Reducing blue-light mediated excitation by red-shifted channelrhodopsins-Proof of concept.

(A) Schematic representation of the approach. A red-shifted channelrhodopsin (R-ChR) is co-expressed with a blue-shifted anion channel (B-ACR). When red light is ON, there is an overall excitation of the cell due to the more dominant R-ChR response. When blue-light is ON, the shunting inhibitory effect of B-ACR reduces the excitation induced by R-ChR to blue light. (B) Validation of the approach in flies expressing Chrimson, Chrimson and GtACR2, or GtACR2 alone . (B1) Experimental setup to record the courtship song of solitary male flies. (B2) Top, Example of a reconstructed neuronal arborization of P1 neurons, Bottom, example of a courtship song induced by LED pulses. (B3) Courtship song production of the solitary male flies during 10-sec of constant illumination with 590 nm (amber) or 470 nm (blue) light. Each dot represents an individual (mean ± SEM)

Comparison of channel properties of light-gated chloride channels and Chrimson variants.

(A) Representative photocurrents of the Chrimson and ACRs variants in whole-cell patch-clamp recordings from HEK293 cells with 590 nm and 470 nm illumination (10mW/mm2). For Chrimson variants, a re-conditioning pulse at 405 nm illumination was applied 15 seconds before 470 nm LED stimulation. The plot shows the off-time constant (top) following 1 sec of 470 nm illumination, and the peak photocurrent (bottom) at -60 mV (mean ± SD, n = 3-13 cells). (B) Typical response of CA1 cells from hippocampal organotypic slices expressing ZipACR to 40 Hz, 5 ms pulses (top black trace) and to an illumination protocol using 40 Hz of 10 ms light pulses at 470 nm (bottom blue trace). (C) Recordings from dentate gyrus granule cells in acute slices expressing ZipACR and vfChrimson. (C1) Representative responses to 500 ms illumination at 470 nm and 635 nm (blue and red traces respectively). Note, 635 nm illumination evoked action potential firing while 470 nm illumination only evoked a single action potential time-locked to the end of the light pulse (arrowhead). Insert, plot showing the number of light-evoked spikes at 635 nm (red bar) and 470 nm (blue bar) illumination. n = 11 cells, paired t-test **p< 0.01; (C2) Example of light-induced firings by 635 nm and 470 nm illumination (red and blue traces respectively, 5 ms pulses at 10 Hz). The arrowheads show consistent APs time-locked to the end of the blue light pulse. Insert, example of a voltage-clamp recording of currents evoked by 5 ms pulses of 635 nm and 470 nm light (red and blue traces respectively). Light-induced currents with 470 nm illumination were initially outward but turned inward immediately following the light offset. Note the late inward current matches the red light induced current, reflecting a slower kinetics for vfChrimson compare to ZipACR.

Characterization of the optimized variants of the ultrafast anion channelrhodopsin ZipACR.

(A) Structure-based design of ZipACR variants. (A1) The retinal binding pocket of GtACR1 and the putative retinal binding pocket of ZipACR homology model. (A2) The alignment of the transmembrane domains (TM) of GtACR1, GtACR2 and ZipACR. The residues surrounding the retinal binding pocket of GtACR1 and the corresponding residues from GtACR2 and ZipACR are highlighted in red. *marks the residues targeted for mutation. The lysine that forms the Schiff base with retinal is indicated in green. The numbers on the left correspond to the number of the first residue in each line. (B) Basic properties of the ZipACR variants compared to Ivf-Chrimson. off-time constant (top) following 1 sec of 470 nm illumination, peak photocurrent (middle) and 590/470 ratio (bottom). The data have been obtained from whole-cell recordings in HEK293 cells. Each dot corresponds to one cell (mean ± SD, n = 2-12).

(C) On-time constant (C1) and normalized photocurrent (C2) of the selected variants Zip(151T) and Zip(151V) as compared to the original ZipACR and IvfChrimson, with 470 nm or 635 nm illumination of various light intensities (in mW/mm2). For the photocurrent, the values have been measured at steady-state. The data points are the mean value ± SEM (n = 6-7). (D) The responses of the selected variants to various wavelengths of the same photon flux. (D1) The action spectra of ZipACR, Zip(151V) and Zip(151T) as measured from 650 nm to 400 nm (D2) For the action spectra, the maximum photocurrent amplitudes measured at each wavelength were normalized to the peak values obtained from the same cell across the spectrum. The data points are the mean value ± SEM (n = 6-7). (E) Validation of the efficiency of the Zip(151T) variant in slices. (E1) The response of dentate gyrus granule cells expressing Zip(151T) variant at 470 nm and 635 nm light pulses delivered through a 63x objective. (E2) Representative traces of the firing induced by current injection at 40 Hz. Overlapping 470 nm but not 635 nm pulses block action potentials. The plot on the right shows the efficiency (in %) of Zip(151T) in blocking individual action potentials at 470 and 635 nm light pulses (mean ± SD, n = 6-10 cells). The traces of the two outliers are presented in supplementary Fig. 5A.

ZipACR variants co-expressed with the red ChR2 IvfChrimson prevent blue-light induced action potentials.

(A) The expression cassette for vfChrimson and bicistronic Zip-IvfChr variants used in rAAV vector (Top). The selected ZipACR variants (Zip(151T) or Zip(151V)) are co-expressed with the red-sensitive IvfChrimson by using the 2A self-cleaving peptide (T2A). All the opsins are soma-targeted with a Kv2.1 peptide. (Bottom) Representatives showing the membrane enriched expression of ZipT-IvfChr and ZipV-IvfChr. Scale bar = 25um. (B) Blue and red light-driven spike fidelity at various light intensities in DG cells (n = 4-10 cells for each opsin; 10 ms pulse width, single pulse). (C) Comparison of the high-frequency red and blue light-induced spiking (C1), and spike probability (C2, n = 6-13 for each opsin), of DG cells expressing vfChrimson or the Zip-IvfChr variants, with 10 and 20 Hz of light pulses (4-7mW/mm2). Holding potential = -60mV. (D) Optical inhibition of spiking in DG cells expressing the Zip-IvfChr variants. Representative traces of the firing induced by current injection at 40 Hz. Overlapping 470 nm but not 635 nm light pulses block action potentials. Holding potential = -60mV. The plots on the right show the efficiency (in %) of ZipT-IvfChr and ZipV-IvfChr in blocking individual action potentials at 470 and 635 nm (n = 9-10 for each opsin).

Red-shifted ChR2 variants are activated by 470 nm and 590 nm pulses of light.

(A) Typical photocurrents (left and middle traces) and on-kinetics (right traces) of Chrimson-ts and IvfChrimson in HEK293 cells with 590 nm and 470 nm illumination at 10 mW/mm2. The traces have been recorded 15-s after a 405 nm reconditioning pulse. (B) Comparison of 590/470 nm ratio for Chrimson-ts and IvfChrimson, measured at peak. Each dot represents a cell (mean ± SD). (C) Blue-light illumination of the Chrimson variants induces firing as efficient as red-light stimulation. This property is represented here with two examples of traces obtained from Chrimson expressing CA1 neurons and vfChrimson expressing DG neurons, in response to 10 Hz of 5 ms pulses at 635 nm and 470 nm.

Membrane trafficking of the improved ultrafast vf-Chrimson, IvfChrimson.

(A) Off-time constant of C-Chrimson-ts and IvfChrimson after 1-s of 635 nm illumination at 10mW/mm2. (B) Photocurrent amplitudes at the peak of C-Chrimson-ts and IvfChrimson, adjusted to membrane fluorescence (left), and comparison of current density and membrane fluorescence (right). The recordings have been obtained from HEK293 cells at -60mV. Each dot represents the values from a single cell. The horizontal black bars represent the mean ± SEM for (A) and mean ± SD for (B). Statistics for panels (A) ****p<0.0001; unpaired t-test and (B) *p<0.05; unpaired t-test (Welch correction).

Channel properties of the ZipACR variants.

(A) 590/470 ratio measured from the ACR variants in HEK293 cells. Each circle corresponds to value obtained from one cell (mean ± SD, n = 3-7). (B) Reversal potential of the selected variants Zip(151T) and Zip(151V) compared to the original ZipACR with cesium (Cs) or potassium gluconate (K) intracellular solution. Each dote corresponds to the value obtained from one cell (mean ± SD, n = 5-6). (C) Typical I/V-relationship for the two variants and ZipACR. The recordings have been done with-or without 470 nm LED illumination.

Photo-induced responses and action spectra of ZipACR variants.

(A) Representative responses of the original ZipACR, Zip(151T) and Zip(151V) in HEK293 cells in response to different light intensities. (B) Normalized photocurrent amplitude in response to 400 nm-650 nm and to 650-400 nm light pulses (as presented in Fig. 3D2). The values have been measured on the photocurrents at the peak. The responses were normalized to the maximum response of each cell. The data points are the mean value ± SEM (n = 6-7).

(A) The traces of the two outliers in Fig.3 E2.

(A1), 470 nm illumination reduced the amplitude of the spikes, but did not abolish the firing. (A2), 470 nm illumination as well as 635 nm light blocked the firing. This cell expressed Zip(151T) channels which was significantly more red-shifted compared to the mean (635/470 nm ratio: 23% for this cell vs 6% for the averaged population). (B) For a range of imposed potentials 470 nm illumination of Zip(151T) does not trigger action potentials. (B1) Typical traces of the DG neurons at various imposed potentials, in response to 10 Hz of 470 nm light illumination. The dotted lines show the potential of the maximal response. (B2) Plot showing the responses in current-clamp of the 10 neurons recorded at -70, -60, -50 and -40 mV imposed potential. Each dot represents a single cell.

Improved membrane trafficking of vfChrimson.

(A) Comparison of trafficking and photocurrent in HEK293 cells at 590 nm illumination for IvfChrimson and ZipV-IvfChr. (B) Comparison of the expression of IvfChrimson-citrine, and ‘2A’. A line profile was drawn across the cell and the membrane values have been averaged and used for calculating the ratio with the mean cytosolic values. Each dot represents the value from a single cell (n = 19-23, mean ± SD). (C) Representative pictures of HEK293 cells expressing the IvfChrimson or ‘2A’.

Excitation spectra of IvfChrimson in HEK293 cells.

(A) Representative responses of IvfChrimson to 1-sec of 470 nm and 590 nm with or without a preconditioning pulse at 405 nm. (B) Normalized responses in voltage-clamp to 590 nm pulses of light, at t = 0, t = 15 and t = 60 sec without 405 nm preconditioning. Due to desensitization IvfChrimson, responses do not recover at least within one minute. Prereconditioning pulse at 405 nm recovers the response.

Temporal recovery of neuronal excitability following light-induced inhibition by the ZipACR variants.

Immediate recovery of ZipV-IvfChr from the inhibition. (A) Experimental paradigm. DG granule cells expressing Zip(151T), ZipT-IvfCh or ZipV-IvfChr have been stimulated by a somatic current injection (5 ms pulse) overlapping or preceded by a 470 nm pulse (5 ms, 4mW/mm2) at 0, 5, 10, 15 or 20 ms intervals. (B) The traces in current-clamp (top) are representative of the responses to current injection for the three constructs. The spike probability (bottom) shows an immediate recovery of ZipV-IvfChr from the off-set of the 470 nm light (n = 7 per construct, mean ± SD).