Analyzed and plotted. Staging and experiments were completed within a blinded fashion and randomized style. Optogenetic behavioral assays. For optogenetic behavior, larvae were grown in darkness on grape agar plates with yeast paste containing five mM all-trans-retinal. Staged third instar larvae (96 h 3 h AEL) had been carefully transferred under low red light conditions to a 2 agar plates with a 1 ml water film. CsChrimson was activated with 625 nm light (760 Wcm for five s. Videos were taken in the course of the experiment and analyzed employing the Fiji cell counter plugin (ImageJ, NIH, Bethesda). Staging, experiments, and analyses had been accomplished in a blinded fashion. Rolling was defined as at the very least a single full 360roll. Bending was defined as a cshape like twitching, usually seen just before rolling behavior, and to not be confused with other described bending behavior47. Turning behavior describes head turning and thereby a direction modifications of locomotion. All behavioral assays and analyses have been performed within a blinded and randomized style. Cold plate assay. For the cold plate assay, 200 larvae (96 h AEL 3 h) were placed on a 7.2 mm 11.2 mm 1 mm brass metal plate, which was coated having a 1 mm 2 agar and 1 ml water film. The plate was put on the heatingcooling block of a PCR Thermo-Cycler (Biometra TGradient, Biomedizinische Analytik GmbH, G tingen, Germany). A plan was established so the temperature on the agar fell from 25 to 3 within 50 s following staying at 25 for 15 s (15 s at 25 , cooling to -3 ). Temperature from the agar surface was monitored and video-captured during the entire experiment (GTH 1170 digital thermometer, Greisinger electronic, Remscheid, Germany). Videos were taken working with a digital camera (Basler ace acA2040-25gm, Basler AG, Ahrensburg, Germany) and FrameGrabber application (StreamPix, NorPix Inc., Montreal, Canada) and analyzed making use of the Fiji cell counter plugin (ImageJ, NIH, Bethesda). Experiments and analyses were completed inside a blinded fashion. Quit and turning behavior describes stopping of standard larval locomotion with subsequent head turning behavior. Contraction behavior was defined by contraction in the larval body in an unbent, straight fashion. Contraction and bending was defined by contraction behavior with simultaneous cshape like bending. Contraction and rolling was defined by contraction behavior with at least a single simultaneous 360roll. Rolling was defined as at the least oneTable 1 Driver lines and transgenes used within this studyLine 27H06-LexA ppk-Gal4 82E12-Gal4 (2nd and 3rd) 82E12-LexA NompC-LexA 82E12-Gal4AD six.14.3-Gal4DBD LexAop-BrpshortmCherry UAS-Drep2-GFP UAS-CD4tdTomato UAS-CD4-tdGFP UAS-TaoRNAi UAS-TaoCA UAS-hTaokwt UAS-hTaok2A135P LexAop-sybspGFP1-10, UASCD4-spGFP-11 UAS-CsChrimson LexAopCsChrimson UAS-GCaMP6m UAS-TNTe LexAop-TNTe-HA UAS-CaMPARI UAS-Ras85DV12 UAS-Rac1V12 Labels C4da C4da A08n A08n C3da, cho A08n split-Gal4 A08n split-Gal4 Presynaptic active zone Aluminum Hydroxide custom synthesis marker Postsynaptic density marker Morphological marker Morphological marker Knockdown of Tao Expression of hyperactivate Tao Expression of hTaok2 wt Expression of hTaok2 ASDlinked variant Labeling synapses with SybGRASP Optogenetic activation Optogenetic activation Calcium imaging Tetanus toxin light chain Tetanus toxin light chain Ratiometric calcium integrator Constitutively active Ras1 Constitutively active Rac1 Supply BDSC# 54751 BDSC# 3207968 BDSC# 40153 and this study BDSC# 54417 BDSC#22 22 31BDSC# 35841 BDSC# 35836 BDSC# 35147 Gift from K. Harvey This study This.
