Simultaneous intracellular recording and calcium imaging in single neurons of hippocampal slices

Fluorescent Ca²⁺ indicator dyes can be introduced into cells through the same microelectrode used for intracellular voltage recording. Simultaneous measurement of cell membrane potential and intracellular Ca²⁺ concentration can be very helpful in interpreting the mechanisms of Ca²⁺ increases. This chapter describes fluorescence image acquisition using a CCD camera and a computer program that also records a synchronized membrane potential trace. The same program allows for preliminary data analysis. More elaborate analyses can be accomplished with commercial programs. We also describe quantitative evaluations of sources of error in the use of the statistic ΔF/F as an indicator of Ca²⁺ concentration. Especially important errors to minimize are changes in background fluorescence and inappropriate autofluorescence corrections. Some improvement of fluorescence images of cells deep within slices may be accomplished by masking. One method is described for making a mask based on the raw fluorescence image. With another method, highly detailed cell morphologies may be conveyed by using masks based on neurobiotin injections and camera lucida drawings.