Abstract
The ability to correlate neuronal morphology and physiology has been greatly advanced by intracellular labeling through the recording pipette. However, visualizing the filled neuron required physically sectioning and reconstructing areas of interest, often resulting in figures that are two-dimensional. We have visualized the three-dimensional morphology of filled neurons reacted with nickel-intensified diaminobenzidine (DAB/Ni) using the confocal scanning laser microscope (CSLM).Neurons in slices of rat hippocampus were filled with biocytin, fixed in 4% paraformaldehyde, incubated in avidin-HRP (1:200) in 0.5% Triton X-100, and reacted with DAB in 0.04% nickel ammonium sulfate. Optical sections and three-dimensional images were recorded by using a Bio Rad MRC-500 CSLM with an argon ion laser.Biocytin filled all aspects of the neuron, including fine axons and spines. Fig. 1 is a conventional micrograph of a single neuron labeled with DAB/Ni. Figs. 2a and b are stereo pairs of the apical and basal dendrites of the neuron in Fig. 1.
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