Peptide immunoreactive neurons in the amygdala and the bed nucleus of the stria terminalis project to the midbrain central gray in the rat.

The central nucleus of the amygdala, bed nucleus of the stria terminalis, and central gray are important components of the neural circuitry responsible for autonomic and behavioral responses to threatening or stressful stimuli. Neurons of the amygdala and bed nucleus of the stria terminalis that project to the midbrain central gray were tested for the presence of peptide immunoreactivity. To accomplish this aim, a combined immunohistochemical and retrograde tracing technique was used. Maximal retrograde labeling was observed in the amygdala and bed nucleus of the stria terminalis after injections of retrograde tracer into the caudal ventrolateral midbrain central gray. The majority of the retrogradely labeled neurons in the amygdala were located in the medial central nucleus, although many neurons were also observed in the lateral subdivision of the central nucleus. Most of the retrogradely labeled neurons in the BST were located in the ventral and posterior lateral subdivisions, although cells were also observed in most other subdivisions. Retrogradely labeled neurotensin, corticotropin releasing factor (CRF), and somatostatin neurons were mainly observed in the lateral central nucleus and the dorsal lateral BST. Retrogradely labeled substance P-immunoreactive cells were found in the medial central nucleus and the posterior and ventral lateral BST. Enkephalin-immunoreactive retrogradely labeled cells were not observed in the amygdala or bed nucleus of the stria terminalis. A few cells in the hypothalamus (paraventricular and lateral hypothalamic nuclei) that project to the central gray also contained CRF and neurotensin immunoreactivity. The results suggest the amygdala and the bed nucleus of the stria terminalis are a major forebrain source of CRF, neurotensin, somatostatin, and substance P terminals in the midbrain central gray.     

Opiocortin and catecholamine input to CRF-immunoreactive neurons in rat forebrain

Neural input to distinct and separate populations of CRF-immunoreactive (ir) neurons in rat forebrain was investigated. The relationship of opiocortin and/or catecholamine fibers to different groups of CRF-containing neurons was elucidated using single and dual labeling immunocytochemical procedures. Antibodies to CRF, ACTH(1–39) and the catecholamine synthesizing enzymes which are tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) were utilized. CRF-ir neuronal populations are localized predominantly in the following regions of rat forebrain: bed nucleus of stria terminalis, medial preoptic area, suprachiasmatic and paraventricular (PVN) nuclei of hypothalamus and central nucleus of amygdala. The present study demonstrates that CRF-ir neuronal groups in rat forebrain are not homogenous in that each population received a characteristic neural input. CRF-ir neurons in the PVN received a dense input of ACTH-, TH-, DBH-, and PNMT-ir fibers. In contrast, CRF-ir neurons in the central nucleus of amygdala are colocalized predominantly with TH-ir fiber/terminals. In the ventral portion of the bed nucleus of stria terminalis, TH-, ACTH- and DBH-ir fibers are demonstrated in close anatomical proximity to CRF-containing perikarya; in the dorsal portion of this nucleus, TH-ir fiber/terminals are colocalized with CRF-ir neurons. In the suprachiasmatic nucleus, neither opiocortin- nor catecholamine-immunostained fibers are observed in association with CRF-ir neurons. Our data suggest that there is a transmitter specificity of neural input to each CRF-ir neuronal population in rat forebrain.     

The organization of prolactin-like-immunoreactive neurons in the rat central nervous system

Summary The localization and distribution of prolactinlike-immunoreactive perikarya and nerve fibers in the rat central nervous system have been studied by a preembedding immunoperoxidase method using well-characterized specific immunsera to rat prolactin. Although the localization of labeled neuronal structures in a number of brain areas correlates with the data of previous immunocytochemical studies, we found prolactin-immunoreactive neurons in various regions not previously reported. In untreated animals, the highest concentrations of prolactinfibers were observed: (i) in the external layers of the median eminence where they exhibited close contact with blood vessels, and (ii) in the bed nucleus of the stria terminalis and in the central nucleus of the amygdala where they closely surrounded unlabeled perikarya. Dense networks of finely varicose prolactin fibers were also observed in the organum vasculosum of the lamina terminalis, in the subfornical organ, and in the dorsolateral regions of the medulla oblongata and the spinal cord. Lastly, a number of large, varicose, intensely immunoreactive fibers were found in the olfactory bulb, the cingulum, and the periventricular regions of the hypothalamus and central gray, whereas isolated fibers could be detected in the caudate nucleus and in the cerebral cortex. In animals treated with colchicine, prolactin-immunoreactive perikarya were essentially located within the periventricular and perifornical regions of the hypothalamus, and within the bed nucleus of the stria terminalis. Although corticotropin (ACTH 17-39)-immunoreactive fibers could be detected in several regions found to contain prolactin fibers, the distribution and organization of both fiber types clearly differed in numerous brain regions, and the regions containing the corresponding perikarya did not overlap. The ultrastructural organization of the prolactin-immunoreactive fibers revealed by electronmicroscopic immunocytochernistry in various brain regions, allowed the characterization of two main types of prolactinergic neurons including: (i) endocrine neurons, whose axons terminated in close vicinity to portal blood vessels in the external median eminence, and (ii) neurons projecting to extrahypothalamic regions, whose axons formed typical synaptic connections with unidentified neuronal units.     

Dopamine- and cyclic AMP-regulated phosphoprotein-immunoreactive neurons activated by acute stress are innervated by fiber terminals immunopositive for pituitary adenylate cyclase-activating polypeptide in the extended amygdala in the rat

The bed nuclei of the stria terminalis (BST) and the central nucleus of the amygdala are highly heterogeneous structures, which form one functional unit, the so-called extended amygdala. Several studies described increased c-fos expression following acute stress in this brain area, confirming its central role in the modulation/regulation of stress responses. The oval nucleus of the BST and the central amygdala exhibit a dense network of pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive (ir) fiber terminals. In addition, several dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32)-immunoreactive neurons were also observed here. Because the extended amygdala plays an important role in the central autonomic regulation during stress and the distribution of PACAP-ir and that of DARPP-32-ir nervous structures overlap, the aims of this study were to investigate the possible activation of DARPP-32-ir neurons following acute systemic stress and to demonstrate synaptic interactions between DARPP-32-ir neurons and fiber terminals immunopositive for PACAP.In summary, this study provided morphological evidence that acute stress resulted in the activation of DARPP-32 neurons, which were innervated by PACAP-ir neuronal structures in the extended amygdala. Furthermore, interaction between neuropeptides/neurotransmitters and phosphoproteins was also demonstrated.     

Immunohistochemical labeling of androgen receptors in the brain of rat and monkey.

Androgen receptor antibodies have recently been developed using fusion proteins containing fragments of human prostatic androgen receptor. We have used a polyclonal antibody raised in rabbits to label androgen receptors in brain sections from male and female rats and monkeys. Free-floating frozen sections were incubated in primary antibody, and processed by the peroxidase-avidin-biotin complex method using biotinylated anti-rabbit IgG. Nickel intensified diaminobenzidine was used as the chromagen, and neurons were labeled in the amygdala, hippocampus, bed nucleus of stria terminalis, septum, preoptic area, in several hypothalamic nuclei including the supraoptic and paraventricular nuclei, in several brain stem motor nuclei and in cerebral cortex. Staining was most intense in cell nuclei but also occurred in cytoplasm and in some neuronal processes. Labeling was more restricted in monkey than in rat brain. Omitting the primary antibody or pre-incubating the primary antibody with rat prostatic cytosol for control purposes demonstrated the specificity of staining.     

REGIONAL DISTRIBUTION OF GLUTAMATE DECARBOXYLASE AND GABA WITHIN THE AMYGDALOID COMPLEX AND STRIA TERMINALIS SYSTEM OF THE RAT

The distribution of glutamate decarboxylase (GAD) and δ-amino butyric acid have been studied in the amygdaloid complex and in the stria terminalis system of the rat. The central and medial nuclei of the amygdala had significantly higher activities of GAD than the lateral olfactory tract nucleus, anterior amygdala, anterior lateral nucleus, posterior lateral nucleus, cortical nucleus, basomedial nucleus, basolateral nucleus, and pyriform cortex. The enzyme activity was about two and a half times higher in the central and medial nuclei than in the pyriform cortex. GABA was also significantly more concentratcd in these nuclei than in the pyriform cortex although this was not true for four other amino acids studied–glutamic acid, aspartic acid, taurine and glycine. GAD activity was also measured in the stria terminalis (the major afferent and efferent pathway of the amygdala) and in its bed nucleus. The enzyme activity was higher in the stria terminalis than in four other fibre tracts studied–the optic tract, anterior commissure, corpus callosum, and fimbria. GAD activity was exceptionally high in the bed nucleus of the stria terminalis particularly in its ventral part. The significance of the results are discussed in terms of what is known about the evolution and anatomy of the amygdala.     

VIP in the rat brain: Evidence for a major pathway linking the amygdala and hypothalamus via the stria terminalis

Summary We report here on the detailed distribution of VIP-like immuno-reactivity in the rat brain by a combined immunological approach using immunocytochemistry and radioimmunoassay. VIP-like immunoreactivity was widely distributed. Cell bodies and fibres were noted principally in the cortex, hippocampus, amygdala, suprachiasmatic nucleus and brain stem. In addition dense areas of immunoreactive fibres and terminals were seen in the stria terminalis and its bed nucleus. The fibres appear to form a major VIP-containing pathway which links the amygdaloid complex with the hypothalamus. Although the functional significance of VIP in the brain is unknown, its presence in the amygdala, the hypothalamus and their linking pathway, as well as its pharmacological actions suggest that is may play a role in neuroendocrine regulation and the modulation of hypothalamic function.     

Neuropeptide Y innervation of amygdaloid and hypothalamic neurons that project to the dorsal vagal complex in rat

The anatomic relationship between neuropeptide Y (NPY)-immunoreactive terminals and forebrain areas in the rat that contain neurons that project to the dorsal vagal complex (DVC) was examined. To accomplish this, the combined retrograde fluorescent tracer and immunofluorescent technique was used. Neurons projecting to the DVC within the parvocellular divisions of the paraventricular nucleus of the hypothalamus were the most heavily innervated of the regions studied. A relatively high density of NPY-immunoreactive terminals innervated regions of the arcuate, dorsomedial and lateral hypothalamic areas that contained DVC efferent cells. Neurons that projected to the DVC within the medial division of the central nucleus of the amygdala and the lateral part of the bed nucleus of the stria terminalis were also innervated by NPY immunoreactive terminals. The results suggest an important role for NPY terminals in the modulation of neurons within the amygdala and hypothalamus that directly influence visceral-autonomic functions of the dorsal vagal complex. The source and possible function of NPY within these regions is discussed.     

Immunocytochemical localization of aromatase-containing neurons in the rat brain during pre- and postnatal development

the present immunohistochemical study demonstrates the ontogenetic appearance of aromatase-immunoreactive neurons in several discrete regions of the hypothalamus and limbic system in the rat brain, using a purified antibody against human placental aromatase cytochrome P450. Immunoreactive cells were first detected in the preoptic area on the 13th day of embryonic life (E 13), and additionally in the bed nucleus of the stria terminalis on E 15. Labeled cells were also found in the medial amygdaloid nucleus and the ventromedial nucleus on E 16, and some were detected in the arcuate nucleus on E 19. As gestation progressed, the number and the immunoreactivity of these cells gradually increased and peaked within definite periods of perinatal life and there-after declined or disappeared. The immunoreactive cells were also found in the central amygdaloid nucleus and the lateral septal nucleus, and in the ventral pallidum, after the 14th day of postnatal life (P 14) and 30th day (P 30), respectively. The distribution of aromatase-immunoreactive neurons was similar between the sexes, while the immunoreactivity was higher in males than in females after late gestational days. No immunoreaction was detectable in other regions of the telencephalon or midbrain at any time periods studied. The aromatase-immunoreactive neurons in the specific regions may be involved in the sexual differentiation of the brain.     

Localization and mechanism of stimulatory feedback action of estrogen: effect of limbic forebrain implantation of estradiol benzoate on advancement of ovulation.

The sites and mechanism of the ovulation-inducing action of estradiol benzoate (EB) were studied by brain implantation of the crystalline steroid through chronically implanted outer cannula at 12:00 on diestrus day 2 in the 5-day cyclic rat. EB implantation in the medial amygdala or the bed nucleus of stria terminalis advanced cyclic changes in vaginal smears, timing of ovulatory LH release, and ovulation by 1 day, resulting in 4-day cycle. When implants in the bed nucleus of stria terminalis were placed for a shorter period of time on diestrus day 2, from 12:00 to 20:00, advancement of these parameters were similarly observed. Serum concentration of FSH and that of prolactin were significantly elevated at 20:00 on the same day in the rats implanted with EB in the medial amygdala or the bed nucleus of stria terminalis, compared with those in the non-treated controls. LH was not affected. The implantation in the arcuate nucleus was also effective to advance ovulation, but the anterior deafferentation prevented the effect. In contrast, EB implantation in the medial septal nucleus, the medial preoptic area, or the medial basal prechiasmatic area was consistently ineffective to advance vaginal cycle and ovulation. Multiunit activity in the arcuate nucleus showed an afternoon elevation on the day of implantation in these areas and as well on the day following, while it did not show such elevation on the day of implantation in the medial preoptic area. It is concluded that EB acts on the medial amygdala and the bed nucleus of stria terminalis in the mid-diestrus in 5-day cycle to stimulate FSH and prolactin release without affecting LH, which changes trigger a chain of reproductive events inducing early release of ovarian steroid responsible for early ovulatory gonadotropin release. The arcuate nucleus in one of the sites of stimulatory action of estrogen, but it requires the neural influence presumably from the medial amygdala and the bed nucleus of stria terminalis via the preoptic area for stimulating the ovulatory hormone release. EB exposure is considered to be endowed with the increase of its responsibility to this neural influence.     

Hypothalamic orexin--a neurons are involved in the response of the brain stress system to morphine withdrawal

Both the hypothalamus-pituitary-adrenal (HPA) axis and the extrahypothalamic brain stress system are key elements of the neural circuitry that regulates the negative states during abstinence from chronic drug exposure. Orexins have recently been hypothesized to modulate the extended amygdala and to contribute to the negative emotional state associated with dependence. This study examined the impact of chronic morphine and withdrawal on the lateral hypothalamic (LH) orexin A (OXA) gene expression and activity as well as OXA involvement in the brain stress response to morphine abstinence. Male Wistar rats received chronic morphine followed by naloxone to precipitate withdrawal. The selective OX1R antagonist SB334867 was used to examine whether orexins' activity is related to somatic symptoms of opiate withdrawal and alterations in HPA axis and extended amygdala in rats dependent on morphine. OXA mRNA was induced in the hypothalamus during morphine withdrawal, which was accompanied by activation of OXA neurons in the LH. Importantly, SB334867 attenuated the somatic symptoms of withdrawal, and reduced morphine withdrawal-induced c-Fos expression in the nucleus accumbens (NAc) shell, bed nucleus of stria terminalis, central amygdala and hypothalamic paraventricular nucleus, but did not modify the HPA axis activity. These results highlight a critical role of OXA signalling, via OX1R, in activation of brain stress system to morphine withdrawal and suggest that all orexinergic subpopulations in the lateral hypothalamic area contribute in this response.     

Neuronal inputs from the hypothalamus and brain stem to the medial preoptic area of the ram: neurochemical correlates and comparison to the ewe

The retrograde tracer, FluoroGold, was used to trace the neuronal inputs from the septum, hypothalamus, and brain stem to the region of the GnRH neurons in the rostral preoptic area of the ram and to compare these imputs with those in the ewe. Sex differences were found in the number of retrogradely labeled cells in the dorsomedial and ventromedial nuclei. Retrogradely labeled cells were also observed in the lateral septum, preoptic area, organum vasculosum of the lamina terminalis, bed nucleus of the stria terminalis, stria terminalis, subfornical organ, periventricular nucleus, anterior hypothalamic area, lateral hypothalamus, arcuate nucleus, and posterior hypothalamus. These sex differences may partially explain sex differences in how GnRH secretion is regulated. Fluorescence immunohistochemistry was used to determine the neurochemical identity of some of these cells in the ram. Very few tyrosine hydroxylase-containing neurons in the A14 group (<1%), ACTH-containing neurons (<1%), and neuropeptide Y-containing neurons (1-5%) in the arcuate nucleus contained FluoroGold. The ventrolateral medulla and parabrachial nucleus contained the main populations of FluoroGold-containing neurons in the brain stem. Retrogradely labeled neurons were also observed in the nucleus of the solitary tract, dorsal raphe nucleus, and periaqueductal gray matter. Virtually all FluoroGold-containing cells in the ventrolateral medulla and about half of these cells in the nucleus of the solitary tract also stained for dopamine beta-hydroxylase. No other retrogradely labeled cells in the brain stem were noradrenergic. Although dopamine, beta-endorphin, and neuropeptide Y have been implicated in the regulation of GnRH secretion in males, it is unlikely that these neurotransmitters regulate GnRH secretion via direct inputs to GnRH neurons.     

Nitric oxide synthase in mating behavior circuitry of male Syrian hamster brain

Chemosensory and hormonal stimuli are essential for mating in the male Syrian hamster. These signals are processed in a neural circuit that includes the medial amygdaloid nucleus (Me), bed nucleus of the stria terminalis (BNST), and medial preoptic area (MPOA). Nitric oxide is implicated in the regulation of male sexual behavior, and nitric oxide synthase (NOS), the enzyme that catalyzes the production of nitric oxide, is present in the limbic system. In this study, the distribution of NOS-containing neurons in mating behavior circuitry of the male Syrian hamster brain was determined using labeling for brain NOS (bNOS) and reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d). bNOS and NADPH-d labeled equivalent populations of neurons. NOS-containing neurons were clustered in specific subnuclei within the Me, BNST, and MPOA. NOS-positive fibers and neurons were seen in the stria terminalis and ventral amygdalofugal pathway, which link the Me with BNST and MPOA. Many NOS-positive neurons in the posterior subdivision of the Me, the medial preoptic nucleus (MPN), and the ventral premammillary nucleus contain androgen receptors. Castration reduced NOS-positive neurons in the MPN, implying a selective regulation of NOS by gonadal steroids. Together, these results suggest that NOS may contribute to the regulation of male sexual behavior by influencing the central neural processing of hormonal and chemosensory signals in the hamster limbic system. © 1996 John Wiley & Sons, Inc.     

Neurons in the brain of fetal rhesus monkeys accumulate 3H-testosterone or its metabolites

Autoradiography was used to map sites in the primate brain at which testosterone may have sexual differentiating actions on brain function and behavior during fetal development. Two female rhesus monkey fetuses were injected in utero on days 112 and 114 of gestation respectively with 3H-testosterone, and were killed 30 and 60 minutes later. Thaw-mount autoradiography of the brains revealed the accumulation of radioactivity, representing 3H-testosterone or its metabolites, in neurons of the medial preoptic-anterior hypothalamic area, bed nucleus (n.) of the stria terminalis, ventromedial hypothalamic n., and corticomedial amygdala. Thus, it appears that steroid receptors are present in a circumscribed system in the brain of the primate fetus at this stage of development.     

Aromatase immunoreactivity in the rat brain: Gonadectomy-sensitive hypothalamic neurons and an unresponsive “limbic ring” of the lateral septum-bed nucleus-amygdala complex

The aromatase (estrogen synthetase) enzyme catalyzes the conversion of androgens to estrogens in peripheral tissues, as well as in the brain. Our study aimed at comparing the brain distribution of aromatase-immunoreactive neurons in male and female, normal and gonadectomized rats. Light microscopic immunostaining was employed using a purified polyclonal antiserum raised against human placental aromatase. Two anatomically separate aromatase-immunoreactive neuronal systems were detected in the rat brain: A “limbic telencephalic” aromatase system was composed by a large population of labeled neurons in the lateral septal area, and by a continuous “ring” of neurons of the laterodorsal division of the bed nucleus of stria terminalis, central amygdaloid nucleus, stria terminalis, and the substantia inominata-ventral pallidum-fundus striati region. The other, “hypothalamic” aromatase system consisted of neurons scattered in a dorsolateral hypothalamic area including the paraventricular, lateral and dorsomedial hypothalamic nuclei, the subincertal nucleus as well as the zona incerta. In addition, a few axon-like processes (unresponsive to gonadectomy) were present in the preoptic-anterior hypothalamic complex, the ventral striatum, and midline thalamic regions. No sexual dimorphism was observed in the distribution or intensity of aromatase-immunostaining. However, 3 days, 2, 3, 8, 16, or 32 weeks after gonadectomy, aromatase-immunoreactive neurons disappeared from the hypothalamus, whereas they were still present in the limbic areas of both sexes. The results indicate the existence of two distinct estrogen-producing neuron systems in the rat brain: (1) a “limbic ring” of aromatase-labeled neurons of the lateral septum-bed nucleus-amygdala complex unresponsive to gonadectomy; and (2) a sex hormone-sensitive “hypothalamic” aromatase neuron system.     

Site-specific changes in brain extra-SCN oscillators during early pregnancy in the rat

The influence of circadian timekeeping systems on behavior and physiology can change substantially as female mammals undergo the transition from a nonpregnant to a pregnant state. Here, we examined the possibility that site-specific changes in extra-SCN oscillators and in local rhythms might coincide with the emergence of new patterns of temporal organization among various behavioral and physiological rhythms. Specifically, we compared daily patterns of immunoreactive FOS and PER2 in 3 brain regions of pregnant and diestrous rats. We found that, in the oval nucleus of the bed nucleus of the stria terminalis (BnST-ov), the peak of the PER2 rhythm occurred approximately 12 hours out of phase in pregnant and diestrous females. In contrast, the phase of the rhythm in FOS was the same, but pregnant rats expressed more FOS in the BnST-ov than diestrous ones. In the central amygdala (CEA) of diestrous females, PER2 expression was arrhythmic, but Fos expression was elevated at night. No rhythms were seen in this region of pregnant females, nor were any rhythms seen in the basolateral amygdala of either group. Overall, the patterns in the BnST-ov and the CEA were consistent with the hypothesis that differential changes in daily rhythms within some extra-SCN brain regions might mediate the changes in the temporal organization of several behavioral and endocrine functions that occur during the transition from a nonpregnant to a pregnant state.     

Cholecystokinin- and dexfenfluramine-induced anorexia compared using devazepide and c-fos expression in the rat brain

It has been proposed that there might be a link between the anorectic actions of cholecystokinin (CCK) and serotonin (5HT). The present study compared the patterns of c-fos protein-like immunoreactivity (FLI) induced in rat brain by CCK and the indirect 5HT agonist dexfenfluramine (DFEN), as well as the ability for devazepide, a CCK-A receptor antagonist, to antagonize both anorexia and FLI induced by these agents. Devazepide reversed the anorectic effect of CCK but not that of DFEN in food deprived rats. The FLI induced by CCK and DFEN occurred in similar brain regions, but in different subdivisions. Such regions included the bed nucleus of the stria terminalis (BST), the lateral central nucleus of the amygdala (CeL), and the lateral parabrachial nucleus (LPB). Devazepide abolished the FLI induced by CCK most of these brain regions, but had no effect on FLI induced by DFEN. These results suggest that the LPB-CeL/BST pathway might be responsible for the anorectic effects of both CCK and DFEN, but different parts or neuronal populations in these structures might be differentially engaged by CCK and DFEN. The putative interaction between CCK and 5HT might happen along this pathway, rather than in the periphery.     

Estrogen receptor and progesterone receptor-immunoreactive cells are not co-localized with gonadotropin-releasing hormone in the brain of the female mink (Mustela vison)

The distribution of gonadal steroid (estrogen, progesterone) receptors in the brain of the adult female mink was mapped by immunocytochemistry. Using a monoclonal rat antibody raised against human estrogen receptor (ER), the most dense collections of ER-immunoreactive (IR) cells were found in the preoptic/anterior hypothalamic area, the mediobasal hypothalamus (arcuate and ventromedial nuclei), and the limbic nuclei (amygdala, bed nucleus of the stria terminalis, lateral septum). Immunoreactivity was mainly observed in the cell nucleus and a marked heterogeneity of staining appeared from one region to another. A monoclonal mouse antibody raised against rabbit uterine progesterone receptor (PR) was used to identify the PR-IR cells in the preoptic/anterior hypothalamic area and the mediobasal hypothalamus (arcuate and ventromedial nuclei). This study also focused on the relationship between cells containing sex-steroid receptors and gonadotropin-releasing hormone (GnRH) neurons on the same sections of the mink brain using a sequential double-staining immunocytochemistry procedure. Although preoptic and hypothalamic GnRH neurons were frequently in close proximity to perikarya containing ER or PR, they did not themselves possess receptor immunoreactivity. The present study provides neuroanatomical evidence that GnRH cells are not the major direct targets for gonadal steroids and confirms for the first time in mustelids the results previously obtained in other mammalian species.