A Role for Blind DN2 Clock Neurons in Temperature Entrainment of the Drosophila Larval Brain

Abstract

Circadian clocks synchronize to the solar day by sensing the diurnal changes in light and temperature. In adult Drosophila, the brain clock that controls rest-activity rhythms relies on neurons showing Period oscillations. Nine of these neurons are present in each larval brain hemisphere. They can receive light inputs through Cryptochrome (CRY) and the visual system, but temperature input pathways are unknown. Here, we investigate how the larval clock network responds to light and temperature. We focused on the CRY-negative dorsal neurons (DN2s), in which light-dark (LD) cycles set molecular oscillations almost in antiphase to all other clock neurons. We first showed that the phasing of the DN2s in LD depends on the pigment-dispersing factor (PDF) neuropeptide in four lateral neurons (LNs), and on the PDF receptor in the DN2s. In the absence of PDF signaling, these cells appear blind, but still synchronize to temperature cycles. Period oscillations in the DN2s were stronger in thermocycles than in LD, but with a very similar phase. Conversely, the oscillations of LNs were weaker in thermocycles than in LD, and were phase-shifted in synchrony with the DN2s, whereas the phase of the three other clock neurons was advanced by a few hours. In the absence of any other functional clock neurons, the PDF-positive LNs were entrained by LD cycles but not by temperature cycles. Our results show that the larval clock neurons respond very differently to light and temperature, and strongly suggest that the CRY-negative DN2s play a prominent role in the temperature entrainment of the network.

Pacemaker-neuron–dependent disturbance of the molecular clockwork by a Drosophila CLOCK mutant homologous to the mouse Clock mutation

“…In control p{dClk-WT};Clk out flies, TIM levels exhibited a large amplitude of oscillation in each subset of pacemaker neurons, with the exception of lLN v s peaking at ZT14 (Fig. 8 E-J), which is earlier than observed under photic cycle conditions (17,19,21,48). In p{dClk-Δ};Clk out flies, TIM levels in both lLN v s and sLN v s were greatly reduced compared with those TIM levels in p{dClk-WT};Clk out flies (Fig.…”

“…Therefore, it might be that DN-generated rhythmic outputs are suppressed in p{dClk-Δ};Clk out flies, resulting in arrhythmic behaviors under the standard (high-intensity light) photic entrainment condition used in this study. It is also important to note that several lines of evidence support the idea that DNs play important roles to control circadian behaviors in temperature entrainment conditions (18)(19)(20)(21). Consistently, in p{dClk-Δ};Clk out flies, relatively intact molecular clockwork in DNs deduced from strong expression of dCLK target genes entrains to the temperature cycle, but with phase delay, and generates the delayed anticipatory behavior of temperature transition, ultimately contributing to improved free-running rhythms compared with the rhythms under photic cycle entrainment conditions.…”

“…When photic and temperature cues are both present, LN v s and LN d s are sensitive to photic transition, whereas DN 1 s, DN 2 s, DN 3 s, and lateral posterior neurons are more sensitive to temperature transitions (18). In addition, blue-light photopigment CRYnegative DN 1 and CRY-null DN 2 are known to play a prominent role in entrainment to temperature cycles (18)(19)(20)(21).…”

Identifying Specific Light Inputs for Each Subgroup of Brain Clock Neurons inDrosophilaLarvae

“…First, PDF sets the DN1s and DN2s to very different phases: the DN2s are set in antiphase with the LNs (Kaneko et al, 1997;Picot et al, 2009), whereas the DN1s are set in phase with the LNs. This suggests that the corresponding signaling cascades differ somewhere downstream from the PDF receptor.…”

“…All experiments, performed at least twice with similar results, were done on whole-mounted third-instar larval brains, which were dissected and labeled as previously described (Malpel et al, 2002). Antibodies were as previously described (Malpel et al, 2002;Picot et al, 2009), except for the use of the GP47 guinea pig anti-CLOCK (Houl et al, 2006), at 1:5000 dilution, or a guinea-pig anti-PDP1 used at 1:10,000 dilution (Benito et al, 2007). Although GP47 cross-reacts with DACHSHUND (Houl et al, 2008), the clock neurons were on average more strongly stained, and could also be distinguished based on size, shape, and location (see Figs.…”