Aromatase activity in the rat brain: Hormonal regulation and sex differences

The intracellular conversion of testosterone to estradiol by the aromatase enzyme complex is an important step in many of the central actions of testosterone. In rats, estrogen given alone, or in combination with dihydrotestosterone, mimics most of the behavioral effects of testosterone, whereas treatment with antiestrogens or aromatase inhibitors block facilitation of copulatory behavior by testosterone. We used a highly sensitive in vitro radiometric assay to analyze the distribution and regulation of brain aromatase activity. Studies using micropunch dissections revealed that the highest levels of aromatase activity are found in an interconnected group of sexually dimorphic nuclei which constitutes a neural circuit important in the control of male sexual behavior. Androgen regulated aromatase activity in many diencephalic nucleic, including the medial preoptic nucleus, but not in the medial and cortical nuclei of the amygdala. Additional genetic evidence for both androgen-dependent and -independent control of brain AA was obtained by studies of androgen-insensitive testicular-feminized rats. These observations suggest that critical differences in enzyme responsiveness are present in different brain areas. Within several nuclei, sex differences in aromatase induction correlated with differences in nuclear androgen receptor concentrations suggesting that neural responsiveness to testosterone is sexually differentiated. Estradiol and dihydrotestosterone acted synergistically to regulate aromatase activity in the preoptic area. In addition, time-course studies showed that estrogen treatment increased the duration of nuclear androgen receptor occupation in the preoptic area of male rats treated with dihydrotestosterone. These results suggest possible ways that estrogens and androgens may interact at the cellular level to regulate neural function and behavior.     

Distribution of gastrin-releasing peptide immunoreactivity in the brain of the collared dove (Streptopelia decaocto)

The distribution of immunoreactivity after applying an antibody against gastrin-releasing peptide (GRP) was studied in the brain of the collared dove (Streptopelia decaocto). In the forebrain GRP-immunoreactive (GRP-ir) cells were found in the hyperstriatum accessorium, medial and lateral parts of the neostriatum, corticoidea dorsolateralis and temporoparieto-occipitalis areas, hippocampus, pre- and parahippocampal areas and prepiriform cortex. In the brainstem, GRP-ir cells were restricted mainly to the substantia nigra and ventral tegmental nucleus. Areas with densely packed GRP-ir clusters of varicosities were the medial intermediate hyperstriatum ventrale and lateral septal nucleus; dense GRP-ir neuropil was found in the parolfactory lobe, and in the dorsal half of the intermediate and caudal archistriatum. The ventral lamina medullaris contained many GRP-ir fibers. Forebrain areas devoid of immunoreactivity were the basal nucleus, ectostriatum, rostral archistriatum, most of the paleostriatum augmentatum and the lateral bed nucleus of the stria terminalis. Moderate densities of GRP-ir elements were found in the other telencephalic areas and further in, among others, the preoptic and hypothalamic region, ventral area of Tsai, cerulean nuclei, parabrachial complex, dorsal glossopharyngeal and vagus motor nuclei and medial nuclei of the solitary complex. The observations are compared with data from the literature and the implications for the definition of specific centers within the avian brain are discussed, with emphasis on systems with a role in visceral and motivational functions and in learning.     

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.     

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.     

The efferent connections of the lateral septal nucleus in the guinea pig: projections to the diencephalon and brainstem

Summary The anterograde Phaseolus vulgaris-leucoagglutinin (PHA-L) tracing technique was used to determine the distribution of efferent fibers originating in the lateral septal nucleus of the guinea pig. For complementary detection of the chemical identity of the target neurons, double-labeling immunocytochemistry was performed with antibodies to PHA-L and to vasopressin, oxytocin, vasoactive intestinal polypeptide, serotonin or dopamine -hydroxylase, respectively. The hypothalamus received the majority of the PHA-L-stained septofugal fibers. Here, a specific topography was observed. (1) The medial and lateral preoptic area, (2) the anterior, lateral, dorsal, posterior hypothalamic and retrochiasmatic area, (3) the supraoptic, paraventricular, suprachiasmatic, dorsomedial, caudal ventromedial and arcuate nuclei, and (4) the tuberomammillary, medial and lateral supramammillary, dorsal and ventral premammillary nuclei always contained PHA-L-labeled fibers. The rostral portion of the ventromedial nucleus and the medial and lateral mammillary nucleus only occasionally showed weak terminal labeling. In other diencephalic areas, termination of PHA-L-labeled fibers was observed in the epithalamus and the nuclei of the midline region of the thalamus. In the mesencephalon, terminal varicosities occurred in the ventral tegmental area, interfascicular and interpeduncular nucleus, and periaqueductal gray. In addition, the dorsal and medial raphe nuclei of the metencephalon, together with the locus coeruleus and the dorsal tegmental nucleus, received lateral septal efferents.     

Estradiol replacement enhances sleep deprivation-induced c-Fos immunoreactivity in forebrain arousal regions of ovariectomized rats

To understand how female sex hormones influence homeostatic mechanisms of sleep, we studied the effects of estradiol (E(2)) replacement on c-Fos immunoreactivity in sleep/wake-regulatory brain areas after sleep deprivation (SD) in ovariectomized rats. Adult rats were ovariectomized and implanted subcutaneously with capsules containing 17beta-E(2) (10.5 microg; to mimic diestrous E(2) levels) or oil. After 2 wk, animals with E(2) capsules received a single subcutaneous injection of 17beta-E(2) (10 microg/kg; to achieve proestrous E(2) levels) or oil; control animals with oil capsules received an oil injection. Twenty-four hours later, animals were either left undisturbed or sleep deprived by "gentle handling" for 6 h during the early light phase, and killed. E(2) treatment increased serum E(2) levels and uterus weights dose dependently, while attenuating body weight gain. Regardless of hormonal conditions, SD increased c-Fos immunoreactivity in all four arousal-promoting areas and four limbic and neuroendocrine nuclei studied, whereas it decreased c-Fos labeling in the sleep-promoting ventrolateral preoptic nucleus (VLPO). Low and high E(2) treatments enhanced the SD-induced c-Fos immunoreactivity in the laterodorsal subnucleus of the bed nucleus of stria terminalis and the tuberomammillary nucleus, and in orexin-containing hypothalamic neurons, with no effect on the basal forebrain and locus coeruleus. The high E(2) treatment decreased c-Fos labeling in the VLPO under nondeprived conditions. These results indicate that E(2) replacement modulates SD-induced or spontaneous c-Fos expression in sleep/wake-regulatory and limbic forebrain nuclei. These modulatory effects of E(2) replacement on neuronal activity may be, in part, responsible for E(2)'s influence on sleep/wake behavior.     

Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system

With the use of an antiserum generated in rabbits against synthetic human calcitonin gene-related peptide (CGRP) the distribution of CGRP-like immunoreactive cell bodies and nerve fibers was studied in the rat central nervous system. A detailed stereotaxic atlas of CGRP-like neurons was prepared. CGRP-like immunoreactivity was widely distributed in the rat central nervous system. CGRP positive cell bodies were observed in the preoptic area and hypothalamus (medial preoptic, periventricular, anterior hypothalamic nuclei, perifornical area, medial forebrain bundle), premamillary nucleus, amygdala medialis, hippocampus and dentate gyrus, central gray and the ventromedial nucleus of the thalamus. In the midbrain a large cluster of cells was contained in the peripeduncular area ventral to the medial geniculate body. In the hindbrain cholinergic motor nuclei (III, IV, V, VI, VII XII) contained CGRP-immunoreactivity. Cell bodies were also observed in the ventral tegmental nucleus, the parabrachial nuclei, superior olive and nucleus ambiguus. The ventral horn cells of the spinal cord, the trigeminal and dorsal root ganglia also contained CGRP-immunoreactivity. Dense accumulations of fibers were observed in the amydala centralis, caudal portion of the caudate putamen, sensory trigeminal area, substantia gelatinosa, dorsal horn of the spinal cord (laminae I and II). Other areas containing CGRP-immunoreactive fibers are the septal area, nucleus of the stria terminalis, preoptic and hypothalamic nuclei (e.g., medial preoptic, periventricular, dorsomedial, median eminence), medial forebrain bundle, central gray, medial geniculate body, peripeduncular area, interpeduncular nucleus, cochlear nucleus, parabrachial nuclei, superior olive, nucleus tractus solitarii, and in the confines of clusters of cell bodies. Some fibers were also noted in the anterior and posterior pituitary and the sensory ganglia. As with other newly described brain neuropeptides it can only be conjectured that CGRP has a neuroregulatory action on a variety of functions throughout the brain and spinal cord.     

Autoradiographic distribution of 125I calcitonin gene-related peptide binding sites in the rat central nervous system

Using autoradiographic method and 125I-Tyro rat CGRP as a ligand, receptor binding sites were demonstrated in the rat central nervous system. Saturation studies and Scatchard analysis of CGRP-binding to slide mounted tissue sections containing primarily cerebellum showed a single class of receptors with a dissociation constant of 0.96 nM and a Bmax of 76.4 fmol/mg protein. 125I-Tyro rat CGRP binding sites were demonstrated throughout the rat central nervous system. Dense binding was observed in the telencephalon (medial prefrontal, insular and outer layers of the temporal cortex, nucleus accumbens, fundus striatum, central and inferior lateral amygdaloid nuclei, most caudal caudate putamen, organum vasculosum laminae terminalis, subfornical organ), the diencephalon (anterior hypothalamic, suprachiasmatic, arcuate, paraventricular, dorsomedial, periventricular, reuniens, rhomboid, lateral thalamic pretectalis and habenula nuclei, zona incerta), in the mesencephalon (superficial layers of the superior colliculus, central nucleus of the geniculate body, inferior colliculus, nucleus of the fifth nerve, locus coeruleus, nucleus of the mesencephalic tract, the dorsal tegmental nucleus, superior olive), in the molecular layer of the cerebellum, in the medulla oblongata (inferior olive, nucleus tractus solitarii, nucleus commissuralis, nuclei of the tenth and twelfth nerves, the prepositus hypoglossal and the gracilis nuclei, dorsomedial part of the spinal trigeminal tract), in the dorsal gray matter of the spinal cord (laminae I-VI) and the confines of the central canal. Moderate receptor densities were found in the septal area, the "head" of the anterior caudate nucleus, medial amygdaloid and bed nucleus of the stria terminalis, the pyramidal layers of the hippocampus and dentate gyri, medial preoptic area, ventromedial nucleus, lateral hypothalamic and ventrolateral thalamic area, central gray, reticular part of the substantia nigra, parvocellular reticular nucleus. Purkinje cell layer of the cerebellum, nucleus of the spinal trigeminal tract and gracile fasciculus of the spinal cord. The discrete distribution of CGRP-like binding sites in a variety of sensory systems of the brain and spinal cord as well as in thalamic and hypothalamic areas suggests a widespread involvement of CGRP in a variety of brain functions.     

The distribution of melatonin binding sites in neuroendocrine tissues of the ewe

Specific high-affinity binding of 2-[125I]iodomelatonin (IMEL) was examined in 20-micrometer sections prepared from intact Suffolk ewes killed during late anestrus or the breeding season. The pars tuberalis contained by far the highest concentration of IMEL binding sites of all areas studied. Within the telencephalon, intense labeling was found in the mediolateral septum, the ventrolateral septal and septohypothalamic nuclei, the entorhinal cortex, the subiculum, and the inner and outer molecular layers of the hippocampus adjacent to the dentate gyrus. Melatonin binding in the medial preoptic nucleus, bed nucleus of the stria terminalis, and medial preoptic area was less striking but still distinct. Among diencephalic regions, melatonin binding sites existed in low concentrations in the anterior hypothalamus, the tuberal medial basal hypothalamus, and the paraventricular thalamic and supramammillary nuclei. Little binding was evident in the suprachiasmatic or ventromedial nuclei. In the midbrain, significant binding was restricted to the ventral raphe complex and the inferior colliculus. Little specific binding was found in the pars distalis or the pineal gland. The distribution of melatonin binding in the sheep brain is discussed in the context of the influence of this pineal hormone upon seasonal changes in neuroendocrine function.     

Noradrenergic afferents and receptors in the medial preoptic area: neuroanatomical and neurochemical links between the regulation of sleep and body temperature

Several studies have shown the importance of the medial preoptic area in the regulation of sleep-wakefulness and of body temperature. The medial preoptic area has a rich noradrenergic innervation, coming mostly from the lateral tegmental noradrenergic system. The accumulating evidences show that the noradrenergic afferents to the medial preoptic area are involved in the induction of sleep. This hypnogenic mechanism operates through the postsynaptic alpha1 and alpha2-adrenergic receptors. Noradrenergic afferents are also involved in the thermoregulatory mechanisms, and the activation of these fibers brings about a fall in body temperature. Though the body temperature changes are brought about by the same receptor subtypes as those involved in hypnogenesis, observations suggest the possibility of separate sets of noradrenergic afferents in the medial preoptic area for sleep regulation and thermoregulation. In this review, we present the compelling evidences, which showed that the noradrenergic afferents of the medial preoptic area bring about a fall in body temperature and other thermoregulatory behavioral alterations associated with sleep.     

Distribution of ADP-ribosylation factor-related protein 1 in mouse brain

ADP-ribosylation factor (Arf)-related protein 1 (ARFRP1) is a membrane-associated GTPase, which inhibits the Arf/Sec7-dependent activation of phospholipase D and belongs to the Arf-like (Arl) GTPases. Although ARFRP1 is involved in post-Golgi membrane trafficking and its lack leads to embryonic lethality, little is known about its possible function in the central nervous system. To obtain more knowledge about ARFRP1, we have characterized its mRNA distribution in adult mouse brain by in situ hybridization and real-time PCR. We observed a widespread distribution of ARFRP1-mRNA, with the highest levels in cerebral cortex, thalamic nuclei, colliculus, substantia nigra and granule cell layer of cerebellum. Moderate levels were observed in some amygdaloid nuclei, CA2 area and dentate gyrus of hippocampus, endopiriform nuclei, globus pallidus, striatum, molecular layer of cerebellum, and locus coeruleus, whereas no expression was detected in hypothalamic nuclei, CA1 and CA3 areas of hippocampus, zona incerta. A significant decrease of ARFRP1-mRNA was observed in cerebral cortex following sleep deprivation, whereas no change was observed in cerebellar cortex, locus courelus, brainstem, hippocampus and pontine nuclei. This study provides the first detailed analysis of the regional distribution of ARFRP1 in the mouse brain and a quantitative view of its changes following sleep deprivation.     

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

Summary 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.Supported by grants from the IBRO/MacArthur Foundation Network Grant, the National Science Foundation (NSF INT 8703030), the Hungarian Academy of Sciences (OTKA 104), the National Institutes of Health (NS 19266), the National Foundation of Technical Development (OKKFT Tt 286/1986) and the Well-come Trust (14685/1.5)     

Autoradiographic distribution of I-galanin binding sites in the rat central nervous system

Galanin (GAL) binding sites in coronal sections of the rat brain were demonstrated using autoradiographic methods. Scatchard analysis of ¹²⁵I-GAL binding to slide-mounted tissue sections revealed saturable binding to a single class of receptors with a Kd of approximately 0.2 nM. ¹²⁵I-GAL binding sites were demonstrated throughout the rat central nervous system. Dense binding was observed in the following areas: prefrontal cortex, the anterior nuclei of the olfactory bulb, several nuclei of the amygdaloid complex, the dorsal septal area, dorsal bed nucleus of the stria terminalis, the ventral pallidum, the internal medullary laminae of the thalamus, medial pretectal nucleus, nucleus of the medial optic tract, borderline area of the caudal spinal trigeminal nucleus adjacent to the spinal trigeminal tract, the substantia gelatinosa and the superficial layers of the dorsal spinal cord. Moderate binding was observed in the piriform, periamygdaloid, entorhinal, insular cortex and the subiculum, the nucleus accumbens, medial forebrain bundle, anterior hypothalamic, ventromedial, dorsal premamillary, lateral and periventricular thalamic nuclei, the subzona incerta, Forel's field H₁ and H₂, periventricular gray matter, medial and superficial gray strata of the superior colliculus, dorsal parts of the central gray, peripeduncular area, the interpeduncular nucleus, substantia nigra zona compacta, ventral tegmental area, the dorsal and ventral parabrachial and parvocellular reticular nuclei. The preponderance of GAL-binding in somatosensory as well as in limbic areas suggests a possible involvement of GAL in a variety of brain functions.     

Differential distribution and regulation of OX1 and OX2 orexin/hypocretin receptor messenger RNA in the brain upon fasting

To further understand the functions of the orexin/hypocretin system, we examined the expression and regulation of the orexin/hypocretin receptor (OX1R and OX2R) mRNA in the brain by using quantitative in situ hybridization. Expression of OX1R and OX2R mRNA exhibited distinct distribution patterns. Within the hypothalamus, expression for the OX1R mRNA was largely restricted in the ventromedial (VMH) and dorsomedial hypothalamic nuclei, while high levels of OX2R mRNA were contained in the paraventricular nucleus, VMH, and arcuate nucleus as well as in mammilary nuclei. In the amygdala, OX1R mRNA was expressed throughout the amygdaloid complex with robust labeling in the medial nucleus, while OX2R mRNA was only present in the posterior cortical nucleus of amygdala. High levels of OX2R mRNA were also observed in the ventral tegmental area. Moreover, both OX1R and OX2R mRNA were observed in the hippocampus, some thalamic nuclei, and subthalamic nuclei. Furthermore, we analyzed the effect of fasting on levels of OX1R and OX2R mRNA in the hypothalamic and amygdaloid subregions. After 20 h of fasting, levels of OX1R mRNA were significantly increased in the VMH and the medial division of amygdala. An initial decrease (14 h) and a subsequent increase (20 h) in OX1R mRNA levels after fasting were observed in the dorsomedial hypothalamic nucleus and lateral division of amygdala. Levels of OX2R mRNA were augmented in the arcuate nucleus, but remained unchanged in the dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus, and amygdala following fasting. The time-dependent and region-specific regulatory patterns of OX1R and OX2R suggest that they may participate in distinct neural circuits under the condition of food deprivation.     

Cardiovascular actions of intrahypothalamic injections of atrial natriuretic peptide

Accumulating evidence implicates atrial natriuretic polypeptide (ANP) as a neurotransmitter in brain. The presence and distribution of ANP, its high affinity binding sites, and the messenger RNA of its precursor have been described in the central nervous system. However, the function(s) of ANP in specific brain areas is largely unknown. We have now determined the cardiovascular effects elicited by microinjection of atriopeptin-III (ANP-III) in hypothalamic and preoptic areas in rats. ANP-III (40 pmol) increased heart rate when injected into the anteromedial preoptic nucleus (AMPO), the medial preoptic area (MPA), the periventricular area, and in two regions of the dorsal hypothalamus. Other nuclei within the hypothalamus were unresponsive. The tachycardic effects elicited by AMPO-MPA injection of ANP-III were abolished by adrenalectomy. These data indicate that ANP-III acts at discrete sites to elicit tachycardia and the mechanism of action for at least one brain site appears to be through central pathways which selectively activate the adrenal gland.     

Exposure to female pheromones stimulates a specific type of neuronal population in the male but not female magnocellular division of the medial preoptic nucleus (MPN mag) of the Syrian hamster

The magnocellular division of the medial preoptic area (MPN mag) integrates pheromonal and hormonal signals to play a critical role in the expression of male typical sex behavior. The MPN mag contains two morphologically distinct neuronal populations; the percentage of each type within the nucleus is sex specific. Males have more neurons with a single nucleolus whereas females have more with multiple nucleoli. To determine which neuronal subtype mediates pheromonal induction of copulation, tissue from male and female hamsters exposed to female pheromones was immunolabeled for the immediate early protein (EGR-1). Subsequently the tissue was counterstained and the number of ERG-1 neurons with one or two nuclei was determined. The results indicate that pheromones stimulate neurons with single nucleoli in males but fail to stimulate either neuronal subtype in females suggesting that synaptic input to the MPN mag is sexually differentiated.