Many animals exhibit morning and evening peaks of locomotor behavior. In Drosophila, two corresponding circadian neural oscillators—M (morning) cells and E (evening) cells—exhibit a corresponding morning or evening neural activity peak. Yet we know little of the neural circuitry by which distinct circadian oscillators produce specific outputs to precisely control behavioral episodes. Here, we show that ring neurons of the ellipsoid body (EB-RNs) display spontaneous morning and evening neural activity peaks in vivo: these peaks coincide with the bouts of locomotor activity and result from independent activation by M and E pacemakers. Further, M and E cells regulate EB-RNs via identified PPM3 dopaminergic neurons, which project to the EB and are normally co-active with EB-RNs. These in vivo findings establish the fundamental elements of a circadian neuronal output pathway: distinct circadian oscillators independently drive a common pre-motor center through the agency of specific dopaminergic interneurons. Liang et al. describe neural outputs from the Drosophila circadian pacemaker network regulating locomotor rhythms: via a specific dopamine relay, the network forms parallel connections to the central complex, the pre-motor area dictating the balance between rest and activity.
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