The γ2 Subunit of the GABAA Receptor is Concentrated in Synaptic Junctions Containing the α1 and β23 Subunits in Hippocampus, Cerebellum and Globus Pallidus

doi:10.1016/S0028-3908(96)00086-X

Neuropharmacology

Volume 35, Issues 9–10, 1996, Pages 1425-1444

https://doi.org/10.1016/S0028-3908(96)00086-X Get rights and content

Abstract

The γ2 subunit is necessary for the expression of the full benzodiazepine pharmacology of GABA_A receptors and is one of the major subunits in the brain. In order to determine the location of channels containing the γ2 subunit in relation to GABA-releasing terminals on the surface of neurons, a new polyclonal antipeptide antiserum was developed to the γ2 subunit and used in high resolution, postembedding, immunoelectron-microscopic procedures. Dual immunogold labelling of the same section for two subunits, and up to three sections of the same synapse reacted for different subunits, were used to characterize the subunit composition of synaptic receptors. The γ2 subunit was present in type 2, “symmetrical” synapses in each of the brain areas studied, with the exception of the granule cell layer of the cerebellum. The γ2 subunit was frequently co-localized in the same synaptic junction with the α1 and β $23$ subunits. The immunolabelling of synapses was coincident with the junctional membrane specialization of the active zone. Immunolabelling for the receptor often occurred in multiple clusters in the synapses. In the hippocampus, the γ2 subunit was present in basket cell synapses on the somata and proximal dendrites and in axo-axonic cell synapses on the axon initial segment of pyramidal and granule cells. Some synapses on the dendrites of GABAergic interneurones were densely labelled for the γ2, α1 and β $23$ subunits. In the cerebellum, the γ2 subunit was present in both distal and proximal Purkinje cell dendritic synapses established by stellate and basket cells, respectively. On the soma of Purkinje cells, basket cell synapses were only weakly labelled. Synapses on interneuron dendrites were more densely labelled for the γ2, α1 and β $23$ subunits than synapses on Purkinje or granule cells. Although immunoperoxidase and immunofluorescence methods show an abundance of the γ2 subunit in granule cells, the labelling of Golgi synapses was much weaker with the immunogold method than that of the other cell types. In the globus pallidus, many type 2 synapses were labelled for the γ2 subunit together with α1 and β $23$ subunits. The results show that γ2 subunit-containing receptor channels are highly concentrated in GABAergic synapses that also contain the α1 and β $23$ subunits. Channels containing the γ2 subunit are expressed in synapses on functionally distinct domains of the same neuron receiving GABA from different presynaptic sources. There are quantitative differences in the density of GABA_A receptors at synapses on different cell types in the same brain area. Copyright © 1996 Elsevier Science Ltd

Section snippets

Light microscopy

Immunohistochemistry was performed as described in Bohlhalter et al. (1996). Four rats were deeply anaesthetized with pentobarbital (50 mg/kg, i.p.) and perfused with 4% paraformaldehyde and ∼0.2% picric acid in phosphate buffer. The brain was removed immediately after the perfusion and postfixed for 4 hr in the same fixative at 4°C. Thereafter, the tissue was processed with a modified antigen-retrieval protocol aimed at optimizing the signal-to-noise ratio in the subsequent immunohistochemical

Control experiments

No immunolabelling was observed in the light microscopic material when the affinity-purified antibodies were omitted or replaced by non-immune rabbit IgG of approximately the same concentration, or by antibody that was preincubated with the antigen.

In the electron microscopic control incubation for double labelling of the same sections, replacement of the monoclonal antibody to the $β2 3$ subunits with an anti-GFAP antibody resulted in selective labelling of glial fibrillary bundles, but never the

Immunolabelling in relation to the synaptic active zone

The synaptic active zone, which is thought to be the site of vesicular transmitter release and action, is recognized in electron microscopic specimens on the basis of the rigid pre- and postsynaptic membrane apposition, the cleft material, the postsynaptic membrane specialization and the presynaptic grid consisting of dense projections (see, for example, Fig. 4). The present study confirms previous reports for other subunits of the GABA_A receptor (Nusser et al. (1995a), Nusser et al. (1995b),

Acknowledgements

We are grateful to Dr Zoltan Nusser for his comments on an earlier version of the manuscript, and to Miss Z. Ahmad, Mr P. Jays and Mr F. Kennedy for their excellent assistance.

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The γ2 Subunit of the GABAA Receptor is Concentrated in Synaptic Junctions Containing the α1 and β23 Subunits in Hippocampus, Cerebellum and Globus Pallidus

Abstract

Section snippets

Light microscopy

Control experiments

Immunolabelling in relation to the synaptic active zone

Acknowledgements

Neuron

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Fedn Eur. Biochem. Socs Lett.

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Brain Res.

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J. Biol. Chem.

Neuroscience

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Neuron

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Brain Res.

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Neuron

Neuroscience

Brain Res.

Immunochemical identification of the α1- and α3-subunits of the GABAA-receptor in rat brain

J. Rec. Res.

Ubiquitous presence of GABAA receptors containing the α1-subunit in rat brain demonstrated by immunoprecipitation and immunohistochemistry

Molec. Neuropharmac.

GABAA-receptor subtypes differentiated by their γ-subunit variants: prevalence, pharmacology and subunit architecture

Neuropharmacology

Laminar compartmentalization of GABAA-receptor subtypes in the spinal cord: an immunohistochemical study

J. Neurosci.

Diverse sources of hippocampal unitary inhibitory postsynaptic potentials and the number of synaptic release sites

Nature

Localization of the GABAA receptor in the rat brain with a monoclonal antibody to the 57,000 Mr peptide of the GABAA receptor/benzodiazepine receptor/Cl− channel complex

J. Neurosci.

Noise analysis of miniature IPSCs in adult rat brain slices: Properties and modulation of synaptic GABAA receptor channels

J. Neurophysiol.

Quantitative immunoprecipitation studies with anti-γ-aminobutyric acidA receptor γ2 1–15 Cys antibodies

J. Neurochem.

Subunit selectivity and epitope characterization of mAbs directed against the GABAA/benzodiazepine receptor

J. Cell Biol.

Five subtypes of type A γ-aminobutyric acid receptors identified in neurons by double and triple immunofluorescence staining with subunit-specific antibodies

Proc. natn. Acad. Sci. U.S.A.

The γ2 Subunit of the GABA_A Receptor is Concentrated in Synaptic Junctions Containing the α1 and β $23$ Subunits in Hippocampus, Cerebellum and Globus Pallidus

Immunochemical identification of the α1- and α3-subunits of the GABA_A-receptor in rat brain

Ubiquitous presence of GABA_A receptors containing the α1-subunit in rat brain demonstrated by immunoprecipitation and immunohistochemistry

GABA_A-receptor subtypes differentiated by their γ-subunit variants: prevalence, pharmacology and subunit architecture

Laminar compartmentalization of GABA_A-receptor subtypes in the spinal cord: an immunohistochemical study

Localization of the GABA_A receptor in the rat brain with a monoclonal antibody to the 57,000 M_r peptide of the GABA_A receptor/benzodiazepine receptor/Cl⁻ channel complex

Noise analysis of miniature IPSCs in adult rat brain slices: Properties and modulation of synaptic GABA_A receptor channels

Quantitative immunoprecipitation studies with anti-γ-aminobutyric acid_A receptor γ2 1–15 Cys antibodies

Subunit selectivity and epitope characterization of mAbs directed against the GABA_A/benzodiazepine receptor