Double immunofluorescence, peroxidase labelling and ultrastructural analysis of interneurones following prolonged electrophysiological recordings in vitro

Inhibitory hippocampal and neocortical interneurones comprise a physiologically, morphologically and neurochemically heterogenous cell population. To identify the roles each class of interneurone plays within a given circuit it is necessary to correlate the electrophysiological properties of individual cells with their neurochemistry and morphology at both the light and electron microscopic level. However, the optimal conditions required for any one part of the protocol typically compromise the results from another. We have developed a protocol which allows the neurochemical content, gross morphology and ultrastructure details of biocytin-filled neurones to be recovered following long, dual intracellular recordings in thick mature slices maintained in an interface recording chamber, helping define sub-populations which could not otherwise be determined. Dual immunofluorescence is performed by incubating the tissue in monoclonal and polyclonal antibodies simultaneously, prior to visualization of biocytin-labelling with precipitation of a peroxidase reaction product. By using a biotinylated anti-avidin D antibody (Vector Laboratories), the intensity of this precipitation can be enhanced further where necessary. It is envisaged that this protocol can not only help determine the neurochemical content of cells recorded in similar in vivo studies, but that the ability to amplify peroxidase labelling in poorly filled cells is also of interest.