All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

All-optical electrophysiology—spatially resolved simultaneous optical perturbation and measurement of membrane voltage—would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk–free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell–derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes

Computational Optogenetics: Empirically-Derived Voltage- and Light-Sensitive Channelrhodopsin-2 Model

Channelrhodospin-2 (ChR2), a light-sensitive ion channel, and its variants have emerged as new excitatory optogenetic tools not only in neuroscience, but also in other areas, including cardiac electrophysiology. An accurate quantitative model of ChR2 is necessary for in silicoprediction of the response to optical stimulation in realistic tissue/organ settings. Such a model can guide the rational design of new ion channel functionality tailored to different cell types/tissues. Focusing on one of the most widely used ChR2 mutants (H134R) with enhanced current, we collected a comprehensive experimental data set of the response of this ion channel to different irradiances and voltages, and used these data to develop a model of ChR2 with empirically-derived voltage- and irradiance- dependence, where parameters were fine-tuned via simulated annealing optimization. This ChR2 model offers: 1) accurate inward rectification in the current-voltage response across irradiances; 2) empirically-derived voltage- and light-dependent kinetics (activation, deactivation and recovery from inactivation); and 3) accurate amplitude and morphology of the response across voltage and irradiance settings. Temperature-scaling factors (Q10) were derived and model kinetics was adjusted to physiological temperatures. Using optical action potential clamp, we experimentally validated model-predicted ChR2 behavior in guinea pig ventricular myocytes. The model was then incorporated in a variety of cardiac myocytes, including human ventricular, atrial and Purkinje cell models. We demonstrate the ability of ChR2 to trigger action potentials in human cardiomyocytes at relatively low light levels, as well as the differential response of these cells to light, with the Purkinje cells being most easily excitable and ventricular cells requiring the highest irradiance at all pulse durations. This new experimentally-validated ChR2 model will facilitate virtual experimentation in neural and cardiac optogenetics at the cell and organ level and provide guidance for the development of in vivo tools

The spatial pattern of light determines the kinetics and modulates backpropagation of optogenetic action potentials

Optogenetics offers an unprecedented ability to spatially target neuronal stimulations. This study investigated via simulation, for the first time, how the spatial pattern of excitation affects the response of channelrhodopsin-2 (ChR2) expressing neurons. First we described a methodology for modeling ChR2 in the NEURON simulation platform. Then, we compared four most commonly considered illumination strategies (somatic, dendritic, axonal and whole cell) in a paradigmatic model of a cortical layer V pyramidal cell. We show that the spatial pattern of illumination has an important impact on the efficiency of stimulation and the kinetics of the spiking output. Whole cell illumination synchronizes the depolarization of the dendritic tree and the soma and evokes spiking characteristics with a distinct pattern including an increased bursting rate and enhanced back propagation of action potentials (bAPs). This type of illumination is the most efficient as a given irradiance threshold was achievable with only 6 % of ChR2 density needed in the case of somatic illumination. Targeting only the axon initial segment requires a high ChR2 density to achieve a given threshold irradiance and a prolonged illumination does not yield sustained spiking. We also show that patterned illumination can be used to modulate the bAPs and hence spatially modulate the direction and amplitude of spike time dependent plasticity protocols. We further found the irradiance threshold to increase in proportion to the demyelination level of an axon, suggesting that measurements of the irradiance threshold (for example relative to the soma) could be used to remotely probe a loss of neural myelin sheath, which is a hallmark of several neurodegenerative diseases

Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine

新規光駆動型イオンチャネルの構造解明と高性能分子ツールの創出 --神経科学に光を当てる--. 京都大学プレスリリース. 2022-02-03.ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology

Recent Advances in Optogenetic Retinal Prostheses

Optogenetics has emerged as a revolutionary technology that enables circuit-specific restoration of neuronal function with millisecond temporal resolution. Restoring vision is one of the most promising and forefront applications of optogenetics. This chapter discusses essential components, mechanisms, present challenges, and future prospects of optogenetic retinal prostheses. The theoretical framework and analysis of optogenetic excitation of retinal ganglion neurons are also presented, which are useful in developing a better understanding and guidance for future experiments. It shows that the newly discovered ChRmine opsin provides control at light powers that are two orders of magnitude smaller than that required with experimentally studied opsins that include ChR2, ReaChR, and ChrimsonR, while maintaining single-spike temporal resolution, in retinal ganglion neurons

Evolution of optogenetic microdevices

Implementation of optogenetic techniques is a recent addition to the neuroscientists\u27 preclinical research arsenal, helping to expose the intricate connectivity of the brain and allowing for on-demand direct modulation of specific neural pathways. Developing an optogenetic system requires thorough investigation of the optogenetic technique and of previously fabricated devices, which this review accommodates. Many experiments utilize bench-top systems that are bulky, expensive, and necessitate tethering to the animal. However, these bench-top systems can make use of power-demanding technologies, such as concurrent electrical recording. Newer portable microdevices and implantable systems carried by freely moving animals are being fabricated that take advantage of wireless energy harvesting to power a system and allow for natural movements that are vital for behavioral testing and analysis. An investigation of the evolution of tethered, portable, and implantable optogenetic microdevices is presented, and an analysis of benefits and detriments of each system, including optical power output, device dimensions, electrode width, and weight is given. Opsins, light sources, and optical fiber coupling are also discussed to optimize device parameters and maximize efficiency from the light source to the fiber, respectively. These attributes are important considerations when designing and developing improved optogenetic microdevices