New illumination technique for IR-video guided patch-clamp recording from neurons in slice cultures on biomembrane.

摘要

Slice cultures on biomembrane are the method of choice for studying Ca2+-dependent plastic changes occurring over several days to weeks. Using IR-differential interference contrast, good visualization of neurons in biomembrane slice cultures has been achieved despite a negative optical effect of the biomembrane, but epifluorescence imaging requires removal of a Wollaston prism and the analyzer. Here, we describe a novel illumination method to overcome this problem. Using optic fiber illumination at a shallow angle from the top of the slice culture, with or without additional illumination from the bottom, we obtained good cellular resolution of neurons in biomembrane slice cultures as well as in acute slices with an infrared-video camera. With this technique, we demonstrate visually guided whole-cell patch-clamp recording of Na+- and K+-currents as well as combination of whole-cell recording with fluorescence imaging of hippocampal and entorhinal cortex neurons in biomembrane slice cultures. Our inexpensive method should prove very useful for studying in vitro effects of long-term manipulations on membrane currents and intracellular Ca2+-signaling.