Neuroligin 2 is present postsynaptically at both GABAergic and cholinergic synapses in the hippocampus.

<p>Electron micrographs from combined immunogold/immunoperoxidase experiments for NLGN2 (immunogold: black particles) and ChAT (DAB: dark, homogenous reaction product) reveal the presence of NLGN2 at ChAT-negative and ChAT-positive type II synapses in the CA1 area. Arrowheads indicate synapse-edges. A, A pyramidal cell body receives a synapse from a ChAT-negative bouton (b_neg) that expresses NLGN2 postsynaptically in a WT mouse. B, C, In contrast, the same type of immunostaining in a NLGN2-KO mice shows no NLGN2-immunoreactive synapses, demonstrating the specificity of the antibody. A GABAergic terminal (b_neg) from str. pyramidale, lacking gold particles at the postsynaptic site is shown (B). An example of a synapse of a ChAT-positive bouton (b₁) on a dendrite (d) in str. radiatum that is immunonegative for NLGN2 in KO mouse (C). D–I: NLGN2 immunogold labeling is present at the postsynaptic site of synapses established by ChAT-positive axon terminals (b_2–5) on dendrites (d) and spines (s) in str. radiatum (D–G) and oriens (H, I) of WT mice. Serial images show the same synapse in D₁ and D₂; E₁ and E₂; F₁ and F₂; G₁ and G₂. E_1–2 demonstrates that some of the presynaptic profiles were small-diameter, intervaricose-like segments of ChAT-positive axons (b₃). In F_1–2 and H, the postsynaptic targets of boutons b₄ and b₆ are putative pyramidal dendrites (Pd) the latter of which is identified by the presence of spines (s). I, Occasionally, we found ChAT-positive presynaptic elements that formed synapses with two postsynaptic targets. Here, bouton b7 forms a synapse with a dendrite and a spine, which receives a type I synapse (black arrowheads). Note, that in many cases, synaptic junctions of ChAT-positive terminals are atypical (E, F, H, I). Scale bar is 200 nm for all images.</p

Cholinergic projection neurons of the basal forebrain and neostriatal cholinergic interneurons express NLGN2 in their inputs synapses.

<p>Images from combined immunogold/immunoperoxidase experiments show that dendrites of cholinergic cells (dChAT, dark, homogenous DAB precipitate) express NLGN2 (intensified gold particles) at postsynaptic membranes of type II synapses (open arrowheads) in the medial septum (A: MS), vertical- and horizontal diagonal band of Broca (B: VDB; C, D: HDB), substantia innominata/ventral pallidum (E: SI/VP) and caudate putamen (F: CPu). In B and C two unlabeled dendrites (d_neg) also express NLGN2 in their type II synapses (black arrowheads). Scale bar is 200 nm for all images.</p

Neuroligin 2 is localized postsynaptically at cholinergic synapses in the neocortex.

<p>Images demonstrate double immunohistochemical reactions for ChAT (dark, homogenous DAB precipitate) combined with NLGN2 (black intensified gold particles) in somatosensory (S1) and prefrontal cortices (PFC). Serial sections of the same synapses are shown in B_1–2, D_1–2 and E_1–2. In both areas, ChAT-positive boutons (b_1–5) form type II synaptic contacts on dendrites (d, A, C, D_1–2) and spines (s, B_1–2, E_1–2) that express NLGN2 at the postsynaptic membranes (open arrowheads label synaptic edges). The innervated spines also received a type I synapse from a ChAT-negative terminal (B_1–2, black arrowheads). In C the postsynaptic dendrite of bouton b₃ receive an additional, type I synaptic input (black arrowheads) from an unlabeled terminal. These type I synapses in B_1–2 and C do not contain NLGN2. In contrast, another ChAT-negative, putative GABAergic bouton (b_neg) establishes a type II, NLGN2-positive synapse (black arrowheads) with a dendrite in D_1–2. Scale bar is 200 nm for all images.</p

Antibodies used in the study.

<p>Antibodies used in the study.</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

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

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

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

Cell loss and degeneration at day 3 post pilocarpine.

<p>HSP72 is heavily expressed in a large population of pyramidal cells (A, B) and in some mossy cells, indicating that they were exposed to excitotoxic insult. In contrast, hardly any granule cells are stained (arrows) (C) compared to 1 day post SE. Gallyas silver impregnation reveals that in the early latent period principal cell loss begins at a large scale (orange cells are alive). Dark silver deposit is present in neurons irreversibly damaged (D, E, F). Argyrophilic cell degeneration can be seen in the vulnerable regions of the hippocampus, like the CA1 pyramidal cell layer (E) and the hilar region (F) (arrows in the CA1, arrowheads in the hilus). (s.o.: str. oriens, s.p. str. pyramidale, s.r.: str. radiatum, s.m.: str. moleculare, s.g.: str. granulosum) Scale: A, D 200 µm, B, E, F, C and F: 50 µm.</p

Table A describes seizure behavioral scoring for each Racine scale value.

<p>Graphs B, C and D show the correlation between seizure intensity (Racine scale value) and cell loss (color code indicates cell loss from 0 to 3, where 0 refers to no cell loss, 1: cell-loss under 10%, 2: cell-loss between 10% and 50%, 3: cell-loss above 50%) in CA1, CA3 and the hilus respectively. Correlation between Racine scale value and cell loss in the regions proved to be significant using Pearson correlation (p<0.05).</p