Article Figures & Data
Figures
- Figure 1.
NMDA-induced cGMP production in acute hippocampal slices. Fluorescent images show cGMP immunolabeling in acute hippocampal slices. A–I, The first column shows labeling in the whole hippocampus, and the second and third columns show only the str. pyramidale of CA1 and CA3ab, respectively. Because in some areas labeling intensity is too weak to show up in the low-power image, high-power images can more closely reflect differences in labeling intensity. A, No immunostained varicosities were detected in control slices, except in CA3ab, where some basket terminals were labeled. B, The NO donor, SNP, increased cGMP levels in all regions, but basket terminal labeling was found only in CA1–3, but not in the DG. Note that, in CA3ab, more terminal labeling was induced by SNP than by NMDA treatment. C, The effect of 5 μm NMDA is region specific: strong in the CA1/CA3c regions, whereas no change is detectable in CA3ab. All terminals in the DG remained negative. D, The NMDAR blockers, MK-801 and d-AP5, nullified the effect of NMDA but did not change labeling in CA3ab. E, Blocking postsynaptic VDCCs (L- and R-type) did not change the effect of NMDA. F, G, The NO receptor blocker, ODQ, and the NOS blocker, l-NAME, prevented the NMDA-induced cGMP production. H, I, In nNOS−/− mice, no neuronal cGMP labeling was detected, and the administration of NMDA had no effect on cGMP production. This shows that the NMDA-induced cGMP production is nNOS dependent. Scale bars: I, first column, 1 mm; I, third column, 10 μm. J, cGMP production was not observed in basket cell terminals of the DG under different experimental conditions (see also str. granulosum in A–C). K, Double labeling in str. pyramidale of CA1 after NMDA treatment showed that cGMP-positive terminals were GAD65 positive, whereas the majority of somatic GAD65-positive terminals were labeled for cGMP. The white arrowheads show examples of colocalization. Scale bar: (in I, third column), J, K, 10 μm.
- Figure 2.
Analysis of the distribution of NMDAR subunits and colocalizations in basket cell terminals. A, Control and NMDA-induced cGMP labeling in CA1 and CA3ab basket terminals tested for GAD65 immunoreactivity. In the CA1 area, terminals were negative for cGMP in controls, whereas the majority of them became strongly positive after NMDA treatment [n = 513 GAD+ terminals (term.), n = 375 cGMP+ term., 3 mice]. In the CA3ab area, some of the terminals were weakly positive for cGMP (n = 330 GAD+ term., n = 118 cGMP+ term., 2 mice), whereas the labeling did not change after NMDA treatment (n = 290 GAD+ term., n = 201 cGMP+ term., 3 mice). B, Measurements from postembedding immunogold experiments show that the density of NMDARs (GluN1 subunits) are highly enriched in inhibitory GABAergic synapses (12.9 times higher), compared with the somatic extrasynaptic membrane. Labeling density is even higher in excitatory synapses (9.7 times that of GABAergic synapses). C, The distribution of NMDAR labeling relative to the postsynaptic membrane of GABAergic synapses showed that NMDARs are associated to the postsynaptic membrane. We found no evidence for presynaptic NMDARs. D–F, Immunogold density for GluN1, GluN2A, and GluN2B subunits, measured on freeze–fracture replica of pyramidal cell somatic membranes. For all subunits, background (measured on the E-face) was negligible, whereas cytoplasmic P-face labeling was enriched in GABAergic synapses (Inhib. syn.), compared with labeling on adjacent somatic membranes (Extrasyn. membr.). G, NMDAR subunit labeling is enriched in the majority of the fully reconstructed somatic GABAergic synapses on freeze–fracture replica. In the pyramidal cell-specific GluN1 knock-out mice, synaptic GluN1 labeling was absent. H, I, Percentages of direct localization of NMDAR subunits in serially reconstructed PV- and vGluT3-positive synapses in immunogold–immunoperoxidase experiments. Note that immunoperoxidase staining for the axon terminal markers decreases the sensitivity of NMDAR labeling. preemb., Pre-embedding; lab., labeling.
- Figure 3.
NMDAR subunits expressed in the active zone of somatic GABAergic synapses of CA1 pyramidal cells. Electron micrographs show postembedding immunogold labeling for NMDAR subunits (black particles). A–C, GluN1, GluN2A, and GluN2B subunits are enriched in the postsynaptic active zones of GABAergic (left) and glutamatergic synapses (right) with large quantitative differences. D, Serial sections of the same synapses, tested for GluN2B and 2A (silver enhanced gold particles). The synapse in the lower half is labeled for both subunits (arrows), and the other synapse was only positive for GluN2A (arrowheads). E, Serial sections of the very same synapse show immunogold labeling for GluN2B, GluN1, as well as GluN2A subunits (arrows). t, Terminal; s, soma; sp, spine head. Scale bar, 100 nm.
- Figure 4.
Delineation of GABAergic synapses on the surface of pyramidal cell somata on replica samples. A, Electron-microscopic image of immunogold labeling on the somatic P-face membrane of a pyramidal cell. The clusters of immunogold labeling for GABAA receptor β3 subunits (10 nm black particles; black arrows) associate well with clusters of IMPs (arrowheads). NMDAR labeling (GluN2A) was also associated with GABAAR labeling (5 nm black particles; white arrows). B, Because several clusters of IMPs occur on the surface of somata without GABAAR labeling, we delineated synaptic areas based on high local GABAAR labeling density. In B, red dots label the position of GABAAR labeling. Grayscale gradients visualize local GABAAR density. After definition of a density threshold (white lines), synapse-associated areas were delineated and these unbiased rules were applied to all measurements. Therefore, the position of NMDAR labeling could not bias the definition of synaptic areas. C, After delineation (white lines), the position of NMDARs were labeled (yellow dots). D, Finally, NMDAR labeling (yellow dots) was quantified inside and outside of synapses. Scale bar, 100 nm.
- Figure 5.
NMDARs colocalize with postsynaptic GABAARs on freeze–fracture replica. Electron micrographs of replica immunogold labeling show the postsynaptic side of GABAergic synapses (delineated with gray lines) on CA1 pyramidal cell somata. A–C, Double immunogold labeling for GABAAR β3 subunit (10 nm gold) and NMDAR subunits (5 nm gold; arrows) revealed a strong association of GluN1 (A), GluN2A (B), and GluN2B (C) subunits to GABAergic synapses. The insets show labeling of presumed pyramidal cell spines for the NMDAR subunit. Scale bar, 100 nm. D, Electron micrograph demonstrates the close association of NMDARs and synaptic GABAAR. Five synaptic areas are shown. IMPs are scattered all over the cytoplasmic P-face of the somatic membrane. Two pieces of extracellular E-face membrane can be seen on the surface of the replica (blue areas). Immunogold labeling for GABAAR β3 subunits (marked with green rings) are enriched over clusters of IMPs. Based on the local density of GABAAR labeling, synaptic areas are delineated by the unbiased rules (pale green areas with gray edges). Then, immunogold labeling for GluN1 subunits was localized as well (marked with red rings). Finally, the density of immunogold labeling for GluN1 subunits was calculated in synapses and extrasynaptically. Scale bar, 200 nm.
- Figure 6.
NMDAR and nNOS colocalize in GABAergic synapses of basket cell terminals. A–C, Electron micrographs demonstrate NMDAR subunit labeling in the postsynaptic active zone of somatic GABAergic synapses using preembedding immunogold single staining for GluN1, GluN2A, and GluN2B (arrows). No presynaptic labeling was found. In A, a pyramidal cell spine (sp) synapse was also labeled. Et, Excitatory terminal; It, inhibitory terminal; Ps, pyramidal cell soma. Scale bar: (in C) A–C, 200 nm. D–K, Preembedding immunogold–immunoperoxidase colocalizations in synapses established by PV- and vGluT3/CCK-positive basket cell terminals. Serial (D, F, H) and single (I–K) sections of somatic GABAergic synapses express GluN1, 2A, and 2B (arrows; gold particles) in synapses of both PV- and vGluT3-immunoreactive terminals (dark precipitation). E, Serial sections show that PV-positive synapses also express nNOS postsynaptically, as reported previously (Szabadits et al., 2007). G, The direct colocalization of nNOS (arrows) in the synapses of vGluT3 terminals is confirmed. Scale bar: (in K) D–K, 250 nm.
