Circadian pacemaker neurons in Drosophila are regulated by two synchronizing signals that are released at opposite times of day, generating a rhythm in intracellular cyclic AMP.

Long term synaptic plasticity requires transcription in response to changes in neuronal activity. While genes induced by neuronal activity have been extensively studied, genes induced by hyperpolarization are largely unknown. We focused on Pura, a Rho1 GEF whose rhythmic expression drives the daily retraction of the projections of Drosophila LN v circadian pacemaker neurons. We found that Pura transcription is repressed by activity and induced by hyperpolarization in LN v s -the opposite of typical activityregulated genes. Pura is repressed by activity-regulated transcriptional factors including Mef2 and Sr (fly Egr-1) and activated by Toy, a Pax6 transcription factor. toy transcription is also induced by inactivity. Thus toy and Pura represent a class of genes induced by hyperpolarization. Highlights• Hyperpolarization activates transcription of Pura, a plasticity gene • This phenomenon occurs in circadian pacemaker neurons and mushroom body neurons • The Pura enhancer integrates recent neuronal activity to regulate transcription • Hyperpolarization activates transcription of toy (Pax-6), which then activates Pura However, neuronal plasticity also includes reducing neuronal excitability and weakening or losing connections. These aspects of neuronal plasticity are probably important in forgetting memories and also in sleep, in which widespread synaptic downscaling has been proposed to reduce overall connectivity in the brain (de Vivo et al., 2017; Diering et al., 2017). However, relatively few molecular mechanisms have so far been described to explain how neurons decrease their connectivity and the role of gene expression in this phenomenon has been largely ignored. One example of how gene expression can weaken neuronal connections comes fromDrosophila circadian pacemaker neurons, which drive ~24hr rhythms in behavior. A subset of pacemaker neurons -the 4 small ventral lateral neurons (s-LN v s) in each brain hemisphere -show predictable and intrinsic daily rhythms in their excitability (Cao and