Tyrosine Hydroxylase lmmunoreactive Neurons Throughout the Hypothalamus Receive Glutamate Decarboxylase lmmunoreactive Synapses: A Double Pre-embedding lmmunocytochemical Study with Particulate Silver and HRP

Silver-intensified gold (SIG) particles were used for lightand electron-microscopic immunocytochemical localization of neuronal antigens, and the SIG method was compared with related heavy-metal methods for the purpose of dual ultrastructural localization of neurotransmitter-related antigens. SIG immunostaining was combined with peroxidase immunostaining to allow simultaneous study of differentially labeled tyrosine hydroxylase and glutamate decarboxylase immunoreactive neurons in the medial hypothalamus. A number of electron-dense markers that might be of use in double immunostaining for light and electron microscopy were examined, either with a simple nitrocellulose dot-blot method or on Formvar-coated slot grids. Of these, silver-intensified 5 nm colloidal gold was the most effective. Silver intensification of colloidal silver and of peroxidase reaction product also showed promise for combined LM and EM double-immunolabeling studies. Since the silver-intensification procedure used here intensifies both gold and peroxidase, in experiments involving double staining, the silver-intensified gold procedure should be used for the first antigen and nonintensified HRP for the second. Presumptive dopaminergic neurons containing the enzyme tyrosine hydroxylase were located throughout the hypothalamus with SIG immunostaining. In the same areas where frequent tyrosine hydroxylase immunoreactive neurons were found, many axons and bouton terminals were also found with antisera against GABA or against the GABA-synthesizing enzyme glutamate decarboxylase. Areas containing cells immunoreactive for tyrosine hydroxylase and stained with SIG and axons immunoreactive for glutamate decarboxylase and stained with peroxidase included the periventricular area (A14), the arcuate nucleus (A12), the dorsomedial hypothalamus/zona incerta area (A13), the posterior hypothalamus (All), the medial paraventricular nucleus, and dorsal to the supraoptic nucleus, in addition to the preoptic area near the third ventricle and dorsally adjacent to the anterior commissure. For comparison, the SIG procedure was also used to stain dopaminergic neurons outside the hypothalamus in the substantia nigra and ventral tegmental area. Double immunocytochemical staining of two different neurotransmitter-related antigens allowed examination with both light