Measurement of neuronal Ca2+ transients using simultaneous microfluorimetry and electrophysiology

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Abstract
Fluorescent indicator molecules, such as fura-2, are useful probes for studying the concentrations of ions in single cells. A key feature of these fluorescent dyes is the shift in their excitation spectra upon binding the ion, thus making alternate excitation from two wavelengths desirable. In this report we describe an inexpensive system for making simultancous electrophysiological and dual excitation fluorescence measurements using equipment much of which is available in a typical biophysical laboratory. In order to synchronize the fluorescence signal with the appropriate excitation wavelength we devised a simple computer program which uses the output of photodiodes placed in the excitation beam to determine which wavelength is illuminating the cell. We also describe the use of a liquid light guide to transmit excitation illumination from the light source to the epifluorescence port of the microscope in order to isolate a perfusion chamber from light, electrical noise and vibration. A sensitive light detection system was assembled using a photomultiplier tube and discriminator that took advantage of sampling single unit events obtained with photon counting rather than the analog of annode current. However, rather than employing a sophisticated and expensive photon counting system, a filter was used to integrate the signal so that an analog output could be presented to a multichannel analog to digital converter commonly used for electrophysiological recordings. This apparatus was sensitive enough to allow the determination of the intracellular free Ca2+ concentration, [Ca2+]i, using fura-2 in the following situations: (a) in single processes of dorsal root ganglion (DRG) neurons grown in primary culture, (b) the release of Ca2+ from intracellular stores in single neurons, (c) the influx of Ca2+ through channels activated by excitatory amino acids and (d) it was also possible to measure [Ca2+]i transients while simultaneously recording Ca2+ currents at precisely controlled membrane potentials in DRG neurons. The instrumentation described here makes use of a number of innovations which investigators developing similar systems may find useful.