Potent optogenetic inhibition of behavior with anion channelrhodopsins

Abstract

Optogenetics employs light exposure to manipulate physiology in genetically modified organisms. There are abundant tools for optogenetic excitation of neuronal activity, but the limitations of current activity photo-inhibitors present an obstacle to demonstrating the necessity of specific neuronal circuits. Here we show that anion channelrhodopsins can be used to specifically and rapidly inhibit a range of systems involved in Drosophila locomotion, wing expansion, memory retrieval and gustation, demonstrating their broad utility to the circuit analysis of behavior. Potent optogenetic inhibition of behaviorPage of 1 24 Potent optogenetic inhibition of behavior Page of 2 24 Figure 1. Guillardia theta anion channelrhodopsins are potent inhibitors of motor function and neuronal spiking. A. Flies expressing GtACR1 in cholinergic neurons (Cha-Gal4>UAS-GtACR1) fell from a vertical surface when illuminated. Light intensities in μW/mm 2 are given in each panel. The proportion of climbing flies is given as a percentage of flies outside the floor area of the chamber. At the highest setting, flies fell in response to green, blue and red light, but responded only to green light at lower intensities. Each solid trace is the mean of 3 experiments (48 flies), line color corresponds to the color of illumination from a projector, error ribbons are 95% confidence intervals. B. Flies expressing GtACR2 (Cha>GtACR2) fell in response to blue and green light at the highest intensity; green light had little effect at 19 μW/mm 2 , blue light (14 μW/mm 2 ) was sufficient to causing falling. C. Representative Muybridge series from a high-speed video (1000 frames per second) during light onset (top row) show that green light illumination (peak 525 nm, 38 μW/mm 2 ) of a fly expressing GtACR1 in cholinergic neurons elicited a rapid onset of immobility. Frames are spaced at 50 ms intervals, numbers indicate time relative to illumination start in ms. Difference images between consecutive frames (bottom row) map the motion. Coloured bar indicates frames where the fly is illuminated. Potent optogenetic inhibition of behavior Page of 3 24 Potent optogenetic inhibition of behavior Page of 7 24 Video acquisition Videos were captured at 30 frames per second using an AVT Guppy F-046B CCD camera (Stemmer Imaging, UK), equipped with a 12 mm CCTV-type lens and connected to a computer via an IEEE 1394 cable. Experiments were conducted under infrared (IR) light; an IR longpass filter (Edmund Optics, Singapore) was used to reduce de-Potent optogenetic inhibition of behavior Page of 14 24Figure 7. GtACRs have minimal toxicity. A1. Central brain cells were observed to express yellow fluorescent protein-tagged GtACR1 (green, endogenous fluorescence) in Crz>GtACR1 fly brains prior to all-trans-retinal (ATR) feeding. A2-3. Antibody staining for Crz protein (α-Crz, red) confirmed GtACR1-expressing cells were Crz + . B1-3. Crz+ cell morphology was normal in Crz>GtACR1 flies after ATR feeding and one day of illumination with 19 μW/mm 2 green light. C1-3. Crz+ cell m...

Insights from intoxicated Drosophila

“…The fly nervous system has also become a leading model in neuroscience with its small, but impressive, central nervous system comprised of ~10,000 larval and ~100,000 adult neurons capable of performing complex behaviors (feeding, sleep, courtship, aggression, memory, etc). The fly's genetic arsenal affords, among many things, the ability to label, ablate, or temporarily silence or activate neural cells of interest (Sivanantharajah and Zhang, 2015;Mohammad et al, 2017).…”