Cerebral Vasospasm After Burr Hole Evacuation of Chronic Subdural Hematoma.

Cerebral vasospasm is a frequent complication of subarachnoid hemorrhage. We report a case of chronic subdural hematoma complicated by cerebral vasospasm after burr hole evacuation. A 74-year-old woman underwent burr hole evacuation of a chronic subdural hematoma. She developed left hemiparesis and disturbance of consciousness on postoperative day 3. Magnetic resonance imaging showed a right parietal infarct and decreased cerebral blood flow signal in the right middle cerebral artery territory. Digital subtraction angiography showed multiple segmental narrowings of the right middle cerebral artery. Her neurological symptoms recovered with conservative treatment. Follow-up angiography showed improvement in the arterial narrowing, which finally led to a diagnosis of cerebral vasospasm. Cerebral vasospasm can occur after burr hole evacuation of chronic subdural hematoma. Magnetic resonance angiography is useful for determining the cause of postoperative neurological worsening in chronic subdural hematoma patients.

Anterior inferior cerebellar artery aneurysm with proximal parent artery stenosis: A case report.

Hemodynamic factors are associated with the progression of cerebral aneurysms. We report a 78-year-old woman with an anterior inferior cerebellar artery aneurysm and proximal stenosis of the anterior inferior cerebellar and basilar arteries. The aneurysm exhibited growth on annual follow-up imaging. Aneurysmal rupture occurred 4 years after diagnosis. Coil embolization resulted in aneurysm occlusion with parent artery preservation. Aneurysms adjacent to a more proximal region of severe stenosis in the parent vessel should be considered at high risk for growth or rupture. Such aneurysms require careful monitoring. Particular attention should be paid to posterior circulation aneurysms that occur at anatomically vulnerable sites.

Sex and interspecies differences in ESR2-expressing cell distributions in mouse and rat brains.

BACKGROUND: ESR2, a nuclear estrogen receptor also known as estrogen receptor ß, is expressed in the brain and contributes to the actions of estrogen in various physiological phenomena. However, its expression profiles in the brain have long been debated because of difficulties in detecting ESR2-expressing cells. In the present study, we aimed to determine the distribution of ESR2 in rodent brains, as well as its sex and interspecies differences, using immunohistochemical detection with a well-validated anti-ESR2 antibody (PPZ0506). METHODS: To determine the expression profiles of ESR2 protein in rodent brains, whole brain sections from mice and rats of both sexes were subjected to immunostaining for ESR2. In addition, to evaluate the effects of circulating estrogen on ESR2 expression profiles, ovariectomized female mice and rats were treated with low or high doses of estrogen, and the resulting numbers of ESR2-immunopositive cells were analyzed. Welch's t-test was used for comparisons between two groups for sex differences, and one-way analysis of variance followed by the Tukey-Kramer test were used for comparisons among multiple groups with different estrogen treatments. RESULTS: ESR2-immunopositive cells were observed in several subregions of mouse and rat brains, including the preoptic area, extended amygdala, hypothalamus, mesencephalon, and cerebral cortex. Their distribution profiles exhibited sex and interspecies differences. In addition, low-dose estrogen treatment in ovariectomized female mice and rats tended to increase the numbers of ESR2-immunopositive cells, whereas high-dose estrogen treatment tended to decrease these numbers. CONCLUSIONS: Immunohistochemistry using the well-validated PPZ0506 antibody revealed a more localized expression of ESR2 protein in rodent brains than has previously been reported. Furthermore, there were marked sex and interspecies differences in its distribution. Our histological analyses also revealed estrogen-dependent changes in ESR2 expression levels in female brains. These findings will be helpful for understanding the ESR2-mediated actions of estrogen in the brain.

Although the brain is a major target organ of estrogens, the distribution of estrogen receptors in the brain is not fully understood. ESR2, also known as estrogen receptor ß, is an estrogen receptor subtype; its localization in the brain has long been controversial because it has traditionally been difficult to detect. In the present study, we analyzed the expression sites of ESR2 in mouse and rat brains using immunohistochemistry with a well-validated antibody, PPZ0506. The immunohistochemical analysis revealed a more localized expression of ESR2 protein in brain subregions than has previously been reported. Additionally, there were clear sex and interspecies differences in the distribution of this protein. We also observed changes in ESR2 expression in the female brain in response to circulating estrogen levels. Our results, which show the precise expression profiles of ESR2 protein in rodent brains, will be helpful for understanding the ESR2-mediated actions of estrogen.

Two-Step Actions of Testicular Androgens in the Organization of a Male-Specific Neural Pathway from the Medial Preoptic Area to the Ventral Tegmental Area for Modulating Sexually Motivated Behavior.

The medial preoptic area (MPOA) is a sexually dimorphic region of the brain that regulates social behaviors. The sexually dimorphic nucleus (SDN) of the MPOA has been studied to understand sexual dimorphism, although the anatomy and physiology of the SDN is not fully understood. Here, we characterized SDN neurons that contribute to sexual dimorphism and investigated the mechanisms underlying the emergence of such neurons and their roles in social behaviors. A target-specific neuroanatomical study using transgenic mice expressing Cre recombinase under the control of Calb1, a gene expressed abundantly in the SDN, revealed that SDN neurons are divided into two subpopulations, GABA neurons projecting to the ventral tegmental area (VTA), where they link to the dopamine system (CalbVTA neurons), and GABA neurons that extend axons in the MPOA or project to neighboring regions (CalbnonVTA neurons). CalbVTA neurons were abundant in males, but were scarce or absent in females. There was no difference in the number of CalbnonVTA neurons between sexes. Additionally, we found that emergence of CalbVTA neurons requires two testicular androgen actions that occur first in the postnatal period and second in the peripubertal period. Chemogenetic analyses of CalbVTA neurons indicated a role in modulating sexual motivation in males. Knockdown of Calb1 in the MPOA reduced the intromission required for males to complete copulation. These findings provide strong evidence that a male-specific neural pathway from the MPOA to the VTA is organized by the two-step actions of testicular androgens for the modulation of sexually motivated behavior.SIGNIFICANCE STATEMENT The MPOA is a sexually dimorphic region of the brain that regulates social behaviors, although its sexual dimorphism is not fully understood. Here, we describe a population of MPOA neurons that contribute to the sexual dimorphism. These neurons only exist in masculinized brains, and they project their axons to the ventral tegmental area, where they link to the dopamine system. Emergence of such neurons requires two testicular androgen actions that occur first in the postnatal period and second in the peripubertal period. These MPOA neurons endow masculinized brains with a neural pathway from the MPOA to the ventral tegmental area and modulate sexually motivated behavior in males.

Mechanical thrombectomy for middle cerebral artery occlusion caused by intracranial internal carotid artery stenosis: A case report.

Tandem internal carotid artery (ICA)/middle cerebral artery (MCA) occlusions are occasionally observed in patients with acute ischemic stroke. Most of them are caused by lesions at the origin of the ICA. In cases of intracranial ICA stenosis, the formation of a large thrombus causing MCA occlusion is extremely rare. Herein We report a case of acute MCA occlusion caused by intracranial ICA stenosis. A 62-year-old female presented with aphasia, right-side weakness, and a National Institute of Health Stroke Scale (NIHSS) score of 5. Magnetic resonance imaging (MRI) showed early ischemic infarction at the precentral gyrus. Left ICA and M1 occlusion were suspected on magnetic resonance angiography. However, the patient had complained of right-side numbness 6 days before the onset. Hence the stroke was assumed to have progressed slowly, and acute occlusion of the left ICA was eliminated as a suspected diagnosis. After admission, the symptoms worsened. MRI showed enlargement of the cerebral infarction. Computed tomography angiography showed complete occlusion of the left M1 and recanalization of the left ICA with severe stenosis of the petrous portion. The etiology of the MCA occlusion was determined to be atherothromboembolism. Percutaneous transluminal angioplasty (PTA) was performed for ICA stenosis, followed by mechanical thrombectomy (MT) for the MCA occlusion. Recanalization of the MCA was achieved. After Seven days, the NIHSS score reduced from a pre-MT assessment of 17-2. PTA followed by MT was safe and effective for treating MCA occlusion caused by intracranial ICA stenosis.

Bilateral vertebral artery dissection extending to the left posterior cerebral artery: A case report.

Intracranial artery dissection accounts for a small percentage (1%-2%) of all ischemic strokes. Vertebral artery dissection sometimes extends to the basilar artery but very rarely to the posterior cerebral artery. We report a case of bilateral vertebral artery dissection extending to the left posterior cerebral artery with the characteristic distribution of intramural hematoma. A 51-year-old woman presented with right hemiparesis and dysarthria 3 days after sudden neck pain. Magnetic resonance imaging on admission revealed infarcts in the left thalamus and temporo-occipital lobe and findings suggestive of bilateral vertebral artery dissection. No infarct was detected in the brainstem. The patient was treated conservatively. Initially, we suspected that infarction in the left posterior cerebral artery territory had been caused by artery-to-artery embolism from the dissected vertebral arteries. However, T1-weighted imaging on day 15 of admission revealed intramural hematoma extending from the left vertebral artery to the left posterior cerebral artery. Therefore, we diagnosed bilateral vertebral artery dissection extending to the basilar artery and the left posterior cerebral artery. The patient's symptoms subsequently improved with conservative treatment, and she was discharged with a modified Rankin Scale score of 1 on day 62 of admission. In this case, intramural hematoma of the basilar artery was found in the anterior vessel wall. Brainstem infarction is less likely when intramural hematoma is located in the anterior vessel wall of the basilar artery in vertebrobasilar artery dissection. T1-weighted imaging is useful for the diagnosis of this rare condition and can predict potentially impaired branches and possible symptoms.

Hypothalamic Overexpression of Neurosecretory Protein GL Leads to Obesity in Male C57BL/6J Mice.

INTRODUCTION: The mechanisms underlying obesity are not fully understood, necessitating the creation of novel animal models for the investigation of metabolic disorders. We have previously found that neurosecretory protein GL (NPGL), a newly identified hypothalamic neuropeptide, is involved in feeding behavior and fat accumulation in rats. However, the impact of NPGL on obesity remains unclear in any animal model. The present investigation sought to elucidate whether NPGL causes obesity in the obesity-prone mouse strain C57BL/6J. METHODS: We overexpressed the NPGL-precursor gene (Npgl) in the hypothalamus using adeno-associated virus in male C57BL/6J mice fed normal chow (NC) or a high-calorie diet (HCD). After 9 weeks of Npgl overexpression, we measured adipose tissues, muscle, and several organ masses in addition to food intake and body mass. To assess the effects of Npgl overexpression on peripheral tissues, we analyzed mRNA expression of lipid metabolism-related genes by quantitative RT-PCR. Whole body energy consumption was assessed using an O2/CO2 metabolism measurement before an apparent increase in body mass. RESULTS: Npgl overexpression increased food intake, body mass, adipose tissues and liver masses, and food efficiency under both NC and HCD, resulting in obesity observable within 8 weeks. Furthermore, we observed fat accumulation in adipose tissues and liver. Additionally, mRNA expression of lipid metabolism-related factors was increased in white adipose tissue and the liver after Npgl overexpression. Npgl overexpression inhibited energy expenditure during a dark period. CONCLUSION: Taken together, the present study suggests that NPGL can act as an obesogenic factor that acts within a short period of time in mice. As a result, this Npgl overexpression-induced obesity can be widely applied to study the etiology of obesity from genes to behavior.

Sexually dimorphic expression of sexual differentiation genes in the internal genital organs of Japanese quail embryos.

Japanese quail (Coturnix japonica) is an avian model used to evaluate the reproductive and developmental toxicity of chemicals. The National Institute for Environmental Studies (NIES) of Japan established a strain of Japanese quail, NIES-L, which may be a better model because of its highly inbred characteristics. To understand sexual differentiation of the reproductive organs and the value of using NIES-L quails for avian toxicity assessment, we profiled estradiol and androgen plasma levels by enzyme-linked immunosorbent assay; the mRNA levels of estrogen receptor-α (ERα), ERß, and androgen receptor (AR) in the gonads, Müllerian ducts, Wolffian ducts; and the mRNA levels of steroidogenic enzymes, cholesterol side chain cleavage enzyme (P450scc), 17α-hydroxylase/C17-20 lyase (P45017α, lyase), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and aromatase (P450arom), anti-Müllerian hormone (AMH), and AMH receptor type 2 (AMHR2) in the gonads of NIES-L Japanese quails on embryonic days 9, 12, and 15 using a real-time quantitative PCR method. The plasma estradiol concentration was higher in females than males on these embryonic days, but no sex difference was found in the plasma androgens. The mRNA levels of all examined steroidogenic enzymes were significantly higher in female than male embryos. In particular, the P450arom mRNA levels showed a striking sex difference: P450arom was expressed in female but not male gonads. In contrast, the AMH and AMHR2 mRNA levels in the gonads were higher in males than females. The ERα, ERß, and AR mRNA levels increased in the left female gonad and peaked on embryonic day 15, but not in the left and right male gonads; therefore, there was a female-biased sex difference. The ERα, ERß, and AR mRNA levels in the left Müllerian duct, but not in the right Müllerian duct, of females increased and peaked on embryonic day 15, which resulted in asymmetric mRNA levels. The Wolffian ducts expressed ERα, ERß, and AR in both sexes, and no sex difference or asymmetry of mRNA levels was found. The information obtained from this study helps elucidate the molecular endocrinological basis of sexual dimorphism formation of reproductive organs and clarify the value of NIES-L quails for toxicity assessment.

Neuronal activation of the sexually dimorphic nucleus of the preoptic area in female and male rats during copulation and its sex differences.

The medial preoptic area, which plays an essential role in the control of sexual behavior in rats, contains a sexually dimorphic nucleus that consists of neurons expressing calbindin-D28 K (Calb) that is referred to as the CALB-SDN. The CALB-SDN is larger and contains more Calb neurons in males than in females. The physiological functions of the CALB-SDN are not fully understood; however, CALB-SDN neurons are activated during sexual behavior in males, suggesting that the male CALB-SDN is involved in regulation of sexual behavior. However, no information exists about the physiological functions of the female CALB-SDN. In the present study, we performed an immunohistochemical analysis of c-Fos, a neuronal activity marker, in the CALB-SDN of female and male rats that had copulated with conspecifics of the opposite sex to determine whether neurons of the female CALB-SDN are activated during copulation and whether the neuronal activity of the CALB-SDN differs between sexes. The numbers of c-Fos-immunoreactive cells with or without Calb-immunoreactivity (c-Fos+/Calb+ and c-Fos+/Calb- cells) were greater in the CALB-SDN of rats that had copulated than in rats that had not copulated in each sex. Although the number of Calb+ cells in the CALB-SDN was smaller in females than in males, the increase in the number of c-Fos+/Calb+ cells in the female CALB-SDN with copulation was comparable to that in the male CALB-SDN with copulation. The increase in the number of c-Fos+/Calb- cells in the CALB-SDN with copulation was more prominent in males than in females. These results suggest that CALB-SDN neurons are activated during copulation in both sexes. The patterns of neuronal activation in the CALB-SDN during copulation may differ between sexes.

Target Embolization of Dilated Post-PICA Segment for Ruptured PICA-Involved Type Vertebral Artery Dissecting Aneurysm.

Objective: In parent artery occlusion (PAO) for ruptured vertebral artery dissecting aneurysms (RVADA), target embolization using coils in a short segment to occlude only the vasodilated area containing the rupture point is selected as a first-choice procedure at our institute. We focused on RVADA involving the posterior inferior cerebellar artery (PICA) and evaluated the treatment results. Methods: This study consisted of eight cases with RVADA involving the PICA which were treated between October 2007 and January 2020. Based on radiological findings such as the bleb, the rupture points were located at the affected vertebral artery (VA) distal to PICA in all cases. Target embolization, by which only coiling at the dilated segment distal to the VA was performed. We aimed to preserve blood flow to the PICA. The incidence and extent of medullary infarctions, and neurological outcome were retrospectively assessed. Results: Regarding the diameter of bilateral VA, there were no differences in six cases while the affected VA with RVADA were larger in the remaining two cases. PICA was preserved in all cases but one in which occlusion of complementary PICA was observed. Postoperative medullary infarction was not noted. There was no rebleeding during the follow-up period. However, recanalization of the VA was observed in four cases and additional coil embolization was performed. All patients were discharged with a good outcome (modified Rankin Scale [mRS] 0; seven patients, mRS 2; one patient). Conclusion: Target embolization preserving the PICA in PICA-involved type RVADA was considered to be an effective treatment method for cases whose rupture point was located in the VA distal to PICA orifice.

Sexually Dimorphic Formation of the Preoptic Area and the Bed Nucleus of the Stria Terminalis by Neuroestrogens.

Testicular androgens during the perinatal period play an important role in the sexual differentiation of the brain of rodents. Testicular androgens transported into the brain act via androgen receptors or are the substrate of aromatase, which synthesizes neuroestrogens that act via estrogen receptors. The latter that occurs in the perinatal period significantly contributes to the sexual differentiation of the brain. The preoptic area (POA) and the bed nucleus of the stria terminalis (BNST) are sexually dimorphic brain regions that are involved in the regulation of sex-specific social behaviors and the reproductive neuroendocrine system. Here, we discuss how neuroestrogens of testicular origin act in the perinatal period to organize the sexually dimorphic structures of the POA and BNST. Accumulating data from rodent studies suggest that neuroestrogens induce the sex differences in glial and immune cells, which play an important role in the sexually dimorphic formation of the dendritic synapse patterning in the POA, and induce the sex differences in the cell number of specific neuronal cell groups in the POA and BNST, which may be established by controlling the number of cells dying by apoptosis or the phenotypic organization of living cells. Testicular androgens in the peripubertal period also contribute to the sexual differentiation of the POA and BNST, and thus their aromatization to estrogens may be unnecessary. Additionally, we discuss the notion that testicular androgens that do not aromatize to estrogens can also induce significant effects on the sexually dimorphic formation of the POA and BNST.

Detection and Characterization of Estrogen Receptor Beta Expression in the Brain with Newly Developed Transgenic Mice.

Two types of nuclear estrogen receptors, ERα and ERß, have been shown to be differentially involved in the regulation of various types of behaviors. Due to a lack of tools for identifying ERß expression, detailed anatomical distribution and neurochemical characteristics of ERß expressing cells and cellular co-expression with ERα remain unclear. We have generated transgenic mice ERß-RFPtg, in which RFP was inserted downstream of ERß BAC promotor. We verified RFP signals as ERß by confirming: (1) high ERß mRNA levels in RFP-expressing cells collected by fluorescence-activated cell sorting; and (2) co-localization of ERß mRNA and RFP proteins in the paraventricular nucleus (PVN). Strong ERß-RFP signals were found in the PVN, medial preoptic area (MPOA), bed nucleus of the stria terminalis, medial amygdala (MeA), and dorsal raphe nucleus (DRN). In the MPOA and MeA, three types of cell populations were identified; those expressing both ERα and ERß, and those expressing exclusively either ERα or ERß. The majority of PVN and DRN cells expressed only ERß-RFP. Further, ERß-RFP positive cells co-expressed oxytocin in the PVN, and tryptophan hydroxylase 2 and progesterone receptors in the DRN. In the MeA, some ERß-RFP positive cells co-expressed oxytocin receptors. These findings collectively suggest that ERß-RFPtg mice can be a powerful tool for future studies on ERß function in the estrogenic regulation of social behaviors.

Actions of Peripubertal Gonadal Steroids in the Formation of Sexually Dimorphic Brain Regions in Mice.

The calbindin-sexually dimorphic nucleus (CALB-SDN) and calbindin-principal nucleus of the bed nucleus of the stria terminalis (CALB-BNSTp) show male-biased sex differences in calbindin neuron number. The ventral part of the BNSTp (BNSTpv) exhibits female-biased sex differences in noncalbindin neuron number. We previously reported that prepubertal gonadectomy disrupts the masculinization of the CALB-SDN and CALB-BNSTp and the feminization of the BNSTpv. This study aimed to determine the action mechanisms of testicular androgens on the masculinization of the CALB-SDN and CALB-BNSTp and whether ovarian estrogens are the hormones that have significant actions in the feminization of the BNSTpv. We performed immunohistochemical analyses of calbindin and NeuN, a neuron marker, in male mice orchidectomized on postnatal day 20 (PD20) and treated with cholesterol, testosterone, estradiol, or dihydrotestosterone during PD20-70, female mice ovariectomized on PD20 and treated with cholesterol or estradiol during PD20-70, and PD70 mice gonadectomized on PD56. Calbindin neurons number in the CALB-SDN and CALB-BNSTp in males treated with testosterone or dihydrotestosterone, but not estradiol, was significantly larger than that in cholesterol-treated males. Noncalbindin neuron number in the BNSTpv in estradiol-treated females was significantly larger than that in cholesterol-treated females. Gonadectomy on PD56 had no significant effect on neuron numbers. Additionally, an immunohistochemical analysis revealed the expression of androgen receptors in the CALB-SDN and CALB-BNSTp of PD30 males and estrogen receptors-α in the BNSTpv of PD30 females. These results suggest that peripubertal testicular androgens act to masculinize the CALB-SDN and CALB-BNSTp without aromatization, and peripubertal ovarian estrogens act to feminize the BNSTpv.

Estrogenic action by tris(2,6-dimethylphenyl) phosphate impairs the development of female reproductive functions.

Developmental exposure to environmental chemicals with estrogen-like activity is suspected to permanently impair women's health. In this study, a mouse model was used to evaluate whether tris(2,6-dimethylphenyl) phosphate (TDMPP), a chemical with a putative estrogen-like action, impairs sexual differentiation of the brain. Either TDMPP and 17ß-estradiol (E2) as positive controls or sesame oil as a negative control were administered subcutaneously to dams from gestational day (GD) 14 to parturition, and to pups from postnatal day (PND) 0 to 9. Precocious puberty, irregular estrous cycles, and a lowered lordosis response were found in the TDMPP- and E2-treated groups. A certain amount of TDMPP and its metabolites in the perinatal brain and the masculinization of sexual dimorphic nuclei in the hypothalamus of female mice after treatment were also detected. The experimental evidence demonstrates that TDMPP directly enters the fetal and neonatal brain, thereby inducing changes of sex-related brain structures and impairing female reproductive functions.

Region-specific effects of copulation on dendritic spine morphology and gene expression related to spinogenesis in the medial preoptic nucleus of male rats.

The medial preoptic nucleus (MPN) plays an essential role in the control of male sexual behavior. In rats, the central part of the MPN (MPNc) contains a sexually dimorphic nucleus exhibiting male-biased morphological sex differences. Although it has been suggested that the MPNc of male rats functions to induce sexual arousal, the mechanisms by which male rats are sexually aroused to successfully achieve copulation are poorly understood. We recently showed that increased neuronal activity in the MPNc of male rats during copulation is higher at their first copulation compared with later copulations, indicating that a plastic change in excitatory synaptic transmission occurs with copulatory experience. In this study, we tested the hypothesis that changes to dendritic spines at structural and molecular levels occur following copulatory experience. First, we examined the effects of at least two copulations on the morphology of dendrites and spines in the MPNc and in the lateral and medial parts of the MPN (MPNlm) of male rats. In the MPNc, the total number of dendrites and their branches, and the surface area of dendrites were not significantly affected by copulation. However, the copulatory experience, specifically experience of ejaculation, significantly reduced the density of mushroom spines but not of filopodia, thin or stubby spines in the MPNc. In the MPNlm, the copulatory experience, specifically experience of ejaculation, significantly increased the surface area of dendrites, although there was no significant effect of copulation on spine density. Next, we measured the mRNA levels of genes encoding actin-binding proteins related to spinogenesis after male rats had copulated for their first and second times. Copulatory stimuli, especially stimuli from ejaculation, significantly reduced the mRNA levels of drebrin A and spinophilin in the MPNc but not in the MPNlm. These results indicate that copulatory experiences, especially experience of ejaculation, reduce spine density in the MPNc of male rats, which may result, in part, from downregulation of genes encoding actin-binding proteins.

Correction to: Female-biased sexual dimorphism of corticotropin-releasing factor neurons in the bed nucleus of the stria terminalis.

Following publication of the original article [1], we noticed a number of errors.

Female-biased sexual dimorphism of corticotropin-releasing factor neurons in the bed nucleus of the stria terminalis.

BACKGROUND: The bed nucleus of the stria terminalis (BNST) contains the highest density of corticotropin-releasing factor (CRF)-producing neurons in the brain. CRF-immunoreactive neurons show a female-biased sexual dimorphism in the dorsolateral BNST in the rat. Since CRF neurons cannot be immunostained clearly with available CRF antibodies in the mouse, we used a mouse line, in which modified yellow fluorescent protein (Venus) was inserted to the CRF gene, and the Neo cassette was removed, to examine the morphological characteristics of CRF neurons in the dorsolateral BNST. Developmental changes of CRF neurons were examined from postnatal stages to adulthood. Gonadectomy (GDX) was carried out in adult male and female mice to examine the effects of sex steroids on the number of CRF neurons in the dorsolateral BNST. METHODS: The number of Venus-expressing neurons, stained by immunofluorescence, was compared between male and female mice over the course of development. GDX was carried out in adult mice. Immunohistochemistry, in combination with Nissl staining, was carried out, and the effects of sex or gonadal steroids were examined by estimating the number of Venus-expressing neurons, as well as the total number of neurons or glial cells, in each BNST subnucleus, using a stereological method. RESULTS: Most Venus-expressing neurons co-expressed Crf mRNA in the dorsolateral BNST. They constitute a group of neurons without calbindin immunoreactivity, which makes a contrast to the principal nucleus of the BNST that is characterized by calbindin immunostaining. In the dorsolateral BNST, the number of Venus-expressing neurons increased across developmental stages until adulthood. Sexual difference in the number of Venus-expressing neurons was not evident by postnatal day 5. In adulthood, however, there was a significant female predominance in the number of Venus expressing neurons in two subnuclei of the dorsolateral BNST, i.e., the oval nucleus of the BNST (ovBNST) and the anterolateral BNST (alBNST). The number of Venus-expressing neurons was smaller significantly in ovariectomized females compared with proestrous females in either ovBNST or alBNST, and greater significantly in orchiectomized males compared with gonadally intact males in ovBNST. The total number of neurons was also greater significantly in females than in males in ovBNST and alBNST, but it was not affected by GDX. CONCLUSION: Venus-expressing CRF neurons showed female-biased sexual dimorphism in ovBNST and alBNST of the mouse. Expression of Venus in these subnuclei was controlled by gonadal steroids.

Temporal Expression Patterns of Genes Related to Sex Steroid Action in Sexually Dimorphic Nuclei During Puberty.

Sex steroids play a major role in sexually dimorphic brain development during not only the perinatal period but also the pubertal period. We previously showed that, in male mice, the estrogen receptor-α (Esr1) and aromatase (Cyp19a1) genes are essential to the sexually dimorphic formation of the anteroventral periventricular nucleus (AVPV) and the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), but the estrogen receptor-ß (Esr2) gene is not necessary. We also showed that the androgen receptor (Ar) gene is essential to the sexually dimorphic formation of the BNSTp. These genes are expressed in the AVPV and BNSTp of perinatal mice. However, it remains unknown whether these genes are expressed in the AVPV and BNSTp during puberty, and whether the expression, if any, differs by sex, age, and brain region. Here, we dissected the AVPV and BNSTp from Nissl-stained brain sections of male and female mice on postnatal day (PD) 20 (prepuberty), PD30 (puberty onset in females), PD40 (puberty onset in males), and PD60 (young adult) using a laser microdissection system. We then examined the mRNA levels of Esr1, Esr2, Cyp19a1, and Ar in these brain regions. In the AVPV, Esr1 mRNA levels were greater in females than males during PD20-60. Esr2 and Ar mRNA expressions did not differ between sexes. Ar mRNA levels were higher at PD30 than PD20. Cyp19a1 mRNA was not detected in the AVPV at PD20-60. In the BNSTp, Esr1 and Esr2 mRNA levels were higher in females than in males during PD20-60, although the mRNA levels of Cyp19a1 and Ar did not differ between sexes. Additionally, we revealed that orchiectomy at PD20 induced a failure of normal formation of the male BNSTp and testosterone replacement in the prepubertal period rescued the effect of orchiectomy at PD20. Taken together, it is suggested that pubertal testosterone transported to the AVPV is not converted to estradiol there and does not act via ESR1 and ESR2. By contrast, the formation of the male BNSTp may be affected by testicular testosterone during puberty via AR and/or via ESR1 after conversion to estradiol by CYP19A1.