Severe behavioural deficits in psychiatric diseases such as autism and schizophrenia have been hypothesized to arise from elevations in the cellular balance of excitation and inhibition (E/I balance) within neural microcircuitry. This hypothesis could unify diverse streams of pathophysiological and genetic evidence, but has not been susceptible to direct testing. Here we design and use several novel optogenetic tools to causally investigate the cellular E/I balance hypothesis in freely moving mammals, and explore the associated circuit physiology. Elevation, but not reduction, of cellular E/I balance within the mouse medial prefrontal cortex was found to elicit a profound impairment in cellular information processing, associated with specific behavioural impairments and increased high-frequency power in the 30–80 Hz range, which have both been observed in clinical conditions in humans. Consistent with the E/I balance hypothesis, compensatory elevation of inhibitory cell excitability partially rescued social deficits caused by E/I balance elevation. These results provide support for the elevated cellular E/I balance hypothesis of severe neuropsychiatric disease-related symptoms.

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, “RCaMPs,” engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca2+-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca2+]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.

The capture and utilization of light is an exquisitely evolved process. The single-component microbial opsins, although more limited than multicomponent cascades in processing, display unparalleled compactness and speed. Recent advances in understanding microbial opsins have been driven by molecular engineering for optogenetics and by comparative genomics. Here we provide a Primer on these light-activated ion channels and pumps, describe a group of opsins bridging prior categories, and explore the convergence of molecular engineering and genomic discovery for the utilization and understanding of these remarkable molecular machines.

The introduction of two microbial opsin-based tools, channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), to neuroscience has generated interest in fast, multimodal, cell type-specific neural circuit control. Here we describe a cation-conducting channelrhodopsin (VChR1) from Volvox carteri that can drive spiking at 589 nm, with excitation maximum red-shifted ~70 nm compared with ChR2. These results demonstrate fast photostimulation with yellow light, thereby defining a functionally distinct third category of microbial rhodopsin proteins.Microbial proteins that can be rapidly activated by light have been adapted for research in neuroscience, including ChR2 and NpHR, which permit millisecond-precision optical control of genetically defined cell types in intact neural tissue 1-6 . Because ChR2 is a blue light-gated cation channel and NpHR is a yellow light-driven chloride pump, the combination of these two proteins allows independent neural excitation and inhibition in the same preparation. However, there has been enormous interest in developing a hypothetical third major optogenetic tool, namely a second cation channel with an action spectrum that is substantially red-shifted relative to ChR2, to allow tests of the interaction of cell types in circuit computation or behavior.Although efforts to develop a distinct light-activated excitatory protein have been focused on molecular engineering of ChR2, another approach would be to identify previously unknown microbial channelrhodopsins using genomic tools. One ChR2-related sequence from the spheroidal alga Volvox carteri (Fig. 1a) has been described, but the absorption spectrum of the protein and the photocycle dynamics are virtually identical to those of ChR2 (refs. 7 ,8 ). Therefore, we searched the genome database from the US Department of Energy Joint Genome Institute, discovered a second Volvox ChR (VChR1) that is more related to ChR1 (ref. 9) from Chlamydomonas reinhardtii, explored its properties in heterologous expression systems and functionally tested the codon-optimized opsin gene in mammalian neurons.We expressed VChR1 in Xenopus oocytes and HEK293 cells and observed evoked photocurrents similar to those of ChR1 from Chlamydomonas 9,10 . The photocurrents were graded with light intensity and showed inactivation from a fast peak toward a reduced stationary plateau (Fig. 1b) (Fig. 1b) 9 , and currents showed an inwardly rectifying current-voltage relationship (Fig. 1c).Certain primary structural differences between VChR1 and the Chlamydomonas ChRs suggested that the properties of VChR1 would be distinct from those of the other ChRs (Fig. 1d). On the basis of electrostatic potential and quantum mechanical-molecular mechanical calculations for bacteriorhodopsin and relatives, the counterion complex of the all-trans retinal Schiff base (RSB; Fig. 1d) should be critical for color tuning 11,12 , but these residues are conserved in both ChR1 and VChR1 (blue sequence, Fig. 1d). On the other hand, calculations and mutational experiments 11,12 predict that fo...

We investigated the efficacy of optogenetic inhibition at presynaptic terminals using halorhodopsin, archaerhodopsin and chloride-conducting channelrhodopsins. Precisely timed activation of both archaerhodopsin and halorhodpsin at presynaptic terminals attenuated evoked release. However, sustained archaerhodopsin activation was paradoxically associated with increased spontaneous release. Activation of chloride-conducting channelrhodopsins triggered neurotransmitter release upon light onset. Our results indicate that the biophysical properties of presynaptic terminals dictate unique boundary conditions for optogenetic manipulation.