Region-specific regulation of glutamic acid decarboxylase (GAD) mRNA expression in central stress circuits

Abstract

Neurocircuit inhibition of hypothalamic paraventricular nucleus (PVN) neurons controlling hypothalamo-pituitary-adrenocortical (HPA) activity prominently involves GABAergic cell groups of the hypothalamus and basal forebrain. In the present study, stress responsiveness of GABAergic regions implicated in HPA inhibition was assessed by in situ hybridization, using probes recognizing the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD65 and GAD67 isoforms). Acute restraint preferentially increased GAD67 mRNA expression in several stress-relevant brain regions, including the arcuate nucleus, dorsomedial hypothalamic nucleus, medial preoptic area, bed nucleus of the stria terminalis (BST) and hippocampus (CA1 and dentate gyrus). In all cases GAD67 mRNA peaked at 1 hr after stress and returned to unstimulated levels by 2 hr. GAD65 mRNA upregulation was only observed in the BST and dentate gyrus. In contrast, chronic intermittent stress increased GAD65 mRNA in the anterior hypothalamic area, dorsomedial nucleus, medial preoptic area, suprachiasmatic nucleus, anterior BST, perifornical nucleus, and periparaventricular nucleus region. GAD67 mRNA increases were only observed in the medial preoptic area, anterior BST, and hippocampus. Acute and chronic stress did not affect GAD65 or GAD67 mRNA expression in the caudate nucleus, reticular thalamus, or parietal cortex. Overall, the results indicate preferential upregulation of GAD in central circuitry responsible for direct (hypothalamus, BST) or multisynaptic (hippocampus) control of HPA activity. The distinct patterns of GAD65 and GAD67 by acute versus chronic stress suggest stimulus duration-dependent control of GAD biosynthesis. Chronic stress-induced increases in GAD65 mRNA expression predict enhanced availability of GAD65 apoenzyme after prolonged stimulation, whereas acute stress-specific GAD67 upregulation is consistent with de novo synthesis of active enzyme by discrete stressful stimuli.

Fig. 1.

Localization of GAD65 mRNA in central stress circuits. GAD65 mRNA is expressed in numerous forebrain stress-relevant nuclei, including the anteromedial (am), anterolateral (al), posteromedial (pm), and posterointermediate (pi) subdivisions of the bed nucleus of the stria terminalis; the medial and lateral preoptic area (POA); the suprachiasmatic nucleus (SCN); the dorsomedial hypothalamic nucleus (DMH); the arcuate nucleus (ARC); and the hippocampal formation (HPC). GAD65 mRNA is also localized to additional regions not implicated in stress regulation, such as the reticular thalamic nucleus (RET). Scale bar, 1 mm.

Fig. 2.

Localization of GAD65 (A) and GAD67 (B) mRNAs in the immediate surround of the PVN. Note that very few GAD mRNA-expressing neurons are present in the medial parvocellular (mp) or posterior magnocellular (pm) PVN; however, aggregates of GAD-positive neurons can be observed to cluster just outside the PVN proper (A, B) and in the neighboring perifornical region. Higher power photomicrographs indicate high levels of GAD65 (C, D) and GAD67 (data not shown) mRNA in scattered neurons in both regions. fx, Fornix. Scale bars: A, B, 200 μm;C, D, 100 μm.

Fig. 3.

Semiquantitative assessment of the effect of acute restraint on GAD65 mRNA expression in forebrain stress-relevant nuclei.A, No changes in GAD65 mRNA expression were observed in the anterior hypothalamic nucleus (AHA), arcuate nucleus (ARC), dorsomedial hypothalamic nucleus (DMH), medial preoptic area (POA), suprachiasmatic nucleus (SCN), caudate nucleus (CAU), and reticular thalamic nucleus (RET). GAD65 mRNA is elevated 60 min after stress in the anteromedial subnucleus (AM) of the bed nucleus of the stria terminalis but is not altered in the anterodorsal (AD), posterolateral (PL), or posteromedial and posterointermediate subnuclei (PM/PI). B, Grain density analysis was used to assess GAD65 mRNA expression in the peri-PVN region (PePVN) and the perifornical nucleus (PeF). Reduced grain density was observed over neurons in the peri-PVN region 120 min after stress exposure.C, Densitometric analysis of GAD65 mRNA in the hippocampus revealed significant elevation in dentate gyrus (DG) 60 min after stress exposure. CTX, Parietal cortex; Unstr, Unstressed.

Fig. 4.

Semiquantitative assessment of the effect of acute restraint on GAD67 mRNA expression in forebrain stress-relevant nuclei.A, In contrast with GAD65 mRNA, GAD67 mRNA was elevated in the ARC, DMH, medialPOA, and anterodorsal and anteromedial BST 60 min after initiation of stress. In all cases, GAD67 mRNA levels returned to baseline by 120 min. B, No changes in GAD67 grain density were observed over neurons in the peri-PVN region orPeF after acute stress. C, GAD67 mRNA was elevated in the hippocampal subfield CA1 andDG 60 min after stress initiation. Abbreviations are given in the Figure 3 legend.

Fig. 5.

Semiquantitative assessment of GAD65 mRNA expression in forebrain stress-relevant nuclei after chronic stress or handling. A, GAD65 mRNA expression was significantly increased in the AHA, DMH,POA, SCN, and the AD andAM divisions of the BST of rats exposed to chronic stress. B, A significant increase in GAD65 mRNA expression/cell was seen in both the peri-PVN region and the perifornical nucleus by grain density analysis. C, GAD65 mRNA expression in the hippocampus was unaffected by chronic stress.Han, Handled; Str, stressed. Other abbreviations are given in the Figure 3 legend.

Fig. 6.

Semiquantitative assessment of GAD67 mRNA expression in forebrain stress-relevant nuclei after chronic stress or handling. A, Chronic stress induction of GAD65 mRNA expression was observed only in the medial POA and theAD and AM divisions of the BST of rats.B, No changes in GAD67 mRNA were seen in the peri-PVN region or perifornical nucleus by grain density analysis.C, Chronic stress exposure increased GAD67 mRNA expression in subfield CA3 of the hippocampus and in theDG. Abbreviations are given in the legends of Figures 3and 5.