Electrical silencing of PDF neurons advances the phase of non-PDF clock neurons in Drosophila

Drosophila clock neurons exhibit self-sustaining cellular oscillations that rely in part on rhythmic transcriptional feedback loops. We have previously determined that electrical silencing of the pigment dispersing factor (PDF)expressing lateral-ventral (LNV) pacemaker subset of fly clock neurons via expression of an inward-rectifier K+ channel (Kir2.1) severely disrupts free-running rhythms of locomotor activity - most flies are arrhythmic and those that are not exhibit weak short-period rhythms - and abolishes LNV molecular oscillation in constant darkness. PDF is known to be an important LNV output signal. Here we examine the effects of electrical silencing of the LNV pacemakers on molecular rhythms in other, nonsilenced, subsets of clock neurons. In contrast to previously described cell-autonomous abolition of free-running molecular rhythms, we find that electrical silencing of the LNV pacemakers via Kir2.1 expression does not impair molecular rhythms in LND, DN1, and DN2 subsets of clock neurons. However, free-running molecular rhythms in these non-LNV clock neurons occur with advanced phase. Electrical silencing of LN(V)s phenocopies PDF null mutation ( pdf(01)) at both behavioral and molecular levels except for the complete abolition of free-running cellular oscillation in the LN(V)s themselves. LNV electrically silenced or pdf(01) flies exhibit weak free-running behavioral rhythms with short period, and the molecular oscillation in non-LNV neurons phase advances in constant darkness. That LNV electrical silencing leads to the same behavioral and non-LNV molecular phenotypes as pdf(01) suggests that persistence of LNV molecular oscillation in pdf(01) flies has no functional effect, either on behavioral rhythms or on non- LNV molecular rhythms. We thus conclude that functionally relevant signals from LN(V)s to non- LNV clock neurons and other downstream targets rely both on PDF signaling and LNV electrical activity, and that LN(V)s do not ordinarily send functionally relevant signals via PDF-independent mechanisms.