Lack of Bmal1 leads to changes in rhythmicity and impairs motivation towards natural stimuli

Abstract

Maintaining proper circadian rhythms is essential for coordinating biological functions in mammals. This study investigates the effects of daily arrhythmicity using Bmal1-knockout (KO) mice as a model, aiming to understand behavioural and motivational implications. By employing a new mathematical analysis based on entropy divergence, we identified disrupted intricate activity patterns in mice derived by the complete absence of BMAL1 and quantified the difference regarding the activity oscillation's complexity. Changes in locomotor activity coincided with disturbances in circadian gene expression patterns. Additionally, we found a dysregulated gene expression profile particularly in brain nuclei like the ventral striatum, impacting genes related to reward and motivation. Further investigation revealed that arrhythmic mice exhibited heightened motivation for food and water rewards, indicating a link between circadian disruptions and the reward system. This research sheds light on how circadian clock alterations impact the gene expression regulating the reward system and how this, in turn, can lead to altered seeking behaviour and motivation for natural rewards. In summary, the present study contributes to our understanding of how reward processing is under the regulation of circadian clock machinery.

Keywords: Bmal1; circadian disruption; daily rhythms; natural reinforcer; reward system.

Figure 1.

Assessment of daily arrhythmicity of Bmal1-KO mice. (a). VAE values as a function of the number of Fourier components M used in their calculation. (b) Reconstruction of the time series of locomotor activity in wild-type and Bmal1-KO mice using only the first M* = 110 Fourier components (n = 11 mice per group). (c) Kronos analyses of locomotor activity data of WT and Bmal1-KO mice. Activity counts were collapsed to 1 h and mean activity along the seven recorded days was calculated for each individual animal. (d). Graphical representation of time-course gene expression data of Per2, Cry2, Rev-erba and Clock genes within the hypothalamus (n = 3 mice per group and time point). Graphs resulting from Kronos software show the cosine-fitted curves and SD from WT or Bmal1-KO mice. Period = 24 h, 12 h light (white zone), 12 h darkness (grey zone). ZT, Zeitgeber time, where ZT0 lights turn on. VAE, variation of accumulated entropy.

Figure 2.

Differentially expressed genes of Bmal1-KO mice compared to WT mice. Comparisons of the expression of 52 genes assessed in OpenArray analysis of mRNA isolated from (a) HT, (b) vSTR and (c) mPFC from WT mice (n = 6) and Bmal1-KO mice (n = 7). The volcano plot displays the relationship between fold change and significance between the two groups, applying a Student’s t‐test. The y-axis depicts the negative log¹⁰ of p-values of the t-tests (the horizontal slider corresponds to a p-value of 0.05) and the x-axis is the difference in expression between the two experimental groups as Ln (fold changes) (vertical sliders indicate mRNAs as either up-regulated (right area, purple dots) or down-regulated (left area, yellow dots) from a fold change (FC) of 1.3. The genes that exhibited both an absolute change greater than FC1.3 in either direction with a p‐value<0.05 were exposed as the differently expressed genes. HT, hypothalamus; mPFC, medial prefrontal cortex; vSTR, ventral striatum.

Figure 3.

Mice operant training and motivation tests for caloric and non-caloric rewards. (a) Schematic outline of the operant training behaviour process for food rewards. (b). Active and inactive nose-pokes during FR1 and FR3 for food reward (WT n = 22; Bmal1-KO n = 14). (c) Total pellets delivered through the 15 days of self-administration (SA) procedure. (d) Total active nose-pokes and breakpoint (last ratio reached) in the PR test for food. (e) Behavioural economic analysis of the demand task and their exponential curve representation of Log of demanded pellets as a function of price. Extra sum-of-squares, ***p < 0.001. (f) Schematic outline of the operant training behaviour process for water rewards. (g) Active and inactive nose-pokes during FR1 and FR3 for water reward (WT n = 14; Bmal1-KO n = 14). (h) Total water deliveries, obtained through the entire SA procedure. (i) Total active nose-pokes and breakpoint (last ratio reached) in the PR test in water SA. Data are represented as mean ± SEM. Two-way repeated measure ANOVA for each FR phase, ***p < 0.001. Student’s t‐test, *p < 0.05; **p < 0.01; ***p < 0.001. See the electronic supplementary material, table S2, for a detailed statistical analysis.

Figure 4.

Bmal1-KO mice show altered expression of genes related to food intake control and the reward system in the HT and vSTR, respectively. Gene expression analysis assessed by qPCR in basal conditions (WT n = 6; Bmal1-KO n = 6) in (a) HT and (b) vSTR; and after caloric restriction period (WT n = 15; Bmal1-KO n = 9) in (c) HT and (d) vSTR. Data are expressed as mean  ±  SEM. Student’s t‐test (*p < 0.05; **p < 0.01; ***p < 0.001). HT, hypothalamus; vSTR, ventral striatum.