Visualization of neurons filled with biotinylated-lucifer yellow following identification of efferent connectivity with retrograde transport.
A new method is described that allows identification of neurons by retrograde transport, intracellular injection of biotin-labeled Lucifer Yellow, and a histochemical reaction of the biotin. Cells in the inferior colliculus that project to the ipsilateral thalamus are identified by injection of rhodamine-labeled latex beads into the medial geniculate body and retrograde transport in vivo. Several days later, the brains are fixed with aldehydes, and the inferior colliculus is cut on a vibratome. The rhodamine-labeled cells are observed with epi-fluorescent optics, impaled with intracellular pipettes under visual control, and injected with 9% Lucifer Yellow dilithium salt and 1% Lucifer Yellow cadaverine biotin-X. The biotinylated Lucifer Yellow is visualized by incubation of the slice overnight in avidin-biotin-HRP complex in the presence of 0.1% Triton X-100 at 4 degrees C. A diaminobenzidine reaction with simultaneous cobalt and nickel intensification follows. This method produces well-filled neuronal cell bodies, dendrites, and spines for light microscopic analysis. The non-fluorescent reaction product in these intracellularly filled cells may permit transmitter immunohistochemistry and synaptic fine structure to be studied in combination with neural connections and dendritic morphology.
Hill
S J
SJ
Department of Anatomy, University of Connecticut Health Center, Farmington 06030.
Oliver
D L
DL
D000818:Animals; D001306:Auditory Pathways / Q000648:ultrastructure; D001710:Biotin; D001933:Brain Stem / Q000648:ultrastructure; D002415:Cats; D003712:Dendrites / Q000648:ultrastructure; D004566:Electrodes; D005453:Fluorescence; D006651:Histocytochemistry; D007245:Inferior Colliculi / Q000648:ultrastructure; D007546:Isoquinolines; D008845:Microinjections; D008863:Microspheres; D009434:Neural Pathways / Q000648:ultrastructure; D009476:Neurons, Efferent / Q000648:ultrastructure*; D010477:Perfusion; D013788:Thalamus / Q000648:ultrastructure; D016707:Tissue Fixation
D007546:Isoquinolines; D001710:Biotin; C017475:lucifer yellow