Vasoactive intestinal polypeptide requires parallel changes in adenylate cyclase and phospholipase C to entrain circadian rhythms to a predictable phase

Abstract

Circadian oscillations in the suprachiasmatic nucleus (SCN) depend on transcriptional repression by Period (PER)1 and PER2 proteins within single cells and on vasoactive intestinal polypeptide (VIP) signaling between cells. Because VIP is released by SCN neurons in a circadian pattern, and, after photic stimulation, it has been suggested to play a role in the synchronization to environmental light cycles. It is not known, however, if or how VIP entrains circadian gene expression or behavior. Here, we tested candidate signaling pathways required for VIP-mediated entrainment of SCN rhythms. We found that single applications of VIP reset PER2 rhythms in a time- and dose-dependent manner that differed from light. Unlike VIP-mediated signaling in other cell types, simultaneous antagonism of adenylate cyclase and phospholipase C activities was required to block the VIP-induced phase shifts of SCN rhythms. Consistent with this, VIP rapidly increased intracellular cAMP in most SCN neurons. Critically, daily VIP treatment entrained PER2 rhythms to a predicted phase angle within several days, depending on the concentration of VIP and the interval between VIP applications. We conclude that VIP entrains circadian timing among SCN neurons through rapid and parallel changes in adenylate cyclase and phospholipase C activities.

Fig. 1.

VIP phase shifts Period (PER)2::LUCIFERASE (LUC) rhythms in the suprachiasmatic nucleus (SCN). A: representative PER2::LUC traces with 10 μM VIP (black trace) or vehicle (gray trace) treatment at CT12 (arrow). Each PER2::LUC rhythm was normalized to the peak before treatment. B: single-plotted actograms of the bioluminescence traces from A. The acrophase (black circle) of PER2::LUC expression was the daily peak of a sine function fit to each cycle's bioluminescence trace (Herzog et al. 2004). Phase shifts were measured as the time difference between linear fits to the acrophases before and after the treatment. C: dose-dependent phase delays induced by VIP applied at CT12. Above 100 nM VIP and below 10 μM VIP, the delay of the PER2::LUC rhythm increased linearly with logarithmic increases in the VIP concentration. Data were fitted with a logistic function (black line). D: phase-response curves (PRCs) for 10 μM or 100 nM VIP (n = 63 and n = 33, respectively) as a function of circadian time of VIP application. Phase delays and advances are shown as negative and positive values, respectively. The PRC for 10 μM VIP was fitted with a fast Fourier transform and adjacent-point average (line). Note that the PRC is dominated by delays and unlike the PRC to light. E: concentration-response curve for the transient induction of PER2 by VIP. Data were fitted with a logistic function (black line).

Fig. 2.

Blockade of cAMP and phospholipase C (PLC) signaling was required to reduce VIP-induced phase shifts. A–C: representative actograms of PER2::LUC rhythms with treatment of 1 μM VIP alone (A), VIP with 2 μM MDL-12,300a [MDL; inhibitor of adenylate cyclase (AC)] + 10 μM U-73122 (inhibitor of PLC) (B), and VIP with 100 μM 9-(tetrahydro-2furyl)-adenosine (THFA; inhibitor of AC) + 10 μM U-73122 (C), respectively. The inhibitor cocktails were applied 1 h before the VIP application at CT12. D: the phase shift (means ± SE) of PER2::LUC rhythms by VIP applied at CT12 was significantly attenuated by the combination of AC and PLC inhibitors (*P < 0.05 and **P < 0.01, one-way ANOVA with Scheffé post hoc test). E: in the absence of VIP, the inhibitors induced little to no phase shifts (n = 25, compared with vehicle-treated cultures, P > 0.05). The number of cultures recorded is shown for each treatment.

Fig. 3.

VIP increased cAMP in SCN neurons. A: a representative SCN neuron expressing the ICUE2 reporter (inset image) showed a reduced Förster resonance energy transfer (FRET) ratio (535/480 nm) after treatment with forskolin (FSK; 20 μM) and 3-isobutyl-1-methylxanthine (IBMX; 75 μM), indicative of increased intracellular cAMP. Data were normalized to the ratio at the initiation of treatment. B: treatment with VIP (1 μM) reduced the FRET ratio in SCN neurons. Each line represents a different neuron. C: pretreatment (2 min) with THFA (100 μM) attenuated the VIP (1 μM)-induced reduction in the FRET ratio. D: plot of mean ± SE responses to VIP (black, n = 10; see B) and THFA + VIP (gray, n = 11; see C) showing that THFA treatment significantly reduced cAMP induction by VIP (unpaired two-tailed t-test, *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.005).

Fig. 4.

VIP entrained PER2::LUC rhythms. A–C: representative actograms of SCN PER2::LUC rhythms during 5 consecutive days of treatment (▾) with either vehicle (A), 10 nM VIP (B), or 25 nM VIP (C). Note that the daily peak of PER2::LUC rhythms shifted to follow the daily VIP pulse by ∼10 h and free ran from that phase after the last VIP treatment. D: the daily peak of PER2::LUC rhythms (means ± SE) of SCN cultures treated with VIP or vehicle indicated that VIP entrained circadian rhythms in the cultured SCN (vehicle, n = 2; 10 nM VIP, n = 3; and 25 nM VIP, n = 3). Similar results were found in two additional replications of this experiment. E: representative Rayleigh plots showing the phase angle of PER2::LUC expression (gray triangles) from cultures treated with vehicle (n = 7), 10 nM VIP (n = 6), or 25 nM VIP (n =7) on the fifth day of treatment. The time between the peak phase of the SCN cultures and the time of treatment is the phase angle of entrainment. Note that cultures treated with VIP had similar times of peak PER2::LUC bioluminescence, whereas vehicle-treated cultures did not tend to peak at similar times. F: the daily peak of PER2::LUC rhythms (means ± SE) over 10 days showed that SCN cultures (▵) synchronized their phase and period to 25 nM VIP applied every 25 h (▾) for 5 consecutive days and free ran (○) when treated with vehicle.