Independent optical excitation of distinct neural populations

Abstract

Optogenetic tools enable examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the study of how different synapses or pathways interact to encode information in the brain. Here we describe two channelrhodopsins, Chronos and Chrimson, discovered through sequencing and physiological characterization of opsins from over 100 species of alga. Chrimson's excitation spectrum is red shifted by 45 nm relative to previous channelrhodopsins and can enable experiments in which red light is preferred. We show minimal visual system-mediated behavioral interference when using Chrimson in neurobehavioral studies in Drosophila melanogaster. Chronos has faster kinetics than previous channelrhodopsins yet is effectively more light sensitive. Together these two reagents enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.

Figure 1. Novel channelrhodopsin spectral classes discovered through algal transcriptome sequencing

(a) Representative GFP (left), tdTomato (middle) and phase contrast (right) images of a tdTomato and opsin-GFP fusion transfected neuron. Yellow line indicates the mask boundary used to quantify soma fluorescence. Scale bar is 10 μm. (b–d) Maximum photocurrents in cultured neurons in response to far-red (660 nm), green (530 nm) and blue (470 nm) light; blue and green photon fluxes were matched, with illumination conditions defined as follows: 1 s pulse at 10 mW/mm² for red, 5 ms pulse at 3.66 mW/mm² for green, and 5 ms pulse at 4.23 mW/mm² for blue. n = 5 – 12 cells for channelrhodopsins with nonzero photocurrent for at least one color; n = 2 – 6 cells for opsins that did not exhibit any photocurrent. See Supplementary Fig. 6 for individual cell data. Plotted is mean ± standard error of the mean (s.e.m) throughout. (e) Phylogeny tree based on transmembrane helix alignments. Scale is # of substitutions per site. (f) Representative voltage-clamp traces in cultured neurons as measured under the screening conditions in b–d (the longer red light pulse was used to ensure we did not miss any red-sensitive channelrhodopsins in our screen). (g) Channelrhodopsin action spectra (HEK293 cells; n = 6 – 8 cells; measured using equal photon fluxes, ~2.5 × 10²¹ photons/s/m²). (h–j) Channelrhodopsin kinetic properties as measured in cultured neurons. Off-kinetics (h) were measured under the same conditions as b–d; on- (i) and recovery-kinetics (j) were measured with 1 s pulse at 5 mW/mm². All opsins were illuminated near their respective peak wavelength, which was either blue or green for all opsins except Chrimson, which was characterized at 625 nm (n = 5 – 12 cells for all kinetic comparisons). (h) τ_off is the monoexponential fit of photocurrent decay. (i–j) Raw traces are shown in Supplementary Fig. 7, and further details in Supplementary Fig. 8. (j) Peak current recovery ratios determined from three 1 s light pulses, with the first pulse response used as the baseline for peak current recovery ratio calculations for both second (1 s in dark after first pulse) and third pulse response (30 s in dark after second pulse). Opsin/genus/species names are: VChR1 (Volvox carteri), C1V1_TT (VChR1/ChR1 chimaera), ChR1 (Chlamydomonas reinhardtii), CsChR (Chloromonas subdivisa), AgChR (Asteromonas gracillis-B), ChR2 (Chlamydomonas reinhardtii), CoChR (Chloromonas oogama), NsChR (Neochlorosarcina sp.), ShChR (Stigeoclonium helveticum; also called Chronos), MvChR1 (Mesostigma viride), SdChR (Scherffelia dubia), TsChR (Tetraselmis striata), TcChR (Tetraselmis cordiformis), BsChR (Brachiomonas submarina), CnChR (Chlamydomonas noctigama; also called Chrimson), HdChR (Haematococcus droebakensis), CbChR (Chlamydomonas bilatus-A), PsChR (Proteomonas sulcata). Statistics for panels b, c, d, h, and i: ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001; ANOVA with Dunnett’s post hoc test, with ChR2 as the reference in d, h, i, and C1V1_TT as the reference in b and c. Plotted is mean ± standard error of the mean (s.e.m.) throughout.

Figure 2. Comparison of optical spiking in cultured neurons

(a–c) Side-by-side comparison of green light-driven spiking fidelity. All green light spiking protocols used a train of 40 pulses, 2 ms pulse width, at 530 nm, and at the indicated powers (n = 5 – 8 cells for each opsin). (a) Representative green light driven spiking traces at the indicated frequencies at 5 mW/mm². (b) Green light driven spike probability over a range of frequencies. Dashed line in b is the electrical spiking control from Chronos-expressing neurons (electrical control consisted of a train of 40 pulses at the indicated frequencies; each current injection pulse was 5 ms long and was varied from 200 – 800 pA depending on each neuron’s spike threshold). (c) Spike latencies calculated for 5 Hz trains at 5 mW/mm² (latency is defined as the time between light pulse onset to the spike peak). (d–f) Comparison of red light (625 nm) spiking. (d) Representative current-clamp traces of red light response and spike fidelity (n = 5 – 8 cells for each opsin; 5 ms pulses, 5 Hz, 5 mW/mm²). (e) Comparison of wildtype Chrimson and Chrimson K176R mutant (a.k.a. ChrimsonR) high frequency red light spiking (n = 10 cells for Chrimson, n = 4 cells for ChrimsonR; 40 pulse train, 2 ms pulse width, 5 mW/mm²). (f) Representative off-kinetics traces for Chrimson vs. ChrimsonR. Statistics for panel c: ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001; ANOVA with Dunnett’s post hoc test with C1V1_TT.

Figure 3. Chrimson evokes action potentials in larval Drosophila motor neurons, and triggers stereotyped behavior in adult Drosophila

Larval motor axons expressing Chrimson fires in response to blue, orange, and red light pulses. (a–c) Intracellular recordings from m6 muscles in 3rd instar larvae expressing Chrimson in motor neurons. Responses to 470 nm, 617 nm, and 720 nm light pulses of indicated power and increasing duration are shown. Short (1–2 ms pulses) of either 470 nm or 617 nm light trigger single excitatory junction potentials (EJPs), longer pulses (4–16 ms) evoke barrages of EJPs. Long duration, high intensity 720nm light pulses also trigger EJPs. Dashes in each subpanel indicate −50 mV. (d) Probability of light-evoked EJPs after 1, 2, 4, 8, 16 ms pulses in response to 470 nm and 617 nm light and after 10, 20, 40, 80, 160 ms pulses in response to 720 nm light. Sample size: n = 6 muscles in 3 animals, for all larvae experiments. (e) Mean ± s.e.m number of EJPs evoked in response to light pulses. (f–h) Behavioral response of flies to light (n = 5 flies in each case for the adult fly experiments). (f) Proboscis extension reflex (PER) of flies (pUAS-Chrimson-mVenus in attP18/w-;Gr64f-Gal4/+;Gr64f-Gal4/+, shown as Gr64f x Chrimson) to 25 pulses of lights at 470 nm, 617 nm, 720 nm (see Methods for PER scoring). (g) Left: PER of Gr64f x Chrimson flies to pulsed lights in darkness (D) or in a visual arena with flowing blue random dots (A). Right: Startle response of control flies (pUAS-Chrimson-mVenus in attP18/+;+/+;+/+, shown as WTB x Chrimson) to the same visual stimuli as left. Startle score is defined as the number of moving legs after stimulation. ^***P < 0.001, ^**P < 0.01.

Figure 4. Characterization of channelrhodopsin blue light (470 nm) sensitivities for two-color excitation in cultured neurons

(a) Current-clamp traces of representative Chrimson-expressing neuron under pulsed vs. continuous illumination. (b) Chrimson blue light induced crosstalk voltages vs. irradiances for individual cells under pulsed illumination (5 ms, 5 Hz, n = 5 cells). (c) Photocurrent vs. blue irradiances (5 ms pulses; n = 4 cells for Chrimson, n = 8 – 10 cells for others). Vertical dotted lines indicate halfway points up the curves for ChR2 and Chronos, as fitted, analogous to “EC50”. (d) Turn-on kinetics (1 s pulse; n = 4 – 7 cells; see Supplementary Fig. 17b,c for raw traces). (e–g) Comparison between ChR2 and Chronos spike probability over three logs of blue irradiance. All pulsed illuminations used 10 pulses, 5 Hz, 5 ms pulse width. (e) Representative spiking traces at the indicated irradiances. (f) Spike probability vs. blue light irradiance, plotted for individual Chronos- or ChR2-expressing neurons and minimum irradiance threshold for 100% spiking (MIT₁₀₀) as a function of GFP fluorescence. (g) Neuron spike threshold and resting potentials (n = 16 – 23 cells).

Figure 5. Independent optical excitation of neural populations in mouse cortical slice using Chrimson and Chronos

(a–e) Spike and crosstalk characterization in opsin-expressing cells. a, f, and j indicate experimental optical configurations. (b) Chrimson and Chronos action spectra emphasizing (vertical shaded bars) the blue (470 nm) and red (625 nm) wavelengths used in this figure. (c–e) Current-clamp characterizations of Chrimson or Chronos expressing neurons in slice to determine optimal irradiance range for two-color excitation. Chrimson-GFP and Chronos-GFP were independently expressed in cortical layer 2/3 neurons in separate mice. 5 ms, 5 Hz light pulses were used; n = 7 cells from 3 animals for Chrimson; n = 11 cells from 4 animals for Chronos. (c) Red light spike probability vs. irradiance. (d) Blue light spike probability vs. irradiance. The blue vertical shaded bar represents the blue irradiance range where Chronos drove spikes at 100% probability and no crosstalk spike was ever observed for any Chrimson neurons. (e) Chrimson subthreshold crosstalk voltage in individual neurons vs. blue irradiances; compare to Fig. 4b. (f–i) Post-synaptic currents (PSC) in non-opsin-expressing neurons downstream of Chrimson and Chronos expressing neurons in brain slice with both opsins introduced into separate neural populations. 0.3 mW/mm² for blue, 4 mW/mm² for red, 5 ms pulses; 6 neurons from 3 animals. All synaptic transmission slice experiments were done using widefield illumination (Supplementary Fig. 18). (g) Triple plasmid electroporation scheme for mutually exclusive Chrimson and Chronos expression in different sets of layer 2/3 cortical pyramidal cells. (h) Histology of intermingled Chrimson- (red) and Chronos-expressing (blue) neurons in layer 2/3 (left) and their axons (right). Scale bar is 100 μm and 20 μm for the left and right images respectively. (i) PSCs in response to optical Poisson stimulation with blue and red light; shown are raw voltage traces (gray) with average trace (black) from a single neuron experiencing blue (top), red (middle) or both (bottom) light pulses. See Supplementary Fig. 18e for PSC traces from 5 different neurons downstream of mutually exclusive Chrimson and Chronos expressing neurons in response to blue or red light. (j–m) PSCs in non-opsin-expressing neurons downstream of Chronos- or Chrimson-expressing neurons. Same illumination conditions as in f–i, except pulses delivered at 0.2 Hz. n = 7 cells from 2 animals for Chronos; n = 12 cells from 4 animals for Chrimson. Black trace is the averaged response, grey traces are individual trials, throughout. (k) Chronos driven PSCs under blue or red light, obtained from a representative neuron (left), with population data (right). (l) Chrimson driven PSCs under blue or red light traces, obtained from a representative neuron (left), with population data (right). (m) Chrimson driven PSC amplitudes (top) and the probability of observing a PSC at all (bottom) vs. blue irradiances.