Influence of estradiol on NADPH diaphorase/neuronal nitric oxide synthase activity and colocalization with progesterone or type II glucocorticoid receptors in ovine hypothalamus

Nitric oxide (NO) has been shown to play an important role in both the neuroendocrine reproductive and stress axes, which are closely linked. Because progesterone (P4) receptors (PRs) and glucocorticoid receptors (GRs) are not found in GnRH neurons and the NOergic system has been implicated in the control of GnRH secretion, this study aimed to ascertain whether steroids altered the NOergic system. Our first objective was to map the distribution of NO synthase (NOS) cells in the ovine preoptic area (POA) and hypothalamus and to determine whether NOS activity is enhanced by estradiol (E2) treatment. Using NADPH diaphorase (NADPHd) histochemistry, we found that NADPHd-positive neurons were spread throughout the ovine POA and hypothalamus, and that all NADPHd cells were immunoreactive for NOS. In response to estradiol, a significant increase in the number of NADPHd cells was noted only in the ventrolateral region of the ventromedial nucleus (VMNvl), with no significant difference in the POA or arcuate nucleus. Progesterone and glucocorticoid receptors were colocalized with NADPHd reactive neurons in the POA, arcuate nucleus, and VMNvl of ewes in both treatment groups. In ewes receiving estradiol, the number of NADPHd-positive cells containing steroid receptors in the POA (PR, 81%; GR, 79%) and arcuate nucleus (PR, 89%; GR, 84%) was similar, but in the VMNvl, fewer NADPHd-positive cells contained GR (PR, 88%, GR, 31%). These data show that estradiol up-regulates NOS activity in a site-specific manner and that the influence and possible interaction of progesterone and corticosteroids on NO producing cells may differ according to the neural location.     

Somatostatin-14 neurons in the ovine hypothalamus: colocalization with estrogen receptor alpha and somatostatin-28(1-12) immunoreactivity,and activation in response to estradiol

Pituitary gland growth hormone (GH) secretion is influenced by two hypothalamic neuropeptides: growth hormone-releasing hormone (GHRH) and somatostatin. Recent data also suggest that estrogen modulates GH release, particularly at the time of the preovulatory luteinizing hormone surge, when a coincident surge of GH is observed in sheep. The GHRH neurons do not possess estrogen receptor alpha (ERalpha), suggesting that estrogen does not act directly on GHRH neurons. Similarly, few somatotropes express ERalpha, suggesting a weak pituitary effect of estradiol on GH. It was hypothesized, therefore, that estradiol may affect somatostatin neurons to modulate GH release from the pituitary. Using immunocytochemical approaches, the present study revealed that although somatostatin neurons were located in several hypothalamic sites, only those in the arcuate nucleus (13% +/- 2%) and ventromedial nucleus (VMN; 29% +/- 1%) expressed ERalpha. In addition, we found that all neurons immunoreactive for somatostatin-14 were also immunoreactive for somatostatin-28(1-12). To determine whether increased GH secretion in response to estradiol is through modulation of GHRH and/or somatostatin neuronal activity, a final study investigated whether c-fos expression increased in somatostatin- and GHRH-immunoreactive cells at the time of the estradiol-induced LH surge in intact anestrous ewes. Estradiol significantly (P < 0.05) increased the percentage of GHRH (estradiol, 75% +/- 3%; no estradiol, 19% +/- 2%) neurons expressing c-fos in the hypothalamus. The percentage of somatostatin-immunoreactive neurons coexpressing c-fos in the estradiol-treated animals was significantly (P < 0.05) higher (periventricular, 44% +/- 3%; arcuate, 72% +/- 5%; VMN, 81% +/- 5%) than in the control animals (periventricular, 22% +/- 1%; arcuate, 29% +/- 3%; VMN, 31% +/- 3%). The present study suggests that estradiol modulates the activity of GHRH and somatostatin neurons but that this effect is most likely mediated through an indirect interneuronal pathway.     

Orphanin FQ in the mediobasal hypothalamus facilitates sexual receptivity through the deactivation of medial preoptic nucleus mu-opioid receptors

Sexual receptivity, lordosis, can be induced by sequential estradiol and progesterone or extended exposure to high levels of estradiol in the female rat. In both cases estradiol initially inhibits lordosis through activation of β-endorphin (β-END) neurons of the arcuate nucleus of the hypothalamus (ARH) that activate μ-opioid receptors (MOP) in the medial preoptic nucleus (MPN). Subsequent progesterone or extended estradiol exposure deactivates MPN MOP to facilitate lordosis. Opioid receptor-like receptor-1 (ORL-1) is expressed in ARH and ventromedial hypothalamus (VMH). Infusions of its endogenous ligand, orphanin FQ (OFQ/N, aka nociceptin), into VMH–ARH region facilitate lordosis. Whether OFQ/N acts in ARH and/or VMH and whether OFQ/N is necessary for steroid facilitation of lordosis are unclear. In Exp I, OFQ/N infusions in VMH and ARH that facilitated lordosis also deactivated MPN MOP indicating that OFQ/N facilitation of lordosis requires deactivation of ascending ARH-MPN projections by directly inhibiting ARH β-END neurons and/or through inhibition of excitatory VMH–ARH pathways to proopiomelanocortin neurons. It is unclear whether OFQ/N activates the VMH output motor pathways directly or via the deactivation of MPN MOP. In Exp II we tested whether ORL-1 activation is necessary for estradiol-only or estradiol + progesterone lordosis facilitation. Blocking ORL-1 with UFP-101 inhibited estradiol-only lordosis and MPN MOP deactivation but had no effect on estradiol + progesterone facilitation of lordosis and MOP deactivation. In conclusion, steroid facilitation of lordosis inhibits ARH β-END neurons to deactivate MPN MOP, but estradiol-only and estradiol + progesterone treatments appear to use different neurotransmitter systems to inhibit ARH-MPN signaling.     

Gonadal hormone regulation of neuronal-glial interactions in the developing neuroendocrine hypothalamus

Recent evidence indicates that, in addition to their well known effects on neurons, gonadal steroids may exert part of their neural effects through astroglia. In adult female rats astroglia participate in the phasic remodelling of synapses that takes place during the estrous cycle in the arcuate nucleus of the hypothalamus under the influence of estradiol. Astroglia also appear to be involved in the genesis of sex differences in synaptic connectivity. Gonadal steroids influence hypothalamic astroglia differentiation in vitro and in vivo. In monolayer mixed neuronal-glial cultures from fetal rat hypothalami, estradiol induces a progressive differentiation of astrocytes from a flattened epithelioid morphology to bipolar, radial and stellate shapes. This effect of estradiol on astroglia is dependent on the expression of specific molecules on the neuronal surface, such as the polysialic acid-rich form of the neural cell adhesion molecule. In the rat arcuate nucleus in situ, perinatal androgen influences astroglia gene expression and differentiation, resulting in a sex difference in astroglia organization by postnatal day 20. By this day, the amount of neuronal surface covered by astroglial processes is higher in males than in females. This difference in the coverage of neuronal surface by astroglia may be directly related to the reduced number of synaptic contacts that is established on the soma of male neurons compared to females.     

Progesterone receptor,estrogen receptor alpha,and the type II glucocorticoid receptor are coexpressed in the same neurons of the ovine preoptic area and arcuate nucleus: a triple immunolabeling study

The neuroendocrine reproductive and stress axes are known to be closely linked, but the mechanisms underlying these links remain poorly understood. In the ovine brain, GnRH neurons do not contain type II glucocorticoid (GR), progesterone (PR), or alpha estrogen (ERalpha) receptors. We sought to determine whether PR, ERalpha, and GR coexist within the same hypothalamic neurons. A triple immunocytochemical study, involving antisera raised in three different species, was performed on cryostat sections from ovariectomized ewes treated either with estradiol and progesterone or with progesterone alone. All PR-immunoreactive neurons contained ERalpha, and about 95% of ERalpha were PR immunoreactive in the preoptic area and arcuate nucleus. Although the PR with ERalpha colocalization ratio was not affected by the steroid treatments, immunolabeling for PR was weaker in animals that did not receive estradiol. Numerous PR- and ERalpha-immunoreactive cells contain GR. PR+ERalpha+GR-immunoreactive cells represent 70% of PR, 65% of ERalpha, and 72% of GR in the preoptic area and 70% of PR, 66% of ERalpha, and 63% of GR in the arcuate nucleus. These results suggest that estrogen, progesterone, and glucocorticoids may influence the activity of the same neurons to modulate both reproductive and stress axes.     

Relationship between neuropeptide Y and luteinizing-hormone-releasing hormone immunoreactivities in the hypothalamus and preoptic region

The purpose of this study was to examine the anatomical relationships of perikarya and fibers containing neuropeptide Y (NPY) and luteinizing-hormone-releasing hormone (LHRH) in the hypothalamus and preoptic region of female rats. In view of our previous report of stimulatory effects of estrogen on LHRH and NPY levels in the median eminence, animals were bilaterally ovariectomized and subsequently implanted subcutaneously with capsules containing estradiol benzoate in oil or vehicle. Following intracerebroventricular injection of colchicine, rats were perfused with fixative and their brains sectioned and processed for immunohistochemical visualization of NPY and LHRH in the same section and in consecutive sections. Estrogen treatment had no discernible effect on the distribution or relationship of these peptides. NPY-immunoreactive fibers were intimately associated with LHRH-labeled primary dendrites and perikarya in the medial preoptic region and horizontal limb of the diagonal band of Broca. Fibers containing NPY or LHRH overlapped extensively in the lateral palisade region of the median eminence and also in the subependymal and internal zones. The external zone of the median eminence displayed relatively less overlap of these peptide systems. LHRH-immunoreactive axons coursed among NPY-labeled perikarya in the arcuate nucleus and appeared to contact these cells. These results suggest that NPY-containing axons may influence LHRH-positive neurons at the cell body and also at the site of axon termination in the median eminence. LHRH-containing axons appear to contact NPY-immunoreactive perikarya in the arcuate nucleus and may interact with terminals in the median eminence. This arrangement may provide a mechanism for communication between NPY and LHRH neurons and for the neuroendocrine coordination of hypothalamic NPY and LHRH secretion before ovulation.     

Steroid modulation of astrocytes in the neonatal brain: implications for adult reproductive function

There is a growing appreciation for the importance of astrocytes, a type of nonneuronal glial cell, to overall brain functioning. The ability of astrocytes to respond to gonadal steroid hormones with changes in morphology has been well documented in the adult brain. It is also apparent that astrocytes of the developing brain are permanently differentiated by the neonatal hormonal milieu, in particular by estradiol, resulting in sexually dimorphic cell morphology, synaptic patterning, and density in males and females. The mechanisms of hormonally mediated astrocyte differentiation are likely to be region specific. In the arcuate nucleus of the hypothalamus, neuron-to-astrocyte signaling appears to play a critical role in estradiol-induced astrocyte differentiation during the first few days of life. Gamma aminobutyric acid (GABA) is an amino acid neurotransmitter that is synthesized and released exclusively by neurons. The levels of GABA are increased in the arcuate nucleus of neonatal males versus females. Preventing the increase in males or mimicking GABA action in females modulates astrocytes accordingly. Speculation about and evidence in support of the functional significance of this dimorphism to adult reproductive functioning is the topic of this review.     

Electron microscopic observations on extraneuronal lipofuscin in the monkey brain

Protein synthesizing activity of the rat hypothalamic arcuate nucleus following partial or total deafferentation of the medial basal hypothalamus was studied by light and electron microscopic autoradiography when administering tritiated leucine into the lateral ventricle. There were significantly more grains over the arcuate nucleus 21 days after disconnection of this hypothalamic region than over the intact nucleus. Isolation of a temporal cortical region induced similar changes in the isolated area, although this effect was not so pronounced as in the arcuate region. Data suggest that the protein synthesizing activity of arcuate neurons increases significantly after interruption of neural connections of the medial basal hypothalamus. It is assumed that the effect is primarily due to transneuronal alteration and/or interruption of inhibitory afferents.     

Ontogenetic studies on the topographical heterogeneity of somatostatin-containing neurons in rat hypothalamus

The ontogeny of the somatostatin-containing neuron system was investigated by light-microscopic immunohistochemistry. During development, immunoreactive somatostatin-containing neurons arise from three discrete regions of the neuroepithelium of the third ventricle and show a chronological difference. The neurons are first evident within the third ventricle floor on day 12.5 of gestation; they move thereafter to the arcuate nucleus. The second generation occurs in the dorsal region of the arcuate nucleus during days 17.5-19.5; these neurons migrate sequentially into the arcuate-ventromedial nuclear region. The third generation is recognized in the neuroepithelial cell layer of the rostral hypothalamus on day 17.5 of gestation; these cells move to the periventricular area. This latter generation is most prominent during days 3-6 after birth, and some of the cells are seen sporadically even up to day 20. The first two generations give rise to the somatostatin neuron system in the arcuate-ventromedial nuclear region, while the latter gives rise to that in the rostral periventricular region in the adult rat hypothalamus.     

Anorectic estrogen mimics leptin's effect on the rewiring of melanocortin cells and Stat3 signaling in obese animals

Metabolic hormones, such as leptin, alter the input organization of hypothalamic circuits, resulting in increased pro-opiomelanocortin (POMC) tone, followed by decreased food intake and adiposity. The gonadal steroid estradiol can also reduce appetite and adiposity, and it influences synaptic plasticity. Here we report that estradiol (E2) triggers a robust increase in the number of excitatory inputs to POMC neurons in the arcuate nucleus of wild-type rats and mice. This rearrangement of synapses in the arcuate nucleus is leptin independent because it also occurred in leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice, and was paralleled by decreased food intake and body weight gain as well as increased energy expenditure. However, estrogen-induced decrease in body weight was dependent on Stat3 activation in the brain. These observations support the notion that synaptic plasticity of arcuate nucleus feeding circuits is an inherent element in body weight regulation and offer alternative approaches to reducing adiposity under conditions of failed leptin receptor signaling.     

Dopamine sensitive neurons of the arcuate region of the hypothalamus and their role in regulating the gonadotropic function of the pituitary]

The effect of the microiontophoretic application of dopamine (DA) into the arcuate region of the hypothalamus on the sensitivity of single neurons and on the plasma and hypophysis LH levels was examined at various stages of the estrous cycle. During the estrous cycle in rats there was no significant differences in the relative number of neurons showing activation and inhibition or non-responsive to DA. However, in the first half of proestrus (P) a significant increase in the number of neurons with the excitative reaction to the iontophoretic application of DA was observed. At all the stages of the cycle, except the second half of P, the excitative reaction of neurons correlated with increased LH level in the plasma. In the hypophysis only in diestrus-2 there was a significant increase of the LH level in response to the iontophoretic application of DA in the arcuate neurons of the hypothalamus.     

Orphanin FQ in the mediobasal hypothalamus facilitates sexual receptivity through the deactivation of medial preoptic nucleus mu-opioid receptors

Sexual receptivity, lordosis, can be induced by sequential estradiol and progesterone or extended exposure to high levels of estradiol in the female rat. In both cases estradiol initially inhibits lordosis through activation of β-endorphin (β-END) neurons of the arcuate nucleus of the hypothalamus (ARH) that activate μ-opioid receptors (MOP) in the medial preoptic nucleus (MPN). Subsequent progesterone or extended estradiol exposure deactivates MPN MOP to facilitate lordosis. Opioid receptor-like receptor-1 (ORL-1) is expressed in ARH and ventromedial hypothalamus (VMH). Infusions of its endogenous ligand, orphanin FQ (OFQ/N, aka nociceptin), into VMH–ARH region facilitate lordosis. Whether OFQ/N acts in ARH and/or VMH and whether OFQ/N is necessary for steroid facilitation of lordosis are unclear. In Exp I, OFQ/N infusions in VMH and ARH that facilitated lordosis also deactivated MPN MOP indicating that OFQ/N facilitation of lordosis requires deactivation of ascending ARH-MPN projections by directly inhibiting ARH β-END neurons and/or through inhibition of excitatory VMH–ARH pathways to proopiomelanocortin neurons. It is unclear whether OFQ/N activates the VMH output motor pathways directly or via the deactivation of MPN MOP. In Exp II we tested whether ORL-1 activation is necessary for estradiol-only or estradiol + progesterone lordosis facilitation. Blocking ORL-1 with UFP-101 inhibited estradiol-only lordosis and MPN MOP deactivation but had no effect on estradiol + progesterone facilitation of lordosis and MOP deactivation. In conclusion, steroid facilitation of lordosis inhibits ARH β-END neurons to deactivate MPN MOP, but estradiol-only and estradiol + progesterone treatments appear to use different neurotransmitter systems to inhibit ARH-MPN signaling.     

Colocalization of atrial natriuretic factor (ANF) and estradiol in hypothalamic neurons by combined autoradiography-immunohistochemistry

The distribution of estrogen target neurons which contain atrial natriuretic factor (ANF) in female rat hypothalamus was investigated by thaw-mount auto-radiography combined with immunocytochemistry using tritium-labeled estradiol and antibodies against ANF. Colocalization of the two hormones was found in the arcuate nucleus, periventricular nucleus, lateral ventromedial nucleus, ventral premammillar nucleus and lateral basal hypothalamus. The percentage of ANF containing cells which concentrate estradiol varies among the different hypothalamic nuclei with the highest number of ANF-positive cells showing nuclear concentration of 3H-estradiol (80-90%) in the nucleus premammillaris ventralis, but less (5-15%) in the other nuclei. These data, together with topographical correspondence in extrahypothalamic brain regions between sites of action of estradiol and production of ANF, suggest extensive interrelationships and modulatory effects of estradiol on ANF production and secretion in the brain, similar to the atrium of the heart.     

Leptin sensitive neurons in the hypothalamus.

A major paradigm in the field of obesity research is the existence of an adipose tissue-brain endocrine axis for the regulation of body weight. Leptin, the peptide mediator of this axis, is secreted by adipose cells. It lowers food intake and body weight by acting in the hypothalamus, a region expressing an abundance of leptin receptors and a variety of neuropeptides that influence food intake and energy balance. Among the most promising candidates for leptin-sensitive cells in the hypothalamus are arcuate nucleus neurons that co-express the anabolic neuropeptides, neuropeptide Y (NPY) and agouti-related peptide (AGRP), and those that express proopiomelanocortin (POMC), the precursor of the catabolic peptide, alphaMSH. These cell types contain mRNA encoding leptin receptors and show changes in neuropeptide gene expression in response to changes in food intake and circulating leptin levels. Decreased leptin signaling in the arcuate nucleus is hypothesized to increase the expression of NPY and AGRP. Levels of leptin receptor mRNA and leptin binding are increased in the arcuate nucleus during fasting, principally in NPY/AGRP neurons. These findings suggest that changes in leptin receptor expression in the arcuate nucleus are inversely associated with changes in leptin signaling, and that the arcuate nucleus is an important target of leptin action in the brain.     

Testosterone-induced alterations in the reactivity pattern of the neurons in the arcuate nucleus of male mice

The effects of testosterone administration on the hypothalamic arcuate nucleus were studied in adult male mice by means of karyometry. Four animals per group were sacrificed 1, 2, and 3 h after intramuscular injection of 100 micrograms testosterone propionate/100 g body weight. The nuclear diameter of neurons was measured in serial coronal sections. Testosterone induced an increase in the nuclear diameter of neurons located in the dorsal and medial periventricular zones of the arcuate nucleus. The neurons exhibiting the greatest changes in nuclear diameter were situated in the rostral portion of the nuclear area examined. In the central portion of the arcuate nucleus no response to testosterone was found. The present data support previous observations showing mosaically arranged nerve-cell groups in this hypothalamic nucleus.     

A histochemical study of the primary catecholamines in the hypothalamic neurons of the rat in relation to the ontogenetic and sexual differentiation

Summary The alterations in the content of the primary catecholamines in the hypothalamus have been studied with the histochemical technique of para-formaldehyde induced fluorescence.In the adult normal rats, independent of the sex, the fluorescence is located in the cell bodies of a few arcuate neurons, around the perikarya of the arcuate, para-ventricular and supra-optic neurons, and in the nerve endings of the arcuate neurons in the median eminence.The appearance of the primary catecholamines takes place at the 20th day of gestation in the para-ventricular and arcuate-peri-ventricular regions. In the supra-optic nucleus the fluorescent nerve terminals are not seen before birth. In the outer layer of the median eminence the fluorescence develops around the 5th post-natal day. No sexual differences were observed in the maturation of the primary catecholamines during the ontogenic development of the rat.More fluorescent cell bodies and nerve endings are seen in the arcuate neurons during the late diestrus than during estrus. The number and intensity of the catecholamine fluorescent neurons in the arcuate nucleus increases during the pregnancy. Castration increases slightly the number and intensity of the fluorescent cell bodies in the arcuate nucleus, but it diminishes the fluorescence in the median eminence. The changes were compensated by a treatment with testosterone propionate. Hypophysectomy alone has no effect on the fluorescence of the hypothalamic neurons.Supported by a grant from The Finnish Medical Society Duodecim.     

Colocalization of atrial natriuretic factor (ANF) and estradiol in hypothalamic neurons by combined autoradiography-immunohistochemistry

Summary The distribution of estrogen target neurons which contain atrial natriuretic factor (ANF) in female rat hypothalamus was investigated by thaw-mount autoradiography combined with immunocytochemistry using tritium-labeled estradiol and antibodies against ANF. Colocalization of the two hormones was found in the arcuate nucleus, periventricular nucleus, lateral ventromedial nucleus, ventral premammillar nucleus and lateral basal hypothalamus. The percentage of ANF containing cells which concentrate estradiol varies among the different hypothalamic nuclei with the highest number of ANF-positive cells showing nuclear concentration of ³H-estradiol (80–90%) in the nucleus premammillaris ventralis, but less (5–15%) in the other nuclei. These data, together with topographical correspondence in extrahypothalamic brain regions between sites of action of estradiol and production of ANF, suggest extensive interrelationships and modulatory effects of estradiol on ANF production and secretion in the brain, similar to the atrium of the heart.     

Relationship of alpha MSH-specific neurons to the arcuate opiocortin neuronal system as determined by dual antigen immunocytochemical procedures

The cross-immunoreactivity, topography, and fiber projections of the alpha MSH-immunoreactive specific neurons in the forebrain of the rat appear to be distinctly different from that of the neurons in the hypothalamic arcuate opiocortin system. The cell bodies, immunoreactive only to -MSH, have a specific pattern of distribution in the dorsal and lateral hypothalamic regions from the level of the retrochiasmatic region to the premammillary area of the posterior hypothalamus. Immunoreactive fibers of these cells appear to extend into regions of the cerebral cortex and hippocampus. An antomical relationship between the immunostained fibers and/or terminals of the arcuate opiocortin pool of neurons and the -MSH-immunoreactive perikarya is described utilizing the ABC (Avidin-Biotin-Peroxidase Complex) and ABC-GO (Glucose Oxidase) or glucose oxidase-antiglucose oxidase complex methods of immunocytochemistry in which two tissue antigens with contrasting colors are demonstrated in the same tissue section.     

Morphological interaction between galanin-like peptide- and dopamine-containing neurons in the rat arcuate nucleus

Galanin-like peptide (GALP) is a 60-amino acid neuropeptide that plays an important role in the neuronal regulation of feeding, energy balance and reproduction. GALP is produced in the hypothalamic arcuate nucleus, an area containing, amongst other neuron types, two populations of neurons in which we were interested: a population of GALP-containing neurons which regulate energy balance and reproduction, and a second population consisting of tuberoinfundibular dopaminergic neurons which suppress prolactin secretion from the adenohypophysis. To characterize morphologically the relationship between GALP and dopamine-containing neurons in the arcuate nucleus, a double immunofluorescence study was performed on cryosections from rat brain. Immunohistochemical double labeling studies revealed that GALP-immunoreactive nerve fibers made direct contact on tyrosine hydroxylase (TH)-containing neuronal cell bodies in the arcuate nucleus. These results suggest that GALP-containing neurons innervate tuberoinfundibular dopaminergic neurons.