Tyrosine hydroxylase immunoreactive neurons throughout the hypothalamus receive glutamate decarboxylase immunoreactive synapses: A double pre-embedding immunocytochemical study with particulate silver and HRP

Abstract

Silver-intensified gold (SIG) particles were used for light- and 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 mm 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 (A11), 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 and electron microscopy. By virtue of a large silver shell formed around the colloidal gold particle and its adsorbed immunoglobulin or protein A, cross-reactivity of the first set of immunoreagents stained with particulate silver and a second set stained with peroxidase could be reduced or eliminated. To test its versatility, the SIG methodology was used to stain five other putative neurotransmitter-related antigens in the hypothalamus, including glutamate decarboxylase, somatostatin, prolactin, luteinizing hormone releasing hormone, and neurophysin in frozen sections or sections embedded in plastic, polyethylene glycol, or paraffin. In all areas examined ultrastructurally, including the arcuate nucleus (A12), the periventricular area (A14), the medial paraventricular nucleus, and the dorsomedial hypothalamus, glutamate decarboxylase immunoreactive boutons stained with peroxidase were found in direct contact with tyrosine hydroxylase immunoreactive dendrites and perikarya stained with the silver-intensified gold procedure. As glutamate decarboxylase immunoreactive boutons were found in synaptic contact with tyrosine hydroxylase immunoreactive neurons throughout the hypothalamus, the inhibitory amino acid transmitter GABA may play a widespread role in modulating the electrical activity of dopamine neurons.