Activation of RubyACR-EYFP variants in larval motor neurons drives rapid and robust hyperpolarization.

A. Expression of ACR variants at the neuromuscular junction. Green shows staining for the ACR, magenta is the membrane localized myr::tdTomato and white represents colocalization; all images are maximum intensity projections (scale bar = 10 μm). Columns correspond to ACR variants: A1ACR1 (left), HfACR1 (middle), and GtACR1 (right). This organization is maintained across panels B–F.

B. Membrane potential responses to 660 nm light (270 μW/mm²) at varying pulse durations (1, 10, 100, 500, and 1000 ms, as indicated in the legend). Red vertical line marks illumination onset; colored dotted lines represent illumination offset for each stimulus duration.

C. Mean membrane voltage responses to 500 msec stimulation across 25 intensities ranging from 4 to 274 μW/mm² in 11 μW/mm² increments.

D. Mean change in membrane potential in response to stimulation at different wavelengths (488 nm blue, 530 nm green, and 660 nm red) and intensities. Line color indicates wavelength; shading represents standard error.

E. Mean half onset time, defined as the time (ms) at which the membrane potential crosses the halfway point between pre-illumination baseline and the minimum voltage during illumination.

F. Mean half offset time, defined as the time (ms) at which the membrane potential crosses the halfway point between the voltage at illumination offset and the return to baseline voltage.

Sample sizes through A-F: A1ACR1-EYFP: n = 10 recordings; HfACR1-EYFP: n = 10; GtACR1-kir: n = 11.

Measuring RubyACR driven inhibition in Mi1 neurons.

A. Maximum intensity projection over time from two-photon imaging of Mi1 neurons expressing GCaMP6s and A1ACR1-EYFP. Fluorescence changes were measured in medulla layers 8–10, outlined in yellow.

B. ΔF/F₀ traces from individual animals. The red shaded region indicates the 30-second period of 660 nm illumination at 88 μW/mm².

C. The ΔF/F₀ response time course as each ChR is illuminated with increasing 660 nm light intensities over the 20 sec period (shaded in red). Legend order corresponds to testing order. Light intensity was increased sequentially across trials, except for the final trial (brown), in which intensity was returned to 88 μW/mm².

D. Mean ΔF/F₀ values over the 20 sec stimulation period from the data in C. Intensity order follows the order in which the illumination intensities were applied. All responses from A1ACR1- and HfACR1-expressing flies were statistically significant (p<0.05 from Kruskal– Wallis test followed by pairwise Mann–Whitney U tests). The only exception to this was HfACR1 at 1100 μW/mm². This observation, together with the U-shape of both RubyACR curves is an indication of channel desensitization at higher intensities. This was confirmed by the smaller amplitude of the final response to the repeat of 88 μW/mm2 stimulation (A1ACR1 Kruskal–Wallis test p = 9.7E-10; Wilcoxon U test: p = 3.9E-3, HfACR1 Kruskal-Wallis test p = 0.02: p = 0.088).

Sample sizes through A-D: A1ACR1-EYFP: n = 12; HfACR1-EYFP: n = 10; GtACR1-kir: n = 6; CsChrimson-KIR2.1: n = 4, and Chrimson88: n = 3.

Optogenetically inhibiting spontaneous motor behavior.

A. Example image from the Fly Bowl showing fly positions and their trajectories. Blue circle marks the initial position of a fly. Black traces represent movement during the 10 sec baseline period, and red traces show movement during the 10 sec testing period under 660 nm LED illumination at 29 μW/mm². Flies shown are VGlut MI04979-Gal4 driving 20XUAS-A1ACR1.

B. Stimulation protocol: each trial consists of a 10-second baseline period (black) followed by a 10-second testing period (red), during which the chamber is illuminated with 660 nm light at 29 μW/mm². This cycle is repeated for 21 trials.

C. Walking speed during the baseline period of the first trial, prior to any LED stimulation. Asterisks indicate driver–effector genotypes with significantly lower speed than their empty-driver controls (p < 0.05, Kruskal-Wallis test with post hoc Mann-Whitney U test). A plus symbol (+) denotes a significant difference between GtACR1 and other ChRs expressed with the same driver (p < 0.05).

D. Mean velocity for each trial. Black lines show average velocity during baseline; red lines show velocity during the subsequent illuminated testing period. Each row corresponds to a specific ChR variant, and each column to a specific driver (columns continue into panel E). Asterisks as markers denote cases where Kruskal-Wallis analysis found a significant difference (p < 0.05), followed by a Wilcoxon signed-rank test comparing baseline to testing periods (p < 0.05). Numbers of flies tested are indicated in the right top corner of each plot.

E. Mean velocity measured per second during trials 1, 3, 5, 7, 9, and 11, as indicated in the legend. The red shaded region indicates the illumination during the testing period. ChR variants are arranged along the rows, columns match the drivers shown in panel C.

Percent Survival of Flies Expressing Different Optogenetic Actuators

Optogenetic activation of RubyACRs and CsChrimson drives opposing outcomes in associative conditioning.

A. MB320C labels punishment-signaling dopaminergic neurons innervating the γ1pedc compartment of the MB. Expression pattern of CsChrimson-mVenus (green) shown with neuropil counterstaining of Brp (magenta).

B. Schematic of the optogenetic olfactory learning assay (adapted from (Aso and Rubin, 2016)). Flies underwent two rounds of odor-light pairing using 629 nm LED illumination at 13 and 26 μW/mm². Each pairing was followed by a test for preference between the LED-paired odor (conditioned stimulus, CS+) and a control odor (CS-).

C. Preference index (PI) of flies expressing different optogenetic inhibitors (ACRs) or activators (CsChrimson) under MB320C. Positive PI values indicate appetitive memory; negative values indicate aversive memory. Each group includes n = 6 replicates (dots). Black bars show the mean, and gray bars indicate the median. Asterisks denote trials with a significant response relative to zero (p = 6.0E-06, Kruskal-Wallis test across groups followed by one-sample Wilcoxon signed-rank tests within groups).

Activating RubyACRs in pIP10 neurons suppresses courtship pulse song

A. Left: Diagram of the courtship assay. Singing male flies are recorded using a microphone while the chamber is illuminated with a 629 nm LED. Right: Schematic of the pIP10 neuron within the central nervous system.

B. Example audio recording from a single fly expressing HfACR1 in pIP10. Red shading indicates periods of LED illumination. Illumination intensity increases from left to right as indicated. Courtship song elements (sine and pulse) are labeled above the trace.

C. Mean rate of song pulses for individual courting pairs over trial stages. Each row represents a particular driver-effector combination, as indicated at left. Stages are 5-second epochs of: baseline (b) prior to illumination; testing (t) when illuminated and post-testing (p). Illumination intensity increased with each successive trial as indicated by the shading. Gray lines represent data from individual courting pairs (n = 16), black bars indicate mean over all pairs. Asterisks denote significant differences between the testing period and either the baseline or post period (p < 0.05, Kruskal-Wallis test with post-hoc Wilcoxon signed-rank test).

D. Percent change in pulse song frequency from baseline to testing (left) and from baseline to post (right), based on data in C. Driver-effector combinations for each row continue from C. Violin plots reflect the data distribution; black bars indicate the mean and gray bars indicate the median. Trials had to have a minimum of 1 pulse/s during baseline to be included in this dataset. Asterisks denote significant differences between driver and non-driver controls at each stimulation intensity (p < 0.05, Kruskal-Wallis test followed by a one-tailed Mann-Whitney U test, comparing driven to empty lines at the same intensity).

Sample sizes through A-D: n = 16 flies of each genotype.

RubyACR-EYFP expression in larval motor neuron cell bodies.

Green shows staining for the ACR variants and Chrimson, magenta is the membrane localized myr::tdTomato and white represents colocalization; all images are maximum intensity projections (scale bar = 10 μm). Rows correspond to different variants, driver is RRa-GAL4. Puncta are visible in all cases except HfACR1-EYFP.

Optogenetically inhibiting spontaneous locomotor activity with LexAop2-RubyACR variants

A. Protocol spans 21 trials with each trial having baseline (black) and testing (red) periods when the chamber is illuminated at 29 µW/mm2 with a 660 nm LED. Both baseline and testing period occurs over 10 sec periods. OR Stimulation protocol: each trial consists of a 10-second baseline period (black) followed by a 10-second testing period (red), during which the chamber is illuminated with 660 nm light at 29 μW/mm². This cycle is repeated for 21 trials.

B. Walking velocity during the baseline period of the first trial, prior to any LED stimulation. Asterisks indicate driver–effector genotypes with significantly lower velocity than their empty-driver controls (p < 0.05, Kruskal-Wallis test with post hoc Mann-Whitney U test). A plus symbol (+) denotes a significant difference between GtACR1 and other ChRs expressed with the same driver (p < 0.05).

C. Mean velocity in each trial. Black lines show average velocity during baseline; red lines show velocity during the subsequent illuminated testing period. As indicated, rows correspond to a specific ChR and columns to a driver. Asterisks as markers denote trials with a significant difference between baseline and stimulation periods (p < 0.05, Kruskal-Wallis test with post hoc Wilcoxon signed-rank test). Numbers of flies tested are indicated in the right top corner of each plot.

D. Velocity measured per second during trials 1, 3, 5, 7, 9, and 11, as indicated in the legend. Red shaded region indicates the illumination during the testing period. ChR variants are arranged along the rows, and drivers along the columns.

The effects of light and opsin expression on pulse song.

A. Mean pulse counts per second of control, empty-driver flies (with or without the ChR transgene) for each trial stage across different stimulation wavelengths and intensities. Stages are: baseline (b) prior to illumination; testing (t) when illuminated and post-testing (p). Stimulation LEDs had peak wavelengths at 475 nm (blue, top row), 530 nm (green, middle row), and 629 nm (red, bottom row). Both blue and red light elicited significant changes in pulse frequency in these control flies (p < 0.05, Kruskal-Wallis test with post-hoc Wilcoxon signed-rank test; n = 64 flies).

B. A1ACR1 and GtACR1 expression increases pulse song. Pulse counts are compared between flies with and without the pIP10 driver (n = 16 per condition). For both baseline and post-stimulation periods, data are pooled across all tested wavelengths and intensities for each genotype. Gray bars represent flies lacking the pIP10 driver; colored bars represent flies with ChR expression driven by pIP10. Asterisks indicate significant differences between empty-driver and pIP10-driver flies (p < 0.05, Kruskal-Wallis test with post-hoc Wilcoxon signed-rank test).

Effects of different stimulation wavelengths and light intensities on pulse song

A. Mean pulse counts per second for each trial stage for different stimulation wavelengths and intensities. LEDs with peak illumination at 475 (blue, top), 530 (green, middle), and 629 nm (red, bottom) were tested. Each row represents a particular driver-effector combination, as indicated at left. Stages are 5-second epochs of: baseline (b) prior to illumination; testing (t) when illuminated and post-testing (p). Illumination intensity increased with each successive trial as indicated by the shading. Gray lines represent data from individual courting pairs (n = 16), black bars indicate mean over all pairs. Asterisks denote significant differences between the testing period and either the baseline or post period (p < 0.05, Kruskal-Wallis test with post-hoc Wilcoxon signed-rank test).

B. Percent change in pulse song frequency from baseline to testing (left) and from baseline to post (right). Driver-effector combinations for each row continue from A. Violin plots reflect the data distribution; black bars indicate the mean and gray bars indicate the median. Trials had to have a minimum of 1 pulse/s during baseline to be included in this dataset. Asterisks denote significant differences between driver and non-driver controls at each stimulation intensity (p < 0.05, Kruskal-Wallis test with post-hoc Mann-Whitney U test).

Sample sizes in A,B: n = 16 flies of each genotype.

Activating RubyACRs in pIP10 does not consistently suppress sine song duration

A. Percent change in sine song duration from baseline to testing (left) and from baseline to post (right) when illuminated with a red (660 nm) LED. As indicated, each row represents a particular driver-effector combination. Red LED intensity increases from left to right. Violin plots reflect the data distribution; black bars indicate the mean and gray bars indicate the median. Asterisks denote significant differences between driver and non-driver controls at each stimulation intensity (p < 0.05, Kruskal-Wallis test with post-hoc Mann-Whitney U test).

B. Percent change in sine song when illuminated with either blue (475 nm), green (530 nm), or red (660 nm) as indicated by background color. Intensities for each color increase from left to right. Percent change between baseline and testing is indicated on the left while percent change from baseline to post is indicated on the right. Asterisks denote significant differences between driver and non-driver controls at each stimulation intensity (p < 0.05, Kruskal-Wallis test with post-hoc Mann-Whitney U test).

Sample sizes in A,B: n = 16 flies of each genotype.