Combining Uncaging Techniques with Patch-Clamp Recording and Optical Physiology

Patch-clamp recording is a powerful approach to monitoring membrane electrical activity with high temporal resolution. However, the spatial resolution of patch-clamp recording in a distributed structure such as a neuron or a brain slice is limited by the fact that each electrode records from just one point, making recording from more than a very small number of points impractical. An approach that allows biological signals to be monitored and manipulated with high spatial resolution is the use of optical methods. For the monitoring of signals, a powerful approach is the use of activity-dependent fl uorescent dyes. In this type of recording, one or more cells are loaded with a fl uorescent dye that is sensitive to some change of interest, such as intracellular calcium or membrane voltage. Loading can be done by including the dye in the patch pipette, by bulk loading of many cells at once with acetoxymethyl (AM) ester dyes (Garaschuk et al., 2006; Sullivan et al., 2005; Tsien, 1999) or voltage-sensitive dyes (Djurisic et al., 2003; Grinvald and Hildesheim, 2004), or by expression of an activity-dependent fl uorescent protein (Bozza et al., 2004; Wang et al., 2003). Regardless of the means of loading, biological signals lead to variations in fl uorescence that can be detected by high-speed fl uorescence microscopy. The combination of electrophysiological and fl uorimetric recording thus allows monitoring of biochemical and electrical signals simultaneously with high temporal and spatial resolution.