Introduction

Channelrhodopsins (ChRs) are light-activated ion channels (Nagel et al., 2003, 2002). They are predominantly found in algae with flagella and eyespot (Rozenberg et al., 2020), where they are used for phototactic movement (Baidukova et al., 2022; Govorunova et al., 2004; Sineshchekov et al., 2005). In optogenetics, cation-conducting ChRs (CCRs) are repurposed to trigger electrical activity in excitable tissue through optically evoked membrane depolarization. To shunt electrical activity in neurons, natural and engineered anion-conducting ChRs (ACRs) are predominantly used to hyperpolarize the membrane potential in neurons (Emiliani et al., 2022). Most recently, potassium-selective CCRs (KCRs) have been discovered in stramenopiles, offering additional potential to silence electrogenic cells (Govorunova et al., 2022; Vierock et al., 2022).

A limiting factor to efficiently control behavior in model organisms is the need for a large spread of light that sufficiently covers a fraction of behavior-promoting neuronal circuits. An activation wavelength (λ_max) in the red spectral range allows penetration in deeper layers than blue light (Ash et al., 2017). Therefore, optogenetic tools that are sensitive to red light are desirable. Currently, RubyACRs from the Labyrinthulea are the most redshifted ACRs reported. However, they exhibit a substantial photocurrent inactivation and a slow photocurrent recovery at high light intensities (Govorunova et al., 2020) caused by the accumulation of long-lasting and non-conducting secondary photointermediates (Sineshchekov et al., 2023). Furthermore, the operational light sensitivity that describes the ability of an optogenetic tool to exert an effect on the cell at a given photon density also defines the volume of activated neuronal tissue. Here, ACRs that combine high expression, high single-channel conductance, and moderate kinetics, resulting in a high operational light sensitivity, are ideal for efficiently silencing larger volumes.

We screened a panel of ChRs across the phylogenetic tree, including previously unexplored ChR clades. Among them, MsACR1, a member of the prasinophyte ACR family (Rozenberg et al., 2020), was the most activated with yellow-green light of 555 nm. Introducing substitutions into the wild-type MsACR1 protein revealed a single mutation that caused a further redshift of the action spectrum and accelerated the photocurrent kinetics. Furthermore, we evaluated the optogenetic usability of MsACR1 and its mutant raACR for “red and accelerated ACR.” As such, we recorded dissociated hippocampal neurons at various light stimulation paradigms and tested the possibility of disrupting neuronal network activity in the primary motor cortex that directly influences locomotion behavior. The ACRs are well-expressed and reliably suppress activity with nano-watt light intensities. Additionally, raACR offers suppression of action potentials at a higher temporal resolution than MsACR1.

Results

Characterization of metagenomically identified ACRs

The expressed opsin candidates represent multiple ChR subfamilies, some coming from previously unexplored clades and include MsACR1 from the mamiellophycean green alga Mantoniella squamata and a member of the prasinophyte ACR family (Rozenberg et al., 2020) GpACR1, a protein from the heterotrophic cryptomonad Goniomonas pacifica unrelated to cryptophyte ACRs and instead putatively related to labyrinthulean ACRs, S16-ACR1, which we discovered in the single amplified genome (SAG) of the uncultured MAST3 stramenopile S16 (Wideman et al., 2020), representing its own deeply-branching clade, TaraACR3, a haptophyte ACR of metagenomic origin, and TaraACR1 and TaraACR2, two related ChR sequences coming from giant viruses (see Supplementary Discussion) and forming a separate ChR subfamily (Govorunova et al., 2021) (Fig. 1A and S1).

Figure 1.

We expressed the opsin domains of the identified putative ChRs in ND7/23 cells to test for electrical activity by voltage-clamp electrophysiology in whole-cell mode. We determined the wavelength sensitivity, photocurrent amplitudes, kinetics, and ion selectivity. All constructs showed bidirectional photocurrents upon illumination, typical for ChRs. However, while MsACR1, TaraACR1, and TaraACR2 showed no or only little photocurrent reduction under continuous illumination (photocurrent inactivation), TaraACR3, S16-ACR1, and GpACR1 showed considerable inactivation. In particular, S16-ACR1 photocurrents showed almost complete inactivation similar to the previously described MerMAIDs and some cryptophyte CCRs (Oppermann et al., 2019; Sineshchekov et al., 2020). This effect was more pronounced at positive holding potentials. GpACR1 photocurrents, on the other hand, elicited a secondary peak photocurrent upon light-off (Fig. 1D) indicative of photointermediate inactivation during illumination (Berndt et al., 2009; Krause et al., 2017).

Most of the tested ChRs showed a λ_max between 470 nm and 510 nm, while MsACR1 exhibited a considerable redshifted λ_max at 555 nm (Fig. 1B, C, and S3A). At –60 mV holding potential, photocurrent amplitudes of up to 10 nA were elicited (Fig. 1E), decaying with an off-kinetic (τ_off) between 3 ms (TaraACR2) and 400 ms (GpACR1) (Fig. 1F). The operative light sensitivity was proportional to τ_off. TaraACR3, GpACR1, and MsACR1, with slow off-kinetics, had a high light sensitivity with half-maximal activation at 20 to 40 µW/mm². In contrast, TaraACR2 and S16-ACR1 photocurrents did not saturate at light intensities up to 4 mW/mm², demonstrating a low operative light sensitivity (Fig. S3B and C).

To determine the ion selectivity of the ChRs, either the external Cl^- ([Cl^-]_ex) was replaced with Asp^- or the external Na⁺ ([Na⁺]_ex) with NMDG⁺. Only the replacement of [Cl^-]_ex, but not [Na⁺]_ex, resulted in photocurrent changes and a substantial shift in the reversal potential (E_rev) close to the Nernst potential (E_Nernst) of Cl^- (Fig. S3D-F). Therefore, all tested ChRs are ACRs.

Single-residue mutations affect the spectral sensitivity of MsACR1

The strong activation of MsACR1 with yellow-green light prompted us to explore its applicability as an optogenetic silencer. Therefore, we tested several point mutations to shift its sensitivity to the red. Generally, a redshift can be achieved by either increasing the polarity close to the β-ionone ring of the retinal chromophore or reducing the polarity in the proximity of the retinal Schiff base (RSB).

First, we tested mutations in the vicinity of the β-ionone ring (Fig. 2A). The exchange of the conserved Proline in this region (HsBR-P186; see Fig. S2 for an alignment) generally causes strong blue-shifts (Lu et al., 2001). However, the converse G186P mutation in MsACR1 also blue-shifts the activity (Fig. 2D), as do all other tested replacements (G186C, G186H, G186T), with a substantial amplitude reduction in the case of G186H (Fig. 2E). Other tested replacements close to the β-ionone ring either did not affect λ_max or resulted in hypsochromic shifts, including mutations mirroring residues unique to the recently described RubyACRs: W142Y, W182Q, and G186I (Fig. 2D) (Govorunova et al., 2020). The only exception was F138N, which showed a small redshift (Fig. 2D) but also strongly increased τ_off (Fig. 2F and S4).

Figure 2.

Next, we focused on the active site close to the retinal Schiff base (RSB). Replacing F185, a residue oriented towards the putative counterion D215 (Fig. 2A), with a Tyrosine (F185Y) also resulted in a blue-shifted λ_max (Fig. 2D) and increased τ_off (Fig. 2F and S4). Substitution of T87 with Alanine led to a minor redshift (Fig. 2D) but decreased photocurrent amplitudes and decelerated τ_off (Fig. 2E and F and S4) (Oda et al., 2018). Introduction of Valine or Serine (T87V and T87S) did not affect the spectral sensitivity (Fig. 2D).

Previously, in Chrimson, the residue A298 next to the retinal-binding Lysine was associated with the redshifted absorption of this ChR (Oda et al., 2018). In MsACR1, the homologous residue is S218. Replacing it with an Alanine (MsACR1-S218A) reduced τ_off 5-fold (Fig. 2F and S4) and shifted λ_max 20 nm towards longer wavelengths (Fig. 2D and S5A). Consequently, the half-maximal activity on the red side of the spectrum also shifted from 605 nm to 635 nm (Fig. 2C and S5B). Additionally, this mutation increased the photocurrent amplitude at 690 nm two-fold in comparison with the wild-type. Relative to GtACR1, the most commonly used ACR for optogenetic application (Mahn et al., 2016; Needham et al., 2019; Tsunoda et al., 2017), MsACR1-S218A exhibits a 19-fold larger photocurrent at 690 nm (Fig. S5C). Due to these advantageous properties, we coined the mutant raACR for “red and accelerated ACR.”

raACR allows efficient shunting in neurons at nano-watt light powers

Next, we explored neuronal applications of both MsACR1 and raACR in an in vitro neuronal preparation. We then transduced dissociated hippocampal neurons with adeno-associated viruses carrying the ACRs under the control of a pan-neuronal human Synapsin I (hSyn1) promoter. After ten days, membrane-localized fluorescence was visible in somatic, axonal, and dendritic compartments without apparent intracellular or vesicular aggregation (Fig. 3A).

Figure 3.

Current-evoked spiking was strongly suppressed in single neurons by 550 nm light at intensities of >200 nW/mm². Excitation of MsACR1 at just 5 nW/mm² light already decreased the spiking probability by 25%. At light intensities between 100 and 7100 nW/mm², evoked activity was further reduced to 30-90% for both ACRs (Fig. 3B and C and S6). To quantify the shunting efficiency at different illumination paradigms, we either applied 550 nm or 635 nm light continuously or pulsed at 10 Hz (2-ms pulse width) during a current-ramp injection (Fig. 3D) (Tsunoda et al., 2017). Notably, the pulsed illumination reduces the photon exposure for the neurons 50-fold compared to continuous illumination. MsACR1 and raACR both completely silenced current-evoked spiking during continuous illumination (Fig. 3E). However, at the on-set of the light pulse, we observed a light-evoked on-set spike (Fig. 3D). This has been similarly described for other ACRs and arises in axonal compartments with a higher intracellular chloride concentration compared to the soma, triggering initial chloride efflux upon ACR-activation that leads to a short depolarization (Mahn et al., 2016).

To avoid misinterpretations arising from an on-set spike during in-vivo optogenetic experiments, we designed and tested soma-targeted (st) versions of MsACR1 and raACR. For this, we fused a short targeting sequence from the voltage-gated potassium channel Kv2.1 to the N-terminus of the ACRs. We determined the on-set spike probability for st-MsACR1 and st-raACR under continuous and pulsed illumination with 550 nm. The latter significantly reduced the on-set spike probability for both variants (Fig. S7C). Furthermore, compared to their untargeted versions, st-raACR showed a significantly reduced on-set spike probability, while st-MsACR1 showed a trend of a reduced on-set spike probability (Fig. S7D). MsACR1 and st-MsACR1 also nearly completely shunt all spikes during pulsed illumination independent of the light color. Activation of raACR, or its soma-targeted version, significantly reduced the number of spikes under all conditions but with a lower potency compared to MsACR1 (Fig. 3E and S7B), likely due to the reduced ion flux during a single photocycle.

We further assessed the minimum duration required to re-evoke a current-induced spike after optically suppressing a preceding action potential (Fig. 3F). raACR allows spike-triggering with a 50% spiking frequency already 50 ms after optical silencing, whereas upon activation of MsACR1, a 50% spiking frequency was reached only 180 ms post-shunting (Fig. 3G).

To assess the potential use of MsACR1 and raACR in vivo, we first modeled the brain tissue volume that could be effectively silenced. We used a Monte Carlo simulation and our in vitro light titration data (Stujenske et al., 2015). The comparison with other commonly used optogenetic attenuators (Jaws (Chuong et al., 2014) and GtACR1 (Govorunova et al., 2015)) showed the superiority of MsACR1 and raACR (Fig. S8). To evaluate the efficiency of MsACR1 and raACR in vivo, we assessed how light-induced transient neuronal network perturbation in the primary motor cortex (M1) translates into locomotion impairments. As such, we injected high-titer viral particles of the ACRs into the M1 of wild-type C57BL/6 mice and implanted a 200-µm optical fiber above the injection site (Fig. 4A).

Figure 4.

Both ACRs exhibit homogenous expression in cortical neurons without visible intracellular aggregates or cell death (Fig. 4A). Two-second illumination bouts with 635-nm light pulsed at 20 Hz led to immediate locomotion arrest in all tested animals (Fig. 4B and C). We quantified these motor arrests by calculating the pixel movement of a body marker between two consecutive frames during periods without and with illumination (Fig. 4D). Activation of either ACR led to significantly reduced velocity. However, the effect was more substantial for MsACR1, likely due to its longer-lasting response compared to raACR (Fig. 3G).

As complete locomotion arrest upon light stimulation points to a complex disruption of M1 circuit function, we decided to dissect this complexity by targeting expression exclusively to parvalbumin-positive inhibitory neurons (PV-Cre) utilizing our soma-targeted versions. Additionally, we implanted a 32-channel single-wire optrode to measure neuronal network responses in vivo upon illumination directly (Anikeeva et al., 2011) (Fig. 5A). Six weeks post-surgery, we measured neuronal responses in the M1 area during a 2-second light pulse while the animals were freely moving in an open field arena. As expected, we observed complex changes in neuronal firing, ranging from an immediate decrease or increase in neuronal spiking, delayed changes in firing rate during light stimulation, and resetting of the firing rate after stimulation (Fig. 5B). To capture the different response modes, we calculated the Z-score for 100-ms binned firing rates for all recorded units during the 2 seconds before, during, and after illumination with blue (473 nm) and red light (645 nm) at both 1 mW and 10 mW (Fig. 5C and D). During the first 100 ms within a blue light stimulation at 1 mW, 9 out of 33 units for MsACR1 showed at least a two-fold change in standard deviation (SD) compared to baseline (2 s before illumination), which increased to 16 units at 10 mW. For illumination with red light at either 1 mW or 10 mW, more than 75% of all units (51 units) responded with a change in firing rate. Interestingly, the fast-cycling raACR shows a superior inhibition at blue and red illumination regimes at 10 mW compared to MsACR1, exhibiting a modulation of neuronal spiking in more than 90% of units. At light intensities of 1 mW for both wavelengths, raACR showed moderate effects on the cortical activity of 35%. In both cases, for MsACR1 and raACR, red light illumination leads to a significant decrease in running speed at 10 mW light intensities. Control animals injected with hSyn1-DIO-eGFP do not show a significant change (Mann-Whitney U, p>0.05 control, p<0.001 for MsACR1 and raACR) (Fig. S9A and B).

Figure 5.

Discussion

Despite the rapidly evolving optogenetic toolbox that offers versatile possibilities to manipulate the electrical activity of excitable cells, highly efficient red-light-activatable tools are still sparse. We identified MsACR1 and tailored a red-light sensitive ACR with large photocurrent amplitudes and fast off-kinetics that we coined raACR. We further demonstrated the applicability and potency of both wildtype and mutant in several optogenetic experiments.

Besides its availability as a useful optogenetic tool, the wild-type MsACR1 may also offer a venue for answering questions about algal phototaxis. Previously, we suggested that in motile prasinophytes, both ACRs and CCRs may cooperate to regulate photo-orientation (Rozenberg et al., 2020). Recently, proton conduction has been shown for CCR1 from Mantoniella squamata (Govorunova et al., 2021). Therefore, M. squamata may be a suitable algae species to investigate the physiological function of proton– and chloride-carried photocurrents.

Furthermore, within the characterized set of ACRs are two of viral origin, TaraACR1 and TaraACR2. The growing number of viral rhodopsins (Bratanov et al., 2019; Needham et al., 2019; Rozenberg et al., 2020; Zabelskii et al., 2020) poses a question about their function during viral infection: Do they have a function during the entry of the viral DNA into the host cell, or are they modifying the phototaxis of the host, or are the rhodopsins disrupting the membrane potential which leads to cell death? Compared to other previously characterized viral ChRs (Rozenberg et al., 2020; Zabelskii et al., 2020), the two described here were well-expressed and showed large photocurrents. Also, TaraACR2 showed fast photocurrent off-kinetics comparable to ZipACR and RapACR (Govorunova et al., 2018, 2017).

While GpACR1 and S16-ACR1 are unsuitable for optogenetic applications due to their photocurrent amplitudes and kinetics, their photocurrents unravel interesting molecular features. GpACR1 photocurrents elicit a second peak upon light shut-off, a behavior seen before for the slow-cycling CrChR2 variant C128S (Berndt et al., 2009), ReaChR (Krause et al., 2017) or a viral ChR (Zabelskii et al., 2020). Its appearance is expected to be caused by the back photoisomerization of photocycle intermediates (Krause et al., 2017). S16-ACR1, on the other hand, shows a substantial voltage-dependent photocurrent inactivation and nearly complete inward rectification similar to MerMAIDs (Oppermann et al., 2019) and may offer further insight into these processes in general.

The action spectrum of MsACR1 shows its highest activity in yellow-green light and a distinct blue-light peak, probably resembling the β-band absorption of the retinal chromophore originating from two-photon absorption (Stavenga et al., 1993). Surprisingly, most of the substitutions in MsACR1 close to the β-ionone ring resulted in a blue shift. Only one mutation (F138N) caused a small redshift, suggesting that this region is already relatively polar. Alternatively, the β-Ionone ring could be twisted, which decouples it from the rest of the polyene chain (Kato et al., 2015). In contrast, the active site around the RSB was more susceptible to modification, although the effect of MsACR1-T87A was less pronounced than for Chrimson (ChrimsonSA) (Oda et al., 2018). However, upon replacing S218 with Alanine, the action spectrum was shifted by 20 nm towards longer wavelengths, mimicking several orange– or red-light-absorbing ChRs (Oda et al., 2018). Similar shifts have been achieved naturally and artificially in animal rhodopsins (Liénard et al., 2021).

Both MsACR1 and raACR reliably shunt spiking in vitro and induced motor arrest in vivo using 635 nm light. Shunting during continuous illumination was complete, though light-evoked on-set spikes observed for both ACRs were minimized with pulsed illumination and soma-targeting of MsACR1 and raACR. Presumably, longer photon exposure is required to evoke this spike, as previously evident for GtACR1 using 10-ms light pulses (Mahn et al., 2016). Therefore, shorter or ramped-on light pulses can reduce the probability of such events. Overall, pulsed illumination and soma-targeting are recommended for artifact-free in vivo optogenetic experiments.

Pulsed illumination was more efficient for MsACR1. This was due to its slower kinetics, resulting in increased ion flux per photocycle. On the other hand, the accelerated kinetics of raACR allows for a higher temporal resolution, enabling current-evoked spiking already 50 ms post-illumination. Ideally, one would slow its kinetics to increase the efficiency of raACR under pulsed illumination. However, most mutations tested here that resulted in slowed kinetics also shifted λ_max to the blue. Potential candidates for combining could be T87A or T87S, as they did not affect λ_max but increased τ_off while the current amplitudes were only marginally reduced. On the other hand, screening mutations of the homologous residues to the DC pair of CrChR2 could be fruitful, as recently demonstrated (Rodriguez-Rozada et al., 2022).

Also, in contrast to other ACRs commonly used as optogenetic silencers, both MsACR1 and raACR do not show substantial photocurrent inactivation during continuous illumination and can be activated again virtually instantly without photocurrent decline. This recommends them as reliable optogenetic actuators. Furthermore, the redshifted raACR reduces potential cross-activation when used with blue-light-sensitive optogenetic actuators or reporter systems (Vierock et al., 2021).

In conclusion, MsACR1 and raACR, due to their good expression and membrane targeting, high chloride conductance and slow τ_off, provide a high operational light sensitivity to neuronal cells, enabling inhibition at light intensities as low as 20 nW/mm². Thus, we believe both ACRs are valuable additions to the optogenetic toolbox when long-lasting inhibition in larger brain volumes is required.

Methods

Identification and phylogenetic analysis

Two of the ChR sequences characterized here have been identified in (Rozenberg et al., 2020): MsACR1 from Mantoniella squamata CCAP 1965/1 (1KP_QXSZ_2010086.p1, extracted from a 1KP transcriptome assembly (2019)) and GpACR1 from Goniomonas pacifica (the originally reported truncated sequence MMETSP0108_DN24423_c0_g1_i1.p1 was extended by transcriptome re-assembly of the raw data from MMETSP0108 and MMETSP0107 (Keeling et al., 2014)). S16-ACR1 was extracted from the single-amplified genome (SAG) S16 assigned to the marine stramenopile clade MAST3g (PRJNA379597-S16_NODE_311_gmes_4206, the coding sequence was predicted with GeneMarkS in the SAG assembly from (Wideman et al., 2020)). Sequences TaraACR1 (from contig SAMEA2622336_1814029), TaraACR2 (contig SAMEA2619952_516956), and TaraACR3 (overlapping contigs SAMEA2621277_718384 and SAMEA2621278_296705) were retrieved from a custom assembly of the Tara Oceans data (Philosof et al., 2017).

For phylogenetic reconstruction, rhodopsin domain sequences of all records included in the Catalog of Natural Channelrhodopsins v. 1.3 (Rozenberg, 2023), covering characterized and uncharacterized proteins, were taken and duplicated domains were excluded. The domain sequences were aligned with mafft v. 7.520 (automatic mode) (Katoh et al., 2002), the alignment was trimmed with trimal v. 1.4.1 (-gt 0.1) (Capella-Gutiérrez et al., 2009), and the phylogeny was reconstructed with iqtree2 v. 2.2.0.3 (-pers 0.2 –nstop 500) with 1000 ultrafast bootstrap replicates (Hoang et al., 2018; Minh et al., 2020).

Structure-supported alignment of the rhodopsin domains for the tested ChRs and reference proteins (BR, ChR2, Chrimson, and GtACR1) was performed with t_coffee v. 13.45.0.4846264 (algorithms sap_pair, mustang_pair, t_coffee_msa and probcons_ms). For the reference proteins available, crystal structures were utilized, while for the ChRs characterized here, structural models were obtained with AlphaFold (Jumper et al., 2021) as implemented in the colabfold workflow (Mirdita et al., 2022). Transmembrane regions of the α-helices of ChR2 (PDB: 6EID) were taken from the OPM database (Lomize et al., 2012).

The bioinformatics workflow is available from the GitHub repository https://github.com/BejaLab/ACRs.

Molecular Biology

For electrophysiological analysis, human/mouse codon-optimized genes coding for the identified ChRs were ordered (GenScript, Piscataway, NJ or Integrated DNA Technologies, Coralville, IA) and cloned in frame with mCherry, mScarlet, or mCerulean fluorophores into pEGFP-C1 or pcDNA3.1 vectors using Gibson cloning (Gibson et al., 2009) or restriction digest (FastDigest NheI and AgeI, Thermo Fisher Scientific, Waltham, MA). The membrane localization of GpACR1 and S16-ACR1 was improved using a membrane trafficking sequence and endoplasmic reticulum release sequence, as previously used (Grimm et al., 2018; Rozenberg et al., 2020). Single residues in MsACR1 were replaced by site-directed mutagenesis using Pfu polymerase (Agilent Technologies, Santa Clara, CA). A structural model of MsACR1 was generated using AlphaFold (Jumper et al., 2021), and the retinal chromophore was added from BR (PDB: 2AT9).

MsACR1 and raACR, in frame with a mCerulean and separated by a P2A site, were subcloned into pAAV2 for expression in neurons under control of the Calmodulin kinase II promoter using Gibson cloning (Gibson et al., 2009) and further subcloned into pAAV2 under the control of the human synapsin promoter and flanked by lox71 and lox66 sites using EcoRI and BamHI restriction sites (FastDigest, Thermo Fisher Scientific). The constructs were soma-targeted using the Kv2.1 targeting domain.

Molecular cloning was planned using NEBuilder v. 2.2.7+ (New England Biolabs Inc., Ipswich, MA) and SnapGene v. 5.0+ (GSL Biotech LLC, Chicago, IL). Plasmids of all constructs used in this study were deposited at Addgene: MsACR1-pmCerulean3-N1 (#204958), raACR-pmCerulean3-N1 (#204959), TaraACR1-pmCherry-C1 (#204960), TaraACR2-pmCherry-C1 (#204961), TaraACR3-pmCherry-C1 (#204962), S16-ACR1-pcDNA3.1-mScarlet (#204963), GpACR1-pcDNA3.1-mScarlet (#204964), MsACR1-mCerulean3-pAAV2-hSyn (#204965), raACR-mCerulean3-pAAV2-hSyn (#204966), MsACR1-TS-Kv-P2A-mCerulean3-pAAV-CaMKII (#204970), raACR-TS-Kv-P2A-mCerulean3-pAAV-CaMKII (#204971).

ND7/23 Cell culture

For electrophysiology, measurements of the identified ChRs were expressed in a mouse neuroblastoma and rat neuron hybrid cell line (ND7/23 cells, ECACC 92090903, Sigma-Aldrich, Munich, Germany). Cells were cultured as previously described (Grimm et al., 2017) at 37°C and 5% CO₂ in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 5% fetal bovine serum (FBS) and 100 µg/ml Penicillin/Streptomycin (both Sigma-Aldrich). For experiments, cells were seeded at a density of 0.4×10⁵ cells/ml on poly-D-lysine-coated coverslips and supplemented with 1 µM all-trans-retinal (Sigma-Aldrich). After one day, cells were transiently transfected with 2 µg DNA by lipofection (FuGENEⓇ HD, Promega, Madison, WI).

Electrophysiology in ND7/23 cells

One to two days after transfection, photocurrents were recorded using the patch-clamp method in whole-cell mode at room temperature. The whole-cell configuration was established at ≥1 GΩ membrane resistance, and cells were discarded at access resistance >10 MΩ. Patch-pipettes were pulled from borosilicate glass capillaries (G150F-8P, Warner Instruments, Hamden, CT) to resistances of 1.5 MΩ to 3 MΩ using a micropipette puller (P-1000, Sutter Instruments, Novato, CA) and fire-polished. During measurements, a 140 mM NaCl agar bridge was used as the reference electrode. A 2 kHz Bessel filter was used for signal amplification (AxoPatch200B). Signals were digitized at 10 kHz (DigiData400) and acquired using Clampex 10.4 software (all Molecular Devices, Sunnyvale, CA). A Polychrom V (TILL Photonics, Planegg, Germany) with 7-nm bandwidth was used as the light source and coupled into an Axiovert 100 microscope (Carl Zeiss, Jena, Germany). The light delivery was controlled by a VS25 and VCM-D1 shutter system (Vincent Associates, Rochester, NY). Light intensities were adjusted manually for light titration experiments using neutral density filters. In recordings of action spectra a motorized filter wheel (Newport, Irvine, CA) was used to ensure delivery of equal photon densities. Light intensities in light titration experiments were measured in the sample plane using a P9710 optometer (Gigahertz Optik, Türkenfeld, Germany) and calculated for the illuminated field (0.066 mm²) of the W Plan-Apochromat x40/1.0 DIC objective (Carl Zeiss).

Measurements were always started in standard buffer (Extracellular (in mM): 110 NaCl, 1 KCl, 1 CsCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES; Intracellular (in mM): 110 NaCl, 1 KCl, 1 CsCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES, 10 EDTA). The pH was adjusted to 7 using N-methyl-D-glucamine (NMDG) or citric acid. The osmolarity was measured (Osmomat 3000basic, Gonotec, Berlin, Germany) and adjusted to 32 mOsm (extracellular) or 290 mOSM (intracellular) using glucose. For ion selectivity measurements, NaCl was replaced by 110 mM NaAsp or 110 mM NMDGCl extracellularly. The extracellular buffer was exchanged manually by adding at least 3 ml to the measuring chamber (volume ∼0.5 ml). Excess buffer was removed using a Ringer Bath Handler MPCU (Lorenz Messgerätebau, Katlenburg-Lindau, Germany).

Action spectra were recorded in standard conditions at –60 mV using 10-ms light flashes of low-intensity (typically in the low µW-range at λ_max). λ_max was determined by normalizing recorded action spectra to the respective maximum photocurrent and applying a Gauss function to the data trimmed between ±70 nm and ±90 nm from the maximum. To determine ion selectivities, photocurrents were elicited using high-intensity light close to λ_max applied for 0.5 s to 1 s at –80 mV to +40 mV increased in steps of 20 mV. The liquid junction potential was calculated and corrected during measurements. Light titrations were recorded at –60 mV with light applied for 1 s. The activity of GtACR1, MsACR1, and raACR with green and red light was measured at –60 mV with optical filters (560±10 nm with 82% transmittance and 692±18 nm with 99% transmittance) inserted directly into the light path.

Hippocampal neurons cell culture and electrophysiology

Primary cultured hippocampal neurons were prepared, as described (Karpova et al., 2013). Neurons were plated (25,000 cells on 12 mm coverslip), maintained at 37°C (5% CO₂), and transduced with crude AAV viruses (1-5 x 10¹⁰ mg/ml) three days after seeding. Experiments were conducted ten to twelve days after transduction. Borosilicate glass pipettes (BF100-58-10, Sutter Instruments) were pulled using a micropipette puller (P-2000, Sutter Instruments; 2-3 MΩ). Pipettes were filled using the internal solution as follows (in mM): 135 potassium gluconate, 4 KCl, 2 NaCl, 10 HEPES, 4 EGTA, 4 MgATP, 0.3 NaGTP, pH 7.3 (KOH). Tyrode’s solution was used as the external solution and prepared as follows (in mM): 125 NaCl, 2 KCl, 30 glucose, 2 MgCl₂, 25 HEPES, 2 CaCl₂, pH 7.3 (NaOH).

Whole-cell patch-clamp recordings of the ACRs were carried out under visual control with a 12-bit monochrome CMOS camera (Hamamatsu Model OrcaFlash). Current clamp recordings were performed using a MultiClamp700B amplifier, filtered at 8 kHz and digitized at 10 kHz using a Digidata 1550A digitizer (Molecular Devices). Neurons were held at –60 mV, and spiking was evoked with a ramp (600 pA injection; 500 ms) or rectangle protocol (pA injection considering the rheobase + 50 pA; 700 ms). The inhibition was tested by activating the ACRs using continuous (700 ms) and pulsed (10 Hz, 2 ms) light and two different wavelengths (550 nm and 635 nm). The pE-4000 (CoolLED, UK) was used for light stimulation, and the light intensities were measured using a PM100D console and an S130VC sensor (Thorlabs, US).

Stereotactic injection of viral vectors and optrode implant

Eight-week-old C57BL/6 mice were anesthetized with isoflurane (∼1% in O₂, v/v) and placed into a stereotaxic frame (RWD). A craniotomy (∼1 mm in diameter) AP ML was made above the injection site. Virus suspensions were slowly injected (100 nl min⁻¹) using a 34 G beveled needle (Nanofil syringe, World Precision Instruments). After injection, the needle was left in place for an additional 5 min and then slowly withdrawn. The surgical procedure was continued with the implantation of an optical fiber (Thorlabs 200um). The optical fiber was secured to the skull using Metabond (Parkell) and dental acrylic. The surgical incision was closed with tissue glue, and 5 mg kg⁻¹ Caprofen was subcutaneously injected for postsurgical analgesia. For AAV/DJ-hSyn1-MsACR-mCerulean and AAV/DJ-hSyn1-raACR-mCerulean, we injected 300 nl with a viral titer of 4.5 x 10¹² vg/ml and 1 x 10¹³ vg/ml, respectively.

Self-made optrodes were built according to the design by Anikeeva et al. (2012), using an OpenEphys shuttle drive EIB 32-Channel with an Omnetics socket (product #A79026-001) and a 200 µm diameter fiber (Thorlabs CFML). Optrodes were implanted immediately after viral injection, and recordings were started 4 weeks post-implantation.

Motor cortex behavior

Mice were attached to patch cords before placing them into the middle of the arena (50 cm x 50 cm). Animals were allowed to explore the arena freely for 1 min. Afterward, optical stimulation started with a duration of 2 s at 20 Hz coming from a laser source that was either 473 nm or 635 nm (MBL-III-473-100mW and MRL-III-635L-200mW). We adjusted the light power to 0.5 mW on the 0.03 mm² tip of the fiber. Stimulation bouts were interspersed with 5-sec periods with no light. Stimulation was repeated 25 times before leaving the animal for another 1 min in the arena.

DeepLapCut (Mathis et al., 2018) was used to track animals with several body poses (head, ears, snout, body center, and tail). Additionally, we estimated the position of the patch cord, which yielded a drastically improved overall likelihood for body poses. The average pixel deviation between consecutive frames was average for 2 s during stimulation and 2 s after stimulation. Neural recordings were acquired using the OpenEphys system, which was connected to the aforementioned laser. Spike sorting was performed using the Waveclus algorithm (Chaure et al., 2018). Z-scores were then calculated for each sorted unit based on thirty trials for each wavelength and light intensity. Units were classified as inhibitory, neutral, or excitatory if the standard deviation (SD) of the baseline firing rate differed two-fold compared to the first 100 ms between trials.

Acknowledgements

We thank Sandra Augustin, Celina Dölle, Tharsana Tharmalingam, and Maila Reh for outstanding technical assistance, Jonas Wietek for pre-selection of Tara Oceans candidates, and Enrico Peter for help with AlphaFold to generate the MsACR1 structural model. We are in debt to Ernesto Duran for building and implanting optrodes. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grant SFB 1078 B2 221545957 (P.H.) and Israel Science Foundation grant 3131/20 (O.B.). O.B. holds the Louis and Lyra Richmond Chair in Life Science. P.H. is Hertie Senior Professor for Neuroscience supported by the Hertie Foundation. T.F. was supported by Alexander-von-Humboldt Foundation/CAPES post-doctoral research fellowship (99999.001756/2014-01) and C.G.C. by the EU program “Novel imaging and brain stimulation methods and technologies’’ JPco-FuND – NiPARK). M.P. received funding from the Leibniz Association program “BestMinds’’ MP J28/2017 SheLi, and Align Science Against Parkinson (ASAP-020505).

Author contributions

J.O., P.H., A.R., and O.B. conceived the project. A.R. performed bioinformatic analyses. J.O. and P.H. designed molecular characterization. J.O. acquired and analyzed electrophysiological data in ND7/23 cells. T.F., C.G.C., and M.P. designed neurobiological experiments. T.F. acquired and analyzed data in hippocampal neurons in vitro. C.G.C. acquired data from behavioral experiments, and R.E.S.P. and M.P. performed data analysis. The manuscript was written by J.O., M.P., and A.R. with contributions from all authors.

Declaration of interests

The authors declare no competing interests.

Supplement

Supplementary Discussion

Viral origin of the environmental ChRs TaraACR1 and TaraACR2

The short metagenomic contigs SAMEA2622336_1814029 (1748 bp) and SAMEA2619952_516956 (1239 bp) encoding the ChRs TaraACR1 and TaraACR2, respectively, were initially suspected to be of viral origin due to lack of introns and short intergenic regions to the neighboring genes. The neighboring genes, a DUF285 protein (SAMEA2622336_1814029) and a short fragment of a gene for a Pif1 helicase (SAMEA2619952_516956), are from protein families widespread among, albeit not restricted to, giant viruses. More importantly, the promoter sequence of TaraACR1 contained the classical mimiviral early promoter motif AAAATTGA (Suhre et al., 2005) characteristic to Imitervirales and some other giant viruses, while that of TaraACR2 contained its minor variation AAAAATGA prevalent e.g. in the genome of Aureococcus anophagefferens virus (Imitervirales) (Roitman et al., 2023). Finally, blastn searches against the GVMAGs database (Schulz et al., 2020) yielded genomes belonging to the Imitervirales containing highly similar nucleotide stretches (best hits to GVMAG-M-3300001348-7 [E-value 3.3×10^-80, 72.5% nucleotide identity] and GVMAG-M-3300023184-101 [E-value 8.7×10^-10, 90.2% identity, respectively, both belonging to the Mesomimiviridae (Aylward et al., 2021)). Although, none of them contained the ChR genes themselves. TaraACR1 and TaraACR2 represent a second example of ChR acquisition by giant viruses, alongside prasinophyte ACRs in genomes of viruses closely related to Pyramimonas orientalis virus 01b (Imitervirales family IM_12 (Aylward et al., 2021)) and a putative Algavirales virus (Rozenberg et al., 2020). The appearance of ion-conducting rhodopsins encoded in genomes of giant algal viruses is not restricted to channelrhodopsins, as two unrelated cases of functional rhodopsin cation channels/pumps are known from the Mesomimiviridae (viral rhodopsins (Bratanov et al., 2019; Zabelskii et al., 2020)) and the Coccolithoviridae (proton-conducting heliorhodopsins (Hososhima et al., 2022)).

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