Dual labelling methods were employed for the electron microscopic detection of glutamate decarboxylase (GAD) immunoreactivity, together with vasoactive intestinal peptide (VIP) or neuropeptide Y (NPY) immunoreactivity in the suprachiasmatic nucleus (SCN) of colchicine pretreated and untreated rats. These methods involved the combined use of diaminobenzidine and benzidine dihydrochloride as distinct chromogens to visualize peroxidase-anti-peroxidase (PAP) immunostaining, and a combination of the PAP procedure with a radioimmunocytochemical method employing 125I-labelled secondary antisera. We were thereby able to demonstrate that gamma-aminobutyric acid (GABA) terminals provide an important afferent synaptic input to VIP neurons. Some of these VIP-immunoreactive neurons also exhibited GAD immunoreactivity. Examples of direct appositions between GABA and NPY terminals, and of a convergence of the two types of terminals on to the same postsynaptic targets, were frequently encountered. NPY/GAD colocalization within a few axonal varicosities was also demonstrated. These data provide additional information concerning chemospecific neuronal interactions that could be of functional importance in the regulation of circadian rhythmicity at the level of the SCN.
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