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.

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.

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.

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.