ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation

Abstract

Channelrhodopsins (ChRs) are used to optogenetically depolarize neurons. We engineered a variant of ChR, denoted red-activatable ChR (ReaChR), that is optimally excited with orange to red light (λ ∼590-630 nm) and offers improved membrane trafficking, higher photocurrents and faster kinetics compared to existing red-shifted ChRs. Red light is less scattered by tissue and is absorbed less by blood than the blue to green wavelengths that are required by other ChR variants. We used ReaChR expressed in the vibrissa motor cortex to drive spiking and vibrissa motion in awake mice when excited with red light through intact skull. Precise vibrissa movements were evoked by expressing ReaChR in the facial motor nucleus in the brainstem and illumination with red light through the external auditory canal. Thus, ReaChR enables transcranial optical activation of neurons in deep brain structures without the need to surgically thin the skull, form a transcranial window or implant optical fibers.

Figure 1. Basic properties of ReaChR compared to C-VChR1 (VChR1 with ChIEF N-terminal) and C1V1(E122T)

(a) Schematic design of ReaChR. ReaChR consists of the N-terminal of the ChEF/ChIEF variant, transmembrane domain A-E and G of VChR1, transmembrane domain F of VChR2, and a Leu171Ile point mutation. (b) Examples of C1V1(E122T) and ReaChR expression in HEK293 cells as visualized by fluorescence of fused Citrine. Much of ReaChR expression was at the plasma membrane, whereas C1V1(E122T) expression was more intracellular, with strong aggregation. (c) The relative plasma membrane expression level (left) and plasma membrane/intracellular fluorescence ratio (right) of VChR1, C-VChR1, C1V1, C1V1(E122T) and ReaChR as measured with Citrine fluorescence. (d) The mean photocurrent amplitudes of C-VChR1, C1V1, C1V1(E122T), VCOMET and ReaChR recorded from HEK293 cells. The current amplitudes were measured at the wavelengths that evoked the greatest response of each variant and normalized to cell capacitance. (e) The response of C-VChR1 (top row), C1V1(E122T) (middle row), and ReaChR (bottom row) to 650, 630, 610, 590, 470, and 410 nm light of same photon flux (5.1 × 10¹⁶ photon/mm²/s). (f) The spectra of the maximum response (left) and steady-state/plateau response (right) of C-VChR1 (n =7), C1V1(E122T) (n = 8) and ReaChR (n = 7). The responses were normalized to the maximum response of each cell. For statistical comparisons, Kruskal-Wallis tests were used with post-hoc Dunn’s tests on all pairs of variants. H = 31.63, k = 5, P < 0.0001 for the left panel of (c), H = 41.28, k = 5, P < 0.0001 for the right panel of (c), H = 48.02, k = 7, P < 0.0001 for (d). The statistical tests for (d) also include comparison to oChEF and oChIEF. Only significant differences detected with Dunn’s tests with VCOMET or ReaChR are shown on the graphs (*, **, *** indicate significance levels of < 0.05, P < 0.01 and P < 0.001, respectively). Scale bar in (a): 20 μm. The graphs in (c), (d) and (f) are presented as mean ± S.E.M.

Figure 2. Kinetics of C-VChR1, C1V1(E122T) and ReaChR

(a) Representative responses of a C-VChR1- (left), C1V1(E122T)- (middle) and ReaChR (right)-expressing HEK293 cell to 0.08, 0.19, 0.34, 0.74, 1.93, 4.09, 7.65, and 11.75 mW/mm² of 610 nm light. (b) The same cells in (a) responding to 630 nm light of varying intensities. (c) Channel onset time constants of C-VChR1 (n = 10), C1V1(E122T) (n = 6) and ReaChR (n = 10) to 610 nm (left) and 630 nm (right) light of different intensities. (d) Channel closure time constants of C-VChR1 (n = 11), C1V1 (n = 6), C1V1(E122T) (n = 8), VCOMET (n = 9) and ReaChR (n = 11). For (d), Kruskal-Wallis test was used with post-hoc Dunn’s multiple comparison tests on all pairs of variants (H = 36.76, k =5, P < 0.0001). *, ** and *** indicate P < 0.05, P < 0.01 and P < 0.001 levels of significance as detected by Dunn’s tests, respectively. The graphs in (c) and (d) are presented as mean ± S.E.M.

Figure 3. Characterization and comparison of different channelrhodopsin variants in primary cultured hippocampal neurons

(a) Representative confocal images of neurons expressing C1V1(E122T)-TS-Citrine and ReaChR-Citrine. (b) The quantification of plasma membrane fluorescence (left) and plasma membrane / cytosolic fluorescence ratio (right) of C1V1(E122T)-Citrine, C1V1(E122T)-TS-Citrine and ReaChR-Citrine. (c) Photocurrent amplitudes of primary cultured neurons expressing hChR2(H134R), oChIEF, C1V1(E122T)-TS and ReaChR as measured with voltage-clamping recordings. The hChR2(H134R) and oChIEF-expressing neurons were stimulated with 10 mW/mm² of 470 nm light and C1V1(E122T)-TS and ReaChR-expressing neurons were stimulated with 10 mW/mm² light of 590 nm. (d) Representative recordings of neurons expressing different channelrhodopsin variants to 1 s of 10 mW/mm² of LED light of the indicated wavelength under current-clamp recordings. (e) Quantification of the level of light-induced depolarization in neurons expressing the different variants to increasing intensity of light. (f) Quantification of the action potential latency from the onset of the light pulse to increasing intensity of light. For statistical comparisons, Kruskal-Wallis tests were used with post-hoc Dunn’s multiple comparison tests on all pairs of variants. H = 37.66, k = 3, P < 0.0001 for the left panel of (b), H = 1.09, k = 3, P = 0.58 for the right panel of (b), H = 18.32, k = 4, P = 0.0004 for (c). Scale bar in (a): 20 μm. The graphs in (b) and (c) are presented as mean ± S.E.M. Lighter color traces in (e) and (f) are responses of individual cells and darker color traces are mean ± S.E.M. *, ** and *** indicate 0.05, 0.01 and 0.001 level of significance as detected by Dunn’s test.

Figure 4. The responses of neurons expressing the different channelrhodopsin variants to 10 Hz pulsed light stimulation train

(a) Example recordings of neurons expressing the indicated channelrhodopsin variants to 10 Hz light stimulation at 10 mW/mm² of 1 ms duration. Two 10 pulses trains 250 ms apart were used to simulate two bursting episodes. Current were injected to sustain the resting membrane potential at -65 mV. (b) The percentage of pulses resulting in action potentials of varying light intensity and pulse duration recorded from neurons expressing the different variants. (c) Percentage of pulses resulting in extra action potentials of varying light intensity and pulse duration of the different variants. 5 - 11 cells were tested in each conditions shown in (b) and (c). (d) and (e) Summaries of the mean latency and the standard deviation of light-triggered action potential of each pulse in ReaChR, hChR2(H134R) and oChIEF-expressing neurons to the indicated wavelength, intensity and duration. Pulses failed to trigger action potential were not included in the analysis, each trace is the average of 5-8 cells and each data points is the average of 2 – 8 values from the cells. (b) and (c) are mean ± S.E.M. (d) and (e) are mean ± S.D.

Figure 5. In vivo expression and utilization of ReaChR for cortical control

(a) Schematic of vM1 stimulation and recording. ReaChR was expressed in vM1 and visualized by Citrine fluorescence (green). vM1 was exposed for electrophysiology and ReaChR-expressing neurons activated with 617 nm light. (b) Coronal section through medial (AGm) and lateral (AGl) agranular vM1 of an infected mouse show ReaChR (green) expression. Neurons counter-labelled with a fluorescent marker (NeuroTrace; magenta). Scale bar: 500 μm. (c) ReaChR-expressing neurons in vM1. Scale bar: 10 μm. (d) Photo-activated spikes in a ReaChR-expressing neuron recorded in vivo during anaesthesia (recording depth = 410 μm). Single 2 ms pulses of 617 nm light (orange bar) evoked one or more spikes (indicated by *; n = 12 pulses at 2 Hz). Arrows indicate low amplitude artefacts associated with on- and off of light. Scale bars: 100 μV and 2 ms. (e) Spike interval histogram and waveforms (inset) of same unit, demonstrating the unit as a single neuron. Scale bars: 100 μV and 0.5 ms. (f) ReaChR-expressing neuron activated with 2 ms pulses of 617 nm light at 5, 10, 20 and 30 Hz (12 pulses). Top panel: Voltage trace during a train of 12 pulses (100 μV scale bar). Middle panel: Rasters of spikes during 6 trains. Bottom panel: Peristimulus time histogram (PSTH) of spikes across all trains. (g) Spikes per pulse (normalized to max) and (h) probability of one or more evoked spikes across a population of ReaChR-expressing neurons (n = 8) as a function of pulse duration (2 – 20 ms; pulse rate 2 Hz). (i) Number of spikes per pulse (normalized to max) and (j) the latency to the first spike neurons as a function of stimulus rate (1 – 30 Hz; pulse duration 5 ms). (k) Schematic of in vivo activation of vM1 with ReaChR (green) through intact skin and bone of awake mice. Vibrissae movements were measured with high-speed video. (l) Traces of vibrissae movements in response to single, 100 ms pulses (orange arrows and bar) of 617 nm light emitted by an LED placed 10 mm above the skin. Increasing values denote vibrissae protraction. Scale bars: 40 deg and 1 s. (m) Absolute movement amplitudes evoked by 100 ms pulses of 470, 617, and 655 nm light through intact skin in ReaChR-expressing mice (n = 3 mice, 10 stimulus repetitions per condition/mouse). Additionally, 3 mice injected with viral vehicle solution (mock control) were stimulated through-skin with 617 nm light (black). (n) Boxplots of movements during the first second after vM1 photo-activation through intact skull of mock transduced (black, n = 3 mice; 617 nm illumination) and ReaChR-expressing mice (n = 3 mice; 470 to 655 nm wavelengths). Vertical lines indicate data range, boxes the 25th to 75th percentile ranges and central mark the median. Light intensity in all panels was 100 mW light output.

Figure 6. In vivo expression and utilization of ReaChR for brainstem activation

(a) Horizontal section through FN showing ReaChR-expressing neurons and associated processes (green). Cells were labelled with a fluorescent marker (NeuroTrace; magenta). (b) Confocal images of ReaChR-expressing motoneurons (arrows). Scale bar: 100 μm. R: rostral, L: lateral. (c) Schematic of awake mice expressing ReaChR in FN photo-activated by LEDs placed at the opening of the external auditory canal. Vibrissae movements were recorded with high-speed video. (d) Vibrissae movements evoked by stimulating FN in a ReaChR-expressing mouse. Left: Video frames (top) show vibrissae in the reference protracted position (No-light) and at peak retraction during photo-activation (Light), and traces of movements (bottom) in response to 470, and 617 nm light. Black trace indicates movement of contralateral C2 vibrissa. Right: Movement amplitude as a function of light output power. (e) Vibrissa movements evoked by stimulating FN in another ReaChR-expressing mouse. Left: Frames (top) show vibrissae in the reference retracted position (No-light) and at peak protraction during stimulation (Light), and traces of movements (bottom) in response to 470 and 617 nm light. Right: Movement amplitude as a function of light output power. Vertical gray bars in d-e indicate 100 ms light pulses (100 mW light output), and scale bars 10 degrees and 1 s. (f) Population data of absolute movement amplitudes evoked by 470 and 617 nm through-ear illumination (100 ms pulses at 1 Hz; n = 12 mice). (g) Comparison of movement amplitudes evoked with light between 470 and 655 nm in ReaChR (left, n = 12 mice) and hChR2(H134R) (right, n = 3 mice) expressing mice (100 mW light output). Movement amplitudes at same wavelengths were larger in ReaChR compared to hChR2(H134R) expressing mice (P < 0.001, KS-test). (h) In vivo recording of a single-unit in the FN region (based on stereotaxic coordinates) during photo-activation with 617 nm light (100 mW light output). Left, top: Voltage trace aligned to a 3 ms light pulse (orange bar, n = 12 pulses). Small artefacts are associated with on and off of light pulses (arrows). A single spike was evoked after each pulse (*) with low temporal jitter and short latency (4 ms) suggesting the unit expressed ReaChR and was directly activated. Scale bars: 100 μV and 2 ms. Left, bottom: Overlapped spikes from same unit demonstrating this as a single neuron. The unit was not spontaneously active and fired a single spike in response to the light pulse. Scale bars: 100 μV and 0.5 ms. Right, top: Voltage trace recorded during a single train of 12 light pulses (100 μV scale bar). Right, middle: Rasters of spikes during 6 trains. Right, bottom: PSTH of spikes across all trains.