Immunocytochemical detection of Substance P neurons, their processes and connections by in vivo microinjections of monoclonal antibodies

摘要

A procedure is described for the immunocytochemical detection of Substance P immunoreactive cells in the central and peripheral nervous system by the in vivo injection of characterized monoclonal antibody to Substance P directly into the living nervous tissue. Model studies are conducted in the raphe pallidus of the rat, a region known to contain numerous Substance P immunoreactive neurons that are generally difficult to localize by immunocytochemistry without prior use of axonal transport inhibitors such as colchicine. The raphe pallidus is surgically accessible from a ventral approach. Monoclonal antibody against Substance P is injected into the raphe pallidus of untreated, anesthetized animals directly by pressure through micropipettes. Following fixation, the immunocytochemical procedures for visualizing the antigen-antibody complexes are conducted using the indirect immunofluorescence method or the peroxidase-antiperoxidase method. The material is examined by light microscopy and by electron microscopy. Neurons are identified as positively labeled by the presence of large numbers of lysosomes and prelysosomal particles which mark the somata as well as axons and dendrites. These particles or complexes have intense, specific immunoreactivity to monoclonal Substance P antibody, and are not present in control injections with antibodies preadsorbed with Substance P. The antigen-antobidy lysosomal complexes are carried by axoplasmic transport in anterograde and retrograde fashion e.g. from the dorsal root ganglia to the spinal cord, and vice versa. Thus the method can be utilized for the tracing of chemically specific projections between different areas of the central or peripheral nervous systems.