Periodic current injection (PCI)--a new method to image steady-state membrane potential of single neurons in situ using extracellular voltage-sensitive dyes.

A new method is described which allows to image the steady-state distribution of membrane potential of single neurons in situ. The method consists of staining the tissue with an extracellular voltage-sensitive dye (Di-4-ANEPPS) and impaling a single neuron with a microelectrode. After focusing the imaging system onto the cell a large series of images are taken with a CCD camera at the appropriate excitation wavelength of the voltage-sensitive dye while the neuron's membrane potential is shifted by a periodic current injection (PCI). Afterwards two groups of images are averaged separately: those images while the cell was at rest and those images while the cell was hyperpolarized. After subtraction of these averaged images, the resulting difference image shows only the membrane potential of the cell which was altered periodically. The success of the method is demonstrated on leech cells in intact ganglia. If applied to cells with a basically two-dimensional arborization pattern, the decrease of activity in the difference image in areas further away from the injection site should relate to a decrease in membrane potential according to the passive electrotonic properties of the cell under study.