Parallel holographic illumination enables sub-millisecond two-photon optogenetic activation in mouse visual cortex in vivo

Selective control of action potential generation in individual cells from a neuronal ensemble is desirable for dissecting circuit mechanisms underlying perception and behavior. Here, by using two-photon (2P) temporally focused computer-generated holography (TF-CGH), we demonstrate optical manipulation of neuronal excitability at the supragranular layers of anesthetized mouse visual cortex. Utilizing amplified laser-pulses delivered via a localized holographic spot, our optical system achieves suprathreshold activation by exciting either of the three optogenetic actuators, ReaChR, CoChR or ChrimsonR, with brief illumination ([≤] 10 ms) at moderate excitation power ((in average [≤] 0.2 mW/m2 corresponding to [≤] 25 mW/cell). Using 2P-guided whole-cell or cell-attached recordings in positive neurons expressing respective opsin in vivo, we find that parallel illumination induces spikes of millisecond temporal resolution and sub-millisecond precision, which are preserved upon repetitive illuminations up to tens of Hz. Holographic stimulation thus enables temporally precise optogenetic activation independently of opsins channel kinetics. Furthermore, we demonstrate that parallel optogenetic activation can be combined with functional imaging for all-optical control of a neuronal sub-population that co-expresses the photosensitive opsin ReaChR and the calcium indicator GCaMP6s. Parallel optical control of neuronal activity with cellular resolution and millisecond temporal precision should be advantageous for investigating neuronal connections and further yielding causal links between connectivity, microcircuit dynamics, and brain functions.