Figure 2.

Optical activation of ChR2-eYFP-expressing CFs results in all-or-none PN responses. A, Multiphoton image of ChR2-eYFP-expressing CFs (green) and a PN filled with Alexa 594 during a whole-cell recording (red) in a parasagittal cerebellar slice. Scale bar, 50 μm. B, Diagram illustrating the two methods used to stimulate the same CF input to an individual PN. Blue circle indicates 0.6–1 ms pulses of blue light (470 nm) used to activate ChR2 expressed in CF. Lightning bolt indicates 0.1 ms electrical stimuli used to electrically activate CF in granule cell layer close to the recorded PN. C, Complex spike responses recorded from a current-clamped PN in response to optical (black line) or electrical (red line) stimuli. Illumination intensity is indicated above traces. D, Overlaid voltage responses from C illustrate the similar complex spike waveforms elicited by optical (black) and electrical (red) stimuli. The gray trace illustrates subthreshold optical stimulation. The electrical stimulus artifact has been removed for clarity. E, PN voltage-clamp traces shown here are the result of light activation that was either subthreshold (2.4, 2.6, and 2.8 mW/mm²) or suprathreshold (3 mW/mm²), giving rise to large (nA) CF-like EPSC. Response to 3 mW/mm² stimulus was recorded at V_m = 0 mV, all others were recorded at −70 mV (see Materials and Methods). Responses are averages of 4–10 trials. F, Plot showing that light stimulation leads to all-or-none CF activation in PNs. Currents, like in E, were recorded at −70 mV for subthreshold responses (plotted for the intensity range from 10 to 20 pA) and 0 mV for large CF responses (plotted for the range −1000 to −3000 pA). Solid symbols indicate intensities at which there were both failures and successes in the recorded PN; however, only the failure amplitudes have been averaged and plotted.