To independently test a role for glutamate in the generation of adult rhythms in DD, we misexpressed Gad1, as in larvae ( Figure 5), to reduce presynaptic glutamate. This specifically affects glutamate levels because no adult clock neurons are GABAergic ( Dahdal et al., 2010 and Hamasaka et al., 2005). tim-Gal4; Pdf-Gal80 > Gad1 flies had lower power rhythms than control flies, whereas tim-Gal4; cry-Gal80 > Gad1 flies had robust DD rhythms ( Figures 7D–7F and Table 1). Thus, two independent manipulations of glutamate signaling indicate that glutamate released from CRY+ non-LNv clock neurons is required for

robust locomotor activity rhythms. However, the rhythms of tim > + VGlutRNAi and tim-Gal4; Pdf-Gal80 > Gad1 flies are both stronger AZD2281 cell line than tim-Gal4; Pdf-Gal80

> dORKΔC flies, suggesting that additional signals from non-LNvs contribute to rhythmic behavior. This interpretation makes sense given the diversity of Drosophila adult clock neurons and the incomplete arrhythmicity of even mutants in Pdf, the major circadian neuropeptide ( Renn this website et al., 1999). Taking all of the adult data together, we find evidence that the principles we identified in the larval circadian network may also operate in adult flies. Specifically, our broad manipulations to adult non-LNv clock neurons indicate that non-LNv signals (1) are important for strong adult rhythms, (2) may gate LNv outputs to shape activity at dawn, and (3) include glutamate. We identified some of the network logic Suplatast tosilate that helps generate a simple rhythmic behavior through precise genetic manipulations

of the larval circadian circuit and extended these findings to the more complex adult circadian network. Previous studies have shown that intercellular signaling in clock neuron networks promotes molecular clock synchrony (Lin et al., 2004, Maywood et al., 2006 and Stoleru et al., 2005) and can strengthen genetically weak molecular clocks (Liu et al., 2007). Our study increases the importance of circadian neural networks by finding that non-LNv clock neurons are as important as the “master” pacemaker LNv clock neurons for rhythmic behavior both in larvae and adult flies. However, LNvs can still be considered pacemakers in DD because most manipulations to non-LNv clock neurons do not affect period length. Non-LNv signals appear to gate pacemaker neuron activity. Why is this necessary when LNvs have their own intrinsic excitability rhythms? We propose that the interaction of two oscillators with opposite signs helps reduce the time when LNvs signal. Without signaling from non-LNvs, adult locomotor activity rhythms are weak and activity is distributed throughout the day and night as in tim-Gal4; Pdf-Gal80 > dORKΔC flies. In contrast, in tim-Gal4; Pdf-Gal80 > NaChBac flies, the timing of locomotor activity is narrowed.