To study the morphological substrate for interaction between two chemically distinct neuronal types, two double ultrastructural immunolabeling strategies were employed. In the first, two different electron-dense markers were used to examine simultaneously two different neurotransmitter-related antigens in the hypothalamic supraoptic nucleus in the same thin section. Results obtained with the first method were confirmed with a second approach based on postembedding immunostaining of alternate serial thin sections with different antisera. Antiserum against glutamate decarboxylase, the enzyme responsible for the synthesis of the inhibitory amino acid transmitter gamma-aminobutyric acid (GABA), or antisera against GABA, was used to localize immunoreactive axons in the hypothalamic supraoptic nucleus. With light microscopy, glutamate decarboxylase- and GABA-immunoreactive axon terminals immunostained with peroxidase were found arborizing throughout all areas of the nucleus; terminal boutons were found adjacent to unlabeled somata within the nucleus. Cells containing immunoreactive oxytocin, vasopressin, and neurophysin were localized with peroxidase. Glutamate decarboxylase-immunoreactive axons stained with peroxidase prior to embedding in plastic were demonstrated to contact neurons which contained vesicles immunostained with neurophysin antiserum by a post-embedding immunocytochemical procedure which used immunoglobulins or protein A adsorbed to colloidal gold as a second ultrastructural marker. Quantitative evaluation of post- embedding staining with colloidal gold using a neurophysin primary antiserum indicated a specific antigen localization in neurosecretory vesicles. A critical factor in this double-labeling paradigm was that immunological reagents used in the second series did not cross-react with those used in the first series, regardless of the species of origin of antisera. To provide further verification of GABAergic synapses on neurophysin-containing neurons, alternate serial ultrathin sections were stained with colloidal gold using antisera against either neurophysin or GABA; boutons immunoreactive for GABA made synaptic contact with supraoptic neurons containing neurophysin immunoreactivity. Converging results obtained with these two procedures indicate that GABAergic axons synapse directly on neurons containing oxytocin or vasopressin in the rat hypothalamic supraoptic nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)
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