IMMUNOLABELING OF RETROGRADELY TRANSPORTED FLUOROGOLD - SENSITIVITY AND APPLICATION TO ULTRASTRUCTURAL ANALYSIS OF TRANSMITTER-SPECIFIC MESOLIMBIC CIRCUITRY

Fluorescence microscopy shows extensive filling of perikarya and dista l dendrites following injections of Fluoro-Gold (FG) into their termin al fields. However, elucidation of synaptic contacts onto identified p rojection neurons has been limited by the lack of compatibility betwee n electron-dense markers required for ultrastructural analysis and mor phology preservation. The recent advent of antisera to FG has revealed numerous potential applications for analyzing chemically defined syna ptic circuitry. To take advantage of the high sensitivity of this retr ograde tracer in ultrastructural studies, we extended and detailed the original description of single immunocytochemical labeling of FG by c omparing the advantages of immunodetection of an antiserum against FG using 2 distinct electron-dense markers: (1) avidin-biotin peroxidase (ABC) reacted with 3,3'-diaminobenzidine and darkened with osmium tetr oxide, or (2) silver-intensified 1 nm colloidal gold particles. We sub sequently examined the utility of combining these markers in single se ctions for detection of transmitters (e.g., gamma-aminobutyric acid (G ABA) and 5-hydroxytryptamine (5-HT)) in axon terminals presynaptic to retrogradely labeled neurons. Both analyses were carried out on the we ll-characterized mesolimbic pathway originating from perikarya in the ventral tegmental area (VTA) that project to the nucleus accumbens. In jections of FG were stereotaxically placed in the nucleus accumbens of anesthetized adult rats. From these animals, vibratome sections of al dehyde-fixed brains were examined for light-microscopic detection of F G using: (1) epi-fluorescence without immunocytochemistry, (2) immunop eroxidase, or (3) immunogold-silver. All 3 methods revealed circumscri bed injections in the nucleus accumbens. Additionally, both immunocyto chemical methods appeared to be as sensitive as epi-fluorescence in li ght-microscopic detection of retrogradely labeled perikarya and fine-c aliber dendrites extending for 2-3 branch points beyond the soma. Elec tron microscopy showed that the FG was detectable not only in lysosome s but also throughout the cytoplasmic matrix of perikarya and dendrite s using either immunoperoxidase or immunogold-silver labeling methods. In the second part of this analysis, single sections of tissue were p rocessed for dual labeling using either immunoperoxidase or immunogold -silver for detection of FG in conjunction with the converse label for GABA or 5-HT, respectively. Regardless of the labeling combinations, the peroxidase and gold-silver reactions were readily distinguished wi thin sections examined by light or electron microscopy. Synaptic junct ions from unlabeled or from GABA or 5-HT labeled terminals were most r eadily identified when the targets were lightly immunoreactive for per oxidase or labeled using silver-intensified colloidal gold. The presen t results indicate that in the mesolimbic pathway, (1) FG can be used as a versatile marker using immunoperoxidase or immunogold-silver as a sensitive means for detection of retrogradely labeled neurons and (2) both labels can be used in reversible combinations for identity of tr ansmitters such as GABA and 5-HT in afferent terminals. Two distinct h istochemical procedures are described for optimizing the visualization of chemically identified synaptic contacts onto projection neurons. T he methods demonstrated in this mesolimbic pathway are most likely app licable to other neuronal circuits and can be used for identifying tra nsmitters in either the projection neurons or their afferent axons.