Channelrhodopsins are light-activated ion channels that mediate cation permeation across cell membranes upon light absorption. Red-light-activated channelrhodopsins are of particular interest, because red light penetrates deeper into biological tissues and also enables dual-color experiments in combination with blue-light-activated optogenetic tools. Here we report the crystal structure of the most red-shifted channelrhodopsin from the algae Chlamydomonas noctigama, Chrimson, at 2.6 Å resolution. Chrimson resembles prokaryotic proton pumps in the retinal binding pocket, while sharing similarity with other channelrhodopsins in the ion-conducting pore. Concomitant mutation analysis identified the structural features that are responsible for Chrimson’s red light sensitivity; namely, the protonation of the counterion for the retinal Schiff base, and the polar residue distribution and rigidity of the retinal binding pocket. Based on these mechanistic insights, we engineered ChrimsonSA, a mutant with a maximum activation wavelength red-shifted beyond 605 nm and accelerated closing kinetics.

Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.

Genetic engineering of natural light-gated ion channels has proven a powerful way to generate optogenetic tools for a wide variety of applications. In recent years, blue-light activated engineered anion-conducting channelrhodopsins (eACRs) have been developed, improved, and were successfully applied in vivo. We asked whether the approaches used to create eACRs can be transferred to other well-characterized cation-conducting channelrhodopsins (CCRs) to obtain eACRs with a broad spectrum of biophysical properties. We generated 22 variants using two conversion strategies applied to 11 CCRs and screened them for membrane expression, photocurrents and anion selectivity. We obtained two novel eACRs, Phobos and Aurora, with blue- and red-shifted action spectra and photocurrents similar to existing eACRs. Furthermore, step-function mutations greatly enhanced the cellular operational light sensitivity due to a slowed-down photocycle. These bi-stable eACRs can be reversibly toggled between open and closed states with brief light pulses of different wavelengths. All new eACRs reliably inhibited action potential firing in pyramidal CA1 neurons. In Drosophila larvae, eACRs conveyed robust and specific light-dependent inhibition of locomotion and nociception.

Channelrhodopsins (ChRs) are algal light-gated ion channels widely used as optogenetic tools for manipulating neuronal activity. ChRs desensitize under continuous bright-light illumination, resulting in a significant decline of photocurrents. Here we describe a metagenomically identified family of phylogenetically distinct anion-conducting ChRs (designated MerMAIDs). MerMAIDs almost completely desensitize during continuous illumination due to accumulation of a late non-conducting photointermediate that disrupts the ion permeation pathway. MerMAID desensitization can be fully explained by a single photocycle in which a long-lived desensitized state follows the short-lived conducting state. A conserved cysteine is the critical factor in desensitization, as its mutation results in recovery of large stationary photocurrents. The rapid desensitization of MerMAIDs enables their use as optogenetic silencers for transient suppression of individual action potentials without affecting subsequent spiking during continuous illumination. Our results could facilitate the development of optogenetic tools from metagenomic databases and enhance general understanding of ChR function.

Systemic inflammation is associated with alterations in complex brain functions such as learning and memory. However, diagnostic approaches to functionally assess and quantify inflammation-associated alterations in synaptic plasticity are not well-established. In previous work, we demonstrated that bacterial lipopolysaccharide (LPS)-induced systemic inflammation alters the ability of hippocampal neurons to express synaptic plasticity, i.e., the long-term potentiation (LTP) of excitatory neurotransmission. Here, we tested whether synaptic plasticity induced by repetitive magnetic stimulation (rMS), a non-invasive brain stimulation technique used in clinical practice, is affected by LPS-induced inflammation. Specifically, we explored brain tissue cultures to learn more about the direct effects of LPS on neural tissue, and we tested for the plasticity-restoring effects of the anti-inflammatory cytokine interleukin 10 (IL10). As shown previously, 10 Hz repetitive magnetic stimulation (rMS) of organotypic entorhino-hippocampal tissue cultures induced a robust increase in excitatory neurotransmission onto CA1 pyramidal neurons. Furthermore, LPS-treated tissue cultures did not express rMS-induced synaptic plasticity. Live-cell microscopy in tissue cultures prepared from a novel transgenic reporter mouse line [C57BL/6-Tg(TNFa-eGFP)] confirms that ex vivo LPS administration triggers microglial tumor necrosis factor alpha (TNFα) expression, which is ameliorated in the presence of IL10. Consistent with this observation, IL10 hampers the LPS-induced increase in TNFα, IL6, IL1β, and IFNγ and restores the ability of neurons to express rMS-induced synaptic plasticity in the presence of LPS. These findings establish organotypic tissue cultures as a suitable model for studying inflammation-induced alterations in synaptic plasticity, thus providing a biological basis for the diagnostic use of transcranial magnetic stimulation in the context of brain inflammation.

Genetic engineering of natural light-gated ion channels has proven a powerful way to generate optogenetic tools for a wide variety of applications. In recent years, blue-light activated engineered anion-conducting channelrhodopsins (eACRs) have been developed, improved, and were successfully applied in vivo. We asked whether the approaches used to create eACRs can be transferred to other well-characterized cation-conducting channelrhodopsins (CCRs) to obtain eACRs with a broad spectrum of biophysical properties. We generated 22 variants using two conversion strategies applied to 11 CCRs and screened them for membrane expression, photocurrents and anion selectivity. We obtained two novel eACRs, Phobos and Aurora, with blue-and red-shifted action spectra and photocurrents similar to existing eACRs. Furthermore, step-function mutations greatly enhanced the cellular operational light sensitivity due to a slowed-down photocycle. These bi-stable eACRs can be reversibly toggled between open and closed states with brief light pulses of different wavelengths. All new eACRs reliably inhibited action potential firing in pyramidal CA1 neurons. In Drosophila larvae, eACRs conveyed robust and specific light-dependent inhibition of locomotion and nociception.The discovery of natural anion-conducting channelrhodopsins (nACRs) 1-3 and the development of engineered anion-conducting channelrhodopsins (eACRs) by targeted mutagenesis of cation-conducting channelrhodopsins (CCRs) 4-7 introduced a new class of optogenetic tools 8 . The existing eACRs were derived from either Chlamydomonas reinhardtii channelrhodopsin-2 (CrChR2) 7 or the channelrhodopsin chimera C1C2 5 using two complementary strategies. Exchange of a single glutamate for an arginine in the central gate of CrChR2 was sufficient to invert selectivity from cations to anions. Additional exchange of two glutamate residues in the outer pore and the inner gate completely eliminated residual proton conductance, yielding the highly anion-selective eACR iChloC 6 . In parallel, mutation of several amino acids within C1C2 to render the electrostatic potential of the conducting pore more positive, strongly favored anion conductance 5 . Further improvements led to a second highly anion-selective eACR iC++ and the related step function version SwiChR++ 4 . These improved versions have been successfully used to silence neurons in mice or rats in vivo 4,6,[9][10][11][12][13] . We asked whether the approaches used to create eACRs can be transferred to other known CCRs to obtain eACRs with a broad spectrum of biophysical properties, especially different kinetics and spectral sensitivities. So far, all eACRs show action spectra similar to CrChR2 with maximal activation in the blue spectral range. ACRs with a red-shifted absorption maximum are desirable for three reasons: First, long-wavelength light penetrates deeper into biological tissue due to lower absorption and scattering 14,15 . This enables silencing of larger volumes

We report BiPOLES, an optogenetic tool for balanced excitation and inhibition of neurons with light of two different colors. BiPOLES consists of the blue-light-sensitive anion-conducting channelrhodopsin GtACR2 fused to the red-light-sensitive cation-conducting channelrhodopsin Chrimson in a single, trafficking-optimized tandem protein. BiPOLES enables multiple applications including potent dual-color spiking and silencing of the same neurons in vivo and dual-color optogenetic control of two independent neuronal populations.

Optogenetics are used to stimulate or inhibit neurons. Because optogenetic stimulation is typically static, neurons and circuits can quickly adapt, allowing perturbation, but not true control. To overcome this, we established an optogenetic voltage-clamp (OVC). The genetically encoded voltage-indicator QuasAr2 provides information for fast, closed-loop optical feedback to the bidirectional optogenetic actuator BiPOLES. Voltage-dependent fluorescence is held within tight margins, thus clamping the cell at a distinct potential. We established the OVC in muscles and neurons of Caenorhabditis elegans, and transferred it to hippocampal neurons in rat brain slices. We calibrated fluorescence signals to electrically measured membrane potentials, showed that the OVC reports on homeostatically altered cellular physiology in mutants affecting neurotransmission, and that it can dynamically clamp spiking. The OVC combines non-invasive imaging with the control capabilities of electrophysiology. Its applicability to individual cells facilitates high-throughput contact-less electrophysiology and paves the way for true optogenetic control in behaving animals.