5= 5, data not shown). We next asked whether spatially selective photoactivation could be obtained via optical read-out of calcium imaging. GCaMP6s, we investigated the factors, including illumination shape and intensity, opsin distribution in the target cell, and cell morphology, which affect the GRL0617 spatial selectivity of single-cell and multicell holographic activation. Parallel optical control of neuronal activity with cellular resolution and millisecond temporal precision should make it easier to investigate neuronal connections and find further links between connectivity, microcircuit dynamics, and brain functions. SIGNIFICANCE STATEMENT Recent developments in the field of optogenetics has enabled researchers to probe the GRL0617 neuronal microcircuit with light by optically actuating genetically encoded light-sensitive opsins expressed in the target cells. Here, we applied holographic light shaping and temporal focusing to simultaneously deliver axially confined holographic patterns to opsin-positive cells in the living mouse cortex. Parallel illumination efficiently induced action potentials with high temporal resolution and precision for three opsins of different kinetics. We extended the parallel optogenetic activation at low intensity to multiple neurons and concurrently monitored their calcium dynamics. These results demonstrate fast and temporally precise control of a neuronal subpopulation, opening new opportunities for revealing circuit mechanisms underlying brain functions. two-photon optogenetics, millisecond photoactivation, mouse visual cortex, temporal focusing Introduction The coordinated spike timing among neurons with precision in the range of GRL0617 milliseconds enables various synaptic mechanisms believed to play significant roles in regulating sensation, perception, and cognitive functions. Optogenetics, a field with a growing number of genetic tools (Mattis et al., 2011; Yizhar et al., 2011; Lin et al., 2013; Klapoetke et al., 2014), offers researchers new ways to investigate those mechanisms via fast and precise optical control of neuronal firing (Nagel et al., 2003; Boyden et al., 2005; Adamantidis et al., 2007; Zhang and Oertner, 2007; Li et al., 2013; Emiliani et al., 2015). However, until recently, researchers have been daunted by the challenge of obtaining optical control of excitability in one or several individually selected cells with millisecond resolution and precision into scattering tissue activity manipulation in depth through optical-fiber endoscopes (Adamantidis et al., 2007; Aravanis GRL0617 et al., 2007; Gradinaru et al., 2007). Single-photon patterned illumination, in contrast to wide-field illumination, provides improved spatial selectivity for activating neuronal ensembles, but is limited to shallow depths or transparent animals (Guo et al., 2009; Wyart et al., 2009; Szabo et al., 2014). Two-photon (2P) optogenetic neuronal activation, which provides accurate targeting of neurons and reduced scattering in tissue (Helmchen and Denk, 2005), GRL0617 has been realized by employing one or a hybrid of two approaches: the scanning method (Packer et al., 2012, 2015; Carrillo-Reid et al., 2016; Yang et al., 2018) and the parallel method (Ronzitti et al., 2017a; Chen et al., 2018). Parallel methods for 2P optogenetic activation use phase-modulation techniques, such as computer-generated holography (CGH; Bgue et al., 2013; Dal Maschio et al., 2017), generalized phase contrast (Glckstad, 1996; Papagiakoumou et al., 2010, 2013), and extended Gaussian beams (Rickgauer et al., 2014; Straub et al., 2016; Pegard et al., 2017). Axial confinement of light patterns can be preserved for hundreds of micrometers by integrating these approaches with temporal focusing (TF; Andrasfalvy et al., 2010; Papagiakoumou et al., 2010, 2013; Bgue et al., 2013; Rickgauer et al., 2014; Baker et al., 2016; Straub et al., 2016; Pegard et al., 2017; Mardinly et al., 2018). Until now, 2P optogenetic spike induction by using scanning-based excitation has been reported for the slow opsin C1V1 (Packer et al., 2015; Yang et al., Rabbit polyclonal to DUSP26 2018). Neuronal activation via 2P holographic spots or temporally focused Gaussian beams has been shown for C1V1.