Glutamate‐like Immunoreactivity in Retinal Terminals of the Mouse Suprachiasmatic Nucleus

With a view to identifying the neurotransmitter content of retinal terminals within the mouse suprachiasmatic nucleus, a highly specific antiserum to glutaraldehyde‐coupled glutamate was used in a postembedding immunogold procedure at the ultrastructural level. Retinal terminals were identified by cholera toxin–horseradish peroxidase transported anterogradely from the retina and reacted with tetramethyl benzidine/tungstate/H2O2, or by their characteristically pale and distended mitochondria with irregular cristae. Controls included model ultrathin sections containing high concentrations of various amino acids. Alternate serial sections were labelled with anti‐glutamate and anti‐γ‐aminobutyric acid (GABA). Data were analysed by computer‐assisted image analysis. Density of glutamate labelling (gold particles per μm2) on whole retinal terminals was > 3 times higher than that on postsynaptic dendrites, and > 5 times higher than that on miscellaneous non‐retinal non‐glutamatergic terminals in the suprachiasmatic nucleus. The overall density of gold particles over retinal terminals was ∼ 3 times higher than that over GABAergic terminals, in which glutamate‐like immunoreactivity was mainly mitochondrial. Labelling of vesicles in retinal terminals was almost 5 times greater than the apparent labelling of vesicles in GABAergic terminals, underscoring the location of transmitter glutamate within synaptic vesicles in retinal terminals. In the retino‐recipient region of the suprachiasmatic nucleus there was also a small population of non‐retinal glutamatergic terminals. Their overall immunoreactivity was similar to or exceeded that of retinal terminals, but morphological features clearly distinguished between these two types of glutamate‐containing terminals. The present results indicate that the vast majority of retinal terminals may use glutamate as a transmitter, in keeping with electrophysiological and neuropharmacological data from other sources. The possibility of cotransmitters within retinal terminals, suggested by the presence of dense‐core vesicles among the glutamate‐containing synaptic vesicles, has still to be addressed.