Research reportDistribution of α1, α4, γ2, and δ subunits of GABAA receptors in hippocampal granule cells
Introduction
The GABAA receptor gene family includes seven major classes of subunits with multiple subtypes: six types of α subunit, four types of β subunit, three types of γ subunit, one type of δ, one type each of θ, ɛ, and π subunit [2], [27]. It is known that certain subunits cluster on postsynaptic membranes and mediate fast synaptic inhibition in the brain. For example, electron microscopic examination of GABAergic synapses in cerebellum and hippocampus reveals aggregates of α1, β2/3, and γ2 subunits at GABAergic synapses [16], [19]. Conversely, post-embedding immunogold electron microscopic studies of cerebellar granule cells have demonstrated that α6 and δ subunits are expressed in the extrasynaptic membrane [11], [19].
In addition to cerebellar granule cells, dentate granule cells in the hippocampus also express the δ subunit mRNA and polypeptide [3], [32]. The α6 subunit is not expressed in these neurons and the δ subunit is believed to combine with the α4 subunit to form functional receptors [34]. A recent study of mouse dentate granule cells demonstrated that GABAA receptors containing the δ subunit are expressed in the perisynaptic region [35]. In addition to the α4 and δ subunits, dentate granule cells express α1 and γ2 subunits. Previous studies suggest that the α1 subunit is expressed at synapses [16]. Recent electrophysiological studies in hippocampal dentate granule cells suggest the existence of synaptic and extrasynaptic GABAA receptors in these neurons [14], [33]. These studies suggested that GABAA receptors with distinct subunit compositions are present in either synaptic or extrasynaptic locations. Taken together, these studies suggest that in dentate granule cells, the distribution of α1, γ2 subunit-containing receptors is distinct from that of receptors containing α4, δ subunits; receptors containing α1, γ2 subunits are expressed at synapses, and α4, δ subunits are expressed at extrasynaptic membrane.
While the distribution of α1, α4, γ2, and δ subunits in dentate gyrus has been described using light microscopy, few studies have compared the distribution of these subunits using high-resolution confocal laser scanning microscopy (CLSM) [29]. Furthermore, the colocalization of these subunits with GABAergic synaptic markers, GAD65 and gephyrin, in hippocampal dentate granule cells has not been assessed. We used double-label fluorescence immunohistochemistry of the hippocampus visualized with CLSM, to study the distribution of α1, α4, γ2, and δ subunits in hippocampal dentate granule cells. High-resolution images of granule cell layer in sections double-labeled for various subunits and synaptic markers GAD65 or gephyrin were analyzed quantitatively.
Section snippets
Animal and tissue preparation and immunohistochemistry
Experiments were performed using 11 male adult Sprague–Dawley rats weighing 250–350 g. Six animals were used to study the colocalization of GABAA receptor subunits with GAD; and five animals were used to study colocalization with gephyrin. Animals were housed at 22 °C, two per cage, on a standard light–dark schedule with free access to food and water. Animals were handled according to NIH Animal Care and Use Guidelines and a protocol approved by the University of Virginia Animal Care and Use
Distribution of α1 and γ2 subunits
The distribution of GABAA receptor α1 subunit immunoreactivity in hippocampal dentate gyrus of adult rats was studied using CLSM. Interneurons at the hilar margin of the dentate gyrus were more intensely stained by anti-α1 subunit antibody than neurons in the granule cell layer (Fig. 2A, arrowhead). Previous studies have demonstrated that a subset of GABAergic interneurons expresses the α1 subunit at a higher level than the principal neurons [8], [10]. Only moderately intense immunoreactivity
Discussion
The distribution of GABAA receptor α1, α4, γ2, and δ subunits and synaptic markers GAD65 and gephyrin in rat hippocampal dentate gyrus was described using high-resolution CLSM. Quantitative analysis of high-resolution images of the granule cell layer in sections double-labeled for various subunits and the synaptic markers revealed that a distinct fraction of α1 and γ2 immunoreactivity colocalized with synaptic markers, which was in the form of large clusters. In contrast, the α4 and δ subunit
Acknowledgements
Public Health Service Grants from NINDS (RO1 NS40337, and RO1NS044370) and a grant from the Epilepsy Foundation through the generous support of the American Epilepsy Society and the Milken Family Foundation supported this work. We thank Dr. A Periasamy and the staff of the Keck Center for Cellular Imaging for their assistance with confocal laser microscopy. We thank Drs. Kevin Kelly and Howard Goodkin for reviewing the manuscript.
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