Identification of synaptic interactions of intracellularly injected neurons in fixed brain slices by means of dual‐label electron microscopy

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Abstract
The injection of the dye Lucifer Yellow (LY) into neurons in slices of fixed brain is used to associate cells displaying a particular dendritic geometry with a specific pattern of neuronal connectivity. In the present report we expand on this technique by combining it at the electron microscopic level with immunocytochemistry and/or degeneration for the study of synaptic relationships. As a model we use the projection neurons of nucleus accumbens. These neurons were retrogradely labeled in vivo with injections or a fluorescent tracer. Fast Blue, into the ventral mesencephalon. Using epifluorescent monitoring, these neurons were located in perfusion‐fixed brain slices and intracellularly injected with LY. They were visualized in the light and electron microscope using a peroxidase‐antiperoxidase immunocytochemical method. Certain afferent connections of these neurons were identified in the same tissue through the use of either dual‐label immunocytochemistry or anterograde degeneration combined with a single‐label immunoreaction. In the dual‐label procedure, a silver‐gold intensification of the diaminobenzidine (DAB) reaction product for the first antigen (LY) was contrasted with a nonintensified reaction product for the second antigen (tyrosine hydroxylase [TH]). Ultrastructurally, metallic gold particles appeared to be dispersed over the immunolabeled perikarya, dendrites, and, occasionally, axonal terminals of LY‐injected neurons whereas the flocculent DAB reaction product was present in TH‐containing axons and terminals. Following lesions of the ventral subiculum in the hippocampal formation, degenerating axon terminals were detected in nucleus accumbens along with immunoreacted, LY‐injected neurons. The techniques outlined in this report should prove invaluable for the study of the synaptic interactions of identified neurons. They can be reliably reproduced with a high yield per experiment.

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