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-gamma-aminobutyric acid (GABA). Data were analysed by computer-assisted image analysis. Density of glutamate labelling (gold particles per micron2) 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 approximately 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.