Because Pdf > ClkDN and Pdf > cycDN larvae had similar light avoidance phenotypes as hyperexciting LNvs via NaChBac, we infer that low CLK/CYC activity increases LNv excitability, which in turn promotes light avoidance. Conversely, because expressing ClkDN or cycDN in DN1s has a similar light avoidance phenotype to hyperpolarizing DN1s via dORKΔC or Kir2.1, we infer that low CLK/CYC activity decreases DN1 excitability and consequently increases light avoidance by reducing DN1-mediated inhibition. To test this further, we asked whether the increased light Fulvestrant datasheet avoidance caused by expression of cycDN in LNvs or DN1s could be reduced by altering neuronal electrical excitability.

We found that coexpressing dORKΔC

with cycDN in LNvs ( Figure 3A) or NaChBac with cycDN in DN1s ( Figure 3B) rendered larvae as insensitive to light at 150 lux as wild-type larvae. However, coexpressing NaChBac with cycDN in LNvs ( Figure 3A) Baf-A1 ic50 did not reverse the increased sensitivity caused by expressing cycDN. These results are consistent with low levels of CLK/CYC activity increasing LNv excitability and thus light avoidance levels—and this is rescued by hyperpolarizing LNvs. Conversely, low CLK/CYC activity seems to decrease DN1 excitability, which also increases light avoidance—and this is rescued by hyperexciting DN1s. Because the phenotypes caused by cycDN can be rescued by altering the excitability of LNvs and DN1s, it seems unlikely that the behavioral phenotypes caused by cycDN arise from putative developmental defects caused by reduced CLK/CYC activity during development ( Goda et al., 2011). Furthermore, we found that expressing cycDN in differentiated larval LNvs for only the 24 hr Histone demethylase immediately prior to assaying behavior still increased light avoidance (see Figure S1 available online). The per01 mutation stops the clock with constitutively high levels of CLK/CYC activity, allowing us to test how

high levels of CLK/CYC activity affect LNv and DN1 excitability. Because per01 larvae display low levels of light avoidance at 750 lux ( Mazzoni et al., 2005), we tested whether light avoidance in per01 mutants could be restored to wild-type levels by manipulating LNv and DN1 excitability. We found that hyperexciting LNvs in a per01 background via NaChBac significantly increased levels of light avoidance, whereas hyperpolarizing LNvs through dORKΔC expression had no effect ( Figure 3C), suggesting that per01 LNvs have reduced excitability. Conversely, dORKΔC expression in DN1s of per01 mutants significantly increased light avoidance, whereas NaChBac expression had no effect ( Figure 3C), suggesting that per01 DN1s have increased excitability. From this, we conclude that per01 mutants display low levels of light avoidance because high CLK/CYC activity in per01 mutants simultaneously reduces LNv excitability and increases DN1 excitability.