Extended Data Figure 9: Activity and sleep profiles of flies in which synaptic transmission is blocked in DN1_p or DN2 clock neurons.

a–c, Locomotor activity averaged over four days of a ramping temperature cycle (left) and averaged sleep profiles (right) for flies expressing tetanus toxin (TNT) in a subset of the DN1_ps, DN1_p>UAS-TNT experimental flies (red, n = 100), and DN1_p>UAS-IMP-TNT controls expressing inactive TNT control (blue, n = 32) (a); glass^60j mutants which lack all DN1_ps (red, n = 49). and glass^60j/+ heterozygotes (blue, n = 26) (b), and flies expressing TNT in the DN2s, DN2>TNT (red, n = 40) and DN2>IMP-TNT control (blue, n = 24) (c). Expression of TNT in DN2s did not affect the locomotor or sleep behaviour of flies under ramping temperature cycles under constant darkness. These results are consistent with previous findings that showed that DN2 synaptic output is required for behaviour synchronization in temperature cycles under constant light conditions, but not under constant dark conditions⁸. See Methods for detailed description of the genetic tools used. d, e, Averaged population locomotor activity on the day in which the temperature is transiently decreased or increased is shown for Clk4.1m>IMP-TNT control flies (n = 24) (d) and Clk4.1m>TNT flies (n = 20) (e). Flies were entrained to a 18 °C to 25 °C ramping temperature cycle for a week, and the temperature was decreased from 23 °C to 18 °C for an hour during the middle of the heating phase (blue bar) and increased from 22 °C to 25 °C for an hour in the middle of the cooling phase (red bar) on the eighth day of entrainment. Both the control and experimental flies show normal behavioural responses to unexpected changes in temperature, suggesting that blocking synaptic transmission from Clk4.1m expressing DN1_ps does not have an effect on acute thermal responses. n = number of flies. In all the plots, dark lines indicate mean and shaded regions indicate s.e.m. Black dashed lines in the plots represent temperature.