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J. Zhang, J. S. Diamond; Subunit- and Pathway-Apecific Localization of NMDA Receptors and Scaffolding Proteins at Ganglion Cell Synapses in Rat Retina. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5804.
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© ARVO (1962-2015); The Authors (2016-present)
NMDARs are critical elements in synaptic transmission and plasticity throughout the CNS, but specific roles for NMDARs in retinal ganglion cell (RGC) function remain largely unexplored. Recent physiological work in mouse retina suggest that NMDARs may be primarily extrasynaptic at ON RGC synapses and primarily synaptic at OFF RGC synapses. Here, we examined the subsynaptic localization of different NR1 splice variants, NR2 subunits and scaffolding proteins (membrane associated guanylate kinases, MAGUKs) at ON and OFF synapses onto identified RGC using postembedding immunogold electron microscopy (EM).
Cholera toxin B was injected bilaterally into the superior colliculi of P15 rats to label retrogradely ganglion cell dendrites. 5-7 days later after CTB injection, animals were sacrificed and retinas were processed by the postembedding immunogold EM method. Ultrathin sections were incubated overnight with primary antibodies to either NMDARs (NR1C2, NR1C2`, NR2A, NR2B) or MAGUKs (PSD-95, PSD-93, SAP-102, SAP-97) and CTB and then with 10 nm and 15 nm gold-conjugated secondary antibodies. Sections were photomontaged for quantitiative analysis. No signal was seen when either preadsorption or negative controls were carried out.
(1)The large majority of particles labeling either NR1C2 (92%) or NR2B (96%) was located perisynaptically. NR2B, but not NR1C2, was preferentially expressed at ON synapses. (2) NR1C2` and NR2A subunits were localized primarily within the PSD (81% and 83%, respectively) and preferentially expressed at OFF synapses. (3) The expression for SAP102 was similar to that of NR1C2 and NR2B, whereas those for PSD-95 and PSD-93 was similar to NR1C2` and NR2A. (4) Triple labeling showed that NR2A and PSD-95 were colocalized in the PSD at OFF synapses.
These results provide the first anatomical evidence for preferential association of particular NR1 splice variants, NR2 subunits and MAGUKs at central synapses and suggest that NMDARs may play different roles at functionally distinct synapses in the retinal circuitry.
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