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

Current electrophysiological and imaging methods have begun to target the s ubcellular structure and function of single CNS neurons. Although physiolog ical imaging methods now permit the resolution of activity of single presyn aptic and postsynaptic elements, it has not been possible to unequivocally examine the array of proteins expressed at these same structures. This prob lem arises from the inability of current immunocytochemical techniques to d ifferentiate between a process of the neuron of interest and the surroundin g neuropil belonging to other cells. Thus, we have sought to develop an ant ibody staining method which would restrict reactivity to only a single neur on. 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 hyd rogen peroxide within a cell of interest which catalyses the peroxidase-med iated (coupled to primary antibody) staining reaction. We have used this me thod successfully with antibodies to a number of neuronal markers (neuron-s pecific enolase, neurofilament, microtubule-associated protein and the glut amate receptor GluR2/3). Our staining method enables subcellular resolution of immunocytochemical markers within a single neuron without confounding s taining of neighboring cells. We anticipate that this approach will facilitate the study of neuronal phen otype in fine dendritic processes in electrophysiologically characterized n eurons in specimens with a complex neuropil, such as brain slices or high-d ensity cultures. (C) 1998 IBRO. Published by Elsevier Science Ltd.