Intracellular injection in fixed slices in combination with neuroanatomical tracing techniques and electron microscopy to determine multisynaptic pathways in the brain.

Intracellular Lucifer Yellow filling in fixed tissue has been recently introduced as a novel neuroanatomical approach to reveal the detailed morphology of individual neurons in isolated preparations of the central nervous system. Since dye injections are performed under visual control, the method is characterized by a high degree of inherent staining selectivity, thus circumventing the element of randomness often considered to be the crux of classical golgi-impregnation techniques. Moreover, the opportunity to optically monitor the injection procedure renders fixed slice preparations highly advantageous to be used in combination with retrograde fluorescent tracing. Subsequently, dye-filled neurons may be subjected to a simple photoconversion procedure leading to the intracellular formation of a stable polymer thus obtaining permanent specimens for light microscopy purposes. Due to the osmiophilic nature of the precipitate the photoconverted material is equally suitable for correlated electron microscopy, thus enabling the analysis of neuronal microcircuitry. At the ultrastructural level, sources of afferent input to identified projection neurons may be revealed by lesion-induced anterograde degeneration of synaptic terminals, therefore enabling the direct demonstration of multisynaptic links. Finally, morphologically identified neurons may be immunocytochemically characterized at the pre- and postembedding levels. It is therefore suggested that their methodological versatility and relative technical ease render intracellular fixed-slice injections a promising complement to the catalogue of anatomical techniques.