Genetic deletion of MT1 melatonin receptors alters spontaneous behavioral rhythms in male and female C57BL/6 mice

Abstract

Behaviors vary over the 24h light/dark cycle and these temporal patterns reflect in part modulation by circadian neural circuits and hormones, such as melatonin. The goal of this study was to investigate the involvement of MT1 melatonin receptors in behavioral regulation by comparing male and female C57 wild type (WT) mice with C57 mice with genetic deletion of the MT1 receptor (MT1KO). A comprehensive array of fifteen distinct spontaneous behaviors was recorded continuously in the homecage over multiple days using the HomeCageScan system. Behaviors assessed were activity-like (i.e. come down, hang, jump, walk), exploration-like (i.e. dig, groom, rear up, sniff, stretch), resting-like (i.e. awake, remain low, rest, twitch) and ingestion-like (i.e. drink, eat). Phenotypic array and temporal distribution analysis revealed distinct behavioral rhythms that differed between WT and MT1KO mice. The rhythms were consistent from day to day in males and varied with the estrous cycle in females. We also studied the role of MT1 receptors on depressive and anxiety-like behaviors. Genetic deletion of MT1 receptors increased immobility time in the forced swim test and decreased the number of marbles buried in the marble burying test in both male and female C57 mice. We conclude that MT1 melatonin receptors are involved in neural pathways modulating diurnal rhythms of spontaneous behavior in the homecage as well as pathways regulating depressive and anxiolytic-like behaviors.

Keywords: Behavioral rhythms; C57BL/6 mouse; Estrous cycle; Homecage; MT(1) receptor deletion.

Figure 1. Phenotypic arrays of daily behavior for male WT and MT₁KO mice

Phenotypic arrays illustrate the overall daily pattern of 15 spontaneous behaviors measured in the homecage over a four-day recording period for WT (A) and MT₁KO (B) male mice. Hourly mean values for each behavior (1-15) of WT (n=16) and MT₁KO (n=16) were scaled and plotted over a 24 hr. period; fold increase is indicated by the increase in color intensity from the baseline. The 14/10 light/dark cycle is indicated by the shading of the bar at the top of the arrays with the onset of dark at ZT14 and the onset of light at ZT0. Numbers 1 through 15 indicate, respectively, the behaviors of come down, hang, jump, walk, dig, groom, rear up, sniff, stretch, drink, eat, awake, remain low, rest and twitch.

Figure 2. Comparison of individual homecage behaviors of WT and MT₁KO male mice over the light/dark cycle

Behaviors indicative of Activity (A, E, I, M), Exploration (B, F, J, N), Ingestion (C, G, K, O) and Resting (D, H, L, P) are plotted individually over the light/dark cycle to compare WT (□) and MT₁KO (■) genotypes. The abscissae represents zeitgeber time (ZT) with ZT14 and ZT0 representing respectively the onset of the dark and the light periods. The shaded gray area represents the dark period. Hours spent in each behavior were plotted as a function of ZT and compared using repeated measure two-way ANOVA with genotype and time as main factors. The overall genotype effect on total magnitude of behavior is indicated by the p-value in the upper right corner of each graph. n.s. non-significant. The genotype effect at each ZT was determined by Bonferoni post-test. *p<0.05.

Figure 3. Phenotypic arrays of daily behavior for female WT and MT₁KO over the estrous cycle

Phenotypic arrays illustrate the overall daily pattern of 15 spontaneous behaviors measured in the homecage over a four-day recording period for WT (A) and MT₁KO (B) female mice. For each day, the stage of the estrous cycle was determined using vaginal cytology (A). Proestrus, estrus, metestrus and diestrus stages were characterized respectively by nucleated epithelial, cornified, leucocytes and cornified and leucocytes cells. Mice were grouped by genotype and estrous cycle stage and their behaviors analyzed. Hourly mean values for each behavior (1-15) were scaled and plotted over a 24 hr. period; fold increase is indicated by the increase in color intensity from the baseline. The 14/10 light/dark cycle is indicated by the shading of the bar at the top of the arrays with the onset of dark at ZT14 and the onset of light at ZT0. Numbers 1 through 15 indicate, respectively, the behaviors of come down, hang, jump, walk, dig, groom, rear up, sniff, stretch, drink, eat, awake, remain low, rest, and twitch.

Figure 4. Comparison of temporal variations in individual homecage behaviors of WT and MT₁KO female mice

Behaviors indicative of Activity (A, E, I, M), Exploration (B, F, J, N), Ingestion (C, G, K, O) and Resting (D, H, L, P) are plotted individually over the light/dark cycle at each stage of the estrous cycle. Each plot compares the pattern for WT (□) and MT₁KO (■) genotypes. The abscissae represents zeitgeber time (ZT) with ZT14 and ZT0 representing respectively the onset of the dark period and the light period. The shaded gray area represent the dark period. Hours spent in each behavior were plotted as a function of ZT and compared using repeated measure two-way ANOVA with genotype and time as main factors. The overall genotype effect on total magnitude of behavior is indicated by the p-value in the upper right corner of each graph. n.s. non-significant. The genotype effect at each ZT was determined by Bonferoni post-test. *p<0.05.

Figure 5. Effects of MT₁ receptor deletion on the forced swim test and the marble burying test in male and female mice

Male (A) and female (B) WT and MT₁KO mice were recorded during the Porsolt forced swim test to assess antidepressant-like activity. Bars indicate the amount of time that mice were immobile over a 4 min period for males (WT: 181.5 ± 4.48 s, n=10 and MT₁KO: 202.7 ± 4.71 s, n=14; d=1.33) and females (WT: 173.9 ± 8.00 s, n=14 and MT₁KO: 204.0 ± 6.69s, n=14; d=1.10). The marble burying test was used to assess anxiolytic-like activity in male (C) and female (D) WT and MT₁KO mice. The number of marbles buried in the bedding during a 30 min period is shown for males (WT: 15.10 ± 0.88 marbles, n=10 and MT₁KO: 8.308 ± 1.64 marbles, n=13; d=1.57).and females (WT: 12.34 ± 0.38 marbles, n=16 and MT₁KO: 2.967 ± 0.82 marbles, n=15; d=3.97). All tests were performed during the light period between ZT9 and ZT11,