Light-activated Ion Channels Research Articles

Optogenetic Stimulation of Nociceptive Escape Behaviors in Drosophila Larvae.

In animals, noxious stimuli activate a neural process called nociception. Drosophila larvae perform a rolling escape locomotion behavior in response to nociceptive sensory stimuli. Noxious mechanical, thermal, and chemical stimuli each trigger this same escape response in larvae. The polymodal sensory neurons that initiate the rolling response have been identified based on the expression patterns of genes that are known to be required for nociception responses. The synaptic output of these neurons, known as class IV multidendritic sensory neurons, is required for behavioral responses to thermal, mechanical, and chemical triggers of the rolling escape locomotion. Importantly, optogenetic stimulation of the class IV multidendritic neurons has also shown that the activation of those cells is sufficient to trigger nociceptive rolling. Optogenetics uses light-activated ion channels expressed in neurons of interest to bypass the normal physiological transduction machinery so that the cell may be activated in response to light that is applied by the investigator. This protocol describes an optogenetic technique that uses channelrhodopsin-2 (ChR2) to activate larval nociceptors and trigger nociceptive rolling. First, we explain how to set up the necessary genetic crosses and culture the larval progeny. Next, we describe how to perform the optogenetic nociception assay on third-instar larvae.

Optoelectronic control of cardiac rhythm: Toward shock-free ambulatory cardioversion of atrial fibrillation.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia, progressive in nature, and known to have a negative impact on mortality, morbidity, and quality of life. Patients requiring acute termination of AF to restore sinus rhythm are subjected to electrical cardioversion, which requires sedation and therefore hospitalization due to pain resulting from the electrical shocks. However, considering the progressive nature of AF and its detrimental effects, there is a clear need for acute out-of-hospital (i.e., ambulatory) cardioversion of AF. In the search for shock-free cardioversion methods to realize such ambulatory therapy, a method referred to as optogenetics has been put forward. Optogenetics enables optical control over the electrical activity of cardiomyocytes by targeted expression of light-activated ion channels or pumps and may therefore serve as a means for cardioversion. First proof-of-principle for such light-induced cardioversion came from in vitro studies, proving optogenetic AF termination to be very effective. Later, these results were confirmed in various rodent models of AF using different transgenes, illumination methods, and protocols, whereas computational studies in the human heart provided additional translational insight. Based on these results and fueled by recent advances in molecular biology, gene therapy, and optoelectronic engineering, a basis is now being formed to explore clinical translations of optoelectronic control of cardiac rhythm. In this review, we discuss the current literature regarding optogenetic cardioversion of AF to restore normal rhythm in a shock-free manner. Moreover, key translational steps will be discussed, both from a biological and technological point of view, to outline a path toward realizing acute shock-free ambulatory termination of AF.

Update on gene therapies in pediatric ophthalmology

Rare eye diseases encompass a broad spectrum of genetic anomalies with or without additional extraocular manifestations. Genetic eye disorders in pediatric patients often lead to severe visual impairments. Therefore, a challenge of gene therapy is to provide better vision to these affected children. In recent years, inherited retinal diseases, inherited optic neuropathies, and corneal dystrophies have dominated discussions to establish gene and cell replacement therapies for these diseases. Gene therapy involves the transfer of genetic material to remove, replace, repair, or introduce a gene, or to overexpress a protein, whose activity would have a therapeutic impact. For the majority of anterior segment diseases, these studies are still emerging at a preclinical stage; however, for inherited retinal disorders, translation has been reached, leading to the introduction of the first gene therapies into clinical practice. In the past decade, the first gene therapy for biallelic RPE65-mediated inherited retinal dystrophy has been developed and the FDA and EMA both approved ocular gene therapy. Other promising approaches by intravitreal injection have been investigated such as in CEP290-Leber congenital amaurosis. Various techniques of gene therapies include gene supplementation, CRISPR-based genome editing, as well as RNA modulation and optogenetics. Optogenetic therapies deliver light-activated ion channels to surviving retinal cell types in order to restore photosensitivity. Beyond retinal function, ataluren, a nonsense mutation suppression therapy, enables ribosomal read-through of mRNA containing premature termination codons, resulting in the production of a full-length protein. An ophthalmic formulation was recently evaluated with the aim of repairing corneal damage, pending new clinical studies. However, various congenital disorders exhibit severe developmental defects or cell loss at birth, limiting the potential for viral gene therapy. Therefore mutation-independent strategies seem promising for maintaining the survival of photoreceptors or for restoring visual function. Restoring vision in children with gene therapy continues to be a challenge in ophthalmology. © 2023 Published by Elsevier Masson SAS on behalf of French Society of Pediatrics.

Structure-function analysis suggests that the photoreceptor LITE-1 is a light-activated ion channel

Sensation of light is essential for all organisms. The eye-less nematode Caenorhabditis elegans detects UV and blue light to evoke escape behavior. The photosensor LITE-1 absorbs UV photons with an unusually high extinction coefficient, involving essential tryptophans. Here, we modeled the structure and dynamics of LITE-1 using AlphaFold2-multimer and molecular dynamics (MD) simulations and performed mutational and behavioral assays in C.elegans to characterize its function. LITE-1 resembles olfactory and gustatory receptors from insects, recently shown to be tetrameric ion channels. We identified residues required for channel gating, light absorption, and mechanisms of photo-oxidation, involving a likely binding site for the peroxiredoxin PRDX-2. Furthermore, we identified the binding pocket for a putative chromophore. Several residues lining this pocket have previously been established as essential for LITE-1 function. A newly identified critical cysteine pointing into the pocket represents a likely chromophore attachment site. We derived a model for how photon absorption, via a network of tryptophans and other aromatic amino acids, induces an excited state that is transferred to the chromophore. This evokes conformational changes in the protein, possibly leading to a state receptive to oxidation of cysteines and, jointly, to channel gating. Electrophysiological data support the idea that LITE-1 is a photon and H2O2-coincidence detector. Other proteins with similarity to LITE-1, specifically C.elegans GUR-3, likely use a similar mechanism for photon detection. Thus, a common protein fold and assembly, used for chemoreception in insects, possibly by binding of a particular compound, may have evolved into a light-activated ion channel.

1772-P: Alpha-Cell Heterogeneity Contributes to Phasic Glucagon Release

Glucagon, produced by α-cells of pancreatic islets, is one of the body’s primary tools for elevating blood glucose during hypoglycemia. The exact mechanism linking hypoglycemia to glucagon release by islet α-cells is, however, unclear; we therefore aimed to investigate it using optogenetics. We generated mice harboring the light-activated ion channel channelrhodopsin-2 (ChR2) specifically in α-cells and imaged the effect of optically induced depolarization of plasma membrane (10-ms 470-nm light pulses at 20Hz) at the level of membrane potential, cytosolic Ca2+ ([Ca2+]i) and ATP/ADP ratio. At 1 mmol/L glucose, optostimulation depolarized the electrically active α-cells from -76mV to -8mV, induced a transient surge in [Ca2+]i and elevated glucagon secretion by 200%. In contrast, the glucagonostatic effect of high glucose (>6 mmol/L) was mitigated by optostimulation of α-cells. At 1 mmol/L glucose, the stimulatory effects were abolished by the KATP channel activator diazoxide (100μmol/L) and L-type Ca2+ channel blocker isradipine (10μmol/L). Notably, KATP channel inhibitor tolbutamide (100μmol/L) induced electrical activity in the fraction of α-cells insensitive to low glucose (35%). As stimulation of electrical activity persisted significantly beyond optoactivation, we probed the interplay between electrical activity and energy metabolism. The depolarizing stimulus induced by a cocktail of amino acids ((AA) 2 mmol/L of alanine + arginine +glutamine) or a moderate increase of extracellular K+ (from 4 to 15 mmol/L) reduced ATP/ADP in α-cells exhibiting spontaneous Ca2+ oscillations at low glucose. Surprisingly, in the population of inactive α-cells, the stimulation increased ATP/ADP ratio. These data indicate that α-cells are functionally heterogeneous due to metabolic differences. The observed secretory responses reflect the combination of stimulatory and inhibitory effects in electrically active and silent cells, which may contribute to the phasic nature of AA-induced glucagon secretion. Disclosure C.A.Miranda: None. H.Dou: None. J.Tolö: None. A.I.Tarasov: None. P.Rorsman: None. Funding Svenska Sällskapet för Medicinsk Forskning (PD20-0056); Swedish Vetenskap Rådet

Optogenetic suppression of drug-induced early afterdepolarisations in the zebrafish heart

Early after-depolarisations (EADs) are electrical disturbances of cardiomyocytes that interrupt the repolarisation phase of the cardiac action potential (AP) and can lead to premature excitation and sustained arrhythmias. Optogenetics has the potential to prevent EADs by using genetically-expressed light-activated ion channels to aid in AP repolarisation. Our goal was to investigate the use of optogenetics to modulate membrane potential (Vm) of ventricular cardiomyocytes for the suppression of EADs in the whole zebrafish heart.

Red Light Optogenetics in Neuroscience.

Optogenetics, a field concentrating on controlling cellular functions by means of light-activated proteins, has shown tremendous potential in neuroscience. It possesses superior spatiotemporal resolution compared to the surgical, electrical, and pharmacological methods traditionally used in studying brain function. A multitude of optogenetic tools for neuroscience have been created that, for example, enable the control of action potential generation via light-activated ion channels. Other optogenetic proteins have been used in the brain, for example, to control long-term potentiation or to ablate specific subtypes of neurons. In in vivo applications, however, the majority of optogenetic tools are operated with blue, green, or yellow light, which all have limited penetration in biological tissues compared to red light and especially infrared light. This difference is significant, especially considering the size of the rodent brain, a major research model in neuroscience. Our review will focus on the utilization of red light-operated optogenetic tools in neuroscience. We first outline the advantages of red light for in vivo studies. Then we provide a brief overview of the red light-activated optogenetic proteins and systems with a focus on new developments in the field. Finally, we will highlight different tools and applications, which further facilitate the use of red light optogenetics in neuroscience.

Optogenetic diaphragm muscle activation in mice

Breathing impairments are the leading cause of morbidity and mortality across all forms of neuromuscular disease. When voluntarily diaphragm activation is insufficient, clinical options include mechanical ventilation or electrical stimulation via intramuscular wires. Here we tested the hypothesis that an optogenetic approach would enable light to serve as an adequate stimulus for inducing diaphragm contraction to sustain breathing during neuromuscular impairments. Wild type, C57/bl6 mice (n=10) received an intrapleural injection of an adeno-associated virus (AAV) construct containing the transgene for the light activated ion channel channelrhodopsin-2 (34 µl, AAV9-ACAGW-ChR2-mVenus, titer: 1.8x1013 vg/ml). After allowing 8-10 weeks for transgene expression, electromyography (EMG) and pneumotachography were measured during spontaneous breathing under isoflurane anesthesia. To test optogenetic activation, a high-power, 472 nm, micro-LED was directed at the inferior diaphragm surface. Stimulus pulse durations (PD) ranging from 0.1-1.0 ms and interpulse intervals (IPI) of 0.1, 0.5, 1.0, and 5.0 ms were tested. Directing light at the diaphragm evoked clearly discernable EMG activity in 10 of 10 animals treated with the AAV9 vector. The 5 ms IPI induced the largest EMG activation, occurring at a level comparable to endogenous diaphragm bursts. Stimulation with a 0.1 ms PD / 0.1 ms IPI, and 1 ms PD / 5 ms IPI both were able to generate robust increases in respiratory airflow. Optogenetic stimulation was repeated following an acute unilateral phrenic nerve section which abolished ipsilateral diaphragm EMG. The contralateral diaphragm inspiratory EMG signal was then used to “trigger” the light stimulation of the paralyzed hemi-diaphragm. This procedure enabled a robust, bilateral diaphragm activation in all mice that received the unilateral phrenicotomy (n= 5). Histology confirmed robust expression of mVenus in diaphragm myofibers in mice treated with the vector. Directing light at the diaphragm of non-AAV treated control mice caused no discernible impact on diaphragm EMG activity (n=2). We conclude that intrapleural AAV9-ACAGW-ChR2-mVenus injection effectively drives transgene expression in the diaphragm, and this approach can be used to enable robust optogenetic activation of this critically important respiratory muscle. To our knowledge, this is the first demonstration of light activation of the diaphragm, and this success opens up new directions for therapeutic targeting of diaphragm paralysis and respiratory insufficiency.

The study on optogenetic tachyarrhythmia termination under pathological conditions from the single cell to the whole heart

Abstract Background Ventricular tachyarrhytmias (VTs) are common among patients suffering from cardiac remodeling and cause significant morbidity and mortality. Current research and treatment options for such VTs are suboptimal, hence new strategies are urgently needed. Optogenetics offers efficacious means to control cardiac rhythm, including shock-free VT termination. However, this has not been demonstrated in diseased hearts in vivo, while clinical translation would not only require such demonstration, but also an in-depth understanding of cellular responses. Purpose To assess the optogenetic response at the cardiac cell, tissue, and whole heart level in terms of rhtyhm control under pathological conditions by an integrative experimental platform including in vitro and in vivo models of cardiac disease. Methods Remodeling was induced in neonatal rat ventricular cardiomyocytes (NRVMs) by phenylephrine (PE) exposure. Pathological conditions leading to ventricular remodeling were mimicked by transverse aortic constriction (TAC) surgery in adult rats. The light-activated ion channel ReaChR was ectopically expressed in NRVMs and in hearts of TAC and sham animals by viral vector-based gene delivery. Results Electrical and structural remodeling was evidenced by elongated action potential durations (p<0.05) and increased cell capacitance (p<0.05) in PE-treated, but not in control cells (CTL). Light-induced ionic currents in ReaChR-expressing PE-treated and CTL NRVMs displayed comparable kinetic properties and current densities (p>0.05). Illumination (1 s) caused a sudden shift in membrane potential leading to a plateau at −7.3 mV for PE-treated and −18.9 mV for CTL cells (p>0.05). Hearts explanted from TAC animals showed increased average heart weight to body weight ratio, ventricular fibrosis and expression of hypertrophy markers (ANP, aSkMA, p<0.05), while tissue preparations showed significant APD increase compared to sham. In vivo gene delivery resulted in expression of the ReaChR-citrine transgene in ∼80% of isolated ventricular myocytes (VMs). Photocurrent densities were not different (p>0.05) in VMs from TAC and sham animals, which currents led to comparable shifts in membrane potential (65.3 mV for TAC and 63.9 mV for CTL). In line with this, illumination caused marked depolarization in tissue preparations (from −77.6 to −16.4 mV) in TAC animals as assessed by conventional sharp electrode measurements. Importantly, as anticipated, electrically-induced VT episodes could be terminated in open chest experiments in TAC animals (n=6; 76.3% of cases) by epicardial illumination in vivo. Conclusions Key operational parameters of the optogenetic response remained unaffected in models of cardiac disease, which allowed efficacious optogenetic VT termination in the diseased rat heart exhibiting structural and electrical remodeling. These findings corroborate the translational potential of shock-free therapy of cardiac arrhythmia by optogenetics. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): This work was supported by personal funding from the Netherlands Organization for Scientific Research (NWO, Vidi grant 1714336 to D.A.P.). D.A.P. is also a recipient of the European Research Council (ERC), Starting grant (716509). Additional support was provided by the Netherlands Heart Institute (ICIN grant 230.148-04 to A.A.F.d.V.).

Optogenetics and Optical Tools in Automated Patch Clamping.

Automated patch clamping (APC) has been used for almost two decades to increase the throughput of electrophysiological measurements, especially in preclinical safety screening of drug compounds. Typically, cells are suctioned onto holes in planar surfaces and a stronger subsequent suction allows access to a whole cell configuration for electrical measurement of ion channel activity. The development of optogenetic tools over a wide range of wavelengths (UV to IR) provides powerful tools for improving spatiotemporal control of in vivo and in vitro experiments and is emerging as a powerful means of investigating cell networks (neuronal), single cell transduction, and subcellular pathways.Combining APC and optogenetic tools paves the way for improved investigation and control of cell kinetics and provides the opportunity for collecting robust data for new and exciting applications and therapeutic areas. Here, we present an APC optogenetics capability on the Qube Opto 384 system including experiments on light activated ion channels and photoactivated ligands.

Optogenetics to Interrogate Neuron-Glia Interactions in Pups and Adults.

In just over 10years, the use of optogenetic technologies in neuroscience has become widespread, having today a tremendous impact on our understanding of brain function. An extensive number of studies have implemented a variety of tools allowing for the manipulation of neurons with light, including light-activated ion channels or G protein-coupled receptors, among other innovations. In this context, the proper calibration of photostimulation in vivo remains crucial to dissect brain circuitry or investigate the effect of neuronal activity on specific subpopulations of neurons and glia. Depending on the scientific question, the design of specific stimulation protocols must consider from the choice of the animal model to the light stimulation pattern to be delivered. In this chapter, we describe a detailed framework to investigate neuron-glia interactions in both mouse pups and adults using an optogenetic approach.

Cardiac pacing using transmural multi-LED probes in channelrhodopsin-expressing mouse hearts

Optogenetics enables cell-type specific monitoring and actuation via light-activated proteins. In cardiac research, expressing light-activated depolarising ion channels in cardiomyocytes allows optical pacing and defibrillation. Previous studies largely relied on epicardial illumination. Light penetration through the myocardium is however problematic when moving to larger animals and humans. To overcome this limitation, we assessed the utility of an implantable multi light-emitting diode (LED) optical probe (IMLOP) for intramural pacing of mouse hearts expressing cardiac-specific channelrhodopsin-2 (ChR2).Here we demonstrated that IMLOP insertion needs approximately 20 mN of force, limiting possible damage from excessive loads applied during implantation. Histological sections confirmed the confined nature of tissue damage during acute use. The temperature change of the surrounding tissue was below 1 K during LED operation, rendering the probe safe for use in situ. This was confirmed in control experiments where no effect on cardiac action potential conduction was observed even when using stimulation parameters twenty-fold greater than required for pacing.In situ experiments on ChR2-expressing mouse hearts demonstrated that optical stimulation is possible with light intensities as low as 700 μW/mm2; although stable pacing requires higher intensities. When pacing with a single LED, rheobase and chronaxie values were 13.3 mW/mm2 ± 0.9 mW/mm2 and 3 ms ± 0.6 ms, respectively. When doubling the stimulated volume the rheobase decreased significantly (6.5 mW/mm2 ± 0.9 mW/mm2).We have demonstrated IMLOP-based intramural optical pacing of the heart. Probes cause locally constrained tissue damage in the acute setting and require low light intensities for pacing. Further development is necessary to assess effects of chronic implantation.

Light Stimulation Parameters Determine Neuron Dynamic Characteristics

Optogenetics is a recently developed technique that is widely used to study neuronal function. In optogenetic experiments, neurons encode opsins (channelrhodopsins, halorhodopsins or their derivatives) by means of viruses, plasmids or genetic modification (transgenic lines). Channelrhodopsin are light activated ion channels. Their expression in neurons allows light-dependent control of neuronal activity. The duration and frequency of light stimulation in optogenetic experiments is critical for stable, robust and reproducible experiments. In this study, we performed systematic analyses of these parameters using primary cultures of hippocampal neurons transfected with channelrhodopsin-2 (ChR2). The main goal of this work was to identify the optimal parameters of light stimulation that would result in stable neuronal activity during a repeated light pulse train. We demonstrated that the dependency of the photocurrent on the light pulse duration is described by a right-skewed bell-shaped curve, while the dependence on the stimulus intensity is close to linear. We established that a duration between 10–30 ms of stimulation was the minimal time necessary to achieve a full response. Obtained results will be useful in planning and interpretation of optogenetic experiments.

Immunocytochemical Labeling of Rhabdomeric Proteins in Drosophila Photoreceptor Cells Is Compromised by a Light-dependent Technical Artifact.

Drosophila photoreceptor cells are employed as a model system for studying membrane protein transport. Phototransduction proteins like rhodopsin and the light-activated TRPL ion channel are transported within the photoreceptor cell, and they change their subcellular distribution in a light-dependent way. Investigating the transport mechanisms for rhodopsin and ion channels requires accurate histochemical methods for protein localization. By using immunocytochemistry the light-triggered translocation of TRPL has been described as a two-stage process. In stage 1, TRPL accumulates at the rhabdomere base and the adjacent stalk membrane a few minutes after onset of illumination and is internalized in stage 2 by endocytosis after prolonged light exposure. Here, we show that a commonly observed crescent shaped antibody labeling pattern suggesting a fast translocation of rhodopsin, TRP, and TRPL to the rhabdomere base is a light-dependent antibody staining artifact. This artifact is most probably caused by the profound structural changes in the microvillar membranes of rhabdomeres that result from activation of the signaling cascade. By using alternative labeling methods, either eGFP-tags or the self-labeling SNAP-tag, we show that light activation of TRPL transport indeed results in fast changes of the TRPL distribution in the rhabdomere but not in the way described previously.

Memories light the corners of my mind.

Using light-activated ion channels to stimulate sensory and motivational pathways, Vetere and colleagues constructed fully artificial memories in mice. Mice preferred or avoided an odor they had never smelled before, depending on the pattern of stimulation.

Unifying photocycle model for light adaptation and temporal evolution of cation conductance in channelrhodopsin-2

Although channelrhodopsin (ChR) is a widely applied light-activated ion channel, important properties such as light adaptation, photocurrent inactivation, and alteration of the ion selectivity during continuous illumination are not well understood from a molecular perspective. Herein, we address these open questions using single-turnover electrophysiology, time-resolved step-scan FTIR, and Raman spectroscopy of fully dark-adapted ChR2. This yields a unifying parallel photocycle model integrating now all so far controversial discussed data. In dark-adapted ChR2, the protonated retinal Schiff base chromophore (RSBH+) adopts an all-trans,C=N-anti conformation only. Upon light activation, a branching reaction into either a 13-cis,C=N-anti or a 13-cis,C=N-syn retinal conformation occurs. The anti-cycle features sequential H+ and Na+ conductance in a late M-like state and an N-like open-channel state. In contrast, the 13-cis,C=N-syn isomer represents a second closed-channel state identical to the long-lived P480 state, which has been previously assigned to a late intermediate in a single-photocycle model. Light excitation of P480 induces a parallel syn-photocycle with an open-channel state of small conductance and high proton selectivity. E90 becomes deprotonated in P480 and stays deprotonated in the C=N-syn cycle. Deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light adaptation. Parallel anti- and syn-photocycles now explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools.

An optimized and automated approach to quantifying channelrhodopsin photocurrent kinetics

Channelrhodopsins are light-activated ion channels that enable targetable activation or inhibition of excitable cells with light. Ion conductance can generally be described by a four step photocycle, which includes two open and two closed states. While a complete understanding of channelrhodopsin function cannot be understood in the absence of kinetic modeling, model fitting requires manual fitting, which is laborious and technically complicated for non-experts. To enhance analysis of photocurrent data, this manuscript describes a fitting program where electrophysiology data can be automatically and quantitatively analyzed. Significant improvement in this program when compared to our previous version includes 1) the ability to automatically find the experiment start time using the derivative of the current signal, 2) utilizing the Object Oriented Programing (OPP) paradigm which is significantly more reliable if the code is used by people with little to no programming experience and 3) the distribution of the code is simplified to sharing a single MATLAB file, including rigorous comments throughout. To demonstrate the utility of this program, we show automated fitting of photocurrents from two member proteins: channelrhodopsin-2 and a chimera between channelrhodopsin-1 and channelrhodopsin-2 (C1C2).

Synthetic Light-Activated Ion Channels for Optogenetic Activation and Inhibition.

Optogenetic manipulation of cells or living organisms became widely used in neuroscience following the introduction of the light-gated ion channel channelrhodopsin-2 (ChR2). ChR2 is a non-selective cation channel, ideally suited to depolarize and evoke action potentials in neurons. However, its calcium (Ca2+) permeability and single channel conductance are low and for some applications longer-lasting increases in intracellular Ca2+ might be desirable. Moreover, there is need for an efficient light-gated potassium (K+) channel that can rapidly inhibit spiking in targeted neurons. Considering the importance of Ca2+ and K+ in cell physiology, light-activated Ca2+-permeant and K+-specific channels would be welcome additions to the optogenetic toolbox. Here we describe the engineering of novel light-gated Ca2+-permeant and K+-specific channels by fusing a bacterial photoactivated adenylyl cyclase to cyclic nucleotide-gated channels with high permeability for Ca2+ or for K+, respectively. Optimized fusion constructs showed strong light-gated conductance in Xenopus laevis oocytes and in rat hippocampal neurons. These constructs could also be used to control the motility of Drosophila melanogaster larvae, when expressed in motoneurons. Illumination led to body contraction when motoneurons expressed the light-sensitive Ca2+-permeant channel, and to body extension when expressing the light-sensitive K+ channel, both effectively and reversibly paralyzing the larvae. Further optimization of these constructs will be required for application in adult flies since both constructs led to eclosion failure when expressed in motoneurons.

Crystal structure of the red light-activated channelrhodopsin Chrimson

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.

Effects of drugs of abuse on channelrhodopsin-2 function

Channelrhodopsins are light activated ion channels used extensively over the past decade to probe the function of genetically defined neuronal populations and distinct neural circuits with high temporal and spatial precision. The widely used Channelrhodopsin-2 variant (ChR2) is an excitatory opsin that undergoes conformational changes in response to blue light, allowing non-selective passage of protons and cations across the plasma membrane thus leading to depolarization. In the addiction neuroscience field, opsins such as ChR2 provide a means to disambiguate the overlapping circuitry involved in mediating the reinforcing and aversive effects of drugs of abuse as well as to determine the plasticity that can occur in these circuits during the development of dependence. Although ChR2 has been widely used in animal models of drug and alcohol self-administration, direct effects of drugs of abuse on ChR2 function may confound its use and lead to misinterpretation of data. As a variety of neuronal ion channels are primary targets of various drugs of abuse, it is critical to determine whether ChR2-mediated currents are modulated by these drugs. In this study, we performed whole-cell electrophysiological recordings in HEK293 cells expressing the commonly used ChR2(H134R) variant and examined the effects of various drugs of abuse and other commonly used agents on light-induced currents. We found no differences in ChR2-mediated currents in the presence of 30 μM nicotine, 30 μM cocaine, 100 μM methamphetamine or 3 mM toluene. Similarly, ChR2 currents were insensitive to 30 mM ethanol but higher concentrations (100–300 mM) produced significant effects on the desensitization and amplitude of light-evoked currents. Tetrahydrocannabinol (1–10 μM) and morphine (30–100 μM) significantly inhibited ChR2 currents while the cannabinoid receptor antagonist AM-251 had no effect. The sodium channel blocker tetrodotoxin (5 μM) and the generic channel blocker/contrast agent gadolinium chloride (10 mM) also reduced ChR2 currents while the divalent ion magnesium (10 mM) had no effect. Together, the results from this study highlight the importance of conducting appropriate control experiments when testing new compounds in combination with optogenetic approaches.