Optogenetic tools enable the causal examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the examination of how different synapses or pathways interact to support computation. Here we report two new channelrhodopsins, Chronos and Chrimson, obtained through the de novo sequencing and physiological characterization of opsins from over 100 species of algae. Chrimson is 45 nm red-shifted relative to any previous channelrhodopsin, important for scenarios where red light would be preferred; we show minimal visual system mediated behavioral artifact in optogenetically stimulated Drosophila. Chronos has faster kinetics than any previous channelrhodopsin, yet is effectively more light-sensitive. Together, these two reagents enable crosstalk-free two-color activation of neural spiking and downstream synaptic transmission in independent neural populations in mouse brain slice.

The ability to silence the activity of genetically specified neurons in a temporally precise fashion would open up the ability to investigate the causal role of specific cell classes in neural computations, behaviors, and pathologies. Here we show that members of the class of light-driven outward proton pumps can mediate very powerful, safe, multiple-color silencing of neural activity. The gene archaerhodopsin-31 (Arch) from Halorubrum sodomense enables near-100% silencing of neurons in the awake brain when virally expressed in mouse cortex and illuminated with yellow light. Arch mediates currents of several hundred picoamps at low light powers, and supports neural silencing currents approaching 900 pA at light powers easily achievable in vivo. In addition, Arch spontaneously recovers from light-dependent inactivation, unlike light-driven chloride pumps that enter long-lasting inactive states in response to light. These properties of Arch are appropriate to mediate the optical silencing of significant brain volumes over behaviourally-relevant timescales. Arch function in neurons is well tolerated because pH excursions created by Arch illumination are minimized by self-limiting mechanisms to levels comparable to those mediated by channelrhodopsins2,3 or natural spike firing. To highlight how proton pump ecological and genomic diversity may support new innovation, we show that the blue-green light-drivable proton pump from the fungus Leptosphaeria maculans4 (Mac) can, when expressed in neurons, enable neural silencing by blue light, thus enabling alongside other developed reagents the potential for independent silencing of two neural populations by blue vs. red light. Light-driven proton pumps thus represent a high-performance and extremely versatile class of “optogenetic” voltage and ion modulator, which will broadly empower new neuroscientific, biological, neurological, and psychiatric investigations.

Optogenetic inhibition of the electrical activity of neurons enables the causal assessment of their contributions to brain functions. Red light penetrates deeper into tissue than other visible wavelengths. We present a red-shifted cruxhalorhodopsin, Jaws, derived from Haloarcula (Halobacterium) salinarum (strain Shark) and engineered to result in red light–induced photocurrents three times those of earlier silencers. Jaws exhibits robust inhibition of sensory-evoked neural activity in the cortex and results in strong light responses when used in retinas of retinitis pigmentosa model mice. We also demonstrate that Jaws can noninvasively mediate transcranial optical inhibition of neurons deep in the brains of awake mice. The noninvasive optogenetic inhibition opened up by Jaws enables a variety of important neuroscience experiments and offers a powerful general-use chloride pump for basic and applied neuroscience.

Technologies for silencing the electrical activity of genetically targeted neurons in the brain are important for assessing the contribution of specific cell types and pathways toward behaviors and pathologies. Recently we found that archaerhodopsin-3 from Halorubrum sodomense (Arch), a light-driven outward proton pump, when genetically expressed in neurons, enables them to be powerfully, transiently, and repeatedly silenced in response to pulses of light. Because of the impressive characteristics of Arch, we explored the optogenetic utility of opsins with high sequence homology to Arch, from archaea of the Halorubrum genus. We found that the archaerhodopsin from Halorubrum strain TP009, which we named ArchT, could mediate photocurrents of similar maximum amplitude to those of Arch (∼900 pA in vitro), but with a >3-fold improvement in light sensitivity over Arch, most notably in the optogenetic range of 1–10 mW/mm2, equating to >2× increase in brain tissue volume addressed by a typical single optical fiber. Upon expression in mouse or rhesus macaque cortical neurons, ArchT expressed well on neuronal membranes, including excellent trafficking for long distances down neuronal axons. The high light sensitivity prompted us to explore ArchT use in the cortex of the rhesus macaque. Optical perturbation of ArchT-expressing neurons in the brain of an awake rhesus macaque resulted in a rapid and complete (∼100%) silencing of most recorded cells, with suppressed cells achieving a median firing rate of 0 spikes/s upon illumination. A small population of neurons showed increased firing rates at long latencies following the onset of light stimulation, suggesting the existence of a mechanism of network-level neural activity balancing. The powerful net suppression of activity suggests that ArchT silencing technology might be of great use not only in the causal analysis of neural circuits, but may have therapeutic applications.

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Whole-cell patch clamp electrophysiology of neurons is a gold standard technique for high-fidelity analysis of the biophysical mechanisms of neural computation and pathology but it requires great skill to perform. We have developed a robot that automatically performs patch clamping in vivo, algorithmically detecting cells by analyzing the temporal sequence of electrode impedance changes. We demonstrate good yield, throughput, and quality of automated intracellular recording in mouse cortex and hippocampus.

Light-oxygen-voltage sensitive (LOV) flavoproteins are ubiquitous photoreceptors that mediate responses to environmental cues. Photosensory inputs are transduced into signaling outputs via structural rearrangements in sensor domains that consequently modulate the activity of an effector domain or multidomain clusters. Establishing the diversity in effector function and sensor-effector topology will inform what signaling mechanisms govern light-responsive behaviors across multiple kingdoms of life and how these signals are transduced. Here, we report the bioinformatics identification of over 6,700 candidate LOV domains (including over 4,000 previously unidentified sequences from plants and protists), and insights from their annotations for ontological function and structural arrangements. Motif analysis identified the sensors from ∼42 million ORFs, with strong statistical separation from other flavoproteins and non-LOV members of the structurally related Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim family. Conserved-domain analysis determined putative light-regulated function and multidomain topologies. We found that for certain effectors, sensor-effector linker length is discretized based on both phylogeny and the preservation of α-helical heptad repeats within an extended coiled-coil linker structure. This finding suggests that preserving sensor-effector orientation is a key determinant of linker length, in addition to ancestry, in LOV signaling structure-function. We found a surprisingly high prevalence of effectors with functions previously thought to be rare among LOV proteins, such as regulators of G protein signaling, and discovered several previously unidentified effectors, such as lipases. This work highlights the value of applying genomic and transcriptomic technologies to diverse organisms to capture the structural and functional variation in photosensory proteins that are vastly important in adaptation, photobiology, and optogenetics.photoreceptors | LOV | flavoproteins | optogenetics