Optogenetic Neuronal Silencing in Drosophila during Visual Processing

Abstract

Optogenetic channels and ion pumps have become indispensable tools in neuroscience to manipulate neuronal activity and thus to establish synaptic connectivity and behavioral causality. Inhibitory channels are particularly advantageous to explore signal processing in neural circuits since they permit the functional removal of selected neurons on a trial-by-trial basis. However, applying these tools to study the visual system poses a considerable challenge because the illumination required for their activation usually also stimulates photoreceptors substantially, precluding the simultaneous probing of visual responses. Here, we explore the utility of the recently discovered anion channelrhodopsins GtACR1 and GtACR2 for application in the visual system of Drosophila. We first characterized their properties using a larval crawling assay. We further obtained whole-cell recordings from cells expressing GtACR1, which mediated strong and light-sensitive photocurrents. Finally, using physiological recordings and a behavioral readout, we demonstrate that GtACR1 enables the fast and reversible silencing of genetically targeted neurons within circuits engaged in visual processing.

Segment-Specific Optogenetic Stimulation inDrosophila melanogasterwith Linear Arrays of Organic Light-Emitting Diodes

“…[23][24][25][26] For inhibition, the anion conducting GtACR2 was used, which efficiently suppresses neural activity in response to blue light, 22 and has been used in previous studies to control Drosophila larval locomotion and to study the visual system. 27,28 In contrast to more traditional genetic inhibitors such as tetanus toxin, 29 and temperature sensitive dynamin proteins (shibire TS ), 30 GtACR2 enables fast, reversible inhibition at low light intensities from 2 µW mm -2 upwards. 27 Figure 1c compares the activation spectra of both channelrhodopsins to the electroluminescence spectrum of our OLEDs.…”

“…27,28 In contrast to more traditional genetic inhibitors such as tetanus toxin, 29 and temperature sensitive dynamin proteins (shibire TS ), 30 GtACR2 enables fast, reversible inhibition at low light intensities from 2 µW mm -2 upwards. 27 Figure 1c compares the activation spectra of both channelrhodopsins to the electroluminescence spectrum of our OLEDs. Since CsChrimson is extremely light-sensitive and also shows a significant response to blue light (sensitivity to 470 nm light from 20 µW mm -2 ) 21 , we matched the OLED electroluminescence to the activation spectrum of GtACR2, which cannot be efficiently excited with red light.…”

“…Since CsChrimson is extremely light-sensitive and also shows a significant response to blue light (sensitivity to 470 nm light from 20 µW mm -2 ) 21 , we matched the OLED electroluminescence to the activation spectrum of GtACR2, which cannot be efficiently excited with red light. 27 The experimental setup for optogenetic stimulation of restrained Drosophila larvae with OLED illumination is shown in Fig. 2a We obtained a conservative estimate for the spatial resolution of the illumination from our microstructured OLEDs by imaging the top side of the larvae under illumination with individual OLED pixels, i.e., collecting OLED illumination after light scattering has taken place across the entire cross-section of the larval body ( Fig.…”

“…The following fly strains were used: Canton S (CS) wild-type, 5-40-GAL4, 14 UAS-CsChrimson inserted in the AttP2 landing site, 21 and UAS-GtACR2-EYFP inserted in the VK00005 landing site. 27 Fly crosses were cultured in the dark at 25°C on conventional cornmeal-agar medium supplemented with 0.5 mM all-trans-retinal (ATR). As a control, 5-40-GAL4-, CsChrimson-, and GtACR2-flies were crossed to CS flies and raised as well on ATR-supplemented food.…”

PiVR: an affordable and versatile closed-loop platform to study unrestrained sensorimotor behavior

“…Different optogenetic tools require excitation at different wavelengths ranging from blue to deep red (22,51,52). The modularity of PiVR enables the experimenter to customize the illumination system to any wavelength range.…”