Figure 5.

Wavelength-selective two-photon uncaging of GABA. Compound 6 was bath applied to acutely isolated brain slices at 200 μM. Currents evoked in CA1 pyramidal neuron filled with Alexa-594 (100 μM) were monitored via whole-cell patch-clamp. Uncaging was effected by two femtosecond-pulsed Ti:sapphire lasers tuned to 900 and 720 nm directed close to the cell which was imaged with a third laser at 1075 nm. (a) Two-photon fluorescence image of neuron (2D maximum projection of 15 z-sections, boxed area shown in (b) and (f)). (b) Reference image showing the position of three uncaging points close to the soma. (c) Currents evoked by three 100 mW uncaging events at 900 nm for 5 ms (n = 61) and 1 ms (n = 11). (d) Plot of power dosage versus evoked current was fit with a quadratic function implying uncaging was through two-photon excitation. (e) Current traces evoked by uncaging at 900 or 720 nm. Black traces are an average of 3–5 uncaging trials with the SE shown in gray. (f) Reference image showing the irradiation points 3.5 μm from the cell used to test the lateral uncaging resolution. (g) Summary of the effects of uncaging on and close to the soma with lasers tuned to 900, 800, and 720 nm at defined lateral distances from the cell body.