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Cerebellar 5HT-2A receptor mediates stress-induced onset of dystonia

Serotonin increased by stress mediates onset of dystonia through activation of 5HT-2A receptors in the deep cerebellar nuclei.


INTRODUCTION
Dystonia is a movement disorder characterized by cocontraction of agonist and antagonist muscles, which results in abnormal body postures. Although various genetic and environmental factors are associated with the etiology of dystonia (1)(2)(3), patients commonly experience worse and more frequent motor symptoms under stressful or anxietyinducing conditions (4)(5)(6). Thus, patients are advised to remain relaxed, often by controlling their environments and limiting social activity (7). Botulinum toxin therapy on target muscles and deepbrain stimulation of the basal ganglia have been used to prevent the typically unpredictable attacks of dystonia (8)(9)(10)(11), but we currently lack an effective medication for this condition.
Indirect evidence has suggested that serotonin [5-hydroxytryptamine (5HT)] is involved in the pathophysiology of dystonia: The dorsal raphe nuclei (DRN), where 5HT neurons reside, show increased activity in patients with dystonia (12). Long-term administration of 5HT reuptake inhibitors (SSRIs) for depression can increase muscle tension and lead to dystonia as a side effect (13). DRN neurons send outputs to the basal ganglia and the deep cerebellar nuclei (DCN) (14), the activities of which increase in dystonia (15). Considering these lines of indirect evidence and the well-known role of 5HT in emotional responses to stress (16)(17)(18)(19)(20), we speculated that the 5HT system can be a link between stress and dystonia.
Here, we hypothesized that 5HT inputs to the DCN may underlie a circuit-based mechanism for the stress-dependent onset of dystonia. To test this hypothesis, we used "tottering" mice harboring a point mutation in a P/Q type calcium channel gene (a1A tot/tot ) (21)(22)(23). These mutant mice develop whole-body dystonia under stressful conditions, such as when they are introduced to an unfamiliar environment (21,22). Although inhibitory neurotransmission has been shown to occur at the synapses between cerebellar Purkinje cells and DCN neurons (24), the mechanism that triggers motor symptoms in response to stress remains unknown. By modulating 5HT-DCN inputs using optogenetics in wild-type (a1A +/+ ) and a1A tot/tot mice, we herein sought to validate the role of 5HT inputs in mediating the stress-induced onset of dystonia.
Next, we asked whether the 5HT + DRN inputs to the DCN modulate motor symptoms in tottering mice (a1A tot/tot ), which represent a genetic model for stress-induced dystonia (21)(22)(23). When exposed to the novelty-induced stress of an open-field box, a1A tot/tot mice gradually developed dystonia (movie S2 and fig. S2A): They Optogenetic stimulation of 5HT + DRN-fDCN circuits in ePet1-cre mice. A confocal image, doublelabeled for ChR2 virus (mCherry, red) and tryptophan hydroxylase (TPH) (green). All merged cells (yellow) mean TPH-positive (5HT + ). Inset magnification, ×20. (B) Axons from DRN 5HT + neurons are found more in the fDCN than in the iDCN or dDCN. Inset magnification, ×20. The signal density was measured as mean fluorescent pixel values in the selected area using ImageJ (44). The signal density (normalized to fDCN; 100%) was calculated for brain sections [anterioposterior (AP), −6.24 to −6.36 mm], including the three nuclei. AU, arbitrary units. (C) EMG traces during photostimulation from hind-limb muscles of wild-type mice. The traces represent results obtained for a 5-min (top) and 20-min (bottom) exposure to an open field. The traces at the bottom represent the dystonic state induced by photostimulations. (D) The crosscorrelation of hind-limb muscles is increased in the photostimulation group (blue) but not in the unstimulated group (black). (E) The dystonia score and attack frequency are increased by photostimulations. Dots represent the worst dystonic score a mouse scored during the entire test session. (F) Optogenetic inhibition of 5HT + DRN-fDCN circuits by 561-nm light in a1A tot/tot mice. Inset magnification, ×20. (G) The eNpHR group exhibits reduced cross-correlation among hind-limb muscles. (H) The eNpHR group shows a lower dystonia score and attack frequency compared to the control. Dots represent the worst dystonic score a mouse scored during the entire test session. Data are presented as means ± SD (error bars).
initially showed an increase in locomotion with jerky limb movements and eventually developed dystonia-like postures; this began with the hind limbs, expanded to the whole body over 10 min, and reached peak severity at around 35 min ( fig. S5A) with increased cocontraction between TA and GS muscles ( fig. S5B). Postmortem histological analyses revealed that a1A tot/tot mice that experienced dystonia had increased c-Fos activity in the DRN and fDCN, supporting the potential role of the 5HT system and fDCN neurons in their motor abnormalities ( fig. S6).

5HT + inputs increase the excitability of fDCN neurons
To more precisely address the impact of 5HT-fDCN inputs on the excitability of fDCN neurons, we performed single-unit recordings in ePet1 cre::DIO-ChR2;a1A +/+ mice using tetrodes under a head-fixed condition, as previously described ( fig. S3C) (26). We measured a significant change in firing rate in 81.8% of fDCN neurons (22 cells from five mice) upon 5HT-fDCN photostimulation (increased, 11; decreased, 7; no response, 4) ( Fig. 2A and fig. S3C). We found that 50% of fDCN neurons showed an increased firing rate with kinetics similar to the onset of dystonia (Fig. 2, A and B). The rate was 19 Hz before photostimulation; it increased starting at 10 min of photostimulation and reached a peak average firing rate of 46 Hz after 30 min of photostimulation [F(2, 20) = 8.508, **P = 0.002, 11 cells from 5 mice, one-way repeated-measures (RM) ANOVA; OFF and

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30 min, t = 4.096, **P = 0.002; 10 and 30 min, t = 2.47, *P = 0.045] (Fig. 2C). 5HT-fDCN ChR2 mice showed more severe symptoms at 30 min compared to 10 min and also exhibited higher firing rates of fDCN neurons at 30 min compared to 10 min (Fig. 2C). This suggests that there may be a positive correlation between the firing rate of fDCN neurons and the severity of motor symptoms.
Increased excitability and Ca 2+ signaling in fDCN neurons of a1A tot/tot mice To physiologically verify the increased excitability of the fDCN in a1A tot/tot mice, we used time-correlated single-photon counting fiber optics (27,28). To express the Ca 2+ sensor GCaMP6m in the fDCN,   In an event-based analysis, we found that the increase of Ca 2+ activity in fDCN neurons was associated with bouts of dystonia in vehicle (0.9% saline)-treated control mice (Fig. 3C), rising to 9.5 times the baseline level during dystonia episodes [F(2, 6) = 8.508, **P = 0.006, n = 4, one-way RM ANOVA; resting and dystonia, t = 4.713, *P = 0.01; walking and dystonia, t = 4.255, *P = 0.011; Fig. 3D]. These results support the hypothesis that the excitatory outputs of fDCN neurons are positively correlated with dystonia in a1A tot/tot mice (Fig. 2).
To explore the role of increased intracellular Ca 2+ in the onset of dystonia, we decided to use OptoSTIM1, a designer protein that increases intracellular Ca 2+ regardless of membrane potential via the light-dependent oligomerization of Ca 2+ release-activated Ca 2+ channels (30), in wild-type mice (a1A +/+ ). We transfected lentiviruses harboring pLenti-CamKII-OptoSTIM1-eGFP into the fDCN and implanted a ferrule just above the injection site (Fig. 3E). Under light illumination in an open-field box (473 nm; frequency, 20 Hz; pulse width, 10 ms; intensity, 5 mW), we found that CamKII-fDCN-OptoSTIM1;a1A +/+ mice showed a greater tendency toward dystonia than CamKII-fDCN-eGFP;a1A +/+ mice (Fig. 3F), as measured by an increase in cocontractions (n = 5, *P = 0.01208, U = 0, Mann-Whitney U test; Fig. 3H) and cross-correlation (n = 5; Fig. 3G) between the TA and GS muscles compared to those of control mice transfected with lentiviruses harboring pLenti-CamKII-eGFP. We also found that the severity of their dystonia symptoms was proportional to the duration of photostimulation (n = 5; Fig. 3I). These results suggest that the increase in intracellular Ca 2+ , which likely occurs through activation of 5HT receptors (31) and voltage-gated Ca 2+ channels (Fig. 2), is critical to increasing the excitability of the fDCN neurons that trigger stress-induced dystonia.

DISCUSSION
Patients who suffer from bouts of dystonia with unpredictable onset or whose motor symptoms grow worse under stressful conditions are typically prescribed some form of relaxation therapy (36). Our results reveal a circuit-based mechanism that explains the relationship between stress and dystonia: Stress enhances the excitability of fDCN neurons via the activation of 5HT-2A receptors, leading to increased intracellular Ca 2+ ( fig. S10A). We further found that inhibition of the 5HT-2A receptor via drugs or shRNAs significantly reduced the onset of symptoms in a genetic model of stress-induced dystonia (Fig. 4), signaling hope for patients who suffer from dystonia. One alternative explanation for the therapeutic effect of 5HT-2A receptor blockade on stress-induced dystonia (Fig. 4) is that such treatment instead blocks the negative emotions associated with stress. However, it is 5HT agonism, not antagonism, that has been associated with a relief from emotional stress (37). Treatment with SSRIs (antidepressants that facilitate 5HT signaling) can instead increase the risk of muscular tension abnormalities like dystonia (13,38). In addition, 5HT-2A receptors are highly expressed in motor-related areas such as the DCN and striatum (39). Thus, the 5HT system seems to control emotion and muscular tension via different 5HT receptors expressed in different brain areas. It is worth noting that the fDCN was not previously a primary focus as a neural substrate for dystonia. Several studies reported that, of the three DCN nuclei, the dDCN plays a critical role in dystonia (15,40,41). Knockdown of torsin A, a gene associated with dystonia, in the dDCN was found to induce dystonia (40). The dDCN induces dystonia through a specific neural pathway wherein the excitatory outputs of dDCN neurons activate neurons in the centromedial or centrolateral thalamus (15). Our present study reveals that DRN-5HT neurons send inputs to the fDCN rather than the dDCN (Fig. 1), and the excitation of these fDCN neurons facilitates the onset of dystonia under stressful conditions. fDCN neurons, like dDCN neurons, send outputs to the centromedial or centrolateral thalamus (fig. S10B); the two DCN outputs converge in the thalamus such that coexcitation of dDCN and fDCN neurons may reduce the threshold for dystonia onset. Stress, however, seems to trigger dystonia through the 5HT-fDCN neuronal pathway.
Our study also provides clues regarding the physiological role of the 5HT-cerebellar circuit mechanism. As an adaptive response to potential danger, emotion-dependent muscle tension may facilitate more powerful motor reactions (42). Still, such tension has drawbacks in that it inevitably limits fine motor control and increases the risk of mistakes in sports and daily life (43). It is plausible that the 5HT-fDCN circuit smoothly increases overall muscle tension under physiological conditions, while leading to severe motor abnormalities and muscular aches under pathological conditions. Further studies on the postsynaptic targets of the fDCN are needed to clarify the physiological role of the 5HT-fDCN circuits in regulating muscular tension and coordination.

Animals
All behavioral experiments were performed on 9-to 11-week-old male ePet1-cre, C57BL/6, and a1A tot/tot mice from The Jackson Laboratory (Bar Harbor, ME, USA). We used age-and gender-matched control mice. To make ePet1 CRE mice with the a1A tot/tot genetic background, we crossed male ePet1 cre/−;a1A +/tot mice with female a1A +/tot mice. Mice had free access to food and water and were kept on a 12-hour light-dark cycle at 22°C. All behavioral assays were conducted during the light phase of the light-dark cycle at the same time of day. Institutional Animal Care and Use Committee guidelines were followed with animal subjects. All of the care and handling of mice was performed according to the directives of the Animal Care and Use Committee of Korea Advanced Institute of Science and Technology (KAIST; Daejeon, Korea).

Statistical analysis
No statistical analysis was performed to predetermine sample sizes; instead, we used sample sizes similar to those found in many previous studies that used similar neurochemical, electrophysiological, optogenetic, and behavioral techniques. All data analyses were performed using SigmaPlot (12.0; Systat Software, USA). For parameters that followed a normal distribution (Shapiro-Wilk test, P > 0.05), differences between two groups were analyzed with the Student's t test. The Mann-Whitney U test was used for data that did not follow a normal distribution. Comparisons of three or more groups were performed with ANOVA. The Holm-Sidak method was used to correct for multiple comparisons. All other statistical tests were Student's two-sided t tests. P values <0.05 were considered statistically significant (Tables 1 and 2). Data are expressed as means ± SD (Tables 3 to 5).