Bmal1 Modulates Striatal cAMP Signaling and Motor Learning

Abstract

The circadian rhythm shapes behavioral processes by providing temporal cues for molecular regulation and adaptation in the hypothalamus of the brain. Deeper yet in the striatum of the brain, circadian rhythm also exerts an impact, conditioning diurnal patterns in neurodegenerative-related motor dysfunction. While motor properties are clearly linked to striatal dopamine, the interplay between the circadian rhythm with the key circadian transcription factor Bmal1 and dopamine signal decoding remains unknown. Here, we utilized both sexes of global and local striatal Bmal1 knock-out mice to investigate changes in dopamine-mediated cAMP signaling and motor behavior. By conducting a 24 h time-course study, we first established Bmal1-dependent molecular signatures in striatal dopamine signaling machinery that correlated with cAMP levels. Next, recording real-time signal transduction with a two-photon FRET biosensor in brain slices revealed diminished efficacy of dopamine signaling in the absence of Bmal1. As a final functional outcome, we then found that striatal Bmal1 was necessary for motor learning in mice. Altogether, our data support a strong connection between striatal Bmal1 and dopamine signaling with potential impact in brain-related motor function.

Keywords: Bmal1; GPCR; adenylyl cyclase; cAMP; dopamine; striatum.

Figure 1.

Profiling Bmal1 influence on cAMP signaling components in the mouse striatum. A, Western blot quantification of Bmal1 in dissected striata from Bmal1^+/+, Bmal1^+/−, and Bmal1^−/− mice. Coomassie stain of PVDF membrane used for normalization. n = 4 mice; nonparametric t test, Mann–Whitney U = 7. B, Representative Western blot from mouse striatum of Bmal1^+/− or Bmal1^−/− for Bmal1, Per1, Per2, Gαi, Gβ, AC5, and PDE10A. Samples taken from mice at indicated zeitgeber time (ZT) of light cue (top). Representative Coomassie stain of PVDF membrane used for normalization (bottom). C, Quantification of Western blot data. Data normalized to ZT 0. n = 3 mice per group per ZT. D, Quantification of protein expression at indicated zeitgeber time (ZT). Both groups normalized to ZT 0. (n = 3 per group per ZT, one-way ANOVA followed by Tukey posttest for multiple comparisons).

Figure 2.

Bmal1 influence on total cAMP level in the mouse striatum. A, ELISA determination of total cAMP level in the striatum at indicated zeitgeber time (ZT) of light cue from Bmal1^+/− and Bmal1^−/− mice (n = 4 mice/genotype). B, Quantification of cAMP at indicated zeitgeber time (ZT) of light cue from Bmal1^+/− and Bmal1^−/− mice (n = 4 mice/genotype, one-way ANOVA followed by Tukey posttest for multiple comparisons). C, Acute brain slices (300 micron) were treated with vehicle (DMSO) for 10 min followed by ELISA determination of total cAMP from striatal punches in Bmal1^+/− and Bmal1^−/− mice at indicated zeitgeber time (ZT) of light cue (n = 5 mice/genotype; one-way ANOVA followed by Tukey posttest for multiple comparisons). D, Acute brain slices (300 micron) were treated with IBMX (50 micromolar) for 10 min followed by ELISA determination of total cAMP from striatal punches in Bmal1^+/− and Bmal1^−/− mice at indicated zeitgeber time (ZT) of light cue (n = 5 mice/genotype; one-way ANOVA followed by Tukey posttest for multiple comparisons). E, Acute brain slices (300 micron) were treated with forskolin (10 micromolar) for 10 min followed by ELISA determination of total cAMP from striatal punches in Bmal1^+/− and Bmal1^−/− mice at indicated zeitgeber time (ZT) of light cue (n = 5 mice/genotype; one-way ANOVA). Unless otherwise indicated, all data are presented as mean ± SEM.

Figure 3.

Bmal1 KO reduces motor learning. A, Schematic of motor learning analysis on an accelerating rotarod in Bmal1^+/− and Bmal1^−/− mice. B, Daily accelerating rotarod performance for Bmal1^+/− and Bmal1^−/− mice at zeitgeber time 0 (ZT0) of light cue (left). Mean performance over 5 d (right). n = 5 mice/genotype. C, Daily accelerating rotarod performance for Bmal1^+/− and Bmal1^−/− mice at zeitgeber time 8 (ZT8) of light cue (left). Mean performance over 5 d (right). n = 9 (Het) and 5 (KO) mice. D, Comparison of average latency to fall from the first day in Bmal1^+/− and Bmal1^−/− mice. ZT0: nonparametric t test, p = 0.0079, Mann–Whitney U = 0. ZT8: nonparametric t test, p = 0.0010, Mann–Whitney U = 0. E, Comparison of average latency to fall from the last day in Bmal1^+/− and Bmal1^−/− mice. ZT0: nonparametric t test, p = 0.2222, Mann–Whitney U = 6. ZT8: nonparametric t test, p = 0.0010, Mann–Whitney U = 0. F, Quantification of accelerating rotarod learning rate in Bmal1^+/− and Bmal1^−/− mice. ZT0: nonparametric t test, p = 0.8413, Mann–Whitney U = 11. ZT8: nonparametric t test, p = 0.0190, Mann–Whitney U = 5. All data are presented as mean ± SEM.

Figure 4.

Profiling striatal-specific Bmal1 influence on cAMP signaling components. A, Bmal1^cKO/cKO mice were bilaterally injected with either AAV-dTomato (Control) or AAV-Cre-P2A-dTomato (striatal knockdown; Stri KD) in the dorsal striatum. B, Western blot from striatal tissue punch to quantify levels of Bmal, Per1, Per2, Gαi, Gβ, AC5, and PDE10A. Samples were taken from mice at indicated ZT. Coomassie stain of PVDF membrane used for normalization. Where applicable, arrow indicates correct size of protein band. C, Quantification of Western blot data between Control and Stri KD normalized to ZT0. D, Quantification of protein expression at indicated zeitgeber time (ZT) normalized to Control at ZT0 (n = 3 mice Stri KD, n = 3 mice Control per ZT, one-way ANOVA followed by Tukey posttest for multiple comparisons).

Figure 5.

Striatal-specific knockdown of Bmal1 blunts motor learning. A, Daily accelerating rotarod performance for Control and Stri KD Bmal1^cKO/cKO mice at zeitgeber time 0 (ZT0) over 5 d. n = 6 mice/group. B, Daily accelerating rotarod performance over 5 d for Control and Stri KD Bmal1^cKO/cKO mice at zeitgeber time 8 (ZT8) of light cue. n = 7 mice/group. C, Quantification of accelerating rotarod learning rate Control and Stri KD Bmal1^cKO/cKO mice at zeitgeber time 0 (ZT0) (n = 6 mice/genotype, nonparametric t test, p = 0.6991, Mann–Whitney U = 15) and zeitgeber time 8 (ZT8) of light cue (n = 6 mice/group, nonparametric t test, p = 0.0006, Mann–Whitney U = 0).

Figure 6.

Bmal1 dominant negative influences AC5 level and motor learning. A, Wild-type mice were bilaterally injected with either AAV-Cre-P2A-Bmal1-WT (WT) or AAV-Cre-P2A-Bmal1-DN (DN) in the dorsal striatum. B, Western blot from striatal tissue punch to quantify levels of Bmal, Per1, Per2, Gαi, Gβ, AC5, and PDE10A. Samples were taken from mice at ZT8. Coomassie stain of PVDF membrane used for normalization. Where applicable, arrow indicates correct size of protein band. C, Quantification of Western blot data between Bmal1-WT and Bmal1-DN. Unpaired t test, n = 3 mice WT, n = 4 mice DN. D, Daily accelerating and mean rotarod performance for Bmal-1WT and Bmal1-DN mice at ZT8 over 5 d. n = 8 mice DN, n = 5 mice WT. E, Quantification of accelerating rotarod learning rate Bmal1-WT and Bmal1-DN mice at ZT8 (n = 8 mice DN, n = 5 mice WT, nonparametric t test, p = 0.0016, Mann–Whitney U = 0.).

Figure 7.

Bmal1 regulates magnitude of cAMP signaling in the dorsal striatum. A, Schematic of experimental design to record dorsal striatal cAMP in acute brain slices of CAMPER mice by injection of AAV-Cre-P2A-Bmal1-WT or AAV-Cre-P2A-Bmal1-DN. Representative image of ^TEpac^VV biosensor in striatal neurons. Scale bar represents 100 μm. B, Representative cAMP response of striatal neurons to bath application of IBMX (50 micromolar). Quantification of cAMP amplitude from Bmal1-WT (n = 33 neurons/5 animals) and Bmal1-DN (n = 30 neurons/5 animals): nonparametric t test, p = 0.4580, Mann–Whitney U = 440.5. C, Representative cAMP response of striatal neurons to bath application of forskolin (10 micromolar). Quantification of cAMP amplitude from Bmal1-WT (n = 31 neurons/5 animals) and Bmal1-DN (n = 26 neurons/5 animals): nonparametric t test, p = 0.0019, Mann–Whitney U = 212. All data are presented as mean ± SEM.

Figure 8.

Characterization of dopamine-induced cAMP responses in D1R+ and D2R+ striatal neurons. A, Schematic of CAMPER mouse crossed with Drd1aCre line to achieve biosensor expression in D1R+ neurons in acute brain slices. B, Response following dopamine (10 micromolar) application to acute brain slices from Drd1aCre-CAMPER mice (data from 3 animals). Average traces obtained after application of response amplitude cutoff criterion (2 multiplied by standard deviation of the baseline). C, Response following dopamine (10 micromolar) and SCH23390 (1micromolar) application to acute brain slices from Drd1aCre-CAMPER mice (data from 3 animals). Average traces obtained after application of response amplitude cutoff criterion (2 multiplied by standard deviation of the baseline). D, Schematic of CAMPER mouse crossed with Drd2Cre line to achieve biosensor expression in D2R+ neurons in acute brain slices. E, Response following dopamine (10 micromolar) application to acute brain slices from Drd2Cre-CAMPER mice (data from 3 animals). Average traces obtained after application of response amplitude cutoff criterion (2 multiplied by standard deviation of the baseline). F, Response following dopamine (10 micromolar) and sulpiride (1 micromolar) application to acute brain slices from Drd1aCre-CAMPER mice (data from 3 animals). Average traces obtained after application of response amplitude cutoff criterion (2 multiplied by standard deviation of the baseline). G, Schematic of CAMPER mouse injected with AAV-Cre-P2A-dTomato to achieve biosensor expression in striatal neurons in acute brain slices. H, Response following dopamine (10 micromolar) application to acute brain slices from AAV-Cre-P2A-dTomato injected CAMPER mice (data from 4 animals). Average traces obtained after application of response amplitude cutoff criterion (2 multiplied by standard deviation of the baseline). I, Response following dopamine (10 micromolar) and SCH23390 (1 micromolar) application to acute brain slices from AAV-Cre-P2A-dTomato injected CAMPER mice (data from 4 animals). Average traces obtained after application of response amplitude cutoff criterion (2 multiplied by standard deviation of the baseline). J, Response following dopamine (10 micromolar) and sulpiride (1 micromolar) application to acute brain slices from AAV-Cre-P2A-dTomato injected CAMPER mice (data from 4 animals). Average traces obtained after application of response amplitude cutoff criterion (2 multiplied by standard deviation of the baseline). Bars graphs presented as mean ± SEM.

Figure 9.

Bmal1-DN decreases efficacy of dopamine signaling in dorsal striatal neurons. A, Response following dopamine (10 micromolar) application to acute brain slices from AAV-Cre-P2A-Bmal1-WT injected CAMPER mice (data from 5 animals). Average traces obtained after application of response amplitude cutoff criterion (2 multiplied by standard deviation of the baseline). B, Response following dopamine (10 micromolar) application to acute brain slices from AAV-Cre-P2A-Bmal1-DN injected CAMPER mice (data from 5 animals). Average traces obtained after application of response amplitude cutoff criterion (2 multiplied by standard deviation of the baseline). C, Representative cAMP response from D1R in striatal neurons from bath application of dopamine (10 micromolar). Quantification of cAMP amplitude from Bmal1-WT (n = 23 neurons/5 animals) and Bmal1-DN (n = 28 neurons/5 animals): nonparametric t test, p = 0.0035, Mann–Whitney U = 170. D, Representative cAMP response from D2R in striatal neurons from bath application of dopamine (10 micromolar). Quantification of cAMP amplitude from Bmal1-WT (n = 20 neurons/5 animals) and Bmal1-DN (n = 24 neurons/5 animals): nonparametric t test, p = 0.0406, Mann–Whitney U = 153. All data are presented as mean ± SEM.