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.

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.

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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.

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.

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.

The locus coeruleus (LC) is a key brainstem region involved in arousal and is highly responsive to alerting/stressful stimuli, including those that activate the hypothalamic-pituitary-adrenocortical (HPA) axis. It is currently unclear whether the LC exerts any regulatory influence on the HPA axis and, consequently, on neuroendocrine responses to stress. The present studies were designed to test the hypothesis that the LC promotes HPA axis responses to acute and chronic stress. Adult male rats received bilateral (6-hydroxydopamine) lesions of the LC that produced severe cell loss in the LC and 80% depletion of noradrenaline in medial prefrontal cortex. Notably, lesions did not affect dopamine-beta-hydroxylase protein content in the parvocellular paraventricular nucleus (PVN), indicating a lack of collateral damage to other ascending noradrenergic pathways. LC lesions significantly reduced peak adrenocorticotropic hormone (ACTH) and corticosterone responses to 30 min acute restraint stress. However, LC lesions did not significantly attenuate neuroendocrine or other physiological responses to a 4-week chronic variable stress regimen. LC lesions did not substantially affect basal concentrations of plasma corticosterone or corticotropin-releasing hormone mRNA expression in the hypothalamic paraventricular nucleus following chronic stress. We conclude that the LC is a HPA-excitatory brain region, promoting neuroendocrine and physiological responses primarily to acute stress. However, a potential role for the LC in the induction of HPA axis hyperactivity following chronic stress can not be ruled out.

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.