Brainstem origins of glutamatergic innervation of the rat hypothalamic paraventricular nucleus.

Multiple lines of evidence document a role for glutamatergic input to the hypothalamic paraventricular nucleus (PVH) in stress-induced activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. However, the neuroanatomical origins of the glutamatergic input have yet to be definitively determined. We have previously shown that vesicular glutamate transporter 2 (VGLUT2) is the predominant VGLUT isoform expressed in the basal forebrain and brainstem, including PVH-projecting regions, and that the PVH is preferentially innervated by VGLUT2-immunoreactive terminals/boutons. The present study employed a dual-labeling approach, combining immunolabeling for a retrograde tract tracer, Fluoro-Gold (FG), with in situ hybridization for VGLUT2 mRNA, to map the brainstem and caudal forebrain distribution of glutamatergic PVH-projecting neurons. The present report presents evidence for substantial dual labeling in the periaqueductal gray, caudal portions of the zona incerta and subparafascicular nucleus, and the lateral parabrachial nucleus. The current data also suggest that relatively few PVH-projecting neurons in ascending raphe nuclei, nucleus of the solitary tract, or ventrolateral medulla are VGLUT2 positive. The data reveal multiple brainstem origins of glutamatergic input to PVH that are positioned to play a role in transducing a diverse range of stressful stimuli.

Forebrain origins of glutamatergic innervation to the rat paraventricular nucleus of the hypothalamus: differential inputs to the anterior versus posterior subregions.

The hypothalamic paraventricular nucleus (PVN) regulates numerous homeostatic systems and functions largely under the influence of forebrain inputs. Glutamate is a major neurotransmitter in forebrain, and glutamate neurosignaling in the PVN is known to mediate many of its functions. Previous work showed that vesicular glutamate transporters (VGluTs; specific markers for glutamatergic neurons) are expressed in forebrain sites that project to the PVN; however, the extent of this presumed glutamatergic innervation to the PVN is not clear. In the present study retrograde FluoroGold (FG) labeling of PVN-projecting neurons was combined with in situ hybridization for VGluT1 and VGluT2 mRNAs to identify forebrain regions that provide glutamatergic innervation to the PVN and its immediate surround in rats, with special consideration for the sources to the anterior versus posterior PVN. VGluT1 mRNA colocalization with retrogradely labeled FG neurons was sparse. VGluT2 mRNA colocalization with FG neurons was most abundant in the ventromedial hypothalamus after anterior PVN FG injections, and in the lateral, posterior, dorsomedial, and ventromedial hypothalamic nuclei after posterior PVN injections. Anterograde tract tracing combined with VGluT2 immunolabeling showed that 1) ventromedial nucleus-derived glutamatergic inputs occur in both the anterior and posterior PVN; 2) posterior nucleus-derived glutamatergic inputs occur predominantly in the posterior PVN; and 3) medial preoptic nucleus-derived inputs to the PVN are not glutamatergic, thereby corroborating the innervation pattern seen with retrograde tracing. The results suggest that PVN subregions are influenced by varying amounts and sources of forebrain glutamatergic regulation, consistent with functional differentiation of glutamate projections.

Effect of animal facility construction on basal hypothalamic-pituitary-adrenal and renin-aldosterone activity in the rat.

Although loud noise and intense vibration are known to alter the behavior and phenotype of laboratory animals, little is known about the effects of nearby construction. We studied the effect of a nearby construction project on the classic stress hormones ACTH, corticosterone, renin, and aldosterone in rats residing in a barrier animal facility before, for the first 3 months of a construction project, and at 1 month after all construction was completed. During some of the construction, noise and vibrations were not obvious to investigators inside the animal rooms. Body weight matched for age was not altered by nearby construction. During nearby construction, plasma ACTH, corticosterone, and aldosterone were approximately doubled compared with those of pre- and postconstruction levels. Expression of CRH mRNA in the paraventricular nucleus of the hypothalamus, CRH receptor and POMC mRNA in the anterior pituitary, and most mRNAs for steroidogenic genes in the adrenal gland were not significantly changed during construction. We conclude that nearby construction can cause a stress response without long-term effects on hypothalamic-pituitary-adrenal axis gene expression and body weight.

Functional role of local GABAergic influences on the HPA axis.

Neuronatomical and pharmacological studies have established GABA-mediated inhibition of the HPA axis at the level of the PVN. The origin of this innervation is a series of local hypothalamic and adjacent forebrain regions that project to stress-integrative hypophysiotropic CRH neurons. While a role in tonic inhibition of the stress axis is likely, this system of inhibitory loci is also capable of producing a dynamic braking capacity in the context of the neuroendocrine stress response. The latter function is mediated in large part by glutamatergic forebrain afferents that increase GABA release at the level of the PVN. In addition, this local GABA system can be inhibited by upstream GABAergic projection neurons, producing activation of the HPA axis via removal of GABAergic tone. This PVN projecting GABA network interfaces with a wide range of homeostatic mechanisms, and is capable of biochemical plasticity in response to chronic stress. Collectively, the elements of this system provide for exquisite control of neuroendocrine activation in the face of stressful stimuli, and loss of this regulatory capacity may underlie many stress-related disorders.

Daily cocaine self-administration under long-access conditions augments restraint-induced increases in plasma corticosterone and impairs glucocorticoid receptor-mediated negative feedback in rats.

Cocaine addiction appears to be associated with a drug-induced dysregulation of stressor responsiveness that may contribute to further cocaine use. The present study examined alterations in stressor-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis in rats provided daily access to cocaine for self-administration (SA) under long-access conditions (1.0 mg/kg/infusion; 6 hx14 days). Cocaine self-administering rats displayed reduced basal plasma corticosterone (CORT) levels but showed an augmented restraint-induced percent increase response from baseline compared to saline self-administering controls when measured 24 days after SA testing. This augmented CORT response may have been attributable to impaired glucocorticoid receptor (GR)-mediated feedback regulation of HPA function, since cocaine self-administering rats were also less susceptible to dexamethasone (0.01 mg/kg, i.p.) suppression of plasma CORT levels. GR protein expression measured using Western blot analysis was significantly reduced in the dorsomedial hypothalamus (including the paraventricular nucleus [PVN]) but not in the pituitary gland, ventromedial hypothalamus, dorsal hippocampus, ventral subiculum, medial prefrontal cortex or amygdala in cocaine self-administering rats. Surprisingly, basal corticotropin-releasing hormone (CRH) mRNA or post-restraint increases in CRH mRNA measured at a single (90 min) time-point in the PVN using in situ hybridization did not differ between groups. The findings suggest that cocaine use produces persistent changes in individual responsiveness to stressors that may contribute to the addiction process.

Restraint-induced corticosterone secretion and hypothalamic CRH mRNA expression are augmented during acute withdrawal from chronic cocaine administration.

Stress responses during cocaine withdrawal likely contribute to drug relapse and may be intensified as a consequence of prior cocaine use. The present study examined changes in stressor-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis during acute withdrawal from chronic cocaine administration. Adult male Sprague-Dawley rats received daily administration of cocaine (30 mg/kg, i.p.) or saline for 14 days. Twenty-four hours after the last injection, rats in each group were sacrificed under stress-free conditions or following 30 min of immobilization. Plasma corticosterone (CORT) was measured in trunk-blood using radioimmunoassay, corticotropin-releasing hormone (CRH) mRNA levels in the paraventricular nucleus (PVN) of the hypothalamus were measured using in situ hybridization and glucocorticoid receptor (GR) protein expression in the pituitary gland and dissected brain regions was measured using Western blot analysis. Basal CRH mRNA in the PVN was unaltered as a result of prior cocaine administration. However, a significant increase in CRH mRNA was observed 90 min following the termination of restraint in cocaine withdrawn, but not saline-treated, rats. Basal CORT was also unaffected by prior cocaine administration, but the CORT response measured immediately after restraint was significantly augmented in cocaine-withdrawn rats. Differences in GR protein expression in number of regions implicated in negative feedback regulation of HPA function, including the hypothalamus, were not observed. These findings indicate that the HPA response to stressors is intensified during early withdrawal from cocaine administration and may be independent of changes in GR-mediated negative feedback.

Spatial memory in middle-aged female rats: assessment of estrogen replacement after ovariectomy.

Previous studies have shown that estrogen influences diverse aspects of neuronal function and morphology and modulates acquisition of various memory tasks in young adult female rodents. It is not clear whether estrogen is critical for optimal memory function in middle-aged female animals, i.e. when cyclicity gradually declines. We trained young adult (5 months) and older (10 months) female Long-Evans rats on a win-shift (delay) 12-arm radial maze (7 arms blocked pre-delay). Rats were preoperatively trained to criterion (< or =2 errors/trial for 3 days) with no delay then with a 60 s delay. All rats were ovariectomized when the age groups were 9 (Y) and 14 months (MA), respectively. Following recovery and retraining to criterion, each rat underwent consecutive treatment cycles with vehicle (Oil) or 17-beta-estradiol benzoate (E). Each 6-day acute treatment cycle, modeled after protocols previously shown to induce morphological and electrophysiological plasticity in the hippocampus at 24-48 h after estrogen injection, consisted of two consecutive daily injections of 10 microg E or Oil (0.1 ml subcutaneously) on Days 1-2 (Oil-Oil or E-E), testing on Days 3-4 at 60 s or 6 h delays, with Days 5-6 comprising of washout days. Each rat received a total of 4 treatment cycles, alternating between Oil and E cycles, in counterbalanced order. Estrogen treatment had no effect in either age group on pre-delay or post-delay errors at either 60 s or 6 h delays. The data indicate that the cyclic estrogen replacement regimen does not influence spatial memory function in young or middle-aged animals in the hippocampal-dependent appetitive radial maze task. Discussion of these unexpected results includes consideration of important experimental design factors that differ between our study and some previous reports, such as the extensive training and task experience our subject received prior to testing for estrogen effects.

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.

Role of the paraventricular nucleus microenvironment in stress integration.

The hypothalamic paraventricular nucleus is the primary controller of hypothalamo-pituitary-adrenocortical glucocorticoid release. In performing this function, the paraventricular nucleus summates a variety of information from both external and internal sources into a net secretory signal to the adrenal cortex. In this review, we will provide an overview of neuronal circuit mechanisms governing activation and inhibition of hypophysiotrophic neurons, highlight recent developments in our understanding of nonsynaptic mechanisms regulating paraventricular cellular activity, including dendritic neuropeptide release, direct steroid feedback, cytokine cascades and gaseous neurotransmission, and illustrate the capacity for hypophysiotrophic, neurohypophysial and preautonomic paraventricular effector pathways to work together in control of glucocorticoid release. The current state of knowledge reveals the paraventricular nucleus to be a dynamic entity, capable of integrating diverse classes of signals into control of adrenocortical activation.

Distribution of vesicular glutamate transporter mRNA in rat hypothalamus.

Two isoforms of the vesicular glutamate transporter, VGLUT1 and VGLUT2, were recently cloned and biophysically characterized. Both VGLUT1 and VGLUT2 specifically transport glutamate into synaptic vesicles, making them definitive markers for neurons using glutamate as a neurotransmitter. The present study takes advantage of the specificity of the vesicular transporters to afford the first detailed map of putative glutamatergic neurons in the rat hypothalamus. In situ hybridization analysis was used to map hypothalamic distributions of VGLUT1 and VGLUT2 mRNAs. VGLUT2 is clearly the predominant vesicular transporter mRNA found in the hypothalamus; rich expression can be documented in regions regulating energy balance (ventromedial hypothalamus), neuroendocrine function (preoptic nuclei), autonomic tone (posterior hypothalamus), and behavioral/homeostatic integration (lateral hypothalamus, mammillary nuclei). Expression of VGLUT1 is decidedly more circumspect and is confined to relatively weak labeling in lateral hypothalamic regions, neuroendocrine nuclei, and the suprachiasmatic nucleus. Importantly, dual-label analysis revealed no incidence of colocalization of VGLUT1 or VGLUT2 mRNAs in glutamic acid decarboxylase (GAD) 65-positive neurons, indicating that GABA neurons do not express either transporter. Our data support a major role for hypothalamic glutamatergic neurons in regulation of all aspects of hypothalamic function.

Local circuit regulation of paraventricular nucleus stress integration: glutamate-GABA connections.

Limbic neurocircuits play a central role in regulation of the hypothalamic-pituitary-adrenocortical (HPA) axis. Limbic influences on adrenocortical hormone secretion are mediated by transynaptic activation or inhibition of hypophysiotrophic neurons in the medial parvocellular paraventricular nucleus (PVN). Projections from the ventral subiculum, prefrontal cortex, medial amygdala, lateral septum, paraventricular thalamus and suprachiasmatic nucleus (SN) terminate in the immediate surround of the PVN, an area heavily populated by GABAergic interneurons. As such, these regions are positioned to modulate paraventricular output via excitation or inhibition of interneuronal projections into the PVN. In addition, the same limbic and diencephalic regions have projections to local PVN-projecting hypothalamic and basal telencephalic nuclei, including the dorsomedial and medial preoptic nuclei and the bed nucleus of the stria terminalis. These regions are involved in both inhibitory and excitatory regulation of the stress axis, indicating that they contain heterogeneous neuronal populations whose relative impact on the PVN is determined by the nature of afferent stimuli. Thus, limbic modulation of the pituitary-adrenocortical system appears to be a multisynaptic process integrated at the level of local PVN-projecting neurocircuits. Local circuits are likely the primary integrators of anticipatory stress responses, and may indeed be the focus of HPA dysfunction seen with aging or affective disease.

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.