Medial Parvocellular Paraventricular Nucleus Research Articles

Estrogen receptors ERα and ERβ participation in hypothalamus–pituitary–adrenal axis activation by hemorrhagic stress

The sympato-adrenal-system and hypothalamus–pituitary–adrenal (HPA) axis are anatomically and functionally connected with participation of several brain areas that express estrogen receptors (ERα and ERβ). We assessed the neuronal activity of these areas for FOS expression and the action of PPT (ERα agonist) or DPN (ERβ agonist) in HPA axis activity during hemorrhagic stress. Ovariectomized Wistar rats treated with vehicle (DMSO) or ER agonists were catheterized for blood collection. Animals received (control) or not (hemorrhagic) immediate reposition with the same volume of saline. Immunohistochemistry was performed for FOS, tyrosine hydroxylase (TH) and corticotropin releasing hormone (CRH) in the brain areas. In vehicle-treated animals, hemorrhage enhanced: plasma corticosterone (CORT), oxytocin (OT) and vasopressin (AVP) measured by radioimmunoassay; the expression of TH-FOS co-localized neurons in ventrolateral medulla (A1C1) and FOS expression in medial parvocellular paraventricular nucleus (mpPVN). In controls, PPT decreased: plasma CORT; FOS expression at locus coeruleus (LC); FOS and CRH-FOS at mpPVN, compared to vehicle. After hemorrhage, PPT decreased: plasma CORT; FOS expression at LC and mpPVN; TH-FOS at LC, solitary tract nucleus (NTS), A1C1; CRH-FOS at mpPVN, compared to vehicle. After hemorrhage DPN decreased: plasma CORT; FOS expression at LC and mpPVN; TH-FOS at LC, A1C1; CRH-FOS at mpPVN, compared to vehicle. PPT blocked the increase of OT secretion and increased AVP secretion, after hemorrhage. DPN reduced OT and increased AVP levels, regardless hemorrhage. In hemorrhagic stress, ERα and ERβ reduced the HPA axis activation and neuronal activity in brain areas involved in the HPA axis control.

Tonic, But Not Phasic Corticosterone, Constrains Stress ActivatedExtracellular-Regulated-Kinase 1/ 2 Immunoreactivity Within the Hypothalamic Paraventricular Nucleus

The negative-feedback actions of corticosterone (CORT) depend on both phasic and tonic CORT secretion patterns to regulate hypothalamic-pituitary-adrenal (HPA) axis activity. How these two different CORT secretion pattens influence specific intracellular signal transduction pathway activity within the cellular elements of the HPA axis has not been determined. For example, it is unknown whether CORT has suppressive actions over signal transduction events within medial parvocellular paraventricular nucleus (PVN) corticotrophin-releasing hormone (CRH) neurones, nor whether these suppressive actions are responsible for alterations in PVN transcriptional processes and neurohormone secretion associated with stress. The extracellular-regulated kinase (ERK) is a stress activated intracellular signalling molecule that is potentially subject to glucocorticoid negative-feedback regulation. We tested the ability of CORT to modulate levels of the active (phosphorylated) form of ERK (pERK1/2) in the PVN of rats. Acute psychological stress (restraint) produced a rapid increase in the number of PVN pERK1/2 immunopositive cells within CRH neurones. Absence of tonic CORT via adrenalectomy (ADX) produced no change in basal pERK1/2 cell counts but augmented the increased pERK1/2 cell counts elicited by acute restraint. Treatment of ADX rats with CORT in the drinking water normalised this enhanced pERK1/2 response to stress. By contrast, treatment of ADX rats with a phasic increase in CORT 1 h before restraint had no effect on pERK1/2 cell counts, despite substantially suppressing stress-induced PVN crh gene expression and adrenonocorticotrophic hormone secretion. This tonic CORT inhibition of stress-induced activation of ERK1/2 may involve both alteration of the activity of stress-dependent neural inputs to PVN CRH neurones and alteration within those neurones of stress-dependent intracellular signalling mechanisms associated with ERK activation.

Pregnancy-related changes in connections from the cervix to forebrain and hypothalamus in mice

The transneuronal tracer pseudorabies virus was used to test the hypothesis that connections from the cervix to the forebrain and hypothalamus are maintained with pregnancy. The virus was injected into the cervix of nonpregnant or pregnant mice, and, after 5 days, virus-labeled cells and fibers were found in specific forebrain regions and, most prominently, in portions of the hypothalamic paraventricular nucleus. With pregnancy, fewer neurons and fibers were evident in most brain regions compared to that in nonpregnant mice. In particular, little or no virus was found in the medial and ventral parvocellular subdivisions, anteroventral periventricular nucleus, or motor cortex in pregnant mice. By contrast, labeling of virus was sustained in the dorsal hypothalamus and suprachiasmatic nucleus in all groups. Based upon image analysis of digitized photomicrographs, the area with label in the rostral and medial parvocellular paraventricular nucleus and magnocellular subdivisions was significantly reduced in mice whose cervix was injected with virus during pregnancy than in nonpregnant mice. The findings indicate that connections from the cervix to brain regions that are involved in sensory input and integrative autonomic functions are reduced during pregnancy. The findings raise the possibility that remaining pathways from the cervix to the forebrain and hypothalamus may be important for control of pituitary neuroendocrine secretion, as well as for effector functions in the cervix as pregnancy nears term.

Dopamine–oxytocin interactions in penile erection

Dopamine and oxytocin have established roles in the central regulation of penile erection in rats; however, the neural circuitries involved in a specific erectile context and the interaction between dopamine and oxytocin mechanisms remain to be elucidated. The medial preoptic area (MPOA), supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus may serve as candidate sites because they contain oxytocin cells, receive dopaminergic inputs and have been implicated in mediating masculine sexual behavior. Double immunofluorescence revealed that substantial numbers of oxytocin cells in the MPOA, SON and PVN possess dopamine D(2), D(3) and D(4) receptors. In anaesthetized rats, using intracavernous pressure as a physiological indicator of erection, blockade of lumbosacral oxytocin receptors (UK, 427843) reduced erectile responses to a nonselective dopamine agonist (apomorphine), suggesting that dopamine recruits a paraventriculospinal oxytocin pathway. In conscious males in the absence of a female, penile erection elicited by a D(2)/D(3) (Quinelorane) but not D(4) (PD168077) agonist was associated with activation of medial parvocellular PVN oxytocin cells. In another experiment where males were given full access to a receptive female, a D(4) (L-745870) but not D(2) or D(3) antagonist (L-741626; nafadotride) inhibited penile erection (intromission), and this was correlated with SON magnocellular oxytocin neuron activation. Together, the data suggest dopamine's effects on hypothalamic oxytocin cells during penile erection are context-specific. Dopamine may act via different parvocellular and magnocellular oxytocin subpopulations to elicit erectile responses, depending upon whether intromission is performed. This study demonstrates the potential existence of interaction between central dopamine and oxytocin pathways during penile erection, with the SON and PVN serving as integrative sites.

Distribution of glucagon-like peptide-1 immunoreactivity in the hypothalamic paraventricular and supraoptic nuclei

Glucagon-like peptide-1 (GLP-1) plays a role in modulating neuroendocrine and autonomic function. The hypothalamic paraventricular nucleus (PVN) contains aggregations of GLP-1 fibers and expresses GLP-1 receptors, making it a likely site of action for GLP-1 signaling. The current study was designed to establish domains of GLP-1 action, focusing on axosomatic appositions on different neuroendocrine and autonomic cell populations in the PVN. The data indicate abundant GLP-1-immunoreactive terminal appositions on corticotropin-releasing hormone neurons in the medial parvocellular PVN. GLP-1 positive boutons can also be observed in apposition to oxytocinergic neurons and on retrogradely labeled pre-autonomic neurons projecting to the region of the nucleus of the solitary tract. In contrast, there were very few vasopressinergic neurons with GLP-1 appositions. Overall, the data indicate that the central GLP-1 system preferentially targets neurons in hypophysiotrophic zones of the PVN, consistent with excitatory actions of GLP-1 on adrenocorticotropin release. GLP-1 is also in position to influence oxytocin secretion and control outflow to brainstem cardiovascular relays.

Selective contributions of the medial preoptic nucleus to testosterone-dependant regulation of the paraventricular nucleus of the hypothalamus and the HPA axis

Previous data have consistently demonstrated an inhibitory effect of androgens on stress-induced hypothalamic-pituitary-adrenal (HPA) responses. Several brain regions may influence androgen-mediated inhibition of the HPA axis, including the medial preoptic area. To test the role of the medial preoptic nucleus (MPN) specifically, we examined in high- and low-testosterone-replaced gonadectomized rats bearing discrete bilateral lesions of the MPN basal and stress-induced indexes of HPA function, and the relative levels of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) mRNA in the amygdala. High testosterone replacement decreased plasma adrenocorticotropin hormone (ACTH) and paraventricular nucleus (PVN) Fos responses to restraint exposure in sham- but not in MPN-lesioned animals. AVP-, but not CRH-immunoreactivity staining in the external zone of the median eminence was increased by testosterone in sham animals, and MPN lesions blocked this increment in AVP. A similar interaction between MPN lesions and testosterone occurred on AVP mRNA levels in the medial nucleus of the amygdala. These findings support an involvement of MPN projections in mediating the AVP response to testosterone in both the medial parvocellular PVN and medial amygdala. We conclude that the MPN forms part of an integral circuit that mediates the central effects of gonadal status on neuroendocrine and central stress responses.

Cocaine- and amphetamine-regulated transcript (CART) expression is differentially regulated in the hypothalamic paraventricular nucleus of lactating rats exposed to suckling or cold stimulation

Neural stimuli, such as suckling or cold exposure, increase TRH mRNA in the paraventricular nucleus (PVN) of the rat hypothalamus, yet only suckling induces prolactin secretion. As TRH co-localizes with cocaine- and amphetamine-regulated transcript (CART) in hypophysiotropic neurons of the PVN, and CART inhibits TRH-induced prolactin release but not TRH-induced TSH release in adenohypophyseal cell cultures, we raised the possibility that differential regulation of CART gene expression in the PVN may explain the differences in prolactin secretion following each of the two stimuli. Primiparous female rats were mated and handled daily during the pre- and postpartum periods. After delivery, the litter was adjusted to 8 pups and at mid-lactation, dams were separated from their pups for 8 h and exposed to either 1 h of cold or 30 min of suckling. Long-term effects of suckling were studied by separating pups from their mothers for 24 h, followed by a 12 h period of continuous suckling. Serum TSH levels increased in response to cold exposure, while prolactin levels were increased by suckling and diminished by cold exposure. CART mRNA levels increased in rostral and mid parts of the medial parvocellular PVN following cold exposure but not after suckling stimulation. These data demonstrate a differential regulation of CART gene expression in hypophysiotropic neurons in response to stimuli that increase TRH mRNA levels, and suggest that CART activation in the PVN may contribute to the decrease in PRL release when the thyroid axis is activated by cold exposure.

Activation of Group II Metabotropic Glutamate Receptors Attenuates Both Stress and Cue-Induced Ethanol-Seeking and Modulates c-fosExpression in the Hippocampus and Amygdala

Major precipitating factors for relapse to drug use are stress and exposure to drug-related environmental stimuli. Group II (mGlu(2/3)) metabotropic glutamate receptors (mGluRs) are densely expressed within circuitries mediating the motivating effects of stress and drug cues and, therefore, may participate in regulating drug-seeking linked to both of these risk factors. Thus, we tested the hypothesis that pharmacological activation of group II mGluRs modifies both stress- and cue-induced ethanol-seeking, using reinstatement models of relapse. In parallel, brain c-fos expression was examined to identify neural substrates for the behavioral effects of group II mGluR activation. The selective mGlu(2/3) agonist LY379268 (1R,4R,5S,6R-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate) (0.3, 1.0, and 3.0 mg/kg, s.c.) dose dependently blocked the recovery of extinguished ethanol-seeking induced by either footshock stress or ethanol-associated discriminative stimuli. These effects were accompanied by modulation of c-fos expression in the hippocampus, central nucleus of the amygdala, bed nucleus of the stria terminalis, and medial parvocellular paraventricular nucleus of the hypothalamus. The results implicate group II mGluRs as a shared neuropharmacological substrate for ethanol-seeking elicited by both drug cues and stress and identify group II mGluRs as promising treatment targets for relapse prevention.

Limited brain diffusion of the glucocorticoid receptor agonist RU28362 following i.c.v. administration: Implications for i.c.v. drug delivery and glucocorticoid negative feedback in the hypothalamic–pituitary–adrenal axis

The experiments described herein present a method for tracking diffusion of the glucocorticoid receptor agonist RU28362 in brain following i.c.v. drug administration. A useful property of glucocorticoid receptor is that it is primarily cytoplasmic when unbound and rapidly translocates to the nucleus when bound by ligand. Thus, removal of endogenous glucocorticoids by adrenalectomy allows us to identify brain regions with activated glucocorticoid receptor after i.c.v. glucocorticoid receptor agonist treatment by examining the presence or absence of nuclear glucocorticoid receptor immunostaining. We have previously demonstrated that an i.p. injection of 150 μg/kg RU28362 1 h prior to restraint stress is sufficient to suppress stress-induced hypothalamic–pituitary–adrenal axis hormone secretion [Ginsberg AB, Campeau S, Day HE, Spencer RL (2003) Acute glucocorticoid pretreatment suppresses stress-induced hypothalamic–pituitary–adrenal axis hormone secretion and expression of corticotropin-releasing hormone hnRNA but does not affect c-fos mRNA or fos protein expression in the paraventricular nucleus of the hypothalamus. J Neuroendocrinol 15:1075–1083]. We report here, however, that in rats i.c.v. treatment with a high-dose of RU28362 (1 μg) 1 h prior to stressor onset does not suppress stress-induced hypothalamic–pituitary–adrenal axis activity. We then performed a series of experiments to examine the possible differences in glucocorticoid receptor activation patterns in brain and pituitary after i.c.v. or i.p. treatment with RU28362. In a dose-response study we found that 1 h after i.c.v. injection of RU28362 (0.001, 0.1 and 1.0 μg) glucocorticoid receptor nuclear immunoreactivity was only evident in brain tissue immediately adjacent to the lateral or third ventricle, including the medial but not more lateral portion of the medial parvocellular paraventricular nucleus of the hypothalamus. In contrast, i.p. injection of RU28362 produced a uniform predominantly nuclear glucocorticoid receptor immunostaining pattern throughout all brain tissue. I.c.v. injection of the endogenous glucocorticoid receptor agonist, corticosterone (1 μg) also had limited diffusion into brain tissue. Time-course studies indicated that there was not a greater extent of nuclear glucocorticoid receptor immunostaining present in brain after shorter (10 or 30 min) or longer (2 or 3 h) intervals of time after i.c.v. RU28362 injection. Importantly, time-course studies found that i.c.v. RU28362 produced significant increases in nuclear glucocorticoid receptor immunostaining in the anterior pituitary that were evident within 10 min after injection and maximal after 1 h. These studies support an extensive literature indicating that drugs have very limited ability to diffuse out of the ventricles into brain tissue after i.c.v. injection, while at the same time reaching peripheral tissue sites. In addition, these studies indicate that significant occupancy of some glucocorticoid receptor within the paraventricular nucleus of the hypothalamus and pituitary is not necessarily sufficient to suppress stress-induced hypothalamic–pituitary–adrenal axis activity.

Dissection of peripheral and central endogenous opioid modulation of systemic interleukin-1β responses using c- fos expression in the rat brain

In opiate addicts or patients receiving morphine treatment, it has been reported that the immune system is often compromised. The mechanisms responsible for the adverse effects of opioids on responses to infection are not clear but it is possible that central and/or peripheral opioid receptors may be important. We have utilised an experimental immune challenge model in rats, the systemic administration of the human pro-inflammatory cytokine interleukin-1β (IL-1β) to study the effects of selectively blocking peripheral opioid receptors only (using naloxone methiodide) or after blocking both central and peripheral opioid receptors (using naloxone). Pre-treatment with naloxone methiodide decreased (15%) IL-1β-induced Fos-immunoreactivity (Fos-IR) in medial parvocellular paraventricular nucleus (mPVN) corticotropin-releasing hormone (CRH) neurons but increased responses in the ventrolateral medulla (VLM) C1 (65%) and nucleus tractus solitarius (NTS) A2 (110%) catecholamine cell groups and area postrema (136%). However no effect of blocking peripheral opioid receptors was detected in the central nucleus of the amygdala (CeA) or dorsal bed nucleus of the stria terminalis (BNST). We next determined the effect of blocking both central and peripheral opioid receptors with naloxone and, when compared to the naloxone methiodide pre-treated group, a further 60% decrease in Fos-IR mPVN CRH neurons induced by IL-1β was detected, which was attributed to block of central opioid receptors. Similar comparisons also detected decreases in Fos-IR neurons induced by IL-1β in the VLM A1, VLM C1 and NTS A2 catecholamine cell groups, area postrema, and parabrachial nucleus. In contrast, pre-treatment with naloxone increased Fos-IR neurons in CeA (98%) and dorsal BNST (72%). These results provide novel evidence that endogenous opioids can influence central neural responses to systemic IL-1β and also suggest that the differential patterns of activation may arise because of actions at central and/or peripheral opioid receptors that might be important in regulating behavioural, hypothalamic-pituitary-adrenal axis and sympathetic nervous system responses during an immune challenge.

Organization and regulation of paraventricular nucleus glutamate signaling systems: N‐methyl‐D‐aspartate receptors

Stress activation of the hypothalamo-pituitary-adrenocortical (HPA) axis is mediated in part by glutamatergic neurotransmission. The precise nature of glutamate effects on stress-integrative hypothalamic paraventricular nucleus (PVN) neurons remains to be determined. Therefore, the current study was designed to delineate the organization of glutamate/NMDA receptor systems in the PVN and to assess regulation of PVN glutamate receptor subunit expression by chronic intermittent stress and glucocorticoids. Immunohistochemical studies verified that N-methyl-D-aspartate (NMDA) receptor subunit proteins NR1 and NR2A/2B are expressed in the medial parvocellular PVN, indicating the potential for NMDA receptor regulation of corticotropin-releasing hormone (CRH) release. Dual-label confocal analysis revealed that CRH neurons are apposed by vesicular glutamate transporter 2 (VGLUT2)-containing terminals, consistent with glutamatergic innervation from hypothalamus and/or brainstem. In situ hybridization analysis revealed a significant and selective stress-induced decrease (37%) in NR2B subunit mRNA expression in the CRH-containing region of the PVN. No changes were observed for NR1 or NR2A mRNAs. In contrast, none of the subunits investigated showed altered expression following adrenalectomy with or without low/high-dose corticosterone replacement. Thus, the observed stress regulation is likely mediated by neurogenic mechanisms in the PVN and upstream stress-transducing neurocircuitry. Because a loss of NR2B subunit inclusion in NR receptors would likely confer increased Ca(++) conductance and faster deactivation kinetics, the stress-induced decrease in NR2B mRNA is consistent with enhanced glutamate signaling in the PVN following chronic stress and, perhaps, increased basal HPA activity and more rapid and/or more robust HPA responses to stress.

RU486 blocks fasting-induced decrease of neuronal nitric oxide synthase in the rat paraventricular nucleus

It has been reported that food deprivation decreases expression of neuronal nitric oxide synthase (nNOS) in the hypothalamic paraventricular nucleus (PVN). Food deprivation produces autonomic changes and the PVN nitric oxide has been suggested to be involved in regulation of autonomic functions. In order to understand the molecular mechanism by which food deprivation decreases nNOS expression in the PVN, we examined if plasma glucocorticoids, which reported to be elevated during food deprivation, mediates the fasting-induced down-regulation of the PVN-nNOS. Male Sprague–Dawley rats underwent 48 h of food deprivation, but not water deprivation, with/without subcutaneous RU486, glucocorticoid receptor antagonist, and the brain tissues were processed for immunohistochemistry with specific antibodies against nNOS. Immunoreactivity of phosphorylated cAMP response element-binding protein (pCREB) was also examined in the PVN sections, because nNOS promoter carries cAMP response element (CRE). Food deprivation significantly decreased both nNOS and pCREB immunoreactivity (-ir) in the medial parvocellular PVN, and RU486 blocked this reduction. In the posterior magnocellular PVN, nNOS-ir, but not pCREB-ir, was decreased by food deprivation, and RU486 exerted no effect. These results suggest that glucocorticoid receptor may mediate the fasting-induced down-regulation of nNOS in the parvocellular PVN, but not in the magnocellular PVN.

Neurocircuitry of Stress Integration: Anatomical Pathways Regulating the Hypothalamo-Pituitary-Adrenocortical Axis of the Rat

The hypothalamo-pituitary-adrenocortical (HPA) axis is recruited by the organism in response to real or perceived threats to homeostasis ("stress"). Regulation of this neuroendocrine system is accomplished by modulation of secretory tone in hypophysiotrophic neurons of the medial parvocellular paraventricular nucleus. Excitation of these neurons is mediated by several sources: direct (and perhaps indirect) inputs from brainstem neurons regulating autonomic tone/arousal; circumventricular organs monitoring blood and CSF constituents; and local-circuit neurons within the hypothalamus and basal forebrain. The latter are predominantly GABAergic; notably, these areas are targets for descending GABAergic input from limbic structures, and may promote PVN secretory activity via disinhibition. Neurosecretory paraventricular nucleus neurons are inhibited by glucocorticoid-dependent and -independent mechanisms. Glucocorticoid negative feedback appears to act both locally and in extrahypothalamic loci, and is likely integrated in a region- and stressor-specific manner. Inhibitory input to the medial parvocellular paraventricular nucleus emanate predominantly from the bed nucleus of the stria terminalis and hypothalamus, and are likely regulated by neuroendocrine homeostats. Descending limbic inhibitory information appears to act through excitation of these inhibitory inputs. Overall, integration of stressful information is a multi-faceted process integrating prior experience and real or anticipated homeostatic disruption into appropriate activation and deactivation of the hypothalamo-pituitary-adrenocortical axis.

Chronic stress regulates levels of mRNA transcripts encoding β subunits of the GABA A receptor in the rat stress axis

Semi-quantitative hybridization histochemical analyses were undertaken to determine expression levels of mRNA transcripts encoding the β1–3 subunits of the GABA Areceptor within the rat hypothalamic paraventricular nucleus (PVN) and hippocampal formation following exposure to a chronic non-habituating stress protocol. After delivery of a battery of stressors on a randomized schedule over a 3-week period, expression levels of the β1 subunit of the GABA A receptor were found to be decreased in the medial parvocellular PVN (mpPVN) by 48.3% relative to control animals. Levels of β2 mRNA following chronic stress were also found to be decreased in the mpPVN (29.8%), but increased in hippocampal subfields CA 1 and CA 3 (33.9 and 23.2%, respectively) and increased (24%) in the dentate gyrus. The results suggest that GABA A receptor subunit composition may be altered at a key regulatory site, and may have important implications for studies aimed at understanding GABAergic inhibitory influences upon the hypothalamic–pituitary–adrenocortical (HPA) axis. Hypophysiotropic CRH neurons serve as the origin of the final common pathway for glucocorticoid secretion in response to stressful stimuli, and GABAergic afferents have been implicated in afferent control of these neurons. Regulation of GABA A receptors at these sites may alter the efficacy of a major inhibitory influence upon the stress axis, and thereby modulate stress-induced glucocorticoid secretion.

Expression of ionotropic glutamate receptor subunit mRNAs in the hypothalamic paraventricular nucleus of the rat

The hypopthalamic paraventricular nucleus (PVN) coordinates multiple aspects of homeostatic regulation, including pituitary-adrenocortical function, cardiovascular tone, metabolic balance, fluid/electrolyte status, parturition and lactation. In all cases, a substantial component of this function is controlled by glutamate neurotransmission. In this study, the authors performed a high-resolution in situ hybridization analysis of ionotropic glutamate receptor subunit expression in the PVN and its immediate surround. N-methyl-D-aspartate (NMDA) receptor 1 (NMDAR1), NMDAR2A, and NMDAR2B mRNAs were expressed highly throughout the PVN and its perinuclear region as well as in the subparaventricular zone. NMDAR2C/2D expression was limited to subsets of neurons in magnocellular and hypophysiotrophic regions. In contrast with NMDA subunit localization, AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate)-preferring and kainate (KA)-preferring receptor subunit mRNAs were expressed heterogeneously in the PVN and surround. Glutamate receptor 1 (GluR1) mRNA labeling was most intense in preautonomic subregions, whereas GluR2, GluR4, GluR5, and KA2 were expressed in hypophysiotrophic cell groups. It is noteworthy that GluR5 mRNA expression was particularly robust in the dorsolateral region of the medial parvocellular PVN, suggesting localization in corticotropin-releasing hormone neurons. All four AMPA subunits and GluR6 and GluR7 mRNAs were expressed highly in the perinuclear PVN region and the subparaventricular zone. These data suggest the capacity for multifaceted regulation of PVN function by glutamate, with magnocellular neurons preferentially expressing NMDA subunits, preautonomic neurons preferentially expressing AMPA subunits, and hypophysiotrophic neurons preferentially expressing KA subunits. Localization of all species in the perinuclear PVN suggests that glutamate input to the immediate region of the PVN may modulate its function, perhaps by communication with local gamma-aminobutyric acid neurons.

Differential recruitment of hypothalamic neuroendocrine and ventrolateral medulla catecholamine cells by non-hypotensive and hypotensive hemorrhages

We performed c- fos expression experiments in conscious rats to quantify the threshold and extent of activation of hypothalamic neuroendocrine cells in response to non-hypotensive and hypotensive hemorrhages allowing us to assess whether their pattern of recruitment corresponded to known oxytocin, vasopressin and ACTH release patterns. Also, because previous studies have implicated ventrolateral medulla catecholamine cells in the generation of certain hypothalamic neuroendocrine cell responses, we examined the response of ventrolateral medulla catecholamine cells to non-hypotensive and hypotensive hemorrhages and directly tested their role in regulating neuroendocrine cell responses to hypotensive hemorrhage. Animals were subjected to hemorrhages of 0, 4, 8, 12 or 16 ml/kg BW, the latter two levels being hypotensive. We found that only supraoptic nucleus vasopressin cells were significantly activated by the smallest non-hypotensive hemorrhage (4 ml/kg), which corresponds to reports that only vasopressin is released into the plasma after a small hemorrhage. Hypotensive hemorrhages resulted in significant recruitment of paraventricular and supraoptic oxytocin and vasopressin cells and parvocellular cells of the medial division of the paraventricular nucleus. Vasopressin cells were recruited in much greater numbers than oxytocin cells, which is in agreement with previous findings that there is a greater release of vasopressin than oxytocin into the plasma after hypotensive hemorrhage. In addition, medial parvocellular cells of the paraventricular nucleus, most likely to be tuberoinfundibular-projecting corticotropin-releasing factor cells, were activated by hypotensive hemorrhage only when arterial pressure dropped below 60 mmHg which also corresponds well with the plasma release response of ACTH. Ventrolateral medulla catecholamine cells were only recruited by hypotensive hemorrhages. While caution must be exercised in interpreting an absence of response, this certainly suggests that catecholamine cells are unlikely to have a role in the activation of supraoptic neurosecretory cells in response to non-hypotensive hemorrhages. Unilateral lesions of the ventrolateral medulla catecholamine cell column, corresponding primarily to the location of A1 noradrenergic cells, significantly reduced the hypotensive hemorrhage-induced activation of hypothalamic vasopressin, oxytocin and medial parvocellular paraventricular nucleus cells. This suggests that A1 noradrenergic cells contribute to the activation of these neuroendocrine cell populations, including oxytocin cells, which is an unexpected finding. More significantly, however, because the reduction in responsiveness after A1 lesions was similar for all cell categories, it seems likely that other factors must determine the differential recruitment of hypothalamic neuroendocrine cells in response to a hypotensive hemorrhage.

Hypertensive rats exhibit heightened expression of corticotropin-releasing factor in activated central neurons in response to restraint stress

To test the hypothesis that chronically elevated sympathetic drive is associated with hyperreactiveness of autonomic centers in the brain to stress, adult spontaneously hypertensive rats (SHRs) and two strains of normotensive rats (Wistar Kyoto [WKY] and Sprague Dawley [SD] rats) were acutely exposed to restraint stress; controls from each strain were not stressed. Brain sections were prepared for Fos immunohistochemistry to identify activated neurons in the paraventricular nucleus of the hypothalamus, Barrington's nucleus of the pons, nucleus of the tractus solitarius, and ventrolateral medulla, or for combined Fos immunohistochemistry and corticotropin-releasing factor (CRF) in situ hybridization in the paraventricular nucleus and Barrington's nucleus. Restraint led to increased activation of neurons in all nuclei. Strain differences were found only in the caudal and rostral paraventricular nucleus where restraint resulted in greater numbers of activated neurons in SHRs compared to either normotensive strain. Levels of CRF mRNA in Barrington's nucleus of unrestrained rats were similar among strains. After restraint, mRNA levels and double labeled neurons were increased in Barrington's nucleus of SHRs. In unstressed rats, CRF mRNA levels were elevated in some regions of the paraventricular nucleus in SHRs. After restraint, mRNA levels increased throughout the paraventricular nucleus of SHRs. Significantly greater numbers of double labeled neurons were found in the dorsolateral medial and ventral medial parvocellular paraventricular nucleus of stressed SHRs compared to WKY or SD rats. These data show that chronic elevation in sympathetic activity, present in SHRs, is associated with hyperreactiveness of the paraventricular and Barrington's nucleus including recruitment of neurons to express CRF, and may have important implications for the response of the hypothalamo–pituitary–adrenal axis during stress.

Expression and neuropeptidergic characterization of estrogen receptors (ERalpha and ERbeta) throughout the rat brain: anatomical evidence of distinct roles of each subtype.

The recent cloning of a second estrogen receptor (ER) provided a new tool to investigate and clarify how estrogens are capable of communicating with the brain and influence gene expression and neural function. The purpose of the present study was to define the neuroanatomical organization of each receptor subtype using a side-by-side approach and to characterize the cellular population (s) expressing the ERbeta transcript in the endocrine hypothalamus using immunohistochemistry combined with in situ hybridization. Axonal transport inhibition was accomplished to cause neuropeptide accumulation into the cytoplasm and thus facilitate the detection of all positive luteinizing hormone-releasing hormone (LHRH), corticotropin-releasing factor (CRF), vasopressin (AVP), oxytocin (OT), gastrin-related peptide (GRP), and enkephalin (ENK) neurons. The genes encoding either ERalpha or -beta were expressed in numerous limbic-associated structures, and fine differences were found in terms of intensity and positive signal. Such phenomenon is best represented by the bed nucleus of the stria terminalis (BnST) and preoptic area/anterior hypothalamus, where the expression pattern of both transcripts differed across subnuclei. The novel ER was also found to be expressed quite exclusively in other hypothalamic nuclei, including the supraoptic (SON) and selective compartments (magnocellular and autonomic divisions) of the paraventricular nucleus (PVN). A high percentage of the ERbeta-expressing neurons located in the ventro- and dorsomedial PVN are of OT type; 40% of the OT-ir cells forming the medial magnocellular and ventromedial parvocellular PVN showed a clear hybridization signal for ERbeta mRNA, whereas a lower percentage (15-20%) of OT neurons were positive in the caudal parvocellular PVN and no double-labeled cells were found in the rostral PVN and other regions of the brain with the exception of the SON. Very few AVP-ir neurons expressing ERbeta transcript were found throughout the rat brain, although the medial PVN displayed some scattered double-labeled cells (<5%). Quite interestingly, the large majority of the ERbeta-positive cells in the caudal PVN were colocalized within CRF-ir perikarya. Indeed, more than 60-80% of the CRF-containing cells located in the caudolateral division of the parvocellular PVN exhibited a positive hybridization signal for ERbeta mRNA, whereas very few (<5%) neuroendocrine CRF-ir parvocellular neurons of the medial PVN expressed the gene encoding ERbeta. A small percentage of ERbeta-expressing cells in the dorsocaudal and ventromedial zones of the parvocellular PVN were also ENK positive. The ventral zone of the medial parvocellular PVN also displayed GRP-ir neurons, but no convincing hybridization signal for ERbeta was detected in this neuronal population. Finally, as previously described for the gene encoding the classic ER, LHRH neurons of both intact and colchicine-pretreated animals did not express the novel estrogen receptor. This study shows a differential pattern of expression of both receptors in the brain of intact rats and that ERbeta is expressed at various levels in distinct neuropeptidergic populations, including OT, CRF, and ENK. The influence of estrogen in mediating genomic and neuronal responses may therefore take place within these specific cellular groups in the brains of cycling as well as intact male mammals.

Leptin prevents fasting-induced suppression of prothyrotropin-releasing hormone messenger ribonucleic acid in neurons of the hypothalamic paraventricular nucleus.

Prolonged fasting is associated with a number of changes in the thyroid axis manifested by low serum T3 and T4 levels and, paradoxically, low or normal TSH. This response is, at least partly, caused by suppression of proTRH gene expression in neurons of the hypothalamic paraventricular nucleus (PVN) and reduced hypothalamic TRH release. Because the fall in thyroid hormone levels can be blunted in mice by the systemic administration of leptin, we raised the possibility that leptin may have an important role in the neuroendocrine regulation of the thyroid axis, through effects on hypophysiotropic neurons producing proTRH. Adult male, Sprague-Dawley rats were either fed normally, fasted for 3 days, or fasted and administered leptin at a dose of 0.5 microg/gm BW i.p. every 6 h. Fasted animals showed significant reduction in plasma total and free T4 and T3 levels compared with controls, that were restored toward normal by the administration of leptin. Percent free T4, but not percent free T3, increased during fasting, further suggesting a reduction in plasma transthyretin levels that did not return to fed levels after leptin administration. By semiquantitative analysis of in situ hybridization autoradiograms, proTRH messenger RNA in medial parvocellular PVN neurons was markedly suppressed in the fasting animals but was restored to normal by leptin administration [fed vs. fast vs. fast/leptin (density units x 10(8)): 8.5 +/- 0.4, 3.2 +/- 0.2, 8.1 +/- 0.8]. In contrast, proTRH messenger RNA in adjacent neurons in the lateral hypothalamus that do not have a hypophysiotropic function remained unchanged by any of the experimental manipulations. These findings indicate that leptin has a selective, central action to modulate the hypothalamic-pituitary-thyroid axis by regulating proTRH gene expression in the PVN but does not have peripheral effects on thyroid-binding proteins. We propose that the fall in circulating leptin levels during fasting resets the set point for feedback inhibition by thyroid hormones on the biosynthesis of hypophysiotropic proTRH, thereby allowing adaptation to starvation.

Involvement of medullary catecholamine cells in neuroendocrine responses to systemic cholecystokinin.

Systemic administration of cholecystokinin (CCK) stimulates neurosecretory oxytocin (OT) and tuberoinfundibular corticotrophin releasing factor (CRF) cells of the hypothalamus. Data from previous studies suggest that A2 noradrengeric neurons of the dorsomedial medulla contribute to the OT cell response, but the role of other medullary catecholamine cells remains unclear. Using c-fos expression as a marker for cellular activity, we have found that CCK (100 micrograms/kg, i.p.) activates substantial populations of tyrosine hydroxylase and phenyl-N-methyl-transferase immunoreactive cells in the medulla, consistent with recruitment of overlapped noradrenergic and adrenergic cell populations in both the ventrolateral and dorsomedial medulla. In the ventrolateral medulla there was a particularly prominent activation of C1 adrenergic neurons at the level of the obex. To directly test the contribution of VLM catecholamine cells to hypothalamic neuroendocrine cell responses to CCK, animals were prepared with unilateral VLM lesions corresponding to those areas that had displayed the most marked response to CCK. VLM lesioned animals treated with CCK displayed a significant although small reduction in paraventricular nucleus (PVN) OT cell c-fos expression ipsilateral to the lesion, but no change in the responses of supraoptic nucleus OT cells or in cells of the medial parvocellular PVN, many of which are CRF cells. These findings indicate that VLM catecholamine cells make little contribution to hypothalamic neuroendocrine cell responses to CCK and thus serve to further highlight the role of dorsomedial catecholamine cells. However, it is now apparent that, in addition to A2 noradrenergic cells, CCK treatment also recruits C2 adrenergic cells of the dorsomedial medulla, many of which have previously been shown to project to the PVN.