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.     

Individual differences in estrogen receptor alpha in select brain nuclei are associated with individual differences in aggression

Steroid hormones play an important role in modulating social behavior in many species. Estrogens are thought to act on an interconnected network of hypothalamic and limbic brain areas to affect aggressive behavior, although the specific nuclei unknown remain unspecified. We show that individual variation in estrogen receptor alpha (ERalpha) immunoreactivity in the lateral septum (LS), ventral bed nucleus of the stria terminalis (vBNST), and anterior hypothalamus (AHA) of CD-1 mice is positively correlated with aggressive behavior. When males were treated with fadrozole (an aromatase inhibitor), aggressive behavior was reduced, although castration did not reduce aggression. These results suggest that estrogens modulate aggressive behavior by acting on a circuit that includes the LS, vBNST, and AHA and that the source of estrogens is non-gonadal. Fadrozole also decreased c-fos expression in the lateral septum following aggressive encounters. Although the effects of estrogen on aggression appear to involve regulation of neuronal activity in the LS, additional processes are likely involved. These results suggest that estrogen acts in a specific subset of a complex network of nuclei to affect aggressive behavior.     

Increased estrogen receptor alpha immunoreactivity in the forebrain of sexually satiated rats

Estrogen receptor alpha (ERalpha) participates in the neuroendocrine regulation of male sexual behavior, primarily in brain areas located in the limbic system. Males of many species present a long-term inhibition of sexual behavior after several ejaculations, known as sexual satiety. It has been shown that androgen receptor density is reduced 24 h after a single ejaculation or mating to satiety, in the medial preoptic area, nucleus accumbens and ventromedial hypothalamus. The aim of this study was to analyze if the density of ERalpha was also modified 24 h after a single ejaculation or mating to satiety. Sexual satiety was associated with an increased ERalpha density in the anteromedial bed nucleus of the stria terminalis (BSTMA), ventrolateral septum (LSV), posterodorsal medial amygdala (MePD), medial preoptic area (MPA) and nucleus accumbens core (NAc). A single ejaculation was related to an increase in ERalpha density in the BSTMA and MePD. ERalpha density in the arcuate (Arc) and ventromedial hypothalamic nuclei (VMN), and serum estradiol levels remained unchanged 24 h after one ejaculation or mating to satiety. These data suggest a relationship between sexual activity and an increase in the expression of ERalpha in specific brain areas, independently of estradiol levels in systemic circulation.     

Sex-Steroid Receptor Mechanism Related to Neuronal Aromatase and the Stigmoid Body

Recent immunohistochemical analyses of rat brains have established the presence of neuronal aromatase and stigmoid body in interfaces of a sex-steroid/brain axis. Aromatase P450-immunoreactive (AROM-I) neurons, which are present only during embryonic Day 16 to postnatal Day 2 (E16-P2), are found in the anterior medial preoptic nucleus, the periventricular preoptic nucleus, and the ventromedial hypothalamic nucleus. The largest AROM-I cell-group localized in the medial preoptico-amygdaloid neuronal arc (mPO-AM) shows a peak during E18-P2 and gradually diminishes after the perinatal days (but still retains the immunoreactivity in adults). In adults, other groups of AROM-I neurons emerge in the lateral septum, the central amygdaloid nucleus, and the bed nucleus of the stria terminalis. Male-predominant sex difference for aromatase expression has been detected at least in the young-to-adult mPO-AM, reflecting sexually distinct endocrine and behavioral responses in the rat reproductive functions. These results suggest the presence of distinct brain-aromatases with different regulatory systems. Neuronal aromatase may modulate development of sexual dimorphism in neonates and activation of reproductive functions in adults. The stigmoid bodies, marked by placental aromatase-associated antigen X-P₂ (PAX) antiserum, are also frequent in the sex-steroid targets of rat brains and intimately coexist with estrogen receptors in the mPO-AM of young females. The neuronal inclusion, suspected as a RNA/protein conglomerate, appears to be induced by the decrease of androgen and/or the increase of estrogen, especially as a result of subneuronal aromatic switching into the estrogenous state. The neuronal aromatase and stigmoid body may play important roles in the pre- and post-receptor steps of subneuronal sex-steroids actions for brain sexual differentiation or induction of reproductive functions.     

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.     

Obesity-inducing amygdala lesions: examination of anterograde degeneration and retrograde transport

Small lesions centered in the posterodorsal region of the medial amygdala resulted in excessive weight gains in female rats. Unilateral lesions were nearly as effective as bilateral lesions in the first 48 h after surgery (+21 to +32 g). Assessment of lesion damage was done by both qualitative evaluation and by a quantitative grid-point counting method. The critical sites for weight gain were the intra-amygdaloid bed nucleus of the stria terminalis and the posterodorsal medial amygdaloid nucleus. Incidental damage to the overlying globus pallidus was negatively related to weight gain. The cupric silver method for demonstrating axonal degeneration was applied to brains with obesity-inducing lesions. A dense pattern of degenerating terminals was found in the lateral septum, amygdala, ventral striatum, and ventromedial hypothalamus. Degeneration in the paraventricular nucleus of the hypothalamus was scarce or absent. Small retrograde tracer injections made in either the intra-amygdaloid bed nucleus of the stria terminalis or in the posterodorsal medial amygdaloid nucleus labeled cells in the amygdala, lateral septum, and hypothalamus, reciprocating the anterograde projections from the amygdala to these areas. The data suggest that subdivisions of the posterodorsal amygdala participate in the regulation of feeding in a manner that is similar to the better-known role of this part of the brain in mediating reproductive behavior. Although topographical differences may exist within the amygdaloid and hypothalamic subdivisions regulating these two sexually dimorphic behaviors, the relays engaged by feeding-related connections and those related to reproduction are remarkably parallel.     

Gonadal steroid-dependent neuronal circuitries in avian limbic and preoptic regions.

In the avian limbic and preoptic region, the sexually dimorphic medial preoptic nucleus and nucleus of the stria terminalis are characterized by the presence of a testosterone-dependent aromatase-immunoreactive neuronal population. In situ hybridization studies confirmed that testosterone is modulating the expression of aromatase gene. Both nuclei are also characterized by a sexually dimorphic, testosterone-dependent vasotocin system. Immunocytochemical and in situ hybridization data, demonstrated that dimorphism and testosterone-sensitivity of this system are both dependent by an embryonic organizational effect of the estradiol. Intracerebroventricularly injected vasotocin, has a profound inhibitory effect on the male sexual behaviour, and immunocytochemical investigations revealed close associations among vasotocin fibres and aromatase cell bodies. These data suggest that this neuropeptidergic system could have a key role in the circuitries controlling different aspects of male reproductive behaviour in the Japanese quail.     

Evidence for an absence of estrogen-concentration by CCK-immunoreactive neurons in the hypothalamus of the female rat

It is becoming increasingly clear that the neuropeptide cholecystokinin (CCK), widely distributed in the rat hypothalamus and limbic system, is subject to both organizational and activational influences of steroid hormones. Sex differences in numbers of CCK-immunoreactive elements have been demonstrated in sexually dimorphic structures such as the bed nucleus of the stria terminalis, medial preoptic nucleus, and ventromedial nucleus of the hypothalamus. Steroid activation of CCK has been indicated by findings that hypothalamic CCK levels and binding capacity vary over the estrous cycle. These studies, in combination with evidence of CCK mediation of sexually differentiated functions, prompted us to test for estrogen concentration among CCK-containing cells of the female rat hypothalamus by combining the techniques of immunohistochemistry and autoradiography. A method employing 2-week ovariectomies and perfusion fixation with 4% paraformaldehyde was compatible with the localization of both estrogen-accumulating and CCK-immunoreactive cell bodies. The maintenance of numbers of CCK-positive cells after gonadectomy suggested that expression of this peptide may not be directly regulated by ovarian steroids in female rats. This suggestion was substantiated by the finding that, with rare exceptions, CCK-immunoreactive cells did not concentrate estrogen in tissues collected from the anterior-posterior extent of the bed nucleus of the stria terminalis, medial preoptic nucleus, anterior hypothalamic area, and paraventricular nucleus.     

Immunocytochemical localization of growth hormone releasing factor (GHRF)-containing structures in the rat brain using anti-rat GHRF serum

The distribution of growth hormone releasing factor (GHRF) immunoreactive structures in the rat hypothalmus was studied after colchicine treatment with PAP immunocytochemistry in vibratome sections using an antiserum directed to rat hypothalamic GHRF. The majority of the GHRF-immunoreactive cell bodies were found in the arcuate nucleus, the medial perifornical region, and the ventral premammillary nuclei of the hypothalamus. Scattered cells were seen in the lateral basal hypothalamus, the medial and lateral portions of the ventromedial nucleus, and the dorsomedial and paraventricular nuclei. Immunoreactive fibers were observed in all the regions mentioned above. GHRF terminals were located in the central region of the median eminence. In addition, GHRF-immunoreactive neuronal processes were seen in the ventral region of the dorsomedial nucleus, the medial preoptic and suprachiasmatic regions, dorsal portion of the suprachiasmatic nucleus, bed nucleus of the stria terminals and the hypothalamic portion of the stria terminals. The localization of GHRF-immunoreactive terminals in the median eminence reinforces the view that GHRF plays a physiological role in the regulation of pituitary function. In addition, the localization of GHRF-immunoreactive structures in areas not usually considered to project to the median eminence suggest that GHRF may act as a neuromodulator or neurotransmitter.     

Immunocytochemical localization of somatostatin in human brain

The distribution of somatostatin (SRIF) was examined in normal human forebrain, using thick vibratome cut sections. The unlabeled antibody enzyme method of immunocytochemistry revealed a widespread distribution of SRIF immunoreactive neurons and fibers throughout the septum, diencephalon and corpus striatum. Within the septum SRIF neurons and fibers were observed in the medial and lateral septal nuclei, the nucleus of the diagonal band, the nucleus accumbens and the bed nucleus of the stria terminalis. SRIF neurons and fibers were found in several hypothalamic and anterior thalamic nuclei as well as all regions of the corpus striatum. An interesting collection of SRIF immunoreactive neurons and processes were observed forming a wide band extending anteriorly from the lateral preoptic area through the lateral hypothalamus and substantia innominata posteriorly. This report on the localization of immunoreactive SRIF in the human forebrain extends previous anatomical findings and lends morphological support to recent biochemical studies.     

Expression of corticosteroid-binding protein in the human hypothalamus, co-localization with oxytocin and vasopressin.

Corticosteroid-binding globulin, a specific steroid carrier in serum with high binding affinity for glucocorticoids, is expressed in various tissues. In the present study, we describe the immunocytochemical distribution of this protein in neurons and nerve fibers in the human hypothalamus. CBG immunoreactive perikarya and fibers were observed in the paraventricular, supraoptic, and sexual dimorphic nuclei in the perifornical region, as well as in the lateral hypothalamic and medial preoptic areas, the region of the diagonal band, suprachiasmatic and ventromedial nuclei, bed nucleus of the stria terminalis and some epithelial cells from the choroid plexus and ependymal cells. Stained fibers occurred in the median eminence and infundibulum. Double immunostaining revealed a partial co-localization of corticosteroid-binding globulin with oxytocin and, to a lesser extent, with vasopressin in the paraventricular and the supraoptic nuclei. Double immunofluorescence staining showed coexistence of these substances in axonal varicosities in the median eminence. We conclude that neurons of the human hypothalamus are capable of expressing corticosteroid-binding globulin, in part co-localized with the classical neurohypophyseal hormones. The distribution of CBG immunoreactive neurons, which is widespread but limited to specific nuclei, indicates that CBG has many physiological functions that may include neuroendocrine regulation and stress response.     

The simultaneous quantification of dopamine,norepinephrine and epinephrine in micropunched rat brain nuclei by on-line trace enrichment HPLC with electrochemical detection: Distribution of catecholamines in the limbic system

A method is described for the simultaneous quantification of dopamine, norepinephrine and epinephrine in microdissected rat brain nuclei using on-line trace enrichment high performance liquid chromatography (HPLC) and electrochemical detection (EC). The method is specific, sensitive and rapid with lower limits of detection comparable to those of radioenzymatic and mass fragmentographic techniques. The ideal application of the method to determinations in discrete microdissected brain nuclei is illustrated by mapping the topographic distribution of these three catecholamines in the limbic system of the rat. Dopamine is well represented and remarkably heterogeneously distributed in the amygdaloid nuclei. Norepinephrine concentrations are high in the interstitial (bed) nucleus of the stria terminalis and the paraventricular, periventricular, dorsomedial and medial preoptic hypothalamic nuclei, though generally more evenly distributed among the limbic nuclei than dopamine. Epinephrine was reliably quantified in a minority of the limbic nuclei examined with the highest concentration found in the paraventricular hypothalamic nuclei. The method described is ideally suited for examination of the pharmacological and functional heterogeneity of dopamine-, norepinephrine- and epinephrine-containing neuronal systems at the level of discrete brain nuclei.     

Localization of proopiomelanocortin (POMC) immunoreactive neurons in the forebrain of the pig

The distribution of proopiomelanocortin (POMC)-immunoreactive neurons was examined in the forebrains of nine sexually mature female pigs by indirect biotin-avidin horseradish peroxidase immunocytochemistry. Primary antiserum against ovine beta-endorphin (Bioflex #BF-EP-3-1) yielded positive staining of neuronal perikarya and processes. Adjacent control sections treated either with primary antiserum preabsorbed with beta-endorphin or substituted with normal rabbit serum lacked specific staining. POMC-immunoreactive cells were located in the anterior and intermediate lobe of the pituitary gland. POMC-immunoreactive perikarya were located in the arcuate nucleus and periarcuate area. The pituitary stalk/median eminence contained sparsely distributed POMC-immunoreactive fibers, which were confined to the zona interna. POMC-immunoreactive fibers were located in the arcuate nucleus and extended rostrally from the arcuate nucleus into the telencephalon coursing adjacent to the wall of the third ventricle as well as through the anterior hypothalamus, suprachiasmatic, supraoptic nuclei and preoptic areas to the nucleus accumbens, diagonal band of Broca, olfactory tubercle, bed nucleus of the stria terminalis and the ventro-lateral aspect of the septum. Caudal projections extended along the wall of the third ventricle to the level of the mammillary bodies and also coursed dorsally, passing through the periventricular, paraventricular, and dorsal medial nuclei of the hypothalamus to the midline thalamic nuclei and habenular nucleus. Lateral projections extended from the arcuate nucleus along the dorsal aspect of the optic tract and terminated in the amygdaloid complex. The distribution of POMC-immunoreactive perikarya and fibers is similar to that of the luteinizing hormone-releasing hormone (LHRH) fiber network. Therefore the opportunities exist, anatomically, for interactions between the POMC and the LHRH systems.     

Immunocytochemical localization of the gonadotropin-releasing hormone-associated peptide portion of the LHRH precursor in the hypothalamus and extrahypothalamic regions of the rat central nervous system

Summary The gonadotropin-releasing hormone-associated peptide (GAP) of the LHRH precursor and the decapeptide LHRH were localized in the rat brain by immunocytochemistry in 12 to 18-day-old animals, by use of thick Vibratome sections and nickel intensification of the diaminobenzidinereaction product. Our results indicate that the GAP portion of the LHRH precursor is present in the same population of neurons that contain LHRH in the rat brain. An important difference observed was that the GAP antiserum, in contrast to LHRH antisera, stained several perikarya in the medial basal hypothalamus. GAP-immunoreactive perikarya were observed in the following regions: the olfactory bulb and tubercle, diagonal band of Broca, medial septum, medial preoptic and suprachiasmatic areas, anterior and lateral hypothalamus, and several regions of the hippocampus. In addition to the preoptico-terminal and the septopreoptico-infundibular pathways, we also observed GAPimmunopositive processes in several major tracts and areas of the brain, including the amygdala, stria terminalis, stria medullaris thalami, fasciculus retroflexus, stria longitudinalis medialis, periventricular plexus, periaqueductal gray of the mesencephalon and extra-cerebral regions, such as the nervus terminalis and its associated ganglion. These results confirm the specificity of previous immunocytochemical results obtained with antisera to LHRH. The presence of GAP immunoreactivity in nerve terminals of the rat brain indicates that GAP or a GAP-like peptide is located in the proper site to serve as a hypophysiotropic substance and/or as a neurotransmitter or neuromodulator.Supported by AKA No. 419427, OTKA No. 104, OKKFT 2.1.5.1 and NSF No. INT-8602688     

Ultrastructural analysis of estrogen receptor immunoreactive neurons in the medial preoptic area of the female rat brain

Neurons of the medial preoptic area were studied in the brain of the female rat by means of ultrastructural immunocytochemistry using a monoclonal antibody generated against purified estrogen receptor (ER), in order to delineate the morphological correlates of estrogen feedback mechanisms. In addition to the preoptic area, the bed nucleus of the stria terminalis, the arcuate and ventromedial nuclei of the hypothalamus exhibited an intense labelling for estrogen receptor. At the light microscopic level, the cell nuclei were immunoreactive. No major alterations were detected in the ER expression of medial preoptic neurons sampled during the estrous cycle, but proestrous rats did exhibit a slightly increased intensity of staining. At the ultrastructural level, the ER immunoreactivity was primarily confined to the nuclei and associated with the chromatin. Long term steroid deprivation elicited by either ovariectomy or ovariectomy plus adrenalectomy resulted in a marked intensity of nuclear labelling. This pattern was not influenced by acute estradiol replacement. These morphological data indicate that neurons of the medial preoptic area have the capacity to detect estrogens via receptor mechanisms and that changes in the level of the circulating ligand are manifested in an alteration in the staining for the estrogen receptor. The study also supports the revised concept of estrogen receptor action by demonstrating the presence of receptors in the nuclei of the cells, whether or not they are occupied by their ligand.     

Immunoreactive pancreatic polypeptide (PP) occurs in the central and peripheral nervous system: Preliminary immunocytochemical observations

Summary Pancreatic polypeptide (PP) is a candidate hormone of unknown physiological significance. It is produced by a population of endocrine cells in the pancreas. In the present study a PP-like peptide was found to occur in the mammalian and avian central and peripheral nervous systems. Immunoreactive nerve fibres and nerve cell bodies were widely distributed in the brain. Dense accumulations of nerve fibres occurred in the following areas: nucleus accumbens, interstitial nucleus of the stria terminalis, para- and periventricular hypothalamic nuclei, and medial preoptic area. In addition, nerve fibres were regularly seen in cortical areas. Immunoreactive perikarya were observed in the following regions: cortex, nucleus accumbens, neostriatum and septum. In the gut, immunoreactive nerve fibers were distributed in the myenteric plexus, in smooth muscle, around blood vessels, and in the core of the villi. Immunoreactive perikarya occurred in the submucosal and myenteric plexus, suggesting that PP immunoreactive nerves are intrinsic to the gut.In the species examined, the neuronal PP-like peptide could be demonstrated with an antiserum raised against avian PP, but not with those raised against bovine or human PP. Thus, neuronal PP is distinct from the PP that occurs in pancreatic endocrine cells.     

Androgen receptors are required for full masculinization of nitric oxide synthase system in rat limbic-hypothalamic region

The neuronal nitric oxide synthase (nNOS) is involved in the control of male and female sexual behavior and its distribution in several regions of the limbic–hypothalamic system, as well as its coexistence with gonadal hormones' receptors, suggests that these hormones may play a significant role in controlling its expression. However, data illustrating the role of gonadal hormones in controlling the nNOS expression are, at present, contradictory, even if they strongly suggest an involvement of testosterone (T) in the regulation of nNOS. The action of T may be mediated through androgen (AR) or, after aromatization to estradiol (E₂), through estrogen receptors.To elucidate the role of AR on nNOS expression, we compared male and female rats with a non-functional mutation of AR (Tfm, testicular feminization mutation) to their control littermates. We investigated some hypothalamic and limbic nuclei involved in the control of sexual behavior [medial preoptic area (MPA), paraventricular (PVN), arcuate (ARC), ventromedial (VMH) and stria terminalis (BST) nuclei]. In BST (posterior subdivision), VMH (ventral subdivision), and MPA we detected a significant sexual dimorphism in control animals and a decrease of nNOS positive elements in Tfm males compared to their littermate. In addition, we observed a significant increase of nNOS positive elements in BST (posterior) of Tfm females. No significant changes were observed in the other nuclei. These data indicate that, contrary to current opinions, androgens, through the action of AR may have a relevant role in the organization and modulation of the nNOS hypothalamic system.     

Sucrose self-administration and CNS activation in the rat

We have previously reported that administration of insulin into the arcuate nucleus of the hypothalamus decreases motivation for sucrose, assessed by a self-administration task, in rats. Because the pattern of central nervous system (CNS) activation in association with sucrose self-administration has not been evaluated, in the present study, we measured expression of c-Fos as an index of neuronal activation. We trained rats to bar-press for sucrose, according to a fixed-ratio (FR) or progressive-ratio (PR) schedule and mapped expression of c-Fos immunoreactivity in the CNS, compared with c-Fos expression in handled controls. We observed a unique expression of c-Fos in the medial hypothalamus (the arcuate, paraventricular, retrochiasmatic, dorsomedial, and ventromedial nuclei) in association with the onset of PR performance, and expression of c-Fos in the lateral hypothalamus and the bed nucleus of stria terminalis in association with the onset of FR performance. c-Fos expression was increased in the nucleus accumbens of both FR and PR rats. Our study emphasizes the importance of both hypothalamic energy homeostasis circuitry and limbic circuitry in the performance of a food reward task. Given the role of the medial hypothalamus in regulation of energy balance, our study suggests that this circuitry may contribute to reward regulation within the larger context of energy homeostasis.     

Gonadotropin-releasing hormone (GnRH) neurons and pathways in the rat brain

Summary Gonadotropin-releasing hormone (GnRH) neurons and their pathways in the rat brain were localized by immunocytochemistry in 6-to 18-day-old female animals, by use of thick frozen or vibratome sections, and silver-gold intensification of the diaminobenzidine reaction product. GnRH-immunoreactive perikarya were observed in the following regions: olfactory bulb and tubercle, vertical and horizontal limbs of the diagonal band of Broca, medial septum, medial preoptic and suprachiasmatic areas, anterior and lateral hypothalamus, and different regions of the hippocampus (indusium griseum, Ammon's horn). In addition to the known GnRH-pathways (preoptico-terminal, preoptico-infundibular, periventricular), we also observed GnRH-immunopositive processes in several major tracts and areas of the brain, including the medial and cortical amygdaloid complex, stria terminalis, stria medullaris thalami, fasciculus retroflexus, medial forebrain bundle, indusium griseum, stria longitudinalis medialis and lateralis, hippocampus, periaqueductal gray of the mesencephalon, and extracerebral regions, such as the lamina cribrosa, nervus terminalis and its associated ganglia. By use of the silver-gold intensification method we present Golgi-like images of GnRH perikarya and their pathways. The possible distribution of efferents from each GnRH cell group is discussed.     

Aromatase-immunoreactive cells are present in mouse brain areas that are known to express high levels of aromatase activity

The transformation of testosterone into estradiol in the brain plays a key role in several behavioral and physiological processes, but it has been so far impossible to localize precisely the cells of the mammalian brain containing the relevant enzyme, viz., aromatase. We have recently established an immunohistochemical technique that allows the visualization of aromatase-immunoreactive cells in the quail brain. In this species, a marked increase in the optical density of aromatase-immunoreactive cells is observed in subjects that have been treated with the aromatase inhibitor, R76713 or racemic Vorozole. This increased immunoreactivity, associated with a total blockade of aromatase activity, has been used as a tool in the present study in which the distribution of aromatase-immunoreactive material has been reassessed in the brain of mice pretreated with R76713. As expected, the aromatase inhibitor increases the density of the immunoreactive signal in mice. Strongly immunoreactive cells are found in the lateral septal region, the bed nucleus of the stria terminalis, the central amygdala, and the dorso-lateral hypothalamus. A less dense signal is also present in the medial preoptic area, the nucleus accumbens, several hypothalamic nuclei (e.g., paraventricular and ventromedial nuclei), all divisions of the amygdala, and several regions of the cortex, especially the cortex piriformis. These data demonstrate that, contrary to previous claims, aromatase-immunoreactive cells are present in all brain regions that have been shown previously to contain high aromatase activity.