CRH Neurons Research Articles

A Role for the Forebrain in Mediating Time-of-Day Differences in Glucocorticoid Counterregulatory Responses to Hypoglycemia in Rats

The time of day influences the magnitude of ACTH and corticosterone responses to hypoglycemia. However, little is known about the mechanisms that impart these time-of-day differences on neuroendocrine CRH neurons in the hypothalamic paraventricular nucleus (PVH). Rats received 0-3 U/kg insulin (or 0.9% saline) to achieve a range of glucose nadir concentrations. Brains were processed to identify phosphorylated ERK1/2 (phospho-ERK1/2)-immunoreactive cells in the PVH and hindbrain and CRH heteronuclear RNA in the PVH. Hypoglycemia did not stimulate ACTH and corticosterone responses in animals unless a glucose concentration of approximately 3.15 mM or below was reached. Critically the glycemic thresholds required to stimulate ACTH and corticosterone release in the morning and night were indistinguishable. Yet glucose concentrations below the estimated glycemic threshold correlated with a greater increase in corticosterone, ACTH, and phospho-ERK1/2-immunoreactive neurons in the PVH at night, compared with morning. In these same animals, the number of phospho-ERK1/2-immunoreactive neurons in the medial part of the nucleus of the solitary tract was unchanged at both times of day. These data collectively support a model whereby changes in forebrain mechanisms alter the sensitivity of neuroendocrine CRH to the hypoglycemia-related information conveyed by ascending catecholaminergic afferents. Circadian clock-driven processes together with glucose-sensing elements in the forebrain would seem to be strong contenders for mediating these effects.

Contribution of Noradrenergic and Adrenergic Cell Groups of the Brainstem and Agouti-Related Protein-Synthesizing Neurons of the Arcuate Nucleus to Neuropeptide-Y Innervation of Corticotropin-Releasing Hormone Neurons in Hypothalamic Paraventricular Nucleus of the Rat

CRH-synthesizing neurons in the hypothalamic paraventricular nucleus (PVN) integrate neuronal and hormonal inputs and serve as a final common pathway to regulate the hypothalamic-pituitary-adrenal axis. One of the neuronal regulators of CRH neurons is neuropeptide Y (NPY) contained in axons that densely innervate CRH neurons. The three main sources of NPY innervation of the PVN are the hypothalamic arcuate nucleus and the noradrenergic and adrenergic neurons of the brainstem. To elucidate the origin of the NPY-immunoreactive (NPY-IR) innervation to hypophysiotropic CRH neurons, quadruple-labeling immunocytochemistry for CRH, NPY, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyltransferase was performed. Approximately 63% of NPY-IR varicosities on the surface of CRH neurons were catecholaminergic (22% noradrenergic and 41% adrenergic), and 37% of NPY-IR boutons were noncatecholaminergic. By triple-labeling immunofluorescence detection of NPY, CRH, and agouti-related protein, a marker of NPY axons projecting from the arcuate nucleus, the noncatecholaminergic, NPY-ergic axon population was shown to arise primarily from the arcuate nucleus. When NPY was administered chronically into the cerebral ventricle of fed animals, a dramatic reduction of CRH mRNA was observed in the PVN (NPY vs. control integrated density units, 23.9 +/- 2.7 vs. 77.09 +/- 15.9). We conclude that approximately two thirds of NPY-IR innervation to hypophysiotropic CRH neurons originates from catecholaminergic neurons of the brainstem, whereas the remaining one third arises from the arcuate nucleus. The catecholaminergic NPY innervation seems to modulate the activation of CRH neurons in association with glucoprivation and infection, whereas the NPY input from the arcuate nucleus may contribute to inhibition of CRH neurons during fasting.

Involvement of Neuropeptide Y Y1 Receptors in the Regulation of Neuroendocrine Corticotropin-Releasing Hormone Neuronal Activity

The neuroendocrine parvocellular CRH neurons in the paraventricular nucleus (PVN) of the hypothalamus are the main integrators of neural inputs that initiate hypothalamic-pituitary-adrenal (HPA) axis activation. Neuropeptide Y (NPY) expression is prominent within the PVN, and previous reports indicated that NPY stimulates CRH mRNA levels. The purpose of these studies was to examine the participation of NPY receptors in HPA axis activation and determine whether neuroendocrine CRH neurons express NPY receptor immunoreactivity. Infusion of 0.5 nmol NPY into the third ventricle increased plasma corticosterone levels in conscious rats, with the peak of hormone levels occurring 30 min after injection. This increase was prevented by pretreatment with the Y1 receptor antagonist BIBP3226. Immunohistochemistry showed that CRH-immunoreactive neurons coexpressed Y1 receptor immunoreactivity (Y1r-ir) in the PVN, and a majority of these neurons (88.8%) were neuroendocrine as determined by ip injections of FluoroGold. Bilateral infusion of the Y1/Y5 agonist, [leu(31)pro(34)]NPY (110 pmol), into the PVN increased c-Fos and phosphorylated cAMP response element-binding protein expression and elevated plasma corticosterone levels. Increased expression of c-Fos and phosphorylated cAMP response element-binding protein was observed in populations of CRH/Y1r-ir cells. The current findings present a comprehensive study of NPY Y1 receptor distribution and activation with respect to CRH neurons in the PVN. The expression of NPY Y1r-ir by neuroendocrine CRH cells suggests that alterations in NPY release and subsequent activation of NPY Y1 receptors plays an important role in the regulation of the HPA.

Alcohol Exposure during the Developmental Period Induces β-Endorphin Neuronal Death and Causes Alteration in the Opioid Control of Stress Axis Function

Proopiomelanocortin-producing neurons in the arcuate nucleus of the hypothalamus secrete beta-endorphin (beta-EP), which controls varieties of body functions including the feedback regulation of the CRH neuronal activity in the paraventricular nucleus of the hypothalamus. Whether ethanol exposure in developing rats induces beta-EP neuronal death and alters their influence on CRH neurons in vivo has not been determined. We report here that binge-like ethanol exposures in newborn rats increased the number of apoptotic beta-EP neurons in the arcuate nucleus of the hypothalamus. We also found that immediately after ethanol treatments there was a significant reduction in the expression of proopiomelanocortin and adenylyl cyclases mRNA and an increased expression of several TGF-beta1-linked apoptotic genes in beta-EP neurons isolated by laser-captured microdissection from arcuate nuclei of young rats. Several weeks after the ethanol treatment, we detected a reduction in the number of beta-EP neuronal perikarya in arcuate nuclei and in the number of beta-EP neuronal terminals in paraventricular nuclei of the hypothalamus in the treated rats. Additionally, these rats showed increased response of the hypothalamic CRH mRNA to the lipopolysaccharide challenge. The ethanol-treated animals also showed incompetent ability to respond to exogenous beta-EP to alter the lipopolysaccharide-induced CRH mRNA levels. These data suggest that ethanol exposure during the developmental period causes beta-EP neuronal death by cellular mechanisms involving the suppression of cyclic AMP production and activation of TGF-beta1-linked apoptotic signaling and produces long-term structural and functional deficiency of beta-EP neurons in the hypothalamus.

Differential Responses of Hypothalamus-Pituitary-Adrenal Axis Immediate Early Genes to Corticosterone and Circadian Drive

The hypothalamus-pituitary-adrenal (HPA) axis diurnal cycle of activity is manifest in circadian rhythms of ACTH and corticosterone secretion, which in the rat peak around the onset of the dark period. This cycle is thought to be driven by daily fluctuations in activity of CRH neurons within the paraventricular nucleus of the hypothalamus (PVN), controlled by suprachiasmatic nucleus inputs. In this study we examined whether the circadian drive that regulates ACTH and corticosterone basal secretion in the rat is reflected in PVN immediate early gene expression and, if so, whether different genes respond uniformly or uniquely to circadian stimulatory input. In addition, we examined how circadian drive and acute stress, two categories of stimuli that induce HPA axis activation, comparatively affect gene expression within different components of the HPA axis (c-fos mRNA, CRH heteronuclear RNA, and zif268 mRNA in PVN; c-fos mRNA, proopiomelanocortin heteronuclear RNA, and zinc finger 268 mRNA in anterior pituitary; c-fos mRNA and nerve growth factor I-B mRNA in adrenal cortex). Finally, we examined whether circadian differences in gene expression depend on endogenous glucocorticoids and, if so, whether the dependence is on an acute or permissive influence of the hormone. We found that a circadian drive that regulates HPA axis basal hormone secretion is also manifest on basal c-fos gene expression in the PVN. Moreover, we show that different immediate early genes within the HPA axis anatomical components display different diurnal patterns of gene expression. These differential patterns result, in part, from gene-specific responses to circadian signals and acute and/or permissive glucocorticoid actions.

Hypothalamic and brainstem sources of pituitary adenylate cyclase‐activating polypeptide nerve fibers innervating the hypothalamic paraventricular nucleus in the rat

The hypothalamic paraventricular nucleus (PVN) coordinates major neuroendocrine and behavioral mechanisms, particularly responses to homeostatic challenges. Parvocellular and magnocellular PVN neurons are richly innervated by pituitary adenylate cyclase-activating polypeptide (PACAP) axons. Our recent functional observations have also suggested that PACAP may be an excitatory neuropeptide at the level of the PVN. Nevertheless, the exact localization of PACAP-producing neurons that project to the PVN is not understood. The present study examined the specific contribution of various brain areas sending PACAP innervation to the rat PVN by using iontophoretic microinjections of the retrograde neuroanatomical tracer cholera toxin B subunit (CTb). Retrograde transport was evaluated from hypothalamic and brainstem sections by using multiple labeling immunofluorescence for CTb and PACAP. PACAP-containing cell groups were found to be retrogradely labeled from the PVN in the median preoptic nucleus; preoptic and lateral hypothalamic areas; arcuate, dorsomedial, ventromedial, and supramammillary nuclei; ventrolateral midbrain periaqueductal gray; rostral and midlevel ventrolateral medulla, including the C1 catecholamine cell group; nucleus of the solitary tract; and dorsal motor nucleus of vagus. Minor PACAP projections with scattered double-labeled neurons originated from the parabrachial nucleus, pericoeruleus area, and caudal regions of the nucleus of the solitary tract and ventrolateral medulla. These observations indicate a multisite origin of PACAP innervation to the PVN and provide a strong chemical neuroanatomical foundation for interaction between PACAP and its potential target neurons in the PVN, such as parvocellular CRH neurons, controlling physiologic responses to stressful challenges and other neuroendocrine or preautonomic PVN neurons.

Acute and repeated cocaine induces alterations in FosB/ΔFosB expression in the paraventricular nucleus of the hypothalamus

Apart from activation of the brain reward system, cocaine administration influences the activity of the hypothalamo–pituitary–adrenal (HPA) axis by affecting CRH neurons in the paraventricular nucleus of the hypothalamus (PVN). In order to find a molecular mechanism of cocaine-evoked effects in the PVN, in the present study, we investigated the impact of cocaine on the expression of FosB/ΔFosB transcription factors in the PVN. Using an immunohistochemical method, we found that acute cocaine treatment (25 mg/kg) induced a relatively long-lasting (at least 72 h) expression of FosB/ΔFosB in the PVN, whereas repeated cocaine administration (25 mg/kg, once daily for 5 consecutive days) caused accumulation of FosB/ΔFosB in the PVN. The latter observation was further confirmed by the Western blot technique which revealed that repeated exposure to cocaine specifically increased the expression of a stable isoform of ΔFosB (35 kDa). Using a double-labeling immunofluorescent method, it was established that FosB/ΔFosB proteins induced by repeated cocaine treatment were present in a small population of CRF-immunoreactive neurons of the PVN. Furthermore, it was found that pretreatment with the specific antagonist of dopamine D1-like receptors SCH 23390 (1 mg/kg) attenuated the expression and accumulation of FosB/ΔFosB in the PVN, evoked by repeated cocaine administration. Although functional consequences of the above effects for the process of addiction remain to be established, the obtained results indicate that cocaine administration can produce relatively long-lasting changes in the expression of FosB/ΔFosB transcription factors in PVN neurons (in some populations of CRF-immunoreactive neurons, among others) and that dopamine D1-like receptors are involved in the above effects. Finally, it is proposed that the long-lasting expression as well as the accumulation of ΔFosB in the PVN may constitute a molecular basis underlying adaptive changes occurring in the HPA axis after relatively high doses of cocaine.

Confocal imaging of acute hypothalamic slices maintained in vitro reveals rapid noradrenergic recruitment of p44/42 MAP kinases in CRH neurons of the hypothalamic paraventricular nucleus (PVH): Comparison with central injection studies

Confocal imaging of acute hypothalamic slices maintained in vitro reveals rapid noradrenergic recruitment of p44/42 MAP kinases in CRH neurons of the hypothalamic paraventricular nucleus (PVH): Comparison with central injection studies

Reduced Anorexigenic Efficacy of Leptin, But Not of the Melanocortin Receptor Agonist Melanotan-II, Predicts Diet-Induced Obesity in Rats

Leptin gains access to the central nervous system where it influences activity of neuronal networks involved in ingestive behavior, neuroendocrine activity, and metabolism. In particular, the brain melanocortin (MC) system is important in leptin signaling and maintenance of energy balance. Although leptin or MC receptor insensitivity has been proposed to be associated with obesity, the present study compared central leptin and MC receptor stimulation on some of the above-mentioned parameters and investigated whether these treatments predict proneness to diet-induced obesity (DIO) in outbred Wistar rats. Third-cerebroventricular administration of equi-anorexigenic doses of leptin and of the MC agonist melanotan-II caused comparable increases in plasma ACTH and corticosterone levels and c-Fos-labeling in approximately 70% of paraventricular hypothalamic (PVN) neuronal cell bodies containing CRH. This reinforces involvement of paraventricular CRH neurons in the short-term neuroendocrine and ingestive effects of leptin and melanocortins. In the DIO prediction study, anorexigenic efficacy of melanotan-II was not correlated with any parameter linked to DIO but was highly correlated with MC in situ binding (with labeled [Nle(4),D-Phe(7)]alpha-MSH) as well as CRH immunoreactivity in the PVN of DIO rats. This suggests intricate relationships among MC signaling, the CRH system, and ingestive behavior unrelated to DIO. In the same animals, leptin's anorexigenic efficacy was not correlated with PVN MC in situ binding or CRH immunoreactivity but correlated inversely to post-DIO plasma leptin, liver weight, and abdominal adiposity, the latter being correlated to insulin resistance. Thus, differences in leptin but not MC signaling might underlie DIO, visceral obesity, and insulin resistance.

In vivo action of a new octadecaneuropeptide antagonist on neuropeptide Y and corticotropin-releasing hormone mRNA levels in rat

It has been reported that several of the effects induced by an octadecaneuropeptide (ODN), derived from an 86-amino-acid polypeptide termed diazepam-binding inhibitor, could be mediated by activation of a metabotropic receptor. In order to investigate the role and mechanism of action of ODN in the regulation of corticotropin-releasing factor (CRH) and neuropeptide Y (NPY) expression in the paraventricular nucleus and arcuate nucleus, respectively, we studied the effects of the acute intracerebroventricular administration of ODN (2 μg/rat) and the ODN antagonist to metabotropic receptor, cyclo 1–8[Dleu 5]OP (20 μg/rat), on the gene expression of the two neuropeptides in castrated male rat. ODN administration resulted in a 45% increase in CRH mRNA expression, an effect which was reversed by cyclo 1–8[Dleu 5]OP. When cyclo 1–8[Dleu 5]OP was administered alone, it induced a 19% decrease in CRH mRNA levels. ODN administration induced a 17% decrease in NPY mRNA expression while cyclo 1–8[Dleu 5]OP increased by 21% the hybridization signal. The administration of both ODN and ODN antagonist completely abolished the depressing effect of ODN on NPY mRNA. These data suggest that the effects of ODN on CRH and NPY mRNA might be mediated by interaction with metabotropic receptors. Moreover, since cyclo 1–8[Dleu 5]OP can by itself influence the expression of two peptide mRNAs, it might be suggested that ODN is exerting a tonic influence on NPY and CRH neurons.

Experimentally-induced hyperthyroidism is associated with activation of the rat hypothalamic–pituitary–adrenal axis

Previous studies on the effects of altered thyroid function on the secretion and metabolism of adrenocortical hormones suggest a degree of adrenocortical hyperactivity in hyperthyroidism. We have previously shown that experimentally-induced hyperthyroidism is associated with significant alterations in pituitary-adrenal responsiveness to synthetic ovine corticotropin-releasing hormone (oCRH) that are contingent upon the duration of the altered thyroid function. The purpose of this study was to assess the time-dependent effects of hyperthyroidism on the functional integrity of the hypothalamic-pituitary-adrenal (HPA) axis by in vivo stimulation of the hypothalamic CRH neuron and adrenal cortex. The functional integrity of the HPA axis was examined in vivo in sham-thyroidectomized male Sprague-Dawley rats given placebo or in thyroidectomized rats given 50 mug of thyroxine every day for 7 or 60 days. Responses to insulin-induced hypoglycemia and IL-1alpha stimulation were used to assess the hypothalamic CRH neuron. Adrenocortical reserve was assessed in response to low-dose adrenocorticotropic hormone (ACTH), following suppression of the HPA axis with dexamethasone. Adrenal and thymus tissue weight, in addition to basal plasma ACTH, corticosterone and thyroid indices were also determined. Basal plasma corticosterone and corticosterone binding globulin (CBG) concentrations were significantly increased in short- and long-term hyperthyroid rats, and by 60 days, cerebrospinal fluid (CSF) corticosterone levels were significantly increased. Basal plasma ACTH levels were similar to controls. Although plasma ACTH responses to hypoglycemic stress and IL-1alpha administration in both short- and long-term hyperthyroidism were normal, corticosterone responses to the ACTH release during the administration of these stimuli were significantly increased. The adrenal reserve was significantly elevated in short-term hyperthyroidsim. Long-term hyperthyroidism, however, was associated with a significant reduction in adrenocortical reserve. A significant increase in adrenal weights and a decrease in thymus weights were observed in both short- and long-term hyperthyroidism. The available data confirms that hyperthyroidism is associated with hypercorticosteronemia, although the locus that is principally affected still remains unclear. Despite the sustained hyperactivity of the HPA axis, long-term experimentally-induced hyperthyroidism is associated with diminished adrenal functional reserve. The alterations in HPA function in states of disturbed thyroid function were found to be somewhat more pronounced as the duration of thyroid dysfunction increased.

Origin of Cocaine- and Amphetamine-Regulated Transcript-Containing Axons Innervating Hypophysiotropic Corticotropin-Releasing Hormone-Synthesizing Neurons in the Rat

Cocaine- and amphetamine-regulated transcript (CART) has stimulatory effects on the hypothalamic-pituitary-adrenal axis through direct effects on hypophysiotropic CRH neurons. Recently CART-containing axons have been demonstrated to densely innervate the hypophysiotropic CRH neurons. Based on the sources of the CART-immunoreactive (IR) innervation of the paraventricular nucleus, the putative origins of these CART-containing fibers include neurons of the hypothalamic arcuate nucleus that coexpress alphaMSH and medullary adrenaline-producing neurons. To determine whether these cell groups contribute to the CART innervation of the hypophysiotropic CRH neurons, we performed a quadruple-labeling immunofluorescent study using antisera against CRH, CART, alphaMSH, and phenylethanolamine-N-methyl-transferase (PNMT), the latter as a marker for adrenaline. Consistent with previous observations, PNMT- and CART-IR axons densely innervated all CRH neurons, whereas the alphaMSH-IR innervation was sparse. Although approximately 60% of CART-IR varicosities in juxtaposition to CRH neurons cocontained PNMT, only approximately 18% of them were immunopositive for alphaMSH. All alphaMSH-IR boutons and approximately 90% of PNMT-containing varicosities on the surface of CRH neurons were also labeled for CART. The remaining 22% of CART axon varicosities in contact with CRH neurons contained neither alphaMSH nor PNMT. These results indicate that medullary adrenergic/CART neurons are the major source for the CART innervation of CRH neurons in the paraventricular nucleus; however, to a lesser extent the arcuate nucleus also contributes to the CART-IR innervation of these neurons. The observation that nearly 20% of the CART-IR afferents contain neither alphaMSH nor PNMT, however, suggests that additional sources also contribute to the CART-IR input of hypophysiotropic CRH neurons.

Hypophysiotropic thyrotropin-releasing hormone and corticotropin-releasing hormone neurons of the rat contain vesicular glutamate transporter-2.

TRH and CRH are secreted into the hypophysial portal circulation by hypophysiotropic neurons located in parvicellular subdivisions of the hypothalamic paraventricular nucleus (PVH). Recently these anatomical compartments of the PVH have been shown to contain large numbers of glutamatergic neurons expressing type 2 vesicular glutamate transporter (VGLUT2). In this report we presented dual-label in situ hybridization evidence that the majority (>90%) of TRH and CRH neurons in the PVH of the adult male rat express the mRNA encoding VGLUT2. Dual-label immunofluorescent studies followed by confocal laser microscopic analysis of the median eminence also demonstrated the occurrence of VGLUT2 immunoreactivity within TRH and CRH axon varicosities, suggesting terminal glutamate release from these neuroendocrine systems. These data together indicate that the hypophysiotropic TRH and CRH neurons possess glutamatergic characteristics. Future studies will need to address the physiological significance of the endogenous glutamate content in these neurosecretory systems in the neuroendocrine regulation of thyroid and adrenal functions.

The role of the locus coeruleus in corticotropin-releasing hormone and stress-induced suppression of pulsatile luteinizing hormone secretion in the female rat.

Despite a wealth of evidence for CRH mediating stress-induced suppression of the hypothalamic GnRH pulse generator, and hence reproductive dysfunction, the site and mechanism of action remains elusive. The locus coeruleus (LC), a prominent noradrenergic brain stem nucleus, is innervated by CRH neurons, mediates several behavioral stress responses, and is implicated in the control of pulsatile LH secretion. The aim of this study was to test the hypothesis that LC CRH has a critical role in mediating stress-induced suppression of pulsatile LH secretion in the rat. Ovariectomized rats with 17beta-estradiol or oil-filled s.c. capsules were implanted with bilateral LC and i.v. cannulae. Central administration of CRH (10 ng to 1 microg) resulted in a dose-dependent suppression of LH pulses, which was reversed by a CRH receptor antagonist (alpha-helical CRF(9-41), 1 microg). The induction of c-fos expression in glutamic acid decarboxylase67 immunostained neurons in the preoptic area suggests activation of the secretion of gamma-aminobutyric acid in response to intracoerulear administration of CRH; 17beta-estradiol further increased the percentage of glutamic acid decarboxylase67-positive neurons that expressed fos and augmented suppression of LH pulses. Furthermore, intracoerulear administration of alpha-helical CRF(9-41) completely blocked restraint stress-induced suppression of LH pulses, without affecting the inhibitory response to hypoglycemia. These results suggest that CRH innervation of the LC may play a pivotal, but differential, role in the normal physiological response of stress-induced suppression of the GnRH pulse generator and hence the reproductive system.

Intravenous 2-deoxy-D-glucose injection rapidly elevates levels of the phosphorylated forms of p44/42 mitogen-activated protein kinases (extracellularly regulated kinases 1/2) in rat hypothalamic parvicellular paraventricular neurons.

CRH neurons within the medial parvicellular part of the hypothalamic paraventricular nucleus (PVHmp) can respond to afferent inputs encoding stress-related information by initiating peptide synthesis (signaling cascades, transcription, and translation) and/or peptide release. However, understanding these cellular events is hampered by three outstanding issues: 1) neural inputs that activate CRH neurons remain incompletely identified; 2) the identity and temporal dynamics of signaling pathways within CRH neurons are poorly understood; and 3) the precise coupling of the first two issues has not been established. Here, we report that the phosphorylated forms of p44/p42 MAPKs (pERK1/2) are rapidly detected in PVHmp cells after i.v. infusion of the antimetabolite, 2-deoxy-D-glucose (2-DG). Combined immunocytochemistry and in situ hybridization revealed that pERK1/2 immunoreactivity is detectable 10 min after 2-DG infusion not only within most PVHmp neurons containing CRH mRNA (78.6% of mean total CRH cells counted) but also in many non-CRH neurons (45.5% of mean total sampled cells). In contrast, Fos protein in the PVHmp was not detected within this time period, consistent with the known time course for its translation. Stress associated with halothane exposure also robustly elevated pERK1/2 levels in PVHmp neurons approximately 10 min after exposure. Our results implicate pERK1/2 in stress-induced activation of CRH neurosecretory cells and underscore their utility as indices of rapid cellular activation. Because 2-DG-induced activation of CRH gene transcription in these neurons requires a catecholaminergic input, our data also suggest that pERK1/2 could couple afferent catecholaminergic signals with CRH gene expression in these neurons.

Corticotropin-releasing hormone-mediated pathway of leptin to regulate feeding, adiposity, and uncoupling protein expression in mice.

To examine the functional role of CRH in the regulation of energy homeostasis by leptin, we measured the effects of the CRH antagonist, alpha-helical CRH 8-41 (alphaCRH) on a number of factors affected by leptin activity. These included food intake, body weight, hypothalamic c-fos-like immunoreactivity (c-FLI), weight and histological characterization of white adipose tissue, and mRNA expressions of uncoupling protein (UCP) in brown adipose tissue (BAT) in C57Bl/6 mice. Central infusion of leptin into the lateral cerebroventricle (icv) caused significant induction of c-FLI in the paraventricular nucleus (PVN), ventromedial hypothalamic nucleus (VMH), dorsomedial hypothalamic nucleus, and arcuate nucleus. In all these nuclei, the effect of leptin on expression of cFLI in the PVN and VMH was decreased by treatment with alphaCRH. Administration of leptin markedly decreased cumulative food intake and body weight with this effect being attenuated by pretreatment with alphaCRH. In peripheral tissue, leptin up-regulated BAT UCP1 mRNA expression and reduced fat depositions in this tissue. Those changes in BAT were also decreased by treatment with alphaCRH. As a consequence of the effects on food intake or energy expenditure, treatment with alphaCRH attenuated the leptin-induced reduction of body adiposity, fat cell size, triglyceride contents, and ob mRNA expression in white adipose tissue. Taken together, these results indicate that CRH neurons in the PVN and VMH may be an important mediator for leptin that contribute to regulation of feeding, adiposity, and UCP expression.

Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo–pituitary–adrenocortical responsiveness

Appropriate regulatory control of the hypothalamo–pituitary–adrenocortical stress axis is essential to health and survival. The following review documents the principle extrinsic and intrinsic mechanisms responsible for regulating stress-responsive CRH neurons of the hypothalamic paraventricular nucleus, which summate excitatory and inhibitory inputs into a net secretory signal at the pituitary gland. Regions that directly innervate these neurons are primed to relay sensory information, including visceral afferents, nociceptors and circumventricular organs, thereby promoting ‘reactive’ corticosteroid responses to emergent homeostatic challenges. Indirect inputs from the limbic-associated structures are capable of activating these same cells in the absence of frank physiological challenges; such ‘anticipatory’ signals regulate glucocorticoid release under conditions in which physical challenges may be predicted, either by innate programs or conditioned stimuli. Importantly, ‘anticipatory’ circuits are integrated with neural pathways subserving ‘reactive’ responses at multiple levels. The resultant hierarchical organization of stress-responsive neurocircuitries is capable of comparing information from multiple limbic sources with internally generated and peripherally sensed information, thereby tuning the relative activity of the adrenal cortex. Imbalances among these limbic pathways and homeostatic sensors are likely to underlie hypothalamo–pituitary–adrenocortical dysfunction associated with numerous disease processes.

Exposure to bisphenol A during gestation and lactation causes loss of sex difference in corticotropin-releasing hormone-immunoreactive neurons in the bed nucleus of the stria terminalis of rats

It has been suspected that endocrine disrupters induce abnormal differentiation and development of reproductive organs. In the present study, we examined whether exposure to bisphenol A (BPA), a known endocrine disrupter, during gestation and lactation affects sex difference in the number of corticotropin-releasing hormone-immunoreactive neurons (CRH neurons) in the preoptic area (POA) and the bed nucleus of the stria terminalis (BST). For that purpose, pregnant female Wistar rats ( n=8–11 per treatment group) were treated with either 0.1% ethanol (control group) or 10 mg/l BPA (BPA group) dissolved in their drinking water until their offspring were weaned. In the control group, we confirmed a previous report that the POA of female rats contained significantly more CRH neurons than that of male rats ( p<0.05). This significant sex difference was also evident in the BPA group, indicating that BPA exposure used in the present study had no effect on the sex difference in CRH neurons in the POA. We also found in the control group that the BST of female rats contained significantly more CRH neurons ( p<0.05) than that of male rats. However, this significant sex difference was not observed in the BPA group ( p>0.05), suggesting that BPA exposure affected the sex difference in CRH neurons in the BST. Since there was no statistically significant difference in the number of CRH neurons between the control and the BPA group, irrespective of the sex, the results suggested that a loss of sex difference in CRH neurons was due to both an increase in CRH neurons in male rats and a decrease in CRH neurons in female rats. The present study indicates that there is a significant sex difference in the number of CRH neurons in the BST as well as in the POA and that exposure to BPA during gestation and lactation causes a loss of this sex difference in the rat BST, but not in the POA. We suggest that CRH neurons in the BST are more susceptible to endocrine disrupters than those in the POA, irrespective of the sex.

Distribution of vesicular glutamate transporter-2 messenger ribonucleic Acid and protein in the septum-hypothalamus of the rat.

The excitatory neurotransmitter glutamate is involved in the control of most, perhaps all, neuroendocrine systems, yet the sites of glutamatergic neurons and their processes are unknown. Here, we used in situ hybridization and immunohistochemistry for the neuron-specific vesicular glutamate transporter-2 (VGLUT2) to identify the neurons in female rats that synthesize the neurotransmitter glutamate as well as their projections throughout the septum-hypothalamus. The results show that glutamatergic neurons are present in the septum-diagonal band complex and throughout the hypothalamus. The preoptic area and ventromedial and dorsomedial nuclei are particularly rich in glutamatergic neurons, followed by the supraoptic, paraventricular, and arcuate nuclei, whereas the suprachiasmatic nucleus does not express detectable amounts of VGLUT2 mRNA. Immunoreactive neurites are seen in very high densities in all regions analyzed, particularly in the preoptic region, followed by the ventromedial, dorsomedial, and arcuate nuclei as well as the external layer of the median eminence, whereas the mammillary complex does not exhibit VGLUT2 immunoreactivity. Many VGLUT2 immunoreactive fibers also contained synaptophysin, suggesting that the transporter is indeed localized to presynaptic terminals. Together, the results identify glutamatergic cell bodies throughout the septum-hypothalamus in region-specific patterns and show that glutamatergic nerve terminals are present in very large numbers such that most neurons in these brain regions can receive glutamatergic input. We examined the GnRH system as an example of a typical neuroendocrine system and could show that the GnRH perikarya are closely apposed by many VGLUT2-immunoreactive boutons, some of which also contained synaptophysin. The presence of VGLUT2 mRNA-containing cells in specific nuclei of the hypothalamus indicates that many neuroendocrine neurons coexpress glutamate as neurotransmitter, in addition to neuropeptides. These systems include the oxytocin, vasopressin, or CRH neurons as well as many others in the periventricular and mediobasal hypothalamus. The presence of VGLUT2 mRNA in steroid-sensitive regions of the hypothalamus, such as the anteroventral periventricular, paraventricular, or ventromedial nuclei indicates that gonadal and adrenal steroid can directly alter the functions of these glutamatergic neurons.

Central administration of neuropeptide Y reduces alpha-melanocyte-stimulating hormone-induced cyclic adenosine 5'-monophosphate response element binding protein (CREB) phosphorylation in pro-thyrotropin-releasing hormone neurons and increases CREB phosphorylation in corticotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus.

Neuropeptide Y (NPY) has a potent inhibitory effect on TRH gene expression in the paraventricular nucleus (PVN) and contributes to the fall in circulating thyroid hormone levels during fasting mediated by a reduction in serum leptin levels. Because alpha-MSH activates the TRH gene by increasing the phosphorylation of CREB in the nucleus of these neurons, we raised the possibility that at least one of the mechanisms by which NPY reduces TRH mRNA in hypophysiotropic neurons is by antagonizing the ability of alpha-MSH to phosphorylate CREB. As NPY increases CRH mRNA in the hypothalamus, we further determined whether intracerebroventricular (i.c.v.) administration of NPY regulates the phosphorylation of CREB in hypophysiotropic CRH neurons. NPY [10 micro g in artificial CSF (aCSF)] was administered into the lateral ventricle i.c.v. 30 min before the i.c.v. administration of aCSF or alpha-MSH (10 micro g in aCSF), the latter in a dose previously demonstrated to increase proTRH mRNA and phosphorylate CREB in TRH neurons. By double-labeling immunocytochemistry, only few TRH neurons in the PVN contained phosphoCREB (PCREB) in animals treated only with aCSF (4 +/- 0.2%) or with NPY followed by aCSF (9.7 +/- 2.5), whereas alpha-MSH-infused animals dramatically increased the percentage of TRH neurons containing PCREB (75.3 +/- 6.9%). Pretreatment with NPY before alpha-MSH infusion, however, significantly reduced the percentage of TRH neurons containing PCREB (40.8 +/- 3.5%) compared with alpha-MSH infused animals (P = 0.01). Only 12.2 +/- 0.9% of CRH neurons of the medial parvocellular neurons contained PCREB nuclei in vehicle-treated animals, whereas 30 min following NPY infusion, the number of CRH neurons containing PCREB increased dramatically to 88 +/- 2.9%. Whereas alpha-MSH infusion increased the percentage of CRH neurons that contained PCREB to 56 +/- 2.2% compared with control, animals pretreated with NPY further increased the number of CRH neurons colocalizing with PCREB to 87 +/- 2.5%. These data demonstrate a functional interaction between NPY and alpha-MSH in the regulation of proTRH neurons in the PVN, suggesting that NPY can antagonize alpha-MSH induced activation of the TRH gene by interfering with melanocortin signaling at the postreceptor level, preventing the phosphorylation of CREB. In contrast, NPY infusion increases the phosphorylation of CREB in CRH neurons, indicating that NPY has independent effects on discrete populations of neurons in the PVN, presumably mediated through different signaling mechanisms.