In vivo sub-millisecond two-photon optogenetics with temporally focused patterned light

Temporally precise control of action potential generation of individual cells from a neuronal ensemble is desirable for dissecting circuit mechanisms underlying perception and behavior. Here we demonstrate that such degree of precision is achievable by using two-photon (2P) temporally focused computer-generated holography (TF-CGH) to control neuronal excitability at the supragranular layers of anesthetized and awake visual cortex in both male and female mice. Using 2P-guided whole-cell or cell-attached recordings in positive neurons expressing either of the three opsins ReaChR, CoChR or ChrimsonR we investigate the dependence of spiking activity on the opsin9s channel kinetics and show that in all cases the use of brief illumination (≤ 10 ms) induces spikes of millisecond temporal resolution and sub-millisecond precision, which are preserved upon repetitive illuminations up to tens of Hz. We also demonstrate that using large illumination spot covering the entire cell body and amplified laser at high peak power enable to reach such degree of temporal precision by using low excitation intensity (in average ≤ 0.2 mW/μm 2 ), thus minimizing the risk for nonlinear photodamage effects. Finally, by combining 2P holographic excitation with electrophysiological recordings and calcium imaging using GCaMP6s, we investigate the factors, including illumination shape and intensity, opsin distribution in the target cell, and cell morphology, which affect the spatial selectivity of single- and multi-cell holographic activation. 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. SIGNIFICANCE STATEMENT Recent development of optogenetics allows probing the neuronal microcircuit with light by optically actuating genetically-encoded light-sensitive opsins expressed in the target cells. Here, we apply holographic light shaping and temporal focusing to simultaneously deliver axially-confined holographic patterns to opsin-positive cells situated in the living mouse cortex. Parallel illumination efficiently induces action potentials with high temporal resolution and precision for three opsins of different kinetics. We extend the parallel optogenetic activation at low intensity to multiple neurons and concurrently monitoring their calcium dynamics. These results demonstrate fast and temporally precise in vivo control of a neuronal sub-population, opening new opportunities to reveal circuit mechanisms underlying brain functions.