Immunolabeling of retrogradely transported Fluoro-Gold : sensitivity and application to ultrastructural analysis of transmitter-specific mesolimbic circuitry

Fluorescence microscopy shows extensive filling of perikarya and distal dendrites following injections of Fluoro-Gold (FG) into their terminal fields. However, elucidation of synaptic contacts onto identified projection neurons has been limited by the lack of compatibility between electron-dense markers required for ultrastructural analysis and morphology preservation. The recent advent of antisera to FG has revealed numerous potential applications for analyzing chemically defined synaptic circuitry. To take advantage of the high sensitivity of this retrograde tracer in ultrastructural studies, we extended and detailed the original description of single immunocytochemical labeling of FG by comparing 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 tetroxide, or (2) silver-intensified 1 nm colloidal gold particles. We subsequently examined the utility of combining these markers in single sections for detection of transmitters (e.g., gamma-aminobutyric acid (GABA) and 5-hydroxytryptamine (5-HT)) in axon terminals presynaptic to retrogradely labeled neurons. Both analyses were carried out on the well-characterized mesolimbic pathway originating from perikarya in the ventral tegmental area (VTA) that project to the nucleus accumbens. Injections of FG were stereotaxically placed in the nucleus accumbens of anesthetized adult rats. From these animals, vibratome sections of aldehyde-fixed brains were examined for light-microscopic detection of FG using: (1) epi-fluorescence without immunocytochemistry, (2) immunoperoxidase, or (3) immunogold-silver. All 3 methods revealed circumscribed injections in the nucleus accumbens. Additionally, both immunocytochemical methods appeared to be as sensitive as epi-fluorescence in light-microscopic detection of retrogradely labeled perikarya and fine-caliber dendrites extending for 2-3 branch points beyond the soma. Electron microscopy showed that the FG was detectable not only in lysosomes but also throughout the cytoplasmic matrix of perikarya and dendrites using either immunoperoxidase or immunogold-silver labeling methods. In the second part of this analysis, single sections of tissue were processed 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 within sections examined by light or electron microscopy. Synaptic junctions from unlabeled or from GABA or 5-HT labeled terminals were most readily identified when the targets were lightly immunoreactive for peroxidase or labeled using silver-intensified colloidal gold. The present 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 transmitters such as GABA and 5-HT in afferent terminals. Two distinct histochemical procedures are described for optimizing the visualization of chemically identified synaptic contacts onto projection neurons. The methods demonstrated in this mesolimbic pathway are most likely applicable to other neuronal circuits and can be used for identifying transmitters in either the projection neurons or their afferent axons.