This gating system can only function if LNvs and non-LNvs have di

This gating system can only function if LNvs and non-LNvs have differently phased neuronal activity. However, most Drosophila clock neurons have similarly phased molecular clocks. We propose that molecular clocks in different clock neurons regulate divergent sets of output genes to generate distinct

phases of neuronal excitability. This would be analogous to the mammalian circadian system, in which molecular clocks in different tissues drive tissue-specific outputs (e.g., Storch et al., 2002). In summary, our genetic dissection of a circadian neural circuit reveals an unexpected PI3K activity and essential role for inhibitory signals from non-LNvs (E cells) in shaping activity profiles at dawn and a mechanism for how clock neurons couple together to promote robust

rhythms. For a complete list of fly stocks used in this paper, see Supplemental Experimental Procedures. For LD experiments, larvae were entrained to 5 days of 12:12 LD cycles at 25°C and tested on the sixth day as third-instar larvae. For DD experiments, larvae were entrained to 12:12 LD at 25°C for 3–4 days and tested on the second or third day in DD. Larvae were removed from LD or DD immediately prior to testing. Approximately 15 larvae were placed in the middle of an 8.5-cm-diameter agar-filled Petri dish, and the number of larvae in the light and dark was recorded after 15 min as in Mazzoni et al. (2005), with the following this website minor modifications: (1) to speed up scoring, any larvae visible through the lid of the plate were recorded as being on the light side even if crossing the midline; (2) because larvae could be found on the walls and lid on both the light and dark sides

of the plate, 17-DMAG (Alvespimycin) HCl they were included in the scoring; (3) light intensity was reduced by moving the light source away from the plate rather than by adding filters; and (4) the light source used was a circular fluorescent 22 W GE Cool White bulb. Data are plotted as percentage of larvae in the dark. Each data point is the average of three or more experiments, with each experiment consisting of ∼45 larvae on three plates assayed simultaneously, except when insufficient larvae of the required genotype were obtained from individual crosses. In this case, data from separate experiments were added in chronological order to reach a total of ∼45 larvae. All experiments on larvae in LD were carried out between ZT3 and ZT6 and in DD between CT11.5 and CT13 (CT12) and CT23.5 and CT1 (CT24). For TrpA1 experiments, larvae were entrained to LD cycles at 20°C for 7 days, then moved to DD and tested on the second day in DD. Larvae were at 26°C for only the duration of the assay.

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