Immunohistochemical and neurochemical evidence for GABAA receptor heterogeneity between the hypothalamus and cortex

doi:10.1016/0891-0618(94)90016-7

Journal of Chemical Neuroanatomy

Volume 7, Issue 4, October 1994, Pages 243-252

https://doi.org/10.1016/0891-0618(94)90016-7 Get rights and content

Abstract

This study examined both the function of the GABA_A receptor complex and the expression of its α1, α2 and α3 subunits within the hypothalamus as compared to that of the cerebral cortex. A large number of different GABA_A receptor subunit combinations potentially exist in various brain regions which, presumably, would intimate differing receptor structure and function. Here, we present evidence that the average functional characteristics of GABA_A receptors within the rat hypothalamus are considerably different from those of the cerebral cortex. We assessed two neurochemical measures of GABA_A receptor function: namely, chloride-facilitation of [³H]flunitrazepam binding and GABA-mediated ³⁶chloride uptake. [³H]Flunitrazepam binding in the rat cortex was facilitated by increasing concentrations (12.5–500 mM) of chloride, and this facilitation was responsive to 15 min restraint. Yet, hypothalamic [³H]flunitrazepam binding was not responsive to increasing chloride-concentration in either the basal or restraint conditions. Also, maximal facilitation of GABA-mediated ³⁶chloride uptake was significantly blunted in the hypothalamus relative to cortex (7.4 ± 0.9 versus 35.8 ± 1.5 nmoles/mg protein, respectively). While in vitro addition of 10 μM diazepam shifted GABA-mediated ³⁶chloride uptake curves of the cortex to the left, diazepam addition appeared to be without effect in the hypothalamus. However, the blunted maximal facilitation of GABA on hypothalamic ³⁶chloride uptake made accurate determination of the EC₅₀ for the diazepam-potentiation difficult. In addition to these functional disparities between the regions, differences in subunit expression were also apparent. Distributions of α1, α2 and α3 subunit immunoreactivities within cingulate, parietal and temporal cortices and 8 major hypothalamic regions were assessed. Staining of the α1 subunit was prevalent throughout the hypothalamus and cortex, and dense in both regions. However, the α2 and α3 subunits, while of intermediate density in cortex, were of low density or absent (α3) in the hypothalamus. The α2-immunoreactivity was restricted to cell bodies of the arcuate nucleus, dorsomedial nucleus and overlying dorsal area and to neuropil staining of the median eminence. Thus, functional responsiveness of the GABA_A receptor differs in the hypothalamus relative to the cortex and this would seem related to the presence of different receptor α subunits in homogenate preparations of the two regions.

References (54)

A. Baude et al.
Differential subcellular distribution of the α6 subunit versus the α1 and $β2 3$ subunits of the GABA_A/benzodiazepine receptor complex in granule cells of the cerebellar cortex
Neuroscience
(1992)
J. Benavides et al.
Comparative distribution of central ω (benzodiazepine) modulatory site subtypes with high, intermediate and low affinity for zolpidem and alpidem
Brain Res.
(1993)
R.C. Drugan et al.
Central and peripheral benzodiazepine receptors: involvement in an organism's response to physical and psychological stress
Neurosci. Biobehav. Rev.
(1991)
I. Elekes et al.
Concentrations of GABA and glycine in discrete brain nuclei
Neuropharmacology
(1986)
K. Fuchs et al.
Identification of α2- and α3-subunits of the GABA_A — benzodiazepine receptor complex purified from the brains of young-rats
FEBS Lett.
(1990)
N. Harary et al.
The relationship of benzodiazepine binding sites to the norepinephrine projection in the hypothalamus of the adult rat
Brain Res.
(1989)
J.R. Inglefield et al.
Selective effects on CRF neurons and catecholamine terminals in two stress-responsive regions of adult rat brain after prenatal exposure to diazepam
Brain Res. Bull.
(1993)
J.R. Inglefield et al.
Alterations in behavioral responses to stressors following excitotoxin lesions of dorsomedial hypothalamic regions
Brain Res.
(1994)
C.K. Kellogg et al.
GABA-stimulated chloride uptake and enhancement by diazepam in synaptoneurosomes from rat brain during prenatal and postnatal development
Dev. Brain Res.
(1989)
C.K. Kellogg et al.
Importance of hypothalamic function to stressor-induced responsiveness of the cortical GABA_A receptor in the cerebral cortex: a non-corticosterone influence
Brain Res.
(1993)

C.G. Markgraf et al.

Hypothalamic, midbrain, and bulbar areas involved in the defense reaction in rabbits

Physiol. Behav.

(1991)

M.K. Markwell et al.

Protein determinations in membrane and lipoprotein samples: manual and automated procedures

Methods Enzymol.

(1981)

H. Milani et al.

GABA-benzodiazepine modulation of aversion in the medial hypothalamus of the rat

Pharmacol. Biochem. Behav.

(1987)

H. Monnikes et al.

CRF in the paraventricular nucleus of the hypothalamus induces dose-related behavioral profile in rats

Brain Res.

(1992)

H. Okamura et al.

Neurons containing messenger RNA encoding glutamate decarboxylase in rat hypothalamus demonstrated by in situ hybridization, with special emphasis on cell groups in medial preoptic area, anterior hypothalamic area and dorsomedial hypothalamic nucleus

Neuroscience

(1990)

R.J. Primus et al.

Experience influences environmental modulation of function at the benzodiazepine (BZD)/GABA receptor chloride channel complex

Brain Res.

(1991)

R.D. Schwartz et al.

Acute stress enhances the activity of GABA receptor-gated chloride ion channel in brain

Brain Res.

(1987)

A. Shekhar

GABA receptors in the region of the dorsomedial hypothalamus of rats regulate anxiety in the elevated plus-maze test. I. Behavioral measures

Brain Res.

(1993)

A. Shekhar et al.

GABA receptors in the posterior hypothalamus regulate experimental anxiety in rats

Brain Res.

(1990)

E. Sigel et al.

The effect of subunit composition of rat brain GABA_A receptors on channel function

Neuron

(1990)

R.D. Simmons et al.

Prenatal diazepam exposure in rats: long-lasting, receptor-mediated effects on hypothalamic norepinephrine-containing neurons

Brain Res.

(1984)

J.H. Skerritt et al.

Enhancement of GABA binding by benzodiazepines and related anxiolytics

Eur. J. Pharmacol.

(1983)

L.W. Swanson et al.

Regulation of multiple peptides in CRF parvocellular neurosecretory neurons: implications for the stress response

T.G. Waldrop et al.

Microinjection of GABA antagonists into the posterior hypothalamus elicits locomotor activity and cardiorespiratory activation

Brain Res.

(1988)

C.P. Yardley et al.

The hypothalamic and brainstem areas from which the cardiovascular and behavioral components of the defence reaction are elicited in the rat

J. Auton. Nerv. Syst.

(1986)

J. Zezula et al.

Separation of α1, α2 and α3 subunits of the GABA_A benzodiazepine receptor complex by immunoaffinity chromatography

Brain Res.

(1991)

F. Zimprich et al.

Immunohistochemical localization of the α1, α2 and α3 subunit of the GABA_A receptor in the rat brain

Neurosci. Lett.

(1991)

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Our mouse data are in keeping with most GABAA subunit antibodies and RNA probes studies which have revealed higher levels of α2 subunit throughout the hypothalamus (Backberg et al., 2004; Fritschy and Mohler, 1995; Wisden et al., 1992). However, others have revealed greater α1 levels when compared with α2 mRNAs or proteins (Inglefield et al., 1994). Areas showing moderate to strong levels of α2, α3, and β2 mRNA transcripts included the dorsomedial nucleus, preoptic, anterior, and ventromedial hypothalamic areas.
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In this regard, it is interesting to note that the increase of Bzd binding site expression observed in the ventromedial nucleus coincides with the most intense period of synapse formation in this nucleus in rat [8,47,58]. These differences could also be explained by differential expression of mRNAs coding for subunits of Bzd binding sites [19,26,63]. Indeed, it has been reported that age-related alterations in Bzd binding sites could be a consequence of the altered regulation of GABAA receptor alpha subunit gene transcription [39].
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It is widely accepted that calorie restriction is an effective way of delaying the aging process. Also, there is an indication that the beneficial effects exerted by dietary manipulation may be due to a direct effect at the molecular level like gene expression. The studies were conducted to determine whether calorie restriction prevents any age-related changes in the structural and molecular aspects of the GABA_A–BZ receptor. In aged (24-month old diet ad libitum) rats, the binding of [³⁵S]t-butyl-bicyclophosphorothionate (TBPS) was significantly reduced in the cerebellum. In contrast, [³⁵S]TBPS binding remained unchanged in the cerebellum of calorie restricted old rats. In order to evaluate the molecular basis of these changes, the α sub-unit mRNA levels were measured. The GABA_A receptor α₁ sub-unit mRNA level remained unchanged in both the old groups of rats. The α₂ subunit mRNA level was significantly decreased in the cerebellum of aged rats (24-month old ad libitum), whereas it remained unchanged in the cerebellum of calorie restricted old animals. These findings indicate a selective age and diet related modulation in the stoichiometry of the GABA_A receptor in aging.
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¹: Present address: Department of Pharmacology, Box 3813, Duke University Medical Center, Durham, NC 27710, USA.

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Immunohistochemical and neurochemical evidence for GABAA receptor heterogeneity between the hypothalamus and cortex

Abstract

Neuroscience

Brain Res.

Neurosci. Biobehav. Rev.

Neuropharmacology

FEBS Lett.

Brain Res.

Brain Res. Bull.

Brain Res.

Dev. Brain Res.

Brain Res.

Physiol. Behav.

Methods Enzymol.

Pharmacol. Biochem. Behav.

Brain Res.

Neuroscience

Brain Res.

Brain Res.

Brain Res.

Brain Res.

Neuron

Brain Res.

Eur. J. Pharmacol.

Brain Res.

J. Auton. Nerv. Syst.

Brain Res.

Neurosci. Lett.

Immunohistochemical and neurochemical evidence for GABA_A receptor heterogeneity between the hypothalamus and cortex