Daytime spikes in dopaminergic activity drive rapid mood-cycling in mice.
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.
Affiliation: Department of Psychiatry, University of Pittsburgh Medical School, Pittsburgh, PA, USA.
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.
- Action Potentials/drug effects/genetics*
- CLOCK Proteins/genetics*
- Circadian Rhythm/genetics*
- Dopaminergic Neurons/drug effects/physiology*
- Adaptation, Ocular/drug effects/genetics
- Cell Line, Transformed
- Dopamine Agents/pharmacology
- Food Preferences/drug effects/physiology
- Gene Expression Regulation/drug effects/genetics
- Maze Learning/drug effects/physiology
- Mice, Inbred C57BL
- Mice, Transgenic
- Motor Activity/drug effects/genetics
- Time Factors
- Tyrosine 3-Monooxygenase/genetics/metabolism
- Ventral Tegmental Area/cytology
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Figure 3: Effect of chronic VTA optical stimulation on manic-related behavioursa) Schematic of the Cre-dependent stable step function opsin (SSFO) viral construct tagged with enhanced yellow florescent protein (eYFP) used for long-term optical control of neural activity. Two incompatible LoxP sites surround a double-inverted open reading frame that ensures specificity of viral transduction and SSFO expression in cre-containing cells. (b) Expression of SSFO [AAV5-ChR2(C128S/D156A)-eYFP] in the ventral tegmental area (VTA) of a tyrosine hydroxylase (TH)::Cre mouse showing specificity of SSFO expression (green) in TH+ cells (red). TH::Cre mice lacking cre-recombinase (−/−) were used as a negative control and showed no viral expression in TH+ cells (data not shown). Scale bar: 4X=500μm; 20X=100μm. (c) Left panel: Schematic of unilateral optrode implantation for simultaneous optic stimulation and electrophysiological recording of VTA dopamine neurons. Right panel: Chronic stimulation paradigm: three-four weeks following surgery, mice received 7 days of optic stimulation. On each day, mice were given a 5 sec pulse of 473nm or 447nm light every 15 min for a total of 60 min. Electrophysiological recordings were conducted in a separate cohort of mice that underwent the chronic stimulation paradigm whereby VTA DA recordings were performed in awake behaving mice on Day 1 and again on Day 8 for comparison. (d) Top panel: Single neuron recording on Day 1 of the stimulation paradigm in an SSFO-mouse confirmed increased VTA dopamine neural firing during the 5 sec light pulse and a sustained increase in firing over the 60 min stimulation paradigm (blue lines atop graph represent single 5 sec pulses of blue light delivered 15 min apart). Bottom panel:1 hroptic stimulation of the VTA in SSFO-mice increased locomotor activity throughout the course of stimulation (main effect of treatment: F1,60=5.84, p=0.019), with a significant peak in activity occurring 15–30min (p<0.05) after the start of stimulation (first stimulation occurred at the 15 min mark)(e) Representative 5 min baseline (i.e. independent of concurrent optic stimulation) in vivo recording from an awake freely-moving TH::Cre mouse expressing SSFO confirmed that 7 days of optic stimulation (as depicted above in Figure 3c, right panel) increased the mean baseline firing rate of VTA dopamine neurons on Day 8 (red trace) compared to Day 1 (black trace). Firing rates were averaged across 5 sec time bins.(f) Chronic optical stimulation paradigm for behavioural testing: four weeks following viral-mediated delivery of SSFO or eYFP to the VTA, TH::Cre mice were optically stimulated with 473nm or 447nm light for 1hr/day for 7 days within the ZT 6-10 time window, followed by behavioural testing to assess anxiety-related (cohort 1) and depressive-related (cohort 2) behaviours. Behavioural tests were separated by 48 hours with a “booster” optical stimulation given between test days to sustain alterations in VTA dopamine neural activity. Note that mice did not receive optical stimulation on the day of behavioural testing.(g–i) TH::Cre mice that received chronic optical stimulation exhibited significantly decreased anxiety-related behaviour as evident by increased entries into, and time spent in,(g) the open arms of the elevated plus maze (entries: t8=3.49, p=0.008; time: t8=4.3, p=0.003), (h) centre of the open field (entries: t7=2.55, p=0.038; time: t7=2.42, p=0.046) and (i) light chamber of the light/dark box (entries: p>0.05; time: t6=3.12, p=0.021).(j) Chronic optical stimulation increased sucrose preference in mice expressing SSFO (t10=2.86, p=0.017).(k) Two-way repeated measures ANOVA revealed a main effect of group (F1,14=13.46, p=0.0025) whereby 7 days of chronic optic stimulation lead to a persistent and sustained increase in baseline locomotor activity at both 1 (p<0.01) and 2 weeks (p<0.05) after cessation of optogenetic stimulation. For behavioural experiments, cohort 1: n=5–6/treatment group; cohort 2, n=5–9/group.
For chronic stimulation, TH::Cre mice were individually placed into KinderScientific locomotor boxes (see Behavioural Assays for more details) and simultaneously received a single unilateral 5 sec pulse of blue light every 15 min for 1 hour between ZT 6-10 (daytime) or ZT 18-22 (night time) for 7 consecutive days (Figure 3e). A multi-mode fiber optic black-jacketed patchcord (NA 0.22, 200μm inner core, Doric Lenses Inc. Quebec, Canada) connected a 100mW 473nm diode-pumped solid-state laser or a 100mW 447nm diode laser (OEM Lasers Systems, East Lansing, MI) to permanently implanted fiber optics. Commutators (Doric Lenses Inc. Quebec, Canada) were used to reduce torque on patchcords that occurs with animal movement. A standard power meter (Thorlabs, Newton, NJ) was used to measure the emitted light power prior to fiber tethering to achieve a minimum of 3mW at fiber tip (corresponding power densities of 39-3mw/mm2 at VTA depths of 0.1–0.6m). Laser output was controlled using a Rigol pulse generator (DG1022, Rigol USA, Oakwood Village, OH).