Bimodal activation of different neuron classes with the spectrally red-shifted channelrhodopsin chimera C1V1 in Caenorhabditis elegans.
ABSTRACT: The C. elegans nervous system is particularly well suited for optogenetic analyses of circuit function: Essentially all connections have been mapped, and light can be directed at the neuron of interest in the freely moving, transparent animals, while behavior is observed. Thus, different nodes of a neuronal network can be probed for their role in controlling a particular behavior, using different optogenetic tools for photo-activation or -inhibition, which respond to different colors of light. As neurons may act in concert or in opposing ways to affect a behavior, one would further like to excite these neurons concomitantly, yet independent of each other. In addition to the blue-light activated Channelrhodopsin-2 (ChR2), spectrally red-shifted ChR variants have been explored recently. Here, we establish the green-light activated ChR chimera C1V1 (from Chlamydomonas and Volvox ChR1's) for use in C. elegans. We surveyed a number of red-shifted ChRs, and found that C1V1-ET/ET (E122T; E162T) works most reliable in C. elegans, with 540-580 nm excitation, which leaves ChR2 silent. However, as C1V1-ET/ET is very light sensitive, it still becomes activated when ChR2 is stimulated, even at 400 nm. Thus, we generated a highly efficient blue ChR2, the H134R; T159C double mutant (ChR2-HR/TC). Both proteins can be used in the same animal, in different neurons, to independently control each cell type with light, enabling a further level of complexity in circuit analyses.
Project description:In optogenetics, rhodopsins were established as light-driven tools to manipulate neuronal activity. However, during long-term photostimulation using channelrhodopsin (ChR), desensitization can reduce effects. Furthermore, requirement for continuous presence of the chromophore all-trans retinal (ATR) in model systems lacking sufficient endogenous concentrations limits its applicability. We tested known, and engineered and characterized new variants of de- and hyperpolarizing rhodopsins in Caenorhabditis elegans. ChR2 variants combined previously described point mutations that may synergize to enable prolonged stimulation. Following brief light pulses ChR2(C128S;H134R) induced muscle activation for minutes or even for hours ('Quint': ChR2(C128S;L132C;H134R;D156A;T159C)), thus featuring longer open state lifetime than previously described variants. Furthermore, stability after ATR removal was increased compared to the step-function opsin ChR2(C128S). The double mutants C128S;H134R and H134R;D156C enabled increased effects during repetitive stimulation. We also tested new hyperpolarizers (ACR1, ACR2, ACR1(C102A), ZipACR). Particularly ACR1 and ACR2 showed strong effects in behavioral assays and very large currents with fast kinetics. In sum, we introduce highly light-sensitive optogenetic tools, bypassing previous shortcomings, and thus constituting new tools that feature high effectiveness and fast kinetics, allowing better repetitive stimulation or investigating prolonged neuronal activity states in C. elegans and, possibly, other systems.
Project description:To develop an animal behavioral assay for the quantitative assessment of the functional efficacy of optogenetic therapies.A triple-knockout (TKO) mouse line, Gnat1-/-Cnga3-/-Opn4-/-, and a double-knockout mouse line, Gnat1-/-Cnga3-/-, were employed. The expression of channelrhodopsin-2 (ChR2) and its three more light-sensitive mutants, ChR2-L132C, ChR2-L132C/T159C, and ChR2-132C/T159S, in inner retinal neurons was achieved using rAAV2 vectors via intravitreal delivery. Pupillary constriction was assessed by measuring the pupil diameter. The optomotor response (OMR) was examined using a homemade optomotor system equipped with light-emitting diodes as light stimulation.A robust OMR was restored in the ChR2-mutant-expressing TKO mice; however, significant pupillary constriction was observed only for the ChR2-L132C/T159S mutant. The ability to evoke an OMR was dependent on both the light intensity and grating frequency. The most light-sensitive frequency for the three ChR2 mutants was approximately 0.042 cycles per degree. Among the three ChR2 mutants, ChR2-L132C/T159S was the most light sensitive, followed by ChR2-L132C/T159C and ChR2-L132C. Melanopsin-mediated pupillary constriction resulted in a substantial reduction in the light sensitivity of the ChR2-mediated OMR.The OMR assay using TKO mice enabled the quantitative assessment of the efficacy of different optogenetic tools and the properties of optogenetically restored vision. Thus, the assay can serve as a valuable tool for developing effective optogenetic therapies.
Project description:Channelrhodopsin-2 (ChR2) has become an indispensable tool in neuroscience, allowing precise induction of action potentials with short light pulses. A limiting factor for many optophysiological experiments is the relatively small photocurrent induced by ChR2. We screened a large number of ChR2 point mutants and discovered a dramatic increase in photocurrent amplitude after threonine-to-cysteine substitution at position 159. When we tested the T159C mutant in hippocampal pyramidal neurons, action potentials could be induced at very low light intensities, where currently available channelrhodopsins were unable to drive spiking. Biophysical characterization revealed that the kinetics of most ChR2 variants slows down considerably at depolarized membrane potentials. We show that the recently published E123T substitution abolishes this voltage sensitivity and speeds up channel kinetics. When we combined T159C with E123T, the resulting double mutant delivered fast photocurrents with large amplitudes and increased the precision of single action potential induction over a broad range of frequencies, suggesting it may become the standard for light-controlled activation of neurons.
Project description:Optogenetics offers unprecedented possibilities to investigate cortical networks. Yet, the number of successful optogenetic applications in non-human primates is still low, and the consequences of opsin expression in the primate brain are not well documented. We assessed histologically if we can target cerebrocortical networks with three common optogenetic constructs (AAV2/5-CaMKII?-eNpHR3.0-mCherry, -ChR2-eYFP, -C1V1-mCherry). The frontal eye field or the dorsal premotor area of rhesus macaques were virally injected, and the resulting transduction spread, expression specificity, and opsin trafficking into axons projecting to parietal and visual areas were examined. After variable periods (2-24 months), expression was robust for all constructs at the injection sites. The CaMKII? promoter driven-expression was predominant, but not exclusive, in excitatory neurons. In the case of eNpHR3.0-mCherry and ChR2-eYFP, opsins were present in axonal projections to target areas, in which sparse, retrogradely transduced neurons could also be found. Finally, the intracellular distribution of opsins differed: ChR2-eYFP had almost exclusive membrane localization, while eNpHR3.0-mCherry and C1V1-mCherry showed additional intracellular accumulations, which might affect neuronal survival in the long-term. Results indicate that all three constructs can be used for local neuronal modulation, but axonal stimulation and long-term use require additional considerations of construct selection and verification.
Project description:Optogenetic approaches using light-activated proteins like Channelrhodopsin-2 (ChR2) enable investigating the function of populations of neurons in live Caenorhabditis elegans (and other) animals, as ChR2 expression can be targeted to these cells using specific promoters. Sub-populations of these neurons, or even single cells, can be further addressed by restricting the illumination to the cell of interest. However, this is technically demanding, particularly in free moving animals. Thus, it would be helpful if expression of ChR2 could be restricted to single neurons or neuron pairs, as even wide-field illumination would photostimulate only this particular cell. To this end we adopted the use of Cre or FLP recombinases and conditional ChR2 expression at the intersection of two promoter expression domains, i.e. in the cell of interest only. Success of this method depends on precise knowledge of the individual promoters' expression patterns and on relative expression levels of recombinase and ChR2. A bicistronic expression cassette with GFP helps to identify the correct expression pattern. Here we show specific expression in the AVA reverse command neurons and the aversive polymodal sensory ASH neurons. This approach shall enable to generate strains for optogenetic manipulation of each of the 302 C. elegans neurons. This may eventually allow to model the C. elegans nervous system in its entirety, based on functional data for each neuron.
Project description:Channelrhodopsins (ChRs) are light-gated ion channels in widespread use in neuroscience for mediating the genetically targetable optical control of neurons (optogenetics). ChRs pass multiple kinds of ions, and although nonspecific ChR-mediated conductance is not an issue in many neuroscience studies, conductance of calcium and protons, which can mediate diverse cellular signals, may be undesirable in some instances. Here, we turned our attention to the creation of ChRs that have high cation photocurrent but pass fewer calcium ions and protons. We developed an automated, time-resolved screening method capable of rapidly phenotyping channelrhodopsin-2 (ChR2) variants. We found substitution mutations throughout ChR2 that could boost current while altering ion selectivity and observed that the mutations that reduced calcium or proton conductance have additive effects. By combining four mutations, we obtained a ChR, ChromeQ, with improved photocurrent that possesses order-of-magnitude reductions in calcium and proton conductance and high fidelity in driving repetitive action potentials in neurons. The approach presented here offers a viable pathway toward customization of complex physiological properties of optogenetic tools. We propose that our screening method not only enables elucidation of new ChR variants that affect microbial opsin performance but may also reveal new principles of optogenetic protein engineering.
Project description:Channelrhodopsin-2 (ChR2) from the green alga Chlamydomonas reinhardtii functions as a light-gated cation channel that has been developed as an optogenetic tool to stimulate specific nerve cells in animals and control their behavior by illumination. The molecular mechanism of ChR2 has been extensively studied by a variety of spectroscopic methods, including light-induced difference Fourier transform infrared (FTIR) spectroscopy, which is sensitive to structural changes in the protein upon light activation. An atomic structure of channelrhodopsin was recently determined by x-ray crystallography using a chimera of channelrhodopsin-1 (ChR1) and ChR2. Electrophysiological studies have shown that ChR1/ChR2 chimeras are less desensitized upon continuous illumination than native ChR2, implying that there are some structural differences between ChR2 and chimeras. In this study, we applied light-induced difference FTIR spectroscopy to ChR2 and ChR1/ChR2 chimeras to determine the molecular basis underlying these functional differences. Upon continuous illumination, ChR1/ChR2 chimeras exhibited structural changes distinct from those in ChR2. In particular, the protonation state of a glutamate residue, Glu-129 (Glu-90 in ChR2 numbering), in the ChR chimeras is not changed as dramatically as in ChR2. Moreover, using mutants stabilizing particular photointermediates as well as time-resolved measurements, we identified some differences between the major photointermediates of ChR2 and ChR1/ChR2 chimeras. Taken together, our data indicate that the gating and desensitizing processes in ChR1/ChR2 chimeras are different from those in ChR2 and that these differences should be considered in the rational design of new optogenetic tools based on channelrhodopsins.
Project description:We demonstrate a two-photon optogenetic method that generates action potentials in neurons with single-cell precision, using the red-shifted opsin C1V1(T). We applied the method to optically map synaptic circuits in mouse neocortical brain slices and to activate small dendritic regions and individual spines. Using a spatial light modulator, we split the laser beam onto several neurons and performed simultaneous optogenetic activation of selected neurons in three dimensions.
Project description:Channelrhodopsins (ChRs) are light-activated channels from algae that provide these organisms with fast sensors to visible light for phototaxis. Since its discovery, channelrhodopsin-2 (ChR2) has been used as a research tool to depolarize membranes of excitable cells with light. Subsequent chimeragenesis, mutagenesis and bioinformatic approaches have introduced additional ChR variants, such as channelrhodopsin-2 with H134R mutation (ChR2/H134R), channelrhodopsin-2 with E123T mutation (ChETA), Volvox carteri channelrhodopsin-1 (VChR1), Volvox carteri channelrhodopsin-2 (VChR2), channelrhodopsin-2 with C128 or D156A mutations (ChR2/C128X/D156A), chimera D (ChD), chimera EF (ChEF) and chimera EF with I170V mutation (I170V). Each of these ChR variuants has unique features and limitations, but there are few resources summarizing and comparing these ChRs in a systematic manner. In this review, the seven following key properties of ChRs that have significant influences on their effectiveness as research tools are examined: conductance, selectivity, kinetics, desensitization, light sensitivity, spectral response and membrane trafficking. Using this information, valuable qualities and deficits of each ChR variant are summarized. Optimal uses and potential future improvements of ChRs as optogenetic tools are also discussed.
Project description:Channelrhodopsin-1 and 2 (ChR1 and ChR2) form cation channels that are gated by light through an unknown mechanism. We tested the DC-gate hypothesis that C167 and D195 are involved in the stabilization of the cation-permeable state of ChRWR/C1C2 which consists of TM1-5 of ChR1 and TM6-7 of ChR2 and ChRFR which consists of TM1-2 of ChR1 and TM3-7 of ChR2. The cation permeable state of each ChRWR and ChRFR was markedly prolonged in the order of several tens of seconds when either C167 or D195 position was mutated to alanine (A). Therefore, the DC-gate function was conserved among these chimeric ChRs. We next investigated the kinetic properties of the ON/OFF response of these bi-stable ChR mutants as they are important in designing the photostimulation protocols for the optogenetic manipulation of neuronal activities. The turning-on rate constant of each photocurrent followed a linear relationship to 0-0.12 mW mm(-2) of blue LED light or to 0-0.33 mW mm(-2) of cyan LED light. Each photocurrent of bi-stable ChR was shut off to the non-conducting state by yellow or orange LED light in a manner dependent on the irradiance. As the magnitude of the photocurrent was mostly determined by the turning-on rate constant and the irradiation time, the minimal irradiance that effectively evoked an action potential (threshold irradiance) was decreased with time only if the neuron, which expresses bi-stable ChRs, has a certain large membrane time constant (eg. ?m > 20 ms). On the other hand, in another group of neurons, the threshold irradiance was not dependent on the irradiation time. Based on these quantitative data, we would propose that these bi-stable ChRs would be most suitable for enhancing the intrinsic activity of excitatory pyramidal neurons at a minimal magnitude of irradiance.