The Zfhx3-Mediated Axis Regulates Sleep and Interval Timing in Mice

Abstract

An AT motif-dependent axis, modulated by the transcription factor Zfhx3, influences the circadian clock in mice. In particular, gain of function of Zfhx3 significantly shortens circadian rhythms and alters the transcriptional activity of an important class of neuropeptides that controls intercellular signaling in the suprachiasmatic nucleus (SCN) of the hypothalamus. The ZFHX3/AT axis revealed an important, largely cell-nonautonomous control of the circadian clock. Here, by studying the recently identified circadian mouse mutant Zfhx3(Sci/+), we identify significant effects on sleep homeostasis, a phenomenon that is outside the canonical circadian clock system and that is modulated by the activity of those neuropeptides at a circuit level. We show that the Zfhx3(Sci/+) mutation accelerates the circadian clock at both the hourly scale (i.e., advancing circadian rhythms) and the seconds-to-minutes scale (i.e., anticipating behavioral responses) in mice. The in vivo results are accompanied by a significant presence of sleep targets among protein-protein interactions of the Zfhx3(Sci/+)-dependent network.

Figure 1. Protein-Protein Interactions

(A) Zfhx3 and sleep genes. The blue nodes are differentially expressed Zfhx3 transcripts taken from Parsons et al. (2015). The red nodes are known mouse genes annotated with the “sleep” Gene Ontology term obtained from Amigo. The yellow ring nodes are differentially expressed transcripts containing a predicted AT motif. The circle highlights the densely connected subnetwork containing significant neuropeptides. (B) Results from the pathway analysis are represented by significance levels, from the most significant (top bar) to the least significant (bottom bar). The bar length represents the inverse logarithm of p values. Significant differences are indicated as follows: **p value < 0.01.

Figure 2. Sleep Homeostasis Is Altered in Zfhx3^Sci/+ Compared with Zfhx3^+/+ Mice

Each graph shows the time course of sleep parameters in 1-hr bins, across 48 hr. The first 24 hr represent the baseline of the experiment, followed by 6 hr of sleep deprivation and 18 hr of recovery. (A and B) The number of episodes of (A) NREM and (B) REM sleep are plotted in the first column. (C and D) In the second column, the graphs display the mean duration of the episodes of (C) NREM and (D) REM sleep. (E and F) The overall delta (E) and theta (F) power in NREM and REM sleep epochs are respectively shown. (G and H) The histograms represent total sleep, NREM, and REM sleep duration in Zfhx3^Sci/+ and Zfhx3^+/+ mice during baseline (G) conditions and during the first 6 hr of recovery (H) after sleep deprivation. Graphs are presented as the mean ± SEM. The white and black stripes in each graph indicate light and dark phases, respectively. Filled small black squares within graphs indicate statistically significant differences between Zfhx3^sci/+ mice and their littermate controls. Significant differences are indicated as follows: *p < 0.05.

Figure 3. Zfhx3^Sci/+ Mutant Mice Shorten Both Circadian Period of Peak Nose-Poking Activity and Short-Interval Responses over 24 hr

(A) Representative double-plotted actograms of nose-poking activity performed by Zfhx3^+/+ and Zfhx3^Sci/+ mice in home cages. Mouse nose-poking activity is collapsed into 15-min bins and is represented by black bars. Shaded regions indicate darkness in both the 12:12 light:dark (LD) and dark:dark (DD) conditions. (B) Periodicities in LD and DD are shown for Zfhx3^+/+ and Zfhx3^Sci/+ mice. (C) Representation of the short-interval task in home cages. The cartoon recapitulates how mice can initialize the trial by nose poking in the central hopper of an operant wall; after a FI, a nose poke at the left hopper is rewarded by the release of a food pellet with a probability 0.8. Unrewarded trials (probe trials) have probability of 0.2. (C₁) An example of a raster plot and the distribution of nose-poke activity across the fixed-interval target during unrewarded probe trials. Green and red dots in the raster plot represent the “starts” and “stops” of responses extracted from trial-by-trial nose-poking activity (see Experimental Procedures). (D) Temporal distribution of nose-poking responses in probe trials of Zfhx3^Sci/+ and Zfhx3^+/+ mice in LD and DD conditions. The FI target was 10 s. (E and F) Mean values and SEMs of starts, stops, and peaks (start + stop/2) in LD (E) and DD (F) conditions. Significant differences between the groups are derived via one-way ANOVA. Significant differences are indicated as follows: ***p < 0.0005; **p < 0.005; *p < 0.05.