Vasoactive intestinal polypeptide mediates circadian rhythmicity and synchrony in mammalian clock neurons

Abstract

The mammalian suprachiasmatic nucleus (SCN) is a master circadian pacemaker. It is not known which SCN neurons are autonomous pacemakers or how they synchronize their daily firing rhythms to coordinate circadian behavior. Vasoactive intestinal polypeptide (VIP) and the VIP receptor VPAC(2) (encoded by the gene Vipr2) may mediate rhythms in individual SCN neurons, synchrony between neurons, or both. We found that Vip(-/-) and Vipr2(-/-) mice showed two daily bouts of activity in a skeleton photoperiod and multiple circadian periods in constant darkness. Loss of VIP or VPAC(2) also abolished circadian firing rhythms in approximately half of all SCN neurons and disrupted synchrony between rhythmic neurons. Critically, daily application of a VPAC(2) agonist restored rhythmicity and synchrony to VIP(-/-) SCN neurons, but not to Vipr2(-/-) neurons. We conclude that VIP coordinates daily rhythms in the SCN and behavior by synchronizing a small population of pacemaking neurons and maintaining rhythmicity in a larger subset of neurons.

Figure 1

Mice with disrupted VIP/VPAC₂ signaling express multiple circadian periods. (a,c) Double-plotted actograms showing representative wheel-running activity of wild-type (C57Bl/6), Vip^−/− and Vipr2^−/− mice in a light/dark schedule (days 1–10), a skeleton photoperiod (days 11–20), and constant darkness (days 20–90). Gray shading indicates the time when lights were on. VIP and VPAC₂ mutant mice appeared normal while on a 12 h/12 h light/dark schedule but were active during both dark phases each day in a skeleton photoperiod, whereas wild-type mice were strictly nocturnal. (b,d) Peaks in the corresponding χ² periodogram for each animal show the dominant and secondary period(s) during days 21–90 in constant darkness. Peaks above the diagonal line (indicating the 99.9% confidence level) between 16 and 34 h were considered significant circadian periods. Note that mutants in a and b (representing two-thirds of mutants of both genotypes) expressed multiple circadian periods, whereas those in c and d (representing one-third of mutants) showed more coherent circadian behavior, with a single, shorter, free-running period. (e) Mice of all three genotypes expressed significant circadian periodicity. The percentage of mice expressing a single circadian period (gray) was higher in wild-type mice than in the two mutant genotypes, whereas a large percentage of mutant mice expressed multiple circadian periods (black). (f) The circadian amplitude of rhythms was diminished in both mutants relative to wild-type. Bars show circadian amplitudes (mean ± s.e.m.) at the dominant period as determined by χ² periodogram analysis. Asterisk (*) indicates P < 0.00001, one-way ANOVA with Scheffé post hoc test.

Figure 2

A reduced proportion of Vip^−/− and Vipr2^−/− SCN neurons fires rhythmically in vitro. (a) Representative firing rate traces showing examples of circadian and arrhythmic neurons from each genotype. The χ² periodogram to the left of each plot shows the period estimation of each neuron as in Figure 1. (b) The percentage of SCN neurons with a circadian firing pattern was reduced for both mutants relative to wild-type. (c) The circadian amplitudes (mean ± s.e.m.) of mutant rhythms were lower than in the wild type. Asterisk (*) indicates P < 0.0005, one-way ANOVA with Scheffé post hoc test.

Figure 3

Genetic knockout of Vip or Vipr2 abolishes synchrony among rhythmic SCN neurons in the same culture (a) Representative rhythmic firing rate records from wild-type, Vipr2^−/− or Vip^−/− SCN neurons in the same high density culture. Rhythmic neurons in the wild-type culture expressed robust rhythms with similar periods and phases, whereas those in the mutant cultures had variable periods and phases. (b) Relative circadian phases of all rhythmic neurons (n = 24 wild-type, 19 Vip^−/− and 26 Vipr2^−/−) from the same three cultures as in a. Data are plotted within circles that represent the fourth day of recording. Arrows indicate the average phase of each distribution. Arrow length reflects the r-value of each phase distribution. Wild-type neurons showed statistically significant synchronization (Rayleigh test, P < 0.05, r = 0.36), but Vipr2^−/− and Vip^−/− neurons did not (P > 0.7 and P > 0.9, r = 0.09 and 0.05, respectively). (c) Distribution of periods for the same neurons as in b. Vipr2^−/− and Vip^−/− SCN neurons showed a significantly broader distribution than did wild-type neurons in the same culture (P < 0.05, Brown-Forsythe's and Levene's tests for equal variance).

Figure 4

The distributions of circadian periods of locomotor activity in mice (left) are similar to those of firing rate rhythms in SCN neurons (right) for the three genotypes. Although the average dominant periods (black bars) of Vip^−/− and Vipr2^−/− mutant mice were shorter than those of wild-type mice (P < 0.05, one-way ANOVA with Scheffé post hoc test), the average of all significant circadian periods (gray bars) was similar between genotypes (P > 0.7, Kruskal-Wallis one-way ANOVA with Scheffé post hoc test). Similarly, the average periods of SCN neuronal rhythms did not change with genotype (P > 0.1, Kruskal-Wallis one-way ANOVA). The distributions of all behavioral and firing rate periods were significantly broader in the mutant mice and neurons (P < 0.005 for behavior and P < 0.00005 for neurons, Brown-Forsythe's and Levene's tests for equal variance), indicating a loss of circadian synchrony.

Figure 5

Daily application of VPAC₂ agonist Ro 25-1553 restores rhythmicity to Vip^−/− SCN neurons. (a) Firing rate records of three initially arrhythmic Vip^−/− neurons and a rhythmic neuron treated with 150 nM Ro 25-1553 every 24 h for 6 d (arrows). The top two traces are from representative neurons in which rhythmicity was restored, and the bottom trace is from a neuron that remained arrhythmic. The third trace is from a representative rhythmic neuron entrained by agonist treatment. Rhythmicity persisted for at least 48 h after the last treatment, indicating that cyclic firing was entrained by drug application. (b) Firing patterns of rhythmic neurons, averaged over the last 4 d of treatment, increased in anticipation of the drug treatment, indicating that the daily change in firing rate was not simply an acute response to the agonist. (c) Relative phases of initially rhythmic (n = 16, left) and arrhythmic neurons with restored rhythms (n = 22, right) from a representative Vip^−/− culture on the sixth day of Ro 25-1553 treatment. Arrowheads at hour 6 denote the time of agonist application. Both subsets of neurons showed statistically significant synchronization (Rayleigh test, P < 0.05, r = 0.43 and 0.39 for rhythmic and arrhythmic neurons, respectively) with the mean bathyphase of firing (arrows) 10–12 h after agonist application. (d) The broad distribution of periods for rhythmic Vip^−/− neurons before treatment (left) was significantly narrowed during agonist treatment (right; P < 0.005, Brown-Forsythe's and Levene's tests). (e) The relative proportion of rhythmic (black) to arrhythmic (white) neurons from Vip^−/− cultures was restored to wild-type levels by daily VPAC₂ agonist treatment, but there was little change in the proportion of rhythmic Vipr2^−/− neurons with the same treatment.