NMDAR subunits are expressed postsynaptically in both GABAergic and glutamatergic synapses at postnatal day 6–7.

<p>Electron micrographs show combined immunogold-immunoperoxidase reactions from stratum radiatum of the hippocampal CA1 region. Synapses of vGluT1 positive presynaptic terminals (marked by dark reaction product in A, C, E1 and E2) contain postsynaptic GluN1 (black particles in A, arrows), GluN2B (black particles in C, arrows), and GluN2A subunits (black particles in E1 and E2, arrows). Synapses of GAD67-positive presynaptic terminals (marked by dark reaction product in B, D1, D2, F1, F2 and F3) contain postsynaptic GluN1 (black particles in B, arrows), GluN2B (black particles in D1 and D2, arrows), and GluN2A subunits (black particles on F1, F2 and F3, arrows). Serial images show the same synapse in D1 and D2; E1 and E2; F1, F2 and F3. Scale bar is 300 nm for all images.</p

Schematic drawing shows the proposed mechanism for GABA_AR and NMDAR cooperation during early postnatal development.

<p>Our results that GABA_A and NMDA receptors are co-expressed in GABAergic synapses can provide the anatomical basis for a new model of the generation of SSAs during the postnatal period. The GABA_AR mediated current leads to postsynaptic depolarization in the GABAergic synapse that allows activation of NMDARs located in the same synapse. This homosynaptic receptor activation in GABAergic synapses causes strong local depolarization that leads to a subsequent heterosynaptic activation of NMDARs in otherwise silent (AMPA receptor-lacking) glutamatergic synapses. The schematic drawing illustrates the position of GABAAR and NMDAR at synapses, where their presence has already been proven by others or in our current study.</p

Antibodies used in the study.

<p>Antibodies used in the study.</p

Analysis of the expression of NMDAR subunits in GABAergic and glutamatergic synapses at postnatal day 6–7. A

<p>, Percentage of synapses positive for different NMDAR subunits out of all identified glutamatergic (white columns) and GABAergic synapses (black columns). <b>B</b>, Linear density of labeling (gold particle/µm) for different NMDAR subunits in glutamatergic synapses (white columns), GABAergic synapses (black columns) and extrasynaptic membranes (grey columns), measured on 100 nm thick electron microscopic sections. The extrasynaptic density of labeling was 0.09, 0.03 and 0.02 immunogold particles/µm for GluN1, GluN2B and GluN2A subunits, respectively. <b>C</b>, Size (µm²) of glutamatergic (white column) and GABAergic (black column) synapses. Data shown in A, B and C were measured from preembedding experiments. <b>D</b>, Density of labeling (gold particle/µm) for GluN1 subunits in glutamatergic synapses (white column), GABAergic synapses (black column) and extrasynaptic (E.S.) membranes (grey column, 0.03 gold particles/µm), measured from quantitative post-embedding experiments.</p

Quantitative post-embedding immunogold labeling reveals NMDAR expression levels in GABAergic and glutamatergic synapses at postnatal day 6–7.

<p>The same synapses were reacted with antibodies against different epitopes on adjacent ultrathin sections using the mirror technique. 10 nm immunogold particles label presynaptic glutamatergic (vGluT1 positive) terminals in A1 and B1. Intensified immunogold particles label presynaptic GABAergic (GAD65/67 positive) terminals in C1 and D1. 10 nm immunogold particles (arrows) label GluN1 subunits in A2-3, B2, C2, D2. Note that the postembedding immunoreaction is on the surface of the sections, therefore, the position of gold particles can be on either side of the postsynaptic membrane, even if the labeled epitope is purely postsynaptic. Images from adjacent sections of the same synapses are displayed in A1-3, in B1-2, in C1-2 and in D1-2. Scale bar is 200 nm for all images.</p