Several studies indicate the presence of an inhibitory GABAergic amacrine cell input which modulates dopamine release from the interplexiform cells (IPC) of carp retina. To define this putative neural circuit requires double label analysis of these two neurotransmitter systems at the ultrastructural level. Previous attempts at electron microscopical (EM) double labeling techniques in the retina have coupled EM autoradiography of radioactive neurotransmitter uptake with EM immunohistochemistry. Since uptake of radioactive neurotransmitter is not always a reliable marker for cells using a particular neurotransmitter system, it is necessary to immunologically label neurotransmitter related antigens, such as synthesizing enzymes. The present study is directed at developing methodologies which allow for the simultaneous ultrastructural mapping of two transmitter related antigens in the vertebrate retina. A standard peroxidase-anti-peroxidase (PAP) procedure wil be employed along with a colloidal gold procedure to provide visually distinct markers for each of two antigens. Using these two techniques, glutamic acid decarboxylase (GAD), the GABA (Gamma-aminobutyric acid) synthesizing enzyme and tyrosine hydroxylase (TOH), the rate limiting enzyme in catecholamine biosynthesis will be simultaneously localized in the carp (Cyprinis carpio) retina. This double immunohistochemical label methodology, once defined, will be useful for identifying synaptic connections and neural circuits involving other transmitters and peptides in the central nervous system. Thus, anatomical information can be integrated with physiological and pharmacological data to analyze neural circuits in normal and pathological states.
