Present address: Dept. Pharmacology, Innsbruck Medical Univ., Peter Mayr Strasse 1a, A-6020 Innsbruck, Austria.
Quantitative localisation of synaptic and extrasynaptic GABAA receptor subunits on hippocampal pyramidal cells by freeze-fracture replica immunolabelling
Article first published online: 14 NOV 2010
© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience
Volume 32, Issue 11, pages 1868–1888, December 2010
How to Cite
Kasugai, Y., Swinny, J. D., Roberts, J. D. B., Dalezios, Y., Fukazawa, Y., Sieghart, W., Shigemoto, R. and Somogyi, P. (2010), Quantitative localisation of synaptic and extrasynaptic GABAA receptor subunits on hippocampal pyramidal cells by freeze-fracture replica immunolabelling. European Journal of Neuroscience, 32: 1868–1888. doi: 10.1111/j.1460-9568.2010.07473.x
- Issue published online: 28 NOV 2010
- Article first published online: 14 NOV 2010
- Received 9 July 2010, revised 3 September 2010, accepted 10 September 2010
- dendritic spine;
- electron microscope;
- GABAergic synapse;
Hippocampal CA1 pyramidal cells, which receive γ-aminobutyric acid (GABA)ergic input from at least 18 types of presynaptic neuron, express 14 subunits of the pentameric GABAA receptor. The relative contribution of any subunit to synaptic and extrasynaptic receptors influences the dynamics of GABA and drug actions. Synaptic receptors mediate phasic GABA-evoked conductance and extrasynaptic receptors contribute to a tonic conductance. We used freeze-fracture replica-immunogold labelling, a sensitive quantitative immunocytochemical method, to detect synaptic and extrasynaptic pools of the alpha1, alpha2 and beta3 subunits. Antibodies to the cytoplasmic loop of the subunits showed immunogold particles concentrated on distinct clusters of intramembrane particles (IMPs) on the cytoplasmic face of the plasma membrane on the somata, dendrites and axon initial segments, with an abrupt decrease in labelling at the edge of the IMP cluster. Neuroligin-2, a GABAergic synapse-specific adhesion molecule, co-labels all beta3 subunit-rich IMP clusters, therefore we considered them synapses. Double-labelling for two subunits showed that virtually all somatic synapses contain the alpha1, alpha2 and beta3 subunits. The extrasynaptic plasma membrane of the somata, dendrites and dendritic spines showed low-density immunolabelling. Synaptic labelling densities on somata for the alpha1, alpha2 and beta3 subunits were 78–132, 94 and 79 times higher than on the extrasynaptic membranes, respectively. As GABAergic synapses occupy 0.72% of the soma surface, the fraction of synaptic labelling was 33–48 (alpha1), 40 (alpha2) and 36 (beta3)% of the total somatic surface immunolabelling. Assuming similar antibody access to all receptors, about 60% of these subunits are in extrasynaptic receptors.