.5 via nasal inhalation. Fetal plasma testosterone concentrations determined by enzyme immunoassay were significantly reduced on GD 18.5 after exposure to 0.9 and 9 ppm, but not to 0.09 ppm, of toluene in male, but not in female, fetuses. We measured, using real-time PCR methods, mRNA levels in fetal testes for several steroidogenic enzymes involved in testosterone synthesis and insulin-like 3 (Insl3), a maker of Leydig cell differentiation. The mRNA levels of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) were significantly reduced after exposure to 0.9-ppm toluene. However, the mRNA levels of cytochrome P450 cholesterol side-chain cleavage, cytochrome P450 17alpha-hydroxylase/c17-20 lyase, 17beta-hydroxysteroid dehydrogenase, and Insl3 were not significantly altered by exposure to 0.9-ppm toluene. In addition, immunohistochemical analysis showed reduced 3beta-HSD-immunoreactive areas in the interstitial region of fetal testes after exposure to 0.9 and 9 ppm, but not 0.09 ppm, toluene. These findings indicate that toluene reduced the synthesis and secretion of testosterone in fetal testes from rats possibly as a consequence of reduced 3beta-HSD expression.

Distributions of two chicken bombesin receptors, bombesin receptor subtype-3.5 (chBRS-3.5) and gastrin-releasing peptide receptor (chGRP-R) mRNAS in the chicken telencephalon

Bombesin (BN)-like peptide receptors are known to be essential to the regulation of not only homeostasis, including feeding behavior, but also of emotional systems in mammal. Recently, two novel BN receptors, chicken BN-like peptide receptor subtype-3.5 (chBRS-3.5) and gastrin-releasing peptide receptor (chGRP-R), have been identified. Here, we report the localizations of these receptors mRNAs in the chick brain through development using in situ hybridization. First, chBRS-3.5 mRNA signals were found in the dorsal ventricular ridge at embryonic day (ED) 9. Strong signals were observed in the hyperpallium accessorium, nidopallium and nucleus basorostralis pallii, and moderate signals were found in the hippocampus, cortex piriformis, hyperpallium intercalatum, area temporo-parieto-occipitalis, nucleus striae terminalis lateralis, nucleus olfactorius anterior and organum septi lateralis at ED16. This wide expression in the pallium persisted during posthatch periods. Abundant expressions in the hyperpallium, nidopallium, considered to be similar to the mammalian cortex, as well as in the hippocampus, indicate participation of these molecules in the processing of sensory information, motor function, learning and memory. Telencephalic areas devoid of chBRS-3.5 signals were the entopallium, arcopallium anterius, globus pallidus, nucleus intrapeduncularis, tuberculum olfactorius, nucleus septalis lateralis, hypothalamic and thalamic areas. In contrast to chBRS-3.5, chGRP-R mRNA signals were found in the pallidum at ED5 and 9. At ED16, chGRP-R mRNA signals were localized in the medial striatum and hypothalamus. GRP-R expression in the hypothalamic region was phylogenically conserved. Thus, chBRS-3.5 mRNA signals were distributed in a broader region and were more intense than chGRP-R mRNA. Taken together, chGRP-R and chBRS-3.5 mRNA occurred in similar regions of mammals that express GRP-R. BN/GRP-immunoreactive neurons and varicosities were found mainly in the pallium, especially in the hyperpallium accessorium and nidopallium, and this distribution coincided with that of chBRS-3.5 mRNA. This result suggests that the endogenous ligands for chBRS-3.5 were likely BN-like peptides produced in the pallium.