Achieving temporally precise, noninvasive control over specific neural cell types in the deep brain would advance the study of nervous system function. Here we use the potent channelrhodopsin ChRmine to achieve transcranial photoactivation of defined neural circuits, including midbrain and brainstem structures, at unprecedented depths of up to 7 mm with millisecond precision. Using systemic viral delivery of ChRmine, we demonstrate behavioral modulation without surgery, enabling implant-free deep brain optogenetics.Certain symptoms of neurological and psychiatric disease can be treated by modulation of pathological brain activity in both preclinical and clinical settings [1][2][3][4] . However, existing methods for neuromodulation are not cell type specific (for example, transcranial magnetic or direct current stimulation) and/or are invasive (for example, electrical deep brain stimulation) 2,3 . In basic research settings, optogenetics with microbial channelrhodopsins (ChRs) enables cell type-specific excitation or inhibition of neuronal activity with light, permitting the tuning of cellular activity in terms of levels, ratios and rhythms with improved precision, which in many cases results in symptom-relevant treatment via optical neuromodulation [4][5][6] . However, the delivery of visible light often requires invasive implantation of foreign materials and devices into the brain, which damages tissue and increases infection and ischemia risk 1,7 . To improve long-term biocompatibility, optogenetic effects can be recruited by light delivered external to a thinned skull 8 . However, light attenuation by scattering and absorption in bone and tissue currently limits transmission of sufficient photon densities to stimulate neural activity in deep brain structures, even with fast ChR variants engineered to respond to lower intensity and/or longer wavelengths of light for access to deeper tissue [9][10][11] . Engineered ChRs with slow off-kinetics (for example, stable step-function opsins) can integrate photons for modulation of distal neural populations, but cannot provide the high temporal control enabled by fast opsins 12 .We recently described the potent fast red-shifted opsin ChRmine, which exhibits extremely large photocurrents with hundred-fold improved operational light sensitivity compared with existing fast red-shifted variants and rapid off-kinetics suitable for millisecond-scale control over neural activity 13 (Supplementary Table 1)-photophysical properties that may be suitable for deep transcranial optogenetics. To determine whether ChRmine can enable safe and rapid transcranial deep brain photoactivation, we performed optically paired in vivo extracellular electrophysiology in the ventral tegmental area (VTA), 4.5 mm deep from the skull surface (Fig. 1a,b). We used ChRmine targeted to somata with a Kv2.1 peptide tag to minimize axonal localization and antidromic activation (AAV8-CamKIIα::ChRmine-oScarlet-Kv2.1; Extended Data Fig. 1a). A 400-µm fiber optic was positioned directly above the surface of th...

The naturally occurring channelrhodopsin variant anion channelrhodopsin-1 (ACR1), discovered in the cryptophyte algae Guillardia theta, exhibits large light-gated anion conductance and high anion selectivity when expressed in heterologous settings, properties that support its use as an optogenetic tool to inhibit neuronal firing with light. However, molecular insight into ACR1 is lacking owing to the absence of structural information underlying light-gated anion conductance. Here we present the crystal structure of G. theta ACR1 at 2.9 Å resolution. The structure reveals unusual architectural features that span the extracellular domain, retinal-binding pocket, Schiff-base region, and anion-conduction pathway. Together with electrophysiological and spectroscopic analyses, these findings reveal the fundamental molecular basis of naturally occurring light-gated anion conductance, and provide a framework for designing the next generation of optogenetic tools.

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Highlightsd The cryo-EM structure of a pump-like channelrhodopsin, at 2.0 A ˚resolution d Identification of key features distinguishing ChRmine from other channelrhodopsins d Identification of key features distinguishing ChRmine from ion pump rhodopsins