Sometimes light-evoked activity was detected with two electrodes simultaneously (Fig. 4D and E) but, in most cases, only one electrode in the probe detected light-evoked activity. This is probably due to the relatively large distance between adjacent electrodes in the probe (at least 40 μm apart). To test the spatial resolution of our photostimulation method further, we stimulated various

areas in the endoscopic field of view and recorded Pirfenidone neural activity from the electrodes. Neural activity-generating points in the endoscopic field of view are shown as small dots in Fig. 5. The dots are color-coded according to the electrodes by which spikes were detected. In this experiment, light-induced activities were detected at seven of the 10 electrodes, Crizotinib molecular weight and only one electrode detected light-induced spiking activity at each stimulation point. This result indicates that our method can activate spatially restricted neuronal populations, and also indicates that by stimulating

different positions in the field of view, different sets of neurons can be activated. We next studied the relationship between light intensity and light-induced neural activity. As the intensity of stimulating light increased, the amplitude of neural activity increased (Fig. 6A and B). This result suggests that multiple neurons were activated with high-intensity photostimulation. On the other hand, at minimal light intensity of neural activity generation (0.16 mW), single-unit-like activity was detected (Fig. 6B). Repeated minimal-intensity photostimulation reliably produced single-unit-like activity (Fig. 6D). This activity was specifically evoked when stimulating enough via the specific fiber core in the stimulating site (Fig. 6C and D). In contrast, stimulating via the other two adjacent fiber cores in the stimulation area (Fig. 6C and D) did not evoke neural activity. Moreover, photostimulation at half the scan speed (32 ms/line; Fig. 6D, right) also evoked spiking activities whose shape was similar to that

evoked by normal scan speed (16 ms/line; Fig. 6D, left and center). These observations suggest that the light-evoked spiking activities represent action potential generation rather than subthreshold membrane potential fluctuations. Most of the spiking activity elicited by photostimulation was blocked with tetrodotoxin treatment (Fig. S1). This result also indicates that the recorded activity represents action potential. In order to precisely estimate the spatial specificity of photostimulation, we measured light-induced action potential generation of ChR2-expressing cells in brain slice preparation. The relationship between light intensity and the distance of photostimulation point from recorded cell was measured (Fig. S2).