(A1–3) Expression of Channelrhodpsin2-YFP under control of ppk-GAL4. (A1) pan-neuronal marker anti-HRP (green). (A2) anti-GFP (magenta). (A3) Merge of a1 and a2. (B) Expression levels of UAS-ChR2-YFP lines measured by pixel intensity of confocal images and normalized to staining intensity of line C. (C) Optogenetic activation of various subsets of multidendritic neurons triggers the “accordion” phenotype at high frequency. (D) Optogenetic activation of Class IV multidendritic neurons elicited nocifensive rolling behavior at high frequency. Rolling behavior with activation of classes I-IV was also elicited (15%). (E) ChR2-YFP expression levels are correlated with efficiency of nocifensive behavior. Sample sizes for c and d (Class I neuron driver 2–21-GAL4: atr+ n=54, atr- n=43), (Class I-IV neuron driver md-GAL4: atr+ n=103, atr- n=92 ), (Classes I & II driver c161-GAL4: atr+ n=117, atr- n=21), (Class II & III driver 1003.3-GAL4: atr+ n=46, atr- n= 52), (Class IV ppk-GAL4 atr+ n=181, atr- n=112). Sample sizes for e (Chop2 n=173, Line 1 n=20, Line 2 n=84, Line AB n=181, Line C n=80). UAS-Chop2 is an untagged Channelrhodopsin-2 line from the Fiala laboratory[34] with an insertion on the third chromosome. Error is standard error of the mean.