ISSN:
1059-910X
Keywords:
Intracellular injection
;
Fixed slice
;
Dual-label immunocytochemistry
;
Silver-gold enhancement
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Natural Sciences in General
Notes:
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. © 1993 Wiley-Liss, Inc.
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jemt.1070240105
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