Abstract
Both excitatory and inhibitory synaptic contacts display activity dependent dynamic changes in their efficacy that are globally termed synaptic plasticity. Although the molecular mechanisms underlying glutamatergic synaptic plasticity have been extensively investigated and described, those responsible for inhibitory synaptic plasticity are only beginning to be unveiled. In this framework, the ultrastructural changes of the inhibitory synapses during plasticity have been poorly investigated. Here we combined confocal fluorescence microscopy (CFM) with high resolution scanning electron microscopy (HRSEM) to characterize the fine structural rearrangements of post-synaptic GABAA Receptors (GABAARs) at the nanometric scale during the induction of inhibitory long-term potentiation (iLTP). Additional electron tomography (ET) experiments on immunolabelled hippocampal neurons allowed the visualization of synaptic contacts and confirmed the reorganization of post-synaptic GABAAR clusters in response to chemical iLTP inducing protocol. Altogether, these approaches revealed that, following the induction of inhibitory synaptic potentiation, GABAAR clusters increase in size and number at the post-synaptic membrane with no other major structural changes of the pre- and post-synaptic elements.
Highlights
Increasing body of evidence has demonstrated that phosphorylation of the GABAA Receptors (GABAARs) by the calcium dependent kinase CaMKII is a key determinant for the changes in GABAARs functioning[8] and localization at synapses[9]
backscattered electron (BSE) analysis on primary hippocampal neuron cultures immunolabelled with an anti-GABAAR α1 subunit (GABAARα1) primary antibody followed FluoroNanogoldTM Fab secondary antibody revealed the presence of GABAARα1 gold clusters of variable size on both soma and neurites (Fig. 1 and Fig. S1)
By coupling fluorescence microscopy with the high-resolution power of HRSEM, correlative light - high resolution scanning electron microscopy (CL-HRSEM) is an excellent method to study the spatial distribution of target molecules at the cellular surface at nanometric level[39]
Summary
Increasing body of evidence has demonstrated that phosphorylation of the GABAARs by the calcium dependent kinase CaMKII is a key determinant for the changes in GABAARs functioning[8] and localization at synapses[9]. The images of the detected backscattered electron (BSE) and the secondary electron (SE) signals were simultaneously acquired in order to localize gold nanoparticles, (which provide high compositional contrast in BSE images) while imaging the fine surface morphology by SE signal In another set of experiments we performed ET on hippocampal neurons immuno-labelled for GABAARα1. The expression of such inhibitory plasticity occurred in the absence of major structural rearrangements of the pre- and post-synaptic membranes Those results indicate that, upon the conditions examined here, the expression of inhibitory synaptic potentiation does not require structural plasticity, leaving the modulation of GABAARα1 receptor trafficking and lateral diffusion as the main determinants of iLTP, differently from what happens in excitatory synapses
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