We next tested GCaMP6 in layer (L)2/3 pyramidal neurons in the mouse visual cortex V1 in vivo (Fig. 2a). The majority of V1 neurons can be driven to fire action potentials in response to drifting gratings30. V1 was infected with adeno-associated virus (AAV) expressing GCaMP variants (AAV-hsyn1-GCaMP variant)11. Three weeks after AAV infection, the vast majority of L2/3 neurons showed fluorescence mainly in the neuronal cytoplasm (Supplementary Fig. 6). Sensory stimuli consisted of moving gratings presented in eight directions to the contralateral eye12,16. Two-photon imaging revealed visual stimulus-evoked fluorescence transients in subsets of neurons (Fig. 2a-c). These responses were stable across trials (Supplementary Fig. 8) and tuned to stimulus orientation (Fig. 2a, b and Supplementary Fig. 9). Orientation tuning was similar for GCaMP5G, GCaMP6f, GCaMP6m, and bulk-loaded OGB1-AM5 (Supplementary Fig. 9). Fluorescence transients were faster with GCaMP6f compared to other sensors and faithfully tracked dynamic sensory stimuli (Fig. 2d).
