Restriction of peroxidase-mediated antibody reactivity to single neurons by local hydrogen peroxide production

Current electrophysiological and imaging methods have begun to target the subcellular structure and function of single CNS neurons. Although physiological imaging methods now permit the resolution of activity of single presynaptic and postsynaptic elements, it has not been possible to unequivocally examine the array of proteins expressed at these same structures. This problem arises from the inability of current immunocytochemical techniques to differentiate between a process of the neuron of interest and the surrounding neuropil belonging to other cells. Thus, we have sought to develop an antibody staining method which would restrict reactivity to only a single neuron. Our assay involves preloading a single neuron with the coupling reagent biocytin. Following fixation, the injected biocytin is then complexed with avidin-linked glucose oxidase, providing a means of locally generating hydrogen peroxide within a cell of interest which catalyses the peroxidase-mediated (coupled to primary antibody) staining reaction. We have used this method successfully with antibodies to a number of neuronal markers (neuron-specific enolase, neurofilament, microtubule-associated protein and the glutamate receptor GluR2/3). Our staining method enables subcellular resolution of immunocytochemical markers within a single neuron without confounding staining of neighboring cells. We anticipate that this approach will facilitate the study of neuronal phenotype in fine dendritic processes in electrophysiologically characterized neurons in specimens with a complex neuropil, such as brain slices or high-density cultures.