Intracellular lucifer yellow injection in fixed brain slices combined with retrograde tracing, light and electron microscopy

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The present paper contains a full methodological description of iontophoretic Lucifer Yellow injections in fixed brain slices in mammals. In brief, cortical tissue was either perfused or immersion-fixed in paraformaldehyde. After Vibratome sectioning, tissue slices were transferred to epifluorescence microscopes equipped with long distance objectives. Under visual guidance, neurons were selectively impaled with Lucifer Yellow-filled electrodes and intracellularly injected until all dendrites appeared brightly fluorescent. Excellent dendritic staining was obtained in both perfusion-fixed cat visual cortex and immersion-fixed human brain biopsies. Dendritic spines, varicosities and growth cones could be readily discerned. Filling of axonal collaterals was, however, incomplete. Callosally projecting neurons in cat visual cortex were retrogradely traced with a mixture of the fluorescent tracers Fast Blue and DiI. Subsequently the morphology of labelled cells was determined by intracellular Lucifer Yellow injection. Although the Fast Blue fluorescence had become undetectable in filled neurons the granular red appearance of DiI was still discernible. Hence the neuronal composition of even relatively sparse projections can be demonstrated. To obtain permanent preparations, dye-filled neurons were immersed in a diaminobenzidine solution and irradiated with epifluorescent illumination until all visible fluorescence had faded. Photo-oxidation resulted in the intracellular formation of a homogeneously distributed brown reaction product visible with the light microscope. Brief osmication enhanced the staining contrast, thus providing a Golgi-like image. Subsequent electron microscopy of photo-converted cells showed the fine granular nature of the electron opaque reaction product, thus revealing numerous cytological features. The precipitate was homogeneously distributed throughout the entire cytoplasm and nucleus, extending into dendrites and axon. Any apparent leakage of the label into the extracellular space was not observed. Intracellular staining in fixed tissue yields a high number of neurons with extensive filling of dendritic arbors. Photo-oxidation provides stable, non-fading preparations with the option of subsequent electron microscopy. In addition, the technique can be combined with immunocytochemistry and a variety of fluorescent tracer substances. These features, combined with its high selectivity and relative methodological simplicity, render the method to be a promising alternative to classical neuroanatomical approaches.