Abstract
Abstract: :
Purpose: Although NMDA receptor channels contribute to evoked synaptic excitation of both amphibian and mammalian retina ganglion cells (GCs), NMDA receptors are not activated normally by the glutamate released from a single vesicle. Our recent physiological experiments in the rat retina indicate that NMDA receptors must be located outside the synaptic cleft. The goal of the present study is to use immuno–EM to determine the precise location of NMDA receptors on GC dendrites. Methods: Cholera toxin B (CTB) 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 NR1, NR2B and CTB and then with 10 nm and 15 nm gold–conjugated secondary antibodies. As a positive control, we found that anti–GluR2/3 antibody labeled postsynaptic densities in GCs. No gold labeling was seen when the secondary antibodies were applied alone (negative control). Results: Retrograde labeling with CTB yielded excellent staining of the somata and dendrites of GCs, allowing GC dendrites to be distinguished easily from other processes in the IPL. 26 cone bipolar–GC synapses, each with a clear presynaptic terminal and well–defined postsynaptic density (PSD), were analyzed by counting associated gold particles on the GC dendrite. Immunogold labeling was found mainly outside the PSD: 96% (132) was distributed at extrasynaptic sites along the dendritic plasma membrane, and only 4% (5) was located in the PSD. The mean length of PSD was 186 ± 57 nm (mean ± SE). Almost 60% of the gold particles were found perisynaptically: ∼40% were located 160 to 340 nm from the edge of the PSD (or 250–430 nm from the center); ∼20% were located between 30 and 150 nm of the edge. The remaining particles were located more than 400 nm from the edge of the PS Conclusions: These results suggest that NMDA receptors are localized primarily perisynaptically on ganglion cell dendrites.
Keywords: excitatory amino acid receptors • ganglion cells • microscopy: electron microscopy