5 mm anterior, 1.0 mm lateral from bregma, 0.5 mm deep from brain surface) of the anesthetized mouse. Initially, brief light pulses of several different light intensities (0.06, 0.3, 1.5 and 6 mW at endoscope tip) were used to determine whether any movement was evoked. If movement was detected at a certain light intensity, a light stimulation series (20 steps of light intensity) was applied. Light intensity was increased by 1.1 × at one step, and the stimuli were delivered in ascending order. At each step, light stimulation contained five 40-ms light pulses with 500-ms intervals. Whisker movements were captured at 50 frames/s with a video camera (RM-6740CL; JAI, Copenhagen,
Denmark). We classified trials as
‘single-whisker movement’, EPZ5676 where only one whisker was diffracted or a large (twice) difference was detected between the best and second-best whisker in movement amplitude at threshold. Video images were analysed using ImageJ (http://rsb.info.nih.gov/ij/) and matlab. We describe here a method for ChR2-assisted optical control of neural activity in vivo with high spatio-temporal resolution. A newly designed optical/electrical probe was used to image neurons, deliver stimulating light with high spatial resolution, and record neural activity in living animals (Fig. 2A). The device was composed of three optical fiber bundles (80 or 125 μm diameter) and 10 tungsten microelectrodes (Fig. 2B; Table 1). The probe tip had a 45 º beveled edge for minimizing brain damage. Smaller diameter electrodes (7.6 μm diameter) were gold-plated to reduce electrical impedance. The optical fiber selleck inhibitor bundle, which consisted from of hundreds of
optical fibers, transmitted an image to a remote end (Fig. 2C). Because light propagates bidirectionally in the optical fibers, the bundle could deliver illuminating light to the neural tissue and transmit fluorescent images back to the photodetector (Fig. 2A). Each optical fiber bundle consisted of 1.9-μm-diameter single-mode optical fibers, and the spacing of each fiber was 3.3 μm, which determined the spatial resolution of a transferred image. The numerical aperture of each fiber is 0.41, and the half angle of emission from the fiber in water was approximately 10 º (Fig. 2D). A previous study showed that the spatial resolution of an optical fiber bundle-based endoscope is sufficient to visualize fluorescently labeled neurons at single-cell resolution (Vincent et al., 2006). Stimulating light was deflected by a pair of galvanometer scanners (Fig. 2A), enabling stimulating light to be sent to a single fiber core in the optical fiber bundles (Fig. 2D). This feature is important for controlling neural activity with high spatial resolution (see below). We used an in utero electroporation technique for targeted expression of ChR2 to projection neurons in layer 2/3 of the mouse cerebral cortex.