The mood stabilizer valproic acid opposes the effects of dopamine on circadian rhythms

Highlights

• Dopamine lengthens the period of behavioral and SCN circadian rhythms in mice.

• The mood stabilizer valproic acid (VPA) shortens the period of circadian rhythms.

• VPA normalizes circadian rhythms in fibroblasts from bipolar patients.

• In Drosophila lacking circadian clocks, effects of VPA on hyperactivity are attenuated.

Abstract

Endogenous circadian (∼24 h) clocks regulate key physiological and cognitive processes via rhythmic expression of clock genes. The main circadian pacemaker is the hypothalamic suprachiasmatic nucleus (SCN). Mood disorders, including bipolar disorder (BD), are commonly associated with disturbed circadian rhythms. Dopamine (DA) contributes to mania in BD and has direct impact on clock gene expression. Therefore, we hypothesized that high levels of DA during episodes of mania contribute to disturbed circadian rhythms in BD. The mood stabilizer valproic acid (VPA) also affects circadian rhythms. Thus, we further hypothesized that VPA normalizes circadian disturbances caused by elevated levels of DA. To test these hypotheses, we examined locomotor rhythms and circadian gene cycling in mice with reduced expression of the dopamine transporter (DAT-KD mice), which results in elevated DA levels and mania-like behavior. We found that elevated DA signaling lengthened the circadian period of behavioral rhythms in DAT-KD mice and clock gene expression rhythms in SCN explants. In contrast, we found that VPA shortened circadian period of behavioral rhythms in DAT-KD mice and clock gene expression rhythms in SCN explants, hippocampal cell lines, and human fibroblasts from BD patients. Thus, DA and VPA have opposing effects on circadian period. To test whether the impact of VPA on circadian rhythms contributes to its behavioral effects, we fed VPA to DAT-deficient Drosophila with and without functioning circadian clocks. Consistent with our hypothesis, we found that VPA had potent activity-suppressing effects in hyperactive DAT-deficient flies with intact circadian clocks. However, these effects were attenuated in DAT-deficient flies in which circadian clocks were disrupted, suggesting that VPA functions partly through the circadian clock to suppress activity. Here, we provide in vivo and in vitro evidence across species that elevated DA signaling lengthens the circadian period, an effect remediated by VPA treatment. Hence, VPA may exert beneficial effects on mood by normalizing lengthened circadian rhythm period in subjects with elevated DA resulting from reduced DAT.

Introduction

As a result of regularly recurring daily cycles of sun exposure caused by earth's rotation, most living organisms have evolved circadian (ca. 24 h) clocks to anticipate daily environmental changes and orchestrate daily temporal programs of physiology and behavior. Within cells, autoregulatory clock proteins generate ca. 24 h rhythms by transcriptional-translational negative feedback loops. Core clock genes include Bmal1, Clock, Period (Per1/2) and Cryptochrome (Cry1/2). BMAL1 and CLOCK proteins heterodimerize and induce Per and Cry gene expression. Later in the day, PER and CRY proteins inhibit BMAL1/CLOCK and thereby their own transcription, thus completing the circadian cycle (Koike et al., 2012). In mammals, the master circadian pacemaker is located in the suprachiasmatic nucleus (SCN), in the hypothalamus, but most other tissues also harbor circadian clocks (Albrecht, 2012) which regulate the timing of key processes involved in metabolism, physiology, behavior, and mood regulation. These processes may in turn feedback to impact clock function as well. For example, disruption of circadian rhythms is a hallmark of most neuropsychiatric disorders, including bipolar disorder (BD) (Landgraf et al., 2014a, McCarthy and Welsh, 2012, Wulff et al., 2010), a disabling condition characterized by episodes of depression and mania. The mechanisms by which circadian and mood-regulating processes influence each other are unclear (Landgraf et al., 2014b). In the case of BD, however, manic behavior is associated with elevated levels of dopamine (DA) (van Enkhuizen et al., 2015), a neurotransmitter that also impacts circadian rhythms. For example, timed treatment with a D1 DA receptor agonist shifts circadian phase of fetal hamsters (Viswanathan and Davis, 1997, Viswanathan et al., 1994), possibly due to activation of D1 receptors in the mammalian SCN (Rivkees and Lachowicz, 1997, Weiner et al., 1991). DA also regulates core clock components in retina and striatum (Hood et al., 2010, Imbesi et al., 2009, Yujnovsky et al., 2006). Thus, DA may be crucially involved in both manic behavior and disrupted circadian rhythms in BD.

One possible reason for increased DA levels in BD patients is lower expression of the dopamine transporter (DAT) that removes DA from synaptic clefts. Variants of the DAT gene (SLC6A3) have been associated with BD and reduced DAT expression (Greenwood et al., 2001, Greenwood et al., 2006, Kelsoe et al., 1996). These associations are consistent with lower DAT levels observed in BD patients and thus elevated synaptic DA (Amsterdam and Newberg, 2007). Furthermore, in mice, knockout of DAT (DAT^−/−) increases DA levels and activity in new environments (Giros et al., 1996), and causes deficits in prepulse inhibition (Ralph et al., 2001). However, behavioral analysis of DAT^−/− mice is complicated by poor physical state (Bosse et al., 1997). Mice with constitutive genetic knockdown of DAT (DAT-KD) express 10% of wild-type (WT) DAT levels and also show high DA levels. They display mania-like behaviors such as hyperactivity in a novel environment (Zhuang et al., 2001), replicating hyper-exploration and increased risk-taking behavior of manic BD patients (Perry et al., 2009, van Enkhuizen et al., 2014, Young et al., 2011b) without concomitant developmental defect. For these reasons, DAT-KD mice have been used as a model for mania. In BD patients, valproic acid (VPA) is used as a mood stabilizer, and particularly as an anti-manic agent. Interestingly, observations that VPA also attenuates the hyper-exploration of DAT-KD mice without affecting their WT littermates (van Enkhuizen et al., 2013) provided predictive validity for this model of BD mania (Young et al., 2011a).

Since DA regulates circadian clocks, we hypothesized that elevated DA levels contribute to circadian disturbances in manic BD patients. We tested this hypothesis in DAT-KD mice and cultured SCN explants treated with a D1 receptor agonist and found that increased DA signaling lengthens the period of circadian rhythms. Furthermore, we showed that VPA shortens circadian period, both in vitro and in vivo, and that the effects of VPA on behavior are less pronounced in the absence of a circadian clock in DAT-deficient Drosophila melanogaster. These data suggest that the mood-stabilizing properties of VPA in manic BD patients might be partly based on reversing the effects of elevated DA on circadian period.