Channelrhodopsin-2-XXL, a powerful optogenetic tool for low-light applications

Abstract

Channelrhodopsin-2 (ChR2) has provided a breakthrough for the optogenetic control of neuronal activity. In adult Drosophila melanogaster, however, its applications are severely constrained. This limitation in a powerful model system has curtailed unfolding the full potential of ChR2 for behavioral neuroscience. Here, we describe the D156C mutant, termed ChR2-XXL (extra high expression and long open state), which displays increased expression, improved subcellular localization, elevated retinal affinity, an extended open-state lifetime, and photocurrent amplitudes greatly exceeding those of all heretofore published ChR variants. As a result, neuronal activity could be efficiently evoked with ambient light and even without retinal supplementation. We validated the benefits of the variant in intact flies by eliciting simple and complex behaviors. We demonstrate efficient and prolonged photostimulation of monosynaptic transmission at the neuromuscular junction and reliable activation of a gustatory reflex pathway. Innate male courtship was triggered in male and female flies, and olfactory memories were written through light-induced associative training.

Fig. 1.

Characterization of ChR2-XXL in Xenopus oocytes. (A) Structural illustration of a ChR based on PDB ID code 3UG9 (46). (B) Enlarged boxed region of transmembrane helices 3 and 4 showing the hydrogen bond (dotted line) between residues C128 and D156 of ChR2. (C) Representative confocal images showing retinal-dependent expression of ChR2-wt and ChR2-XXL C-terminally tagged with YFP. (Scale bar: 300 µm.) (D) Comparisons of fluorescence intensities describe enhanced expression of ChR2-XXL both with (filled bars) and without (open bars) retinal supplementation. (E) Consistent with an increased retinal affinity of the mutant, steady-state photocurrent amplitudes of ChR2-XXL were greatly increased irrespective of retinal addition. (F) Example photocurrent and on-kinetics in response to 20-s (Left; 473 nm, 8 mW/mm², 2 × 10¹⁸ photons⋅cm⁻²⋅s⁻¹) or 5-ns light pulses (Right; 473 nm, pulse energy density 13 mJ/mm²), respectively. Average values for time constants of channel opening (τ_on), closing (τ_off) and data points in figures are given as mean ± SD. Statistical comparisons were performed with the two-tailed Student t test (***P ≤ 0.001). (G) Action spectra of photocurrents mediated by ChR2-XXL and ReaChR.

Fig. 2.

Photostimulation at the larval Drosophila NMJ. (A) Scheme indicating ChR2 expression (blue) in motor neurons leaving the ventral nerve chord (VNC) to innervate muscles at the NMJ. (B) Larvae expressing ChR2 variants (filled symbols, 100 µM retinal; open symbols, no retinal addition) in motor neurons were immobilized during continuous irradiation. The duration of immobilization scaled with light intensity (blue light stimulation, measured at 460 nm; 623 nm for ReaChR). Data are presented as mean ± SEM (no error bars for <1 s and >1,000 s). (C and D) Antibody staining against ChR2 (green) and HRP (horseradish peroxidase, magenta), a marker of neuronal membranes. (C) In the VNC, ChR2-wt was confined to motor neuron cell bodies (arrow) and absent from the efferent nerves (arrowhead), where ChR2-XXL localized strongly. (D) Whereas ChR2-XXL was present at the NMJ, ChR2-wt was not detected in the periphery. (Scale bars: C, 30 µm; D, 10 µm.)

Fig. 3.

Electrophysiological characterization of light-evoked neuromuscular transmission. (A) ChR2-T159C elicited EPSPs that terminated with the end of the light pulse. The frequency of EPSPs depended on light intensity (Inset, asterisk indicates 29 µW/mm²). (B) With ChR2-XXL, a brief, low-intensity light pulse triggered EPSPs, which persisted after the end of the stimulus and (C) decayed exponentially (mean ± SEM). Both genotypes were fed retinal.

Fig. 4.

Photostimulation of motor control in adult Drosophila. (A) Light-induced immobilization of adult flies expressing ChR2-XXL (circles) or ReaChR (diamonds) in motor neurons. (B) Spectral dependence (0.28–0.4 µW/mm²) of immobilization by ChR2-XXL. Dashed line, action spectrum of ChR2-XXL in oocytes (Fig. 1G) with axis on the right. (C) Snapshots showing light-induced PER with ChR2-XXL and no reaction with ChR2-wt (2 µW/mm² at 460 nm). (D) Dependence of PER on light intensity (at 460 nm; 1-s light pulse; gray, ChR2-T159C plus retinal; black, ChR2-XXL plus retinal) and stimulus frequency (1-s light pulse; ChR2-T159C, 0.32 mW/mm²; ChR2-XXL, 8.58 µW/mm²). Data are presented as mean ± SEM.

Fig. 5.

Light-triggered male courtship behavior. (A) Examples of male courtship modules. (B) Ethogram of courtship behaviors (gray, abdomen bending; dark green, bilateral wing extension; light green, unilateral wing extension; red, proboscis extension) evoked by ChR2-XXL in 10 individual male flies (plus retinal) following an ∼2-s light pulse (vertical blue line; 30 µW/mm² at 460 nm). (Inset) Data as a cumulative plot of courtship behaviors. (C) Example of photostimulated courtship song produced by unilateral wing vibration of a male fly (Lower). The pulse components are similar to the natural courtship song (produced by a male of the same genotype courting a wild-type female in red light (Upper). (D) Ethogram of courtship behaviors evoked by ChR2-XXL in 10 individual female flies (plus retinal) following an ∼2-s light pulse (vertical blue line; 30 µW/mm² at 460 nm). (E) Example of photostimulated courtship song. The pulse components lack male-specific precision.

Fig. 6.

Light-controlled induction of associative olfactory learning. (A) In adult Drosophila, activation of dopaminergic neurons with light induced an odor-associated aversive memory via ChR2-XXL (UAS-chop2^XXL; TH-GAL4), without a requirement for retinal supplementation. Neither control strains (UAS-chop2^T159C, UAS-chop2^XXL, TH-GAL4) nor flies expressing ChR2-T159C in dopaminergic neurons (UAS-chop2^T159C; TH-GAL4) showed an aversive memory after pairing odor and light stimulation. (B) All genotypes associated odors with electric shock punishment, irrespective of retinal addition. n = 8 per experimental group. Learning indices were tested for significant negative differences from 0 using one-tailed Student t test with Bonferroni correction for multiple tests (***P ≤ 0.001).