Intracellular filling and reconstruction of identified neurons in fixed rat brain slices.

A simple technique for the morphological characterization of pre-labelled neurons in fixed brain slices is described. Neurons are retrogradely-labeled with a carbocyanine dye and the tissue is fixed and sliced. Individual labeled cells from the interior of a slice are then visualized on an upright fluorescence microscope and impaled with a micropipet containing rhodamine or fluorescein-peroxidase conjugates. The cells are filled by iontophoresis, postfixed, and the peroxidase is oxidized into a permanent, opaque reaction product. The dendritic morphology of the neurons is then reconstructed under camera lucida and quantified.