Differentially Timed Extracellular Signals Synchronize Pacemaker Neuron Clocks

Synchronized neuronal activity is vital for complex processes like behavior. Circadian pacemaker neurons offer an unusual opportunity to study synchrony as their molecular clocks oscillate in phase over an extended timeframe (24 h). To identify where, when, and how synchronizing signals are perceived, we first studied the minimal clock neural circuit in Drosophila larvae, manipulating either the four master pacemaker neurons (LN(v)s) or two dorsal clock neurons (DN(1)s). Unexpectedly, we found that the PDF Receptor (PdfR) is required in both LN(v)s and DN(1)s to maintain synchronized LN(v) clocks. We also found that glutamate is a second synchronizing signal that is released from DN(1)s and perceived in LN(v)s via the metabotropic glutamate receptor (mGluRA). Because simultaneously reducing Pdfr and mGluRA expression in LN(v)s severely dampened Timeless clock protein oscillations, we conclude that the master pacemaker LN(v)s require extracellular signals to function normally. These two synchronizing signals are released at opposite times of day and drive cAMP oscillations in LN(v)s. Finally we found that PdfR and mGluRA also help synchronize Timeless oscillations in adult s-LN(v)s. We propose that differentially timed signals that drive cAMP oscillations and synchronize pacemaker neurons in circadian neural circuits will be conserved across species.