Nitric oxidergic cells related to ejaculation in gerbil forebrain contain androgen receptor and respond to testosterone

Two clusters of forebrain neurons-one in the posterodorsal preoptic nucleus (PdPN) and one in the lateral part of the posterodorsal medial amygdala (MeApd)-are activated at ejaculation in male rats and gerbils as seen with Fos immunocytochemistry. To understand the functions of these cells and how they respond synchronously, it may be useful to identify their neurotransmitters. Nitric oxide (NO) was of interest because its levels in the preoptic area affect ejaculation, and it could synchronize clustered neurons through paracrine/volume transmission. Thus, we determined whether the ejaculation-related cells produce NO by assessing Fos co-localization with NO synthase (NOS) in recently mated male gerbils. We also studied NOS-Fos co-localization in the medial part of the medial preoptic nucleus (MPNm), where half of the neurons that express Fos after mating reflect ejaculation. We also quantified NOS co-localization with androgen receptor (AR) and NOS sensitivity to androgens at these sites. Without quantification, we extended these analyses throughout the hypothalamus and amygdala. Many mating-activated PdPN, lateral MeApd, and MPNm cells contained NOS (32-54%), and many NOS neurons at these sites expressed Fos (34-51%) or AR (25-69%). PdPN and MPNm NOS cells were sensitive to testosterone but not its androgenic metabolite dihydrotestosterone. The overall distribution of NOS and NOS-AR cells was similar to that in rats. These data suggest that NO may help to synchronize the activation of PdPN and lateral MeApd neurons at ejaculation and that NOS in PdPN and MPNm cells is regulated by testosterone acting via estradiol or without undergoing metabolism.     

Evidence for a ventral non-strial pathway from the amygdala to the bed nucleus of the stria terminalis in the male golden hamster

Male hamsters in which the stria terminalis (ST) had been interrupted either by electrolytic lesions or knife cuts, or normal control males, received iontophoretic injections of horseradish peroxidase in either the bed nucleus of the stria terminalis (BNST) or the medial preoptic-anterior hypothalamic area (MPOAH). Comparison of intact and ST-lesioned brains revealed the existence of a ventral non-strial pathway, from cells in the medial amygdaloid nucleus (M) to the preoptic portion of the BNST but not to the MPOAH. Since bilateral lesions of M completely eliminate male hamster mating behavior, but ST lesions do not, we suggest that the ventral pathway to the BNST may be an important route by which M influences male copulatory behavior.     

Afferent connections of the sexually dimorphic area of the hypothalamus of male and female gerbils

We studied neural inputs to the sexually dimorphic area (SDA) of the gerbil hypothalamus by injecting wheat-germ agglutinin-horseradish peroxidase into its medial or lateral components in males and females. To confirm the topography of SDA afferents, we injected Phaseolus vulgaris-leucoagglutinin into areas where retrograde labeling from the medial and lateral SDA differed. Both methods indicated that the medial SDA received stronger inputs from the medial part of the bed nucleus of the stria terminalis, the ventral part of the lateral septal nucleus, the medial amygdaloid nucleus, and the amygdalohippocampal area, than the lateral SDA does. In contrast, the rostrodorsal part of the lateral septum, the lateral part of the bed nucleus of the stria terminalis, the anterior and posterior hypothalamic areas, and the dorsomedial hypothalamic nucleus project more heavily to the lateral than to the medial SDA. In addition, retrograde labeling suggested that the ventral part of the premammillary nucleus projects more strongly to the medial than to the lateral SDA, whereas the infralimbic area of the cortex and the lateral preoptic area project more strongly to the lateral than to the medial SDA. The densities of cells in the bed nucleus of the stria terminalis and medial amygdaloid nucleus that could be retrogradely labeled from the medial SDA were greater in males than in females. This was not true of labeling in the arcuate nucleus or in the ventral part of the lateral septal nucleus. Since the medial SDA receives strong inputs from areas with many steroid-accumulating cells, it could respond to steroids directly and via these afferents. In contrast, hormonal effects on the lateral SDA are more likely to occur locally.     

Photoperiodic regulation of substance P immunoreactivity in the mating behavior pathway of the male golden hamster

Mating behavior in the male golden hamster is regulated by both gonadal steroids and photoperiod. Gonadal steroids may regulate mating behavior by actions on the medial nucleus of the amygdala, bed nucleus of the stria terminalis, and medial preoptic area. Neurons in these areas actively accumulate gonadal steroids and lesions of these nuclei disrupt mating behavior in male hamsters. Photoperiodic regulation of mating behavior is regulated, at least in part, by decreased responsiveness to gonadal steroids. Therefore, we sought to determine if the changes induced by changes in gonadal steroids would mimic those induced by changes in photoperiod. The number of substance P-containing neurons in these areas decrease following castration and are restored with testosterone treatment suggesting that this peptide may mediate steroidal regulation of male mating behavior. To determine the effect of photoperiod on substance P, peptide containing neurons were counted in (1) enucleates (n = 6), (2) enucleated castrates treated with testosterone (n = 6), (3) castrates treated with testosterone (n = 4), and (4) intact controls (n = 6). Bilateral enucleation caused a decrease in the number of substance P neurons in the medial nucleus, bed nucleus of the stria terminalis, and medial preoptic area (P < 0.05). Testosterone treatment prevented this decrease (P < 0.05). Thus, a decrease in daylength causes a decrease in substance P in the medial nucleus of the amygdala, the medial bed nucleus of the stria terminalis and the medial preoptic area that is mediated by changes in testosterone levels.     

Region-specific expression and sex-steroidal regulation on aromatase and its mRNA in the male rat brain: immunohistochemical and in situ hybridization analyses

The brain has an estrogen-biosynthetic potential resulting from the presence of neuronal aromatase, which controls the intraneural sex-steroidal milieu and is involved in brain sexual differentiation, psychobehavioral regulation, and neuroprotection. In the rat brain, three distinct aromatase-P450-immunoreactive (AromP450-I) neural groups have been categorized in terms of their peak expression time (fetal, fetoneonatal, and young-to-adult groups), suggesting the presence of region-specific regulation on brain AromP450. In the present study, we compared the expressions between AromP450 protein and mRNA by using immunohistochemistry and in situ hybridization with an ovary-derived cRNA probe in serial sections of fetal, fetoneonatal, and adult male rat brains and then performed steroidal manipulations to evaluate the sex-steroidal effects on AromP450 in adult orchiectomized and adrenalectomized (OCX + ADX) male rats. As a result, prominent mRNA signals were detected in the fetal (i.e., the anterior medial preoptic nucleus) and fetoneonatal (i.e., the medial preopticoamygdaloid neuronal arc) groups, although no detectable signal was found in the "young-to-adult" group (i.e., the central amygdaloid nucleus). In addition, the "fetoneonatal" AromP450-I neurons were prominently reduced in number and intensity after OCX + ADX and then were reinstated by the administration of dihydrotestosterone, testosterone, or 17beta-estradiol. In contrast, none of the sex steroids had any significant effects on the young-to-adult group. Several possible explanations were explored for why the young-to-adult group may differ in aromatase expression and regulation, including the possibility that distinct splicing variants or isozymes for aromatase exist in the rat brain.     

GABA and glutamate in mating-activated cells in the preoptic area and medial amygdala of male gerbils

The posterodorsal medial amygdala (MeApd), the posterodorsal preoptic nucleus (PdPN), and the medial cell group of the sexually dimorphic preoptic area (mSDA) contain cells that are activated specifically at ejaculation as assessed by Fos expression. The mSDA also expresses Fos early in the mating context. Because little is known about the neurotransmitters of these activated cells, the possibility that they use gamma-aminobutyric acid (GABA) or glutamate was assessed. Putative glutamatergic cells were visualized with immunocytochemistry (ICC) for glutamate and its neuron-specific transporter. Their distributions were compared with those of GABAergic cells visualized with ICC for the 67-kDa form of glutamic acid decarboxylase (GAD(67)) and in situ hybridization for GAD(67) messenger RNA (mRNA). Colocalization of Fos and GAD(67) mRNA in recently mated males indicated that half of the activated cells in the PdPN, mSDA, and lateral MeApd are GABAergic. Colocalization of Fos and glutamate suggested that a quarter of the activated mSDA and lateral MeApd cells are glutamatergic. The PdPN does not appear to have glutamatergic cells. In the lateral MeApd, the percentage of activated cells that are GABAergic (45%) matches the percentage that project to the principal part of the bed nucleus of the stria terminalis (BST; 43%), and the percentage likely to be glutamatergic (27%) matches the percentage projecting to the mSDA (27%). The latter could help to trigger ejaculation. The distribution of GABAergic and putative glutamatergic cells in the caudal preoptic area, caudal BST, and medial amygdala of male gerbils is also described.     

Projections of the posterodorsal preoptic nucleus and the lateral part of the posterodorsal medial amygdala in male gerbils, with emphasis on cells activated with ejaculation

The posterodorsal preoptic nucleus (PdPN) and the lateral part of the posterodorsal medial amygdala (MeApd) express Fos with ejaculation in male gerbils. Ejaculation-activated cells participate in the PdPN and MeApd projections to each other and to the sexually dimorphic preoptic area (SDA), but those projections involve less than 20% of the activated PdPN cells and less than 50% of the activated MeApd cells. To identify other potential targets of ejaculation-activated cells, we traced PdPN and lateral MeApd outputs using biotinylated dextran amine. The principal part of the bed nucleus of the stria terminalis (BSTpr) and the anteroventral periventricular nucleus (AVPv) were labeled from both sites and were injected with Fluoro-Gold to determine whether PdPN and lateral MeApd cells that express Fos with ejaculation would be retrogradely labeled. Fluoro-Gold was also applied to the dorsomedial hypothalamus (DMH) and retrorubral field (RRF) because such injections label PdPN cells in rats. The PdPN-DMH projection is minimal in gerbils, involving few, if any, ejaculation-related cells. Ejaculation-activated PdPN cells project to the AVPv (43%), dorsal BSTpr (30%), and RRF (12%). Those in the lateral MeApd project to the dorsal BSTpr (43%) and AVPv (18%). When these percentages are combined with those for ejaculation-activated cells involved in the PdPN and lateral MeApd projections to each other and to the medial SDA, the totals reach 100%. Thus, every PdPN and MeApd cell activated with ejaculation may participate in one of these projections. Similar projections may contribute to the similar behavioral effects of the PdPN and MeApd.     

Sexually differentiated and neuroanatomically specific co‐expression of aromatase neurons and GAD67 in the male and female quail brain

Testosterone aromatization into estrogens in the preoptic area (POA) is critical for the activation of male sexual behavior in many vertebrates. Yet, the cellular mechanisms mediating actions of neuroestrogens on sexual behavior remain largely unknown. We investigated in male and female Japanese quail by dual‐label fluorescent in situ hybridization (FISH) whether aromatase‐positive (ARO) neurons express glutamic acid decarboxylase 67 (GAD67), the rate‐limiting enzyme in GABA biosynthesis. ARO cells and ARO cells double labeled with GAD67 (ARO‐GAD67) were counted at standardized locations in the medial preoptic nucleus (POM) and the medial bed nucleus of the stria terminalis (BST) to produce three‐dimensional distribution maps. Overall, males had more ARO cells than females in POM and BST. The number of double‐labeled ARO‐GAD67 cells was also higher in males than in females and greatly varied as a function of the specific position in these nuclei. Significant sex differences were however present only in the most caudal part of POM. Although both ARO and GAD67 were expressed in the VMN, no colocalization between these markers was detected. Together, these data show that a high proportion of estrogen‐synthesizing neurons in POM and BST are inhibitory and the colocalization of GAD67 with ARO exhibits a high degree of anatomical specificity as well as localized sex differences. The fact that many preoptic ARO neurons project to the periaqueductal gray in male quail suggests possible mechanisms through which locally produced estrogens could activate male sexual behavior.     

Neuronal aromatase expression in preoptic,strial, and amygdaloid regions during late prenatal and early postnatal development in the rat

Brain aromatase has been considered to be an important clue in elucidating the actions of androgen on brain sexual differentiation. Using highly specific anti-P450arom antiserum, the regional and subcellular distributions were immunohistochemically evaluated in the preoptic, strial, and amygdaloid regions of developing rat brains. Aromatase-immunoreactive (AROM-I) neurons were classified into three groups. The first, in which immunostaining occurs only during certain pre- or neonatal days (E16–P2), included the anterior medial preoptic nucleus, the periventricular preoptic nucleus, neurons associated with the strial part of the preoptic area, and the rostral portion of the medial preoptic nucleus. The second is a striking AROM-I cell group in the “medial preopticoamygdaloid neuronal arc,” which extends from the medial preoptic nucleus to the principal nucleus of the bed nucleus of the stria terminals and the posterodorsal part of the medial amygdaloid nucleus. The AROM-I neurons appeared by E16, reaching a peak in staining intensity between E18 and P2 and diminishing after the perinatal stage. After P14, a third group of AROM-I neurons emerged in the lateral septal nucleus, the oval nucleus of the bed nucleus of the stria terminalis, and the central amygdaloid nucleus. The second group was thought to be the major aromatization center in developing rat brains, while the center might partly shift to the third group of neurons after the late infantile stage. The distribution and developmental patterns were basically similar in males and females, suggesting that the neonatally prominent aromatase is not induced by male-specific androgen surges occurring around birth. On immunoelectron microscopy, subneuronal aromatase was predominantly localized on the nuclear membrane and endoplasmic reticulum, which appeared to be appropriate for the efficient conversion of androgen into estrogen just prior to blinding to the nuclear receptors. © 1994 Wiley-Liss, Inc.     

Sex and Species Differences in the Vasopressin Innervation of Sexually Naive and Parental Prairie Voles, Microtus ochrogaster and Meadow Voles, Microtus pennsylvanicus

To study whether central systems that are implicated in functions associated with reproduction show different changes in males and females that become parental, the central vasopressin (AVP) innervation was compared in two species of voles: prairie voles, in which males and females provide parental care, and meadow voles, in which only females provide parental care. For both species, the densities of AVP-immunoreactive (AVP-ir) fibers in the lateral septum, lateral habenular nucleus, medial preoptic area and paraventricular nucleus of the thalamus were compared in males and females that were sexually inexperienced or had become parents 6 days before sacrifice. The lateral septum and lateral habenular nucleus presumably receive their projections from the bed nucleus of the stria terminalis and medial amygdaloid nucleus, while the other two areas presumably receive their projections from the suprachiasmatic nucleus. Differences between sexually naive and parental animals were found only in the presumed projections of the bed nucleus of the stria terminalis and medial amygdaloid nucleus. In both species, AVP-ir fiber densities in the lateral habenular nucleus and the lateral septum were much greater in males than in females regardless of parental state. In prairie voles, AVP-ir fiber density in the lateral septum and lateral habenular nucleus was reduced in parental males, while no differences were found in females. In parental meadow voles, the AVP-ir fiber density in the lateral septum did not show changes, while the fiber density in the lateral habenular nucleus was increased. The reduction in AVP-ir fiber density in parental prairie vole males and the absence of such a reduction in meadow vole males may be related to differences in their contribution to parental care.     

Anatomical interrelationships of the medial preoptic area and other brain regions activated following male sexual behavior: A combined Fos and tract-tracing study

The medial preoptic nucleus (MPN) is an essential site for the regulation of male sexual behavior. Previous studies using c-fos as a marker for neural activation have shown that copulation increased c-fos expression in the MPN. Neural activation was also present in brain regions that are connected with the MPN and are involved in male sexual behavior, including the posteromedial bed nucleus of the stria terminalis (BNSTpm), posterodorsal preoptic nucleus (PD), posterodorsal medial amygdala (MEApd), and parvocellular subparafascicular thalamic nucleus (SPFp). The present study investigated whether the copulation-induced, activated neurons in these brain regions are involved in the bidirectional connections with the MPN. Therefore, mating-induced Fos expression was combined with application of anterograde (biotinylated dextran amine) or retrograde (cholera toxin B subunit) tracers in the MPN. The results demonstrated that neurons in the BNSTpm, PD, MEApd, and SPFp that project to the MPN were activated following copulation. However, in males that displayed sexual behavior but did not achieve ejaculation, few double-labeled neurons were evident, although both retrogradely labeled neurons and Fos-immunoreactive cells were present. In addition, retrograde neurons that expressed Fos were located in discrete subdivisions within the brain regions studied, where Fos is induced after ejaculation. Likewise, anterogradely labeled fibers originating from the MPN were not distributed homogeneously but were particularly dense in these discrete subdivisions. These results demonstrate that copulation-induced Fos-positive neurons in specific subdivisions of the BNSTpm, PD, MEApd, and SPFp have bidirectional connections with the MPN. Taken together with previous findings, this supports the existence of a discrete subcircuit within a larger neural network underlying male sexual behavior. J. Comp. Neurol. 397:421–435, 1998. © 1998 Wiley-Liss, Inc.     

Projections from Ventrolateral Hypothalamic Neurons Containing Progestin Receptor- and Substance P-Immunoreactivity to Specific Forebrain and Midbrain Areas in Female Guinea Pigs

Many neurons within the ventrolateral hypothalamus in guinea pigs contain estrogen-induced progestin receptors as well as substance P. Retrograde tracing combined with immunocytochemistry was used to determine the specific projections of this subset of steroid- sensitive cells. Unilateral Fluoro-Gold injections into the dorsal midbrain, including the central gray, labeled a large proportion of the ventrolateral hypothalamic neurons immunoreactive for both progestin receptors and substance P (approximately 30%); substantially fewer of these neurons were labeled by unilateral Fluoro-Gold injections into the preoptic area (approximately 6%), medial amygdala (approximately 10%), or the bed nucleus of the stria terminalis (approximately 11 %). The projections of progestin receptor-immunoreactive neurons in the ventrolateral hypothalamus were similar to those of progestin receptor/substance P double-labeled neurons, while a slightly lower percentage of the ventrolateral hypothalamic, substance P-immunoreactive neurons tended to project to each of these areas. These pathways may prove to be components of the neural circuitry underlying a variety of functions influenced by gonadal steroid hormones and substance P, such as female sexual behavior, salt intake, nociception and aggression.     

Peripeduncular lesion alters expression of c-fos induced in the female rat brain by male mounting

In order to investigate the role of the peripeduncular nucleus (PP) in the control of lordosis in female rats, activation of neurons after mounts without intromission was investigated by means of FOS immunoreactivity (FOS-IR). Ovariectomized rats were injected with estradiol and progesterone and submitted to approximately 50 mounts by the male. The vaginal area was covered with masking tape to prevent intromission and vaginocervical stimulation. This limited stimulation produced FOS-IR in the ventrolateral division of the ventromedial hypothalamic nucleus (VMHVL), in the lateral periaqueductal grey (LPAG), in the peripeduncular nucleus (PP), and in the posterior intralaminar thalamic nucleus (PIL). No significant differences were found in the anterodorsal or posterodorsal parts of the medial amygdaloid nucleus, in the medial part of the medial preoptic nucleus, in the dorsomedial periaqueductal grey and in the medial division of the posterointermediate part of the bed nucleus of the stria terminalis. The same experiment was performed in rats with unilateral lesion of the PP. Both VMHVL and LPAG activation were significantly reduced in the ipsilateral PP lesion side, leading to the conclusion that those structures are primary targets for the somatic stimuli that trigger lordotic reflexes and which are relayed in the PP. Taking into account what is known about the function of the target structures, it is proposed that afferences relayed in the PP reaching the VMHVL would contribute to control the long range level of sexual receptivity, whereas stimuli reaching the LPAG would serve to control lordotic responses in a moment to moment fashion.     

Organization of multisynaptic circuits within and between the medial and the central extended amygdala

The central and medial extended amygdala comprises the central (CEA) and medial nuclei of the amygdala (MEA), respectively, together with anatomically connected regions of the bed nucleus of the stria terminalis (BST). To reveal direct and multisynaptic connections within the central and medial extended amygdala, monosynaptic and transneuronal viral tracing experiments were performed in adult male rats. In the first set of experiments, a cocktail of anterograde and retrograde tracers was iontophoretically delivered into the medial CEA (CEAm), anterodorsal MEA (MEAad), or posterodorsal MEA (MEApd), revealing direct, topographically organized projections between distinct amygdalar and BST subnuclei. In the second set of experiments, the retrograde transneuronal tracer pseudorabies virus (PRV) was microinjected into the CEAm or MEAad. After 48 hours of survival, there were no significant differences between monosynaptic and PRV cases in the subnuclear distribution or proportions of retrogradely labeled BST neurons. However, after 60 hours of survival, CEAm‐injected cases displayed an increased proportion of labeled neurons within the anteromedial group of BST subnuclei (amgBST) and within the posterior BST, which do not directly innervate the CEA. MEApd‐injected 60‐hour cases displayed a significantly increased proportion of retrograde labeling in the amgBST compared with monosynaptic and 48‐hour cases, whereas MEAad‐injected cases displayed no proportional changes over time. Thus, multisynaptic circuits within the medial extended amygdala overlap the direct connections making up this anatomical unit, whereas the multisynaptic boundaries of the central extended amygdala extend into BST subnuclei previously identified as part of the medial extended amygdala. J. Comp. Neurol. 521:3406‐3431, 2013. © 2013 Wiley Periodicals, Inc.     

Progestin receptor immunoreactivity within steroid-responsive vasopressin-immunoreactive cells in the male and female rat brain

Progestin receptor immunoreactivity is found in the same regions of the bed nucleus of stria terminalis (BST) and centromedial amygdala (CMA) as steroid-responsive vasopressin immunoreactive (AVP-ir) cells. To test whether AVP-ir cells express progestin receptors, brains of male rats were stained immunocytochemically for arginine vasopressin as well as progestin receptors. In BST and CMA, over 95% of AVP-ir cells contained progestin receptor immunoreactivity. In contrast, none of the AVP-ir cells in the suprachiasmatic, supraoptic and paraventricular nuclei expressed progestin receptor immunoreactivity. To study whether progestin receptor expression in AVP-ir cells in the BST and CMA is responsive to gonadal steroids, male and female rats were castrated and implanted with either empty capsules or capsules filled with testosterone or oestradiol, respectively. Ten days later, brains were processed for AVP and progestin receptor immunoreactivity. Although there was no effect of hormonal status on the percentage of colocalized cells, the level of progestin receptor immunoreactivity was higher in rats that received gonadal steroids than those that did not. The presence of progestin receptor immunoreactivity in steroid responsive AVP-ir cells, and the responsiveness of this expression to gonadal hormones, is consistent with the possibility that the effects of gonadal steroids on AVP-ir expression in the BST and CMA may be mediated at least in part by progestin receptors.     

Anatomical and functional connections among cell groups in the gerbil brain that are activated with ejaculation

Based on Fos expression, four areas of the gerbil brain are activated with ejaculation, i.e., the posterodorsal preoptic nucleus (PdPN), the lateral part of the posterodorsal medial amygdala (MeApd), the medial cell group of the sexually dimorphic preoptic area (medial SDA), and the parvicellular part of the subparafascicular thalamus (SPFp). The SPFp and medial SDA also express Fos earlier in the context of mating. To study connections among these areas, we injected one with FluoroGold and assessed the colocalization of FluoroGold and mating-induced Fos in the others. To determine if any of these areas activates the others, we lesioned one unilaterally and measured mating-induced Fos ipsilaterally and contralaterally in the others. Half of the SPFp cells projecting to the medial SDA, PdPN, and MeApd were activated with mating. SPFp lesions also decreased Fos expression in those areas. However, those areas do not project to the SPFp or affect its Fos expression with mating. Projections from the lateral MeApd to the medial SDA and PdPN, and from the medial SDA to the lateral MeApd, were also activated with mating, but lesions in these areas did not affect Fos expression in the others. Because 32-50% of the mating-activated cells in the SPFp participated in each SPFp projection identified, projections may have been identified for all of the mating-activated cells in the SPFp. In contrast, most of the mating-activated cells in the lateral MeApd, PdPN, and medial SDA do not participate in any projection studied, suggesting that they are either interneurons or project elsewhere.     

Differential Localization of Estrogen Receptors in Various Vasopressin Synthesizing Nuclei of the Rat Brain

Vasopressin (VP) cells in the bed nucleus of the stria terminalis, medial amygdaloid nucleus and supraoptic and paraventricular nuclei are influenced by gonadal steroids. The present paper examined whether VP cells in the bed nucleus of the stria terminalis, medial amygdaloid nucleus, and supraoptic and paraventricular nuclei contain estrogen receptors. Brains from adult short-term castrated, colchicine-treated male rats were fixed with 4% paraformaldehyde and 0.5% glutaraldehyde. In the immunocytochemical double-staining procedure Vibratome sections were first incubated with an estrogen receptor antibody (#H222) and stained with diaminobenzidine-Ni⁺. Following methanol-hydrogen peroxide washes, sections were incubated with anti-neurophysin and stained with diaminobenzidine. Parvocellular cells in the bed nucleus of the stria terminalis and medial amygdaloid nucleus were double-stained with a blue-black nucleus (indicating the estrogen receptors) surrounded by brown cytoplasm (resulting from VP-neurophysin-immunoreactivity). Our results provide the first direct anatomical evidence supporting the hypothesis that gonadal steroids' influence of parvocellular VP cells in the bed nucleus of the stria terminalis and medial amygdaloid nucleus is mediated directly via estrogen receptors localized in nuclei of VP neurons. We were unable to co-localize any estrogen receptors in VP and oxytocin cells of magnocellular size in the supraoptic, paraventricular and anterior commissural nuclei, suggesting that estrogen indirectly affects these magnocellular hypothalamic cells.     

Estrogen receptor-alpha distribution in male rodents is associated with social organization

It has been hypothesized that site-specific reduction of estrogen receptor-alpha (ERalpha) is associated with the expression of male prosocial behaviors. Specifically, highly social males are predicted to express significantly lower levels of ERalpha than females and less social males in brain regions associated with prosocial behavior including the bed nucleus of the stria terminalis (BST) and the medial amygdala (MeA). This hypothesis was tested by comparing ERalpha immunoreactivity (IR) in three species of microtines, the polygynous montane (Microtus montanus) and meadow (M. pennsylvanicus) voles and the monogamous pine vole (M. pinetorum), and two species of cricetines that differ in the extent of social pair-bond formation, Siberian (Phodopus sungorus) and Djungarian (P. campbelli) hamsters. As predicted, ERalpha-IR was sexually dimorphic in the BST and MeA of the highly social species, with females expressing more ERalpha-IR cells than males. Male and female montane voles did not differ. Male and female meadow voles differed in the ventromedial hypothalamus, with females expressing more ERalpha-IR cells. Male pine voles expressed lower levels of ERalpha-IR in the MeA than male montane and meadow voles and in the BST relative to montane males. Male Djungarian hamsters, which show higher levels of parental care, had fewer ERalpha-IR cells in the BST than male Siberian hamsters. Results indicate that the distribution of ERalpha differs relative to the continuum of species-typical affiliative behavior and supports the hypothesis that ERalpha has a significant role in regulating species-specific social organization.     

Morphology and axonal projection pattern of neurons in the telencephalon of the fire-bellied toad Bombina orientalis: an anterograde, retrograde, and intracellular biocytin labeling study

The connectivity and cytoarchitecture of telencephalic centers except dorsal and medial pallium were studied in the fire-bellied toad Bombina orientalis by anterograde and retrograde biocytin labeling and intracellular biocytin injection (total of 148 intracellularly labeled neurons or neuron clusters). Our findings suggest the following telencephalic divisions: (1) a central amygdala-bed nucleus of the stria terminalis in the caudal midventral telencephalon, connected to visceral-autonomic centers; (2) a vomeronasal amygdala in the caudolateral ventral telencephalon receiving input from the accessory olfactory bulb and projecting mainly to the preoptic region/hypothalamus; (3) an olfactory amygdala in the caudal pole of the telencephalon lateral to the vomeronasal amygdala receiving input from the main olfactory bulb and projecting to the hypothalamus; (4) a medial amygdala receiving input from the anterior dorsal thalamus and projecting to the medial pallium, septum, and hypothalamus; (5) a ventromedial column formed by a nucleus accumbens and a ventral pallidum projecting to the central amygdala, hypothalamus, and posterior tubercle; (6) a lateral column constituting the dorsal striatum proper rostrally and the dorsal pallidum caudally, and a ventrolateral column constituting the ventral striatum. We conclude that the caudal mediolateral complex consisting of the extended central, vomeronasal, and olfactory amygdala of anurans represents the ancestral condition of the amygdaloid complex. During the evolution of the mammalian telencephalon this complex was shifted medially and involuted. The mammalian basolateral amygdala apparently is an evolutionary new structure, but the medial portion of the amygdalar complex of anurans reveals similarities in input and output with this structure and may serve similar functions.     

Sex difference and steroidal stimulation of galanin immunoreactivity in the ferret's dorsal preoptic area/anterior hypothalamus

A sexually dimorphic male nucleus (MN) is seen in Nissl-stained sections from the dorsal preoptic area/anterior hypothalamus (dPOA/AH) of male, but not female, ferrets. We used immunohistochemical methods to determine whether particular neuropeptides are found in cells of the MN. A sexually dimorphic cluster of galanin-immunoreactive (IR) cells was found in the dPOA/AH of ferrets killed either on embryonic day (E) 38 or in adulthood. Significantly more galanin-IR cells were distributed in the MN and in other subregions of the dPOA/AH of intact breeding males than estrous females. The density of galanin-IR cells in the dPOA/AH was significantly reduced in adult males by castration and restored to the level of intact breeding males by daily injections of testosterone propionate (TP) for 5 weeks. The same TP treatment failed to augment the density of galanin-IR cells in the dPOA/AH of adult, ovariectomized females. Computer-assisted image analysis and grid-crossing analysis showed that the area and the number of galanin-IR fibers in the dPOA/AH were significantly greater in adult females than in males, regardless of subjects' concurrent steroidal condition. A cluster of galanin-IR cells was present in the dPOA/AH of males, but not females, killed on either E34 or E38. Administration of TP between E28 and E37 significantly increased the density of galanin-IR cells in the dPOA/AH of females killed on E38, up to the level seen in control males. The results suggest that the capacity of cells located in the dPOA/AH to express galanin after adult steroid exposure is sexually differentiated by the fetal action of testosterone, or its metabolite, estradiol, in males. J. Comp. Neurol. 389:277–288, 1997. © 1997 Wiley-Liss, Inc.