Immunocytochemical labeling methods and related techniques for ultrastructural analysis of neuronal connectivity.

We describe a series of techniques based on the use of immunocytochemical labeling methods for the study of the neurochemistry and connectivity of cells within the central nervous system (CNS). According to our current view, neurons communicate to each other mainly at the level of synapses. Although it is now widely accepted that non synaptic neuron-to-neuron communication and neuron-to-glia cross-talk also occur, ultrastructural localization of transmitters/modulators at synapses and analysis of neuronal connections still remain major challenges for a correct understanding of the way in which neuronal networks are organized and operate. This chapter is mainly devoted to the description of a number of immunocytochemical procedures which are performed on material which, as far as possible, has been prepared according to the protocols for conventional electron microscopy, particularly regarding the use of osmium tetroxide as a fixative for the achievement of optimal ultrastructure. We will also briefly consider several techniques which can be used in combinations with the above methods and may be useful to further dissect the complex interactions between the cells of the CNS. In situ hybridization techniques at the ultrastructural level are considered in Chapter 8. A detailed description of the methods employed for combined ultrastructural and electrophysiological analysis of central neurons is given in Chapter 11 and examples of applications of gold labeling procedures to the study of cell proliferation and apoptosis are considered in Chapter 14.