Two novel staining methods for combined functional and structural analysis of single CNS synapses

The great diversity in morphology, receptor composition, and vesicle content of CNS synapses suggests that structural changes are important events in the establishment of neuronal plasticity. We have developed two novel methods for conducting functional/structural correlation studies of single CNS synapses. In the first method, a neuron is filled with biocytin and fluo-3 (a Ca²+ indicator), imaged with a C C D camera for changes in [Ca²+];, reacted with fluorescein-conjugated avidin and imaged by confocal microscopy. Imaging of the [Ca²+]i allows for the functional assessment of a single synapse, and confocal microscopy allows for the measurement of the spine size and its reconstruction in 3D. To permit electron microscopic examination of the same synapse, the cell is reacted with biotinylated anti-avidin antibody and is visualized by a commercially available ABC kit. This technique allows the examination of the structure of a functionally characterized synapse at both light and electron microscopic levels. While physiological imaging methods 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, with our second method, we have sought to develop an antibody staining technique 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 catalyzes the peroxidasemediated (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 AMPA receptor GluR 2/3). Our staining method enables subcellular resolution of immunocytochemical markers within a single neuron without confounding staining of neighboring cells. We anticipate that these two approaches 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. [Scientific formulae used in this abstract could not be reproduced.]Medicine, Faculty ofGraduat