Identification of synaptic interactions of intracellularly injected neurons in fixed brain slices by means of dual-label electron microscopy.
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.
Meredith
G E
GE
Department of Anatomy and Embryology, Faculty of Medicine, Free University, Amsterdam, The Netherlands.
Wouterlood
F G
FG
D000818:Animals; D000950:Antigens, Ly / Q000648:ultrastructure; D001921:Brain / Q000648:ultrastructure*; D003712:Dendrites / Q000648:ultrastructure; D005110:Extracellular Space; D005260:Female; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D007267:Injections; D016253:Microscopy, Immunoelectron / Q000379:methods*; D009474:Neurons / Q000648:ultrastructure*; D051381:Rats; D017208:Rats, Wistar; D013569:Synapses / Q000648:ultrastructure*; D014446:Tyrosine 3-Monooxygenase / Q000648:ultrastructure
D000950:Antigens, Ly; D014446:Tyrosine 3-Monooxygenase