Elucidation of Neuronal Circuitry: Protocol(s) Combining Intracellular Labeling, Neuroanatomical Tracing and Immunocytochemical Methodologies

We describe a protocol combining either intracellular biotinamide staining or anterograde biotinylated dextran amine (BDA) tracing with retrograde horseradish peroxidase (HRP) labeling and immunocytochemistry in order to map physiologically identified neuronal pathways. Presynaptic neurons including their boutons are labeled by either intracellular injection of biotinamide or extracellular injection of BDA while postsynaptic neurons are labeled with HRP via retrograde transport. Tissues are first processed to detect HRP using a tetramethylbenzidine and sodium-tungstate method. Biotinamide or BDA staining is then visualized using an ABC-diaminobenzidine-Ni method and finally the tissue is immunocytochemically stained using choline acetyltransferase (ChAT) or parvalbumin antibodies and a peroxidase-anti-peroxidase method. After processing, biotinamide, BDA, HRP and immunocytochemical staining can readily be distinguished by differences in the size, color and texture of their reaction products. We have utilized this methodology to explore synaptic relationships between trigeminal primary afferent neurons and brainstem projection and motoneurons at both the light and electron microscopic levels. This multiple labeling methodology could be readily adapted to characterize the physiological, morphological and neurochemical properties of other neuronal pathways.