Expression of α5 GABAA receptor subunit in developing rat hippocampus

doi:10.1016/j.devbrainres.2004.04.003

Developmental Brain Research

Volume 151, Issues 1–2, 19 July 2004, Pages 87-98

https://doi.org/10.1016/j.devbrainres.2004.04.003 Get rights and content

Abstract

The GABAergic system plays an important role in the hippocampal development. Here we have studied the developmental expression of the α5 subunit of the GABA_A receptor (from rat hippocampus) by RT-competitive PCR, immunoblot and immunocytochemistry. Our results demonstrated an early induction of the α5 subunit expression (at mRNA and protein levels) during the first postnatal week, peaking at P5 and decreasing after this age. The peak of α5 subunit expression precedes the peak of expression for the synaptophysin, GAD65 and GAD67. Thus, the increase in the α5 GABA_A receptor subunit expression may precede the GABAergic synaptogenesis. Importantly, between P0 and P7, the expression of the α5 subunit was concentrated at the cell somata of the pyramidal and granular cells. After P10, its localization shifted from the cell bodies to the dendritic layers. This developmental pattern is similar to that reported for the Na⁺–K⁺–2Cl⁻ system and it might be correlated with the transition from excitatory to inhibitory GABAergic activity.

Introduction

In the adult hippocampus, the GABAergic cells mediate the major inhibitory control of the principal neurons, promoting the Cl⁻ entry through the GABA_A receptor. However, in the developing hippocampus, GABA may act as a major excitatory neurotransmitter [6]. During this period, the GABAergic system, also interacting through the GABA_A receptor, mediates the cell depolarization and, in consequence, promotes the calcium entry through the voltage dependent Ca²⁺ channel [9], [21] and it is implicated in the generation of synchronized neuronal discharges, known as giant depolarizing potentials [5]. On the other hand, the implication of the neurotransmitter GABA in the differentiation of both principal and GABAergic cells during the first postnatal week is also known (see [5], [24] for reviews). Concomitantly, the expression of the different subunits of the GABA_A receptor also seems to be developmental regulated. Within the different subunits, those belonging to the alpha family are the most affected subunits during this period [35].

Although there is abundant information about the developmental changes in the expression of the GABA_A receptor subunits (see [5], [7], [16], [20], [28]), there is no any detailed study focused on the expression and neuronal localization of the α5 subunit (see Ref. [20]). However, it is noteworthy that, at this early postnatal days, the GABA_A receptor displayed a slow decay kinetic [14], [18] and very low affinity for zolpidem [7], [30], similar to those observed in cortical cells expressing the α5 subunit [14]. These findings suggest the relevance of the α5 subunit in the GABA_A receptor during this important developmental period. Thus, in this work we have analyzed the postnatal maturation of the α5 subunit in the rat hippocampus. To this aim, we have determined the expression (by reverse transcription (RT)-competitive PCR and Western blots) and the neuronal localization (by immunohistochemistry) of this subunit during the postnatal maturation of the rat hippocampus.

Section snippets

Hippocampus isolation

New born (0, 2, 5, 7, 10 and 20 days after birth) and adult (3 months) Wistar rats were killed by decapitation and the hippocampi were rapidly dissected and frozen in liquid nitrogen. The hippocampi were stored at −80 °C until use. For 0 to 5 days after birth, three pools were made using the hippocampi from 10 different pups. For all other ages, three to five different rats were used. All animal experiments were carried out in accordance with the National Institute of Health Guide for the care

Developmental changes in the expression of the α5 subunit

We first quantified the mRNA levels coding for the α5 subunit. As shown in Fig. 1A, at birth the levels of α5 subunit mRNA were similar to that of adults and increased until P5, reaching a peak at this age. After this time point, there was a significant decrease in the expression of this subunit. In fact, the mRNA levels at P10 and P20 were similar to that observed in adults. The values from P2 and P5 were significantly different from adults (ANOVA F(5,19)=41.40, p=0.00001; Tukey p<0.05). The

Discussion

In the present work, we have determined the postnatal maturation of the α5 subunit of the GABA_A receptor by RT-competitive-PCR, Western blots and immunohistochemistry. The results demonstrated that: (i) the α5 subunit expression (mRNA and protein levels) increases during the first postnatal week, peaking at P5. After this postnatal age, the expression of this subunit decreases towards the adult values; (ii) during this postnatal stage (P0–P7), the expression of this subunit protein is

Acknowledgements

We thank Dr. J.M. Fritschy for providing the anti-α5-Nterminal antibody. We also thank S. Jimenez for the preparation and Western blots with the His-fusion proteins. This work was supported by Grants FIS 00/0314 and SAF2002-03448 (to J.V), FIS 03/0214 (to A.G) and FIS PI03/0177 (to D.R.). D.R. is supported by a contract from the Ramon y Cajal program from Ministerio de Ciencia y Tecnologia. B.R. and J.F.L-T. were recipient of fellowships from Ministerio de Ciencia y Tecnologia, and J.V., and

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¹: These two authors contributed equally to this work.

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Research reportExpression of α5 GABAA receptor subunit in developing rat hippocampus

Abstract

Introduction

Section snippets

Hippocampus isolation

Developmental changes in the expression of the α5 subunit

Discussion

Acknowledgements

J. Biol. Chem.

Eur. J. Pharmacol.

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Trends Neurosci.

Trends Neurosci.

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Mol. Cell. Neurosci.

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J. Biol. Chem.

Prog. Brain Res.

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Neuroscience

Brain Res., Brain Res. Protoc.

Native gamma-aminobutyric acid type A receptors from rat hippocampus, containing both α1 and α5 subunits, exhibit a single benzodiazepine binding site with α5 pharmacological properties

J. Pharmacol. Exp. Ther.

Gamma-aminobutyric acid(A) receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development

J. Neurochem.

Intact sorting, targeting, and clustering of aminobutyric acid A receptor subtypes in hippocampal neurons in vitro

J. Comp. Neurol.

Research report
Expression of α5 GABA_A receptor subunit in developing rat hippocampus