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Daytime spikes in dopaminergic activity drive rapid mood-cycling in mice.

Sidor MM, Spencer SM, Dzirasa K, Parekh PK, Tye KM, Warden MR, Arey RN, Enwright JF, Jacobsen JP, Kumar S, Remillard EM, Caron MG, Deisseroth K, McClung CA - Mol. Psychiatry (2015)

Bottom Line: Mood-cycling coincides with abnormal daytime spikes in ventral tegmental area (VTA) dopaminergic activity, tyrosine hydroxylase (TH) levels and dopamine synthesis.To determine the significance of daytime increases in VTA dopamine activity to manic behaviors, we developed a novel optogenetic stimulation paradigm that produces a sustained increase in dopamine neuronal activity and find that this induces a manic-like behavioral state.Finally, we show that CLOCK acts as a negative regulator of TH transcription, revealing a novel molecular mechanism underlying cyclic changes in mood-related behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry, University of Pittsburgh Medical School, Pittsburgh, PA, USA.

ABSTRACT
Disruptions in circadian rhythms and dopaminergic activity are involved in the pathophysiology of bipolar disorder, though their interaction remains unclear. Moreover, a lack of animal models that display spontaneous cycling between mood states has hindered our mechanistic understanding of mood switching. Here, we find that mice with a mutation in the circadian Clock gene (ClockΔ19) exhibit rapid mood-cycling, with a profound manic-like phenotype emerging during the day following a period of euthymia at night. Mood-cycling coincides with abnormal daytime spikes in ventral tegmental area (VTA) dopaminergic activity, tyrosine hydroxylase (TH) levels and dopamine synthesis. To determine the significance of daytime increases in VTA dopamine activity to manic behaviors, we developed a novel optogenetic stimulation paradigm that produces a sustained increase in dopamine neuronal activity and find that this induces a manic-like behavioral state. Time-dependent dampening of TH activity during the day reverses manic-related behaviors in ClockΔ19 mice. Finally, we show that CLOCK acts as a negative regulator of TH transcription, revealing a novel molecular mechanism underlying cyclic changes in mood-related behavior. Taken together, these studies have identified a mechanistic connection between circadian gene disruption and the precipitation of manic episodes in bipolar disorder.

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CLOCK binding at the TH promoter(a) Fold enrichment, as calculated from Ct values, at distal promoter region across ZT time following chromatin immunoprecipitation with a CLOCK-specific antibody in C57BL/6 mice (n =3–4 per time point). (a, right) Comparison of CLOCK binding at the distal TH promoter during the light (averaged values of ZT 4 and ZT 9 from graph, left) versus dark phase (averaged values of ZT 16 and ZT 21 from graph, left) shows a strong trend toward a diurnal variation in promoter occupancy (t13=2.113, p=0.056). (b) Representative agarose gels of q-PCR products from ChIP assay for graphs in (a). (c) Binding at the proximal TH promoter. (c, right) Significant diurnal variation in CLOCK occupancy of the TH proximal promoter (t13=2.713, p=0.02).(d) Representative agarose gels of q-PCR products from ChIP assay for graphs in (b). (e,f) Fold enrichment at distal (e) and proximal promoter region (f) following ChIP with a CLOCK-specific antibody comparing ClockΔ19 mutants to wild-type (WT) controls; (n =4–6 per time point).(g) Relative luciferase activity in PC12 cells transfected with WT TH-luc constructs (250 bp or 1000 bp) or TH-luc constructs containing mutant E-boxes. Mutating the E-boxes significantly increased luciferase activity at the proximal (t16=11.30, p<0.0001) and distal site (t9=3.158, p<0.01).(h) Differential levels of p-CREB (s133) binding at the proximal TH promoter across ZT time with increased binding observed during the beginning of the dark phase (ZT 16) in C57BL/6 mice.
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Figure 4: CLOCK binding at the TH promoter(a) Fold enrichment, as calculated from Ct values, at distal promoter region across ZT time following chromatin immunoprecipitation with a CLOCK-specific antibody in C57BL/6 mice (n =3–4 per time point). (a, right) Comparison of CLOCK binding at the distal TH promoter during the light (averaged values of ZT 4 and ZT 9 from graph, left) versus dark phase (averaged values of ZT 16 and ZT 21 from graph, left) shows a strong trend toward a diurnal variation in promoter occupancy (t13=2.113, p=0.056). (b) Representative agarose gels of q-PCR products from ChIP assay for graphs in (a). (c) Binding at the proximal TH promoter. (c, right) Significant diurnal variation in CLOCK occupancy of the TH proximal promoter (t13=2.713, p=0.02).(d) Representative agarose gels of q-PCR products from ChIP assay for graphs in (b). (e,f) Fold enrichment at distal (e) and proximal promoter region (f) following ChIP with a CLOCK-specific antibody comparing ClockΔ19 mutants to wild-type (WT) controls; (n =4–6 per time point).(g) Relative luciferase activity in PC12 cells transfected with WT TH-luc constructs (250 bp or 1000 bp) or TH-luc constructs containing mutant E-boxes. Mutating the E-boxes significantly increased luciferase activity at the proximal (t16=11.30, p<0.0001) and distal site (t9=3.158, p<0.01).(h) Differential levels of p-CREB (s133) binding at the proximal TH promoter across ZT time with increased binding observed during the beginning of the dark phase (ZT 16) in C57BL/6 mice.

Mentions: We next determined the ability of CLOCK to directly bind E-box elements in the TH promoter (CANNTG) by performing chromatin immunoprecipitation (ChIP) assays on VTA-containing midbrain tissue. Two primer sets were created to amplify distal and proximal regions of the promoter containing putative E-Boxes (Supplementary Table 1; Supplementary Figure 11). ChIP analyses were performed on C57BL/6 mice at 4 time points: daytime = ZT 4 and ZT 9 and night time = ZT 16 and ZT 21. CLOCK was present at both TH promoter sites during the day (distal: Figure 4a; proximal: Figure 4c), with no enrichment observed above background at night. CLOCK binding was then assessed in ClockΔ19 mutants during the day at ZT 4, when binding is expected based on the results in Figures 4a,c. Consistent with the WT data, the CLOCKΔ19 mutated protein was enriched at both TH promoter sites in ClockΔ19 mice (distal: Figure 4e; proximal: 4f). This data indicates that the mutated CLOCK protein is capable of binding DNA, however the missing exon 19 appears critical for conferring proper transcriptional regulation. To test the functional significance of CLOCK binding at the TH promoter, we interfered with CLOCK’s ability to properly bind TH. Luciferase assays were performed with TH-luc reporter plasmids containing either an intact or mutated E-box. Disruption of CLOCK binding through mutation of either E-box site significantly increased TH-luc reporter activity (proximal, p<0.0001 and distal, p<0.01; Figure 4g), demonstrating that the repression of TH transcription is E-box dependent.


Daytime spikes in dopaminergic activity drive rapid mood-cycling in mice.

Sidor MM, Spencer SM, Dzirasa K, Parekh PK, Tye KM, Warden MR, Arey RN, Enwright JF, Jacobsen JP, Kumar S, Remillard EM, Caron MG, Deisseroth K, McClung CA - Mol. Psychiatry (2015)

CLOCK binding at the TH promoter(a) Fold enrichment, as calculated from Ct values, at distal promoter region across ZT time following chromatin immunoprecipitation with a CLOCK-specific antibody in C57BL/6 mice (n =3–4 per time point). (a, right) Comparison of CLOCK binding at the distal TH promoter during the light (averaged values of ZT 4 and ZT 9 from graph, left) versus dark phase (averaged values of ZT 16 and ZT 21 from graph, left) shows a strong trend toward a diurnal variation in promoter occupancy (t13=2.113, p=0.056). (b) Representative agarose gels of q-PCR products from ChIP assay for graphs in (a). (c) Binding at the proximal TH promoter. (c, right) Significant diurnal variation in CLOCK occupancy of the TH proximal promoter (t13=2.713, p=0.02).(d) Representative agarose gels of q-PCR products from ChIP assay for graphs in (b). (e,f) Fold enrichment at distal (e) and proximal promoter region (f) following ChIP with a CLOCK-specific antibody comparing ClockΔ19 mutants to wild-type (WT) controls; (n =4–6 per time point).(g) Relative luciferase activity in PC12 cells transfected with WT TH-luc constructs (250 bp or 1000 bp) or TH-luc constructs containing mutant E-boxes. Mutating the E-boxes significantly increased luciferase activity at the proximal (t16=11.30, p<0.0001) and distal site (t9=3.158, p<0.01).(h) Differential levels of p-CREB (s133) binding at the proximal TH promoter across ZT time with increased binding observed during the beginning of the dark phase (ZT 16) in C57BL/6 mice.
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Figure 4: CLOCK binding at the TH promoter(a) Fold enrichment, as calculated from Ct values, at distal promoter region across ZT time following chromatin immunoprecipitation with a CLOCK-specific antibody in C57BL/6 mice (n =3–4 per time point). (a, right) Comparison of CLOCK binding at the distal TH promoter during the light (averaged values of ZT 4 and ZT 9 from graph, left) versus dark phase (averaged values of ZT 16 and ZT 21 from graph, left) shows a strong trend toward a diurnal variation in promoter occupancy (t13=2.113, p=0.056). (b) Representative agarose gels of q-PCR products from ChIP assay for graphs in (a). (c) Binding at the proximal TH promoter. (c, right) Significant diurnal variation in CLOCK occupancy of the TH proximal promoter (t13=2.713, p=0.02).(d) Representative agarose gels of q-PCR products from ChIP assay for graphs in (b). (e,f) Fold enrichment at distal (e) and proximal promoter region (f) following ChIP with a CLOCK-specific antibody comparing ClockΔ19 mutants to wild-type (WT) controls; (n =4–6 per time point).(g) Relative luciferase activity in PC12 cells transfected with WT TH-luc constructs (250 bp or 1000 bp) or TH-luc constructs containing mutant E-boxes. Mutating the E-boxes significantly increased luciferase activity at the proximal (t16=11.30, p<0.0001) and distal site (t9=3.158, p<0.01).(h) Differential levels of p-CREB (s133) binding at the proximal TH promoter across ZT time with increased binding observed during the beginning of the dark phase (ZT 16) in C57BL/6 mice.
Mentions: We next determined the ability of CLOCK to directly bind E-box elements in the TH promoter (CANNTG) by performing chromatin immunoprecipitation (ChIP) assays on VTA-containing midbrain tissue. Two primer sets were created to amplify distal and proximal regions of the promoter containing putative E-Boxes (Supplementary Table 1; Supplementary Figure 11). ChIP analyses were performed on C57BL/6 mice at 4 time points: daytime = ZT 4 and ZT 9 and night time = ZT 16 and ZT 21. CLOCK was present at both TH promoter sites during the day (distal: Figure 4a; proximal: Figure 4c), with no enrichment observed above background at night. CLOCK binding was then assessed in ClockΔ19 mutants during the day at ZT 4, when binding is expected based on the results in Figures 4a,c. Consistent with the WT data, the CLOCKΔ19 mutated protein was enriched at both TH promoter sites in ClockΔ19 mice (distal: Figure 4e; proximal: 4f). This data indicates that the mutated CLOCK protein is capable of binding DNA, however the missing exon 19 appears critical for conferring proper transcriptional regulation. To test the functional significance of CLOCK binding at the TH promoter, we interfered with CLOCK’s ability to properly bind TH. Luciferase assays were performed with TH-luc reporter plasmids containing either an intact or mutated E-box. Disruption of CLOCK binding through mutation of either E-box site significantly increased TH-luc reporter activity (proximal, p<0.0001 and distal, p<0.01; Figure 4g), demonstrating that the repression of TH transcription is E-box dependent.

Bottom Line: Mood-cycling coincides with abnormal daytime spikes in ventral tegmental area (VTA) dopaminergic activity, tyrosine hydroxylase (TH) levels and dopamine synthesis.To determine the significance of daytime increases in VTA dopamine activity to manic behaviors, we developed a novel optogenetic stimulation paradigm that produces a sustained increase in dopamine neuronal activity and find that this induces a manic-like behavioral state.Finally, we show that CLOCK acts as a negative regulator of TH transcription, revealing a novel molecular mechanism underlying cyclic changes in mood-related behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry, University of Pittsburgh Medical School, Pittsburgh, PA, USA.

ABSTRACT
Disruptions in circadian rhythms and dopaminergic activity are involved in the pathophysiology of bipolar disorder, though their interaction remains unclear. Moreover, a lack of animal models that display spontaneous cycling between mood states has hindered our mechanistic understanding of mood switching. Here, we find that mice with a mutation in the circadian Clock gene (ClockΔ19) exhibit rapid mood-cycling, with a profound manic-like phenotype emerging during the day following a period of euthymia at night. Mood-cycling coincides with abnormal daytime spikes in ventral tegmental area (VTA) dopaminergic activity, tyrosine hydroxylase (TH) levels and dopamine synthesis. To determine the significance of daytime increases in VTA dopamine activity to manic behaviors, we developed a novel optogenetic stimulation paradigm that produces a sustained increase in dopamine neuronal activity and find that this induces a manic-like behavioral state. Time-dependent dampening of TH activity during the day reverses manic-related behaviors in ClockΔ19 mice. Finally, we show that CLOCK acts as a negative regulator of TH transcription, revealing a novel molecular mechanism underlying cyclic changes in mood-related behavior. Taken together, these studies have identified a mechanistic connection between circadian gene disruption and the precipitation of manic episodes in bipolar disorder.

Show MeSH
Related in: MedlinePlus