IP3R1 deficiency in the cerebellum/brainstem causes basal ganglia-independent dystonia by triggering tonic Purkinje cell firings in mice

The type 1 inositol 1,4,5- trisphosphate receptor (IP3R1) is a Ca2+ channel on the endoplasmic reticulum and is a predominant isoform in the brain among the three types of IP3Rs. Mice lacking IP3R1 show seizure-like behavior; however the cellular and neural circuit mechanism by which IP3R1 deletion causes the abnormal movements is unknown. Here, we found that the conditional knockout mice lacking IP3R1 specifically in the cerebellum and brainstem experience dystonia and show that cerebellar Purkinje cell (PC) firing patterns were coupled to specific dystonic movements. Recordings in freely behaving mice revealed epochs of low and high frequency PC complex spikes linked to body extension and rigidity, respectively. Remarkably, dystonic symptoms were independent of the basal ganglia, and could be rescued by inactivation of the cerebellum, inferior olive or in the absence of PCs. These findings implicate IP3R1-dependent PC firing patterns in cerebellum in motor coordination and the expression of dystonia through the olivo-cerebellar pathway.


INTRODUCTION
The inositol 1,4,5-trisphosphate receptors (IP 3 Rs) are intracellular Ca 2+ channels localized at the endoplasmic reticulum and regulate the spatio-temporal change of intracellular Ca 2+ concentration, which are important for diverse physiological phenomena including gene expression, development, growth, neural plasticity, and secretion (Berridge et al., 2000). There are three subtypes of IP 3 R in mammals and each IP 3 R isoform exhibits a distinct expression pattern in vivo. Among the three subtypes, the type 1 IP 3 receptor (IP 3 R1) is a brain dominant subtype (Foskett et al., 2007;Mikoshiba, 2007). We have previously showed that mice lacking the IP 3 R1 receptor (Itpr1 −/− ) exhibit ataxia and seizurelike posture with multiple abnormal movements such as repetitive rigid posture, opisthotonus, tonic contractions of the neck and trunk, and premature death around the third week after birth (Matsumoto et al., 1996). However, since IP 3 R1 is expressed in a wide range of brain regions including cerebellum, cerebral cortex, hippocampus, and striatum, the particular neural activities and circuits causing these involuntary movements in the Itpr1 −/− mice remain unknown.
Dystonia is a neurological disorder in which sustained muscle contractions induce twisting and repetitive movements or abnormal posturing. Simultaneous abnormal contractions of agonistic and antagonistic muscles (co-contractions) are one of the most distinct features of dystonic movements. Because of various phenotypic and genotypic subtypes in dystonia, its pathogenic mechanisms remain elusive. Traditionally, dystonia has been thought to be a basal ganglia (BG) disorder (Marsden and Quinn, 1990;Lenz et al., 1998;Vitek et al., 1999;Zhuang et al., 2004;Chiken et al., 2008;Nambu et al., 2011). In contrast, recent accumulating evidence has further suggested abnormalities of the cerebellum and brainstem in some dystonic patients (Ceballos-Baumann et al., 1995;Eidelberg et al., 1998;Mazziotta et al., 1998;Odergren et al., 1998;Hutchinson et al., 2000). Several animal models of dystonia also exhibit cerebellar abnormalities (Ledoux and Lorden, 2002;Pizoli et al., 2002;Raike et al., 2005;Walter et al., 2006;Chen et al., 2009;Calderon et al., 2011;Ledoux, 2011;Filip et al., 2013) and aberrant cerebellar activities in dystonic model animals were reported (Ledoux and Lorden, 2002;Walter et al., 2006;Chen et al., 2009), however, little is known about firing patterns of Purkinje cell (PC) activity associated with particular dystonic movements of freely moving mice. At the neural circuit level, it was suggested that cerebellar outputs alter BG activity thereby leading to dystonic movements (Neychev et al., 2008;Calderon et al., 2011).
In this study, we showed that genetic deletion of IP 3 R1 within the cerebellum and brainstem is sufficient to cause dystonia in mice. Although in the previous report we described epilepticlike seizures in Itpr1 −/− mice, in the current study we concluded that the behavior of Itpr1 −/− mice is better described as dystonia with severe ataxia because there was no abnormal electroencephalogram activity during the seizure-like posture ( Figure 1A). In addition, we revealed distinct patterns of PC firing that were tightly coupled to the dystonic movements in freely moving mutant mice. We also showed that pharmacological inactivation of the cerebellum or inferior olive (IO), but not BG, and deletion of PCs ameliorate the dyskinesia. Thus, our mutant mice provide a therapeutic dystonia model solely dependent upon abnormal olivocerebellar pathways and provide a coherent mechanism for a specific type of dystonia.

Frontiers in Neural Circuits
www.frontiersin.org October 2013 | Volume 7 | Article 156 | 2 they were hand-fed a nutritionally complete soft diet, DietGel76A (ClearH 2 O), for their entire lives after weaning. All animals were ethically treated according to the guideline of Animal Experiments Committee of RIKEN Brain Science Institute.

CYTOCHROME OXIDASE (CO) STAINING
Frozen 4%PFA-fixed 8-week-old brain sections (100 μm thick) were incubated in 0.1 M phosphate buffer containing 4 g sucrose, 50 mg of cytochrome C, and 50 mg of diaminobenzidine per 100 ml of buffer at 37 • C for 1-2 h. To compare the CO staining intensities among different genotypes of mice, brains were fixed, cut, and reacted with the same solutions, and the digital photographs were taken on a same day.

ADMINISTRATION OF DRUGS INTO THE MOUSE BRAIN (CEREBELLUM, INFERIOR OLIVE, AND BASAL GANGLIA)
Mice (∼2 month-old) were anesthetized with 1.5% halothane anesthesia with N 2 O:O 2 (3:2) ventilation. A guide cannula (C313, inner diameter: 0.39 mm, outer diameter: 0.71 mm, Plastics One) was implanted at the middle of vermis of cerebellum (1.1 mm in depth). After 3 days of recovery, an internal cannula (1.0 mm projection length from the guide cannula) was replaced with the dummy cannula and PBS or CNQX (5 mM in PBS, Tocris) were infused into the cerebellum at the speed of 0.5 μl/min for 20 min. The same guide cannula system was used for lidocaine (4.0% in PBS, MP Biomedicals) injection into the IO (the tip of the cannula was targeted to just above the medial nuclei) and bilateral BG (entopeduncular nucleus, 1.3 mm posterior to the Bregma, lateral to 2.2, 4.5 mm depth).

EXTRACELLULAR RECORDING IN ANESTHETIZED MICE
Recordings were performed in anesthetized mice (1-2 months old) after an intraperitoneal injection of 50 mg/kg Nembutal  (Gordon and Stryker, 1996). The animal's temperature was maintained at 38 • C. The heart rate was monitored continuously. A small hole (1.0 mm) was drilled in the occipital bone above the cerebellar vermis of lobule IV (midline, 4.5 mm caudal from lambda), and the dura was exposed and covered with warm agarose (2.8% in saline). The microelectrode tip (epoxy-coated tungsten microelectrodes, 9-12 M impedance; FHC, ME) was positioned above the small hole and advanced into the cerebellar lobule IV vermis using a stepping motor controlled micromanipulator. Raw signals from the electrodes were amplified, filtered (0.3-5 kHz), digitized at 25 kHz and stored (LabVIEW, National Instrument, Austin, TX). Single and multiunit PC activities (peak heights above the 6 sigma noise level) were isolated by off-line spike sorting (Offline sorter, Plexon, Dallas, TX). Simple spikes (SSs) and complex spikes (CSs) were identified based on their characteristic waveforms.

EEG, EMG RECORDING
A stainless screw electrode for EEG recording was secured over the cerebellar cortex (2.0 mm posterior to the lambda, on the midline) and a reference screw electrode was placed over the somatosensory (1.5 mm lateral to the midline, 1.0 mm posterior to the bregma) or frontal cortex (1.0 mm lateral to the midline, 2.0 mm anterior to the bregma) (Miyamoto et al., 2012). A stainless wire was inserted in the neck muscle for EMG recording. Polygraphic signals (band-pass filtered at 0.7-170 Hz) were amplified by telemetry system (Data Sciences International, St. Paul, MN) and sampled at 500 Hz (SleepSign, KISSEI COMTEC, Japan). Based on polygraph and infra-red camera monitoring, sleep/waking behavioral state and epileptic EEG pattern was explored.

EXTRACELLULAR RECORDING FROM BEHAVING MICE
The tetrodes of four nichrome wires (13 μm) were stereotaxically implanted into the cerebellar vermis of the lobule IV (6.25 mm posterior to the Bregma, on the midline). Signals from each electrode were band-pass filtered (1-6 kHz) and digitized at 25 kHz sampling frequency (Plexon, Dallas, TX). A reference electrode was chosen from electrodes which did not show neuronal activity. Neuronal spike data (firing rate, autocorrelogram, interspike interval) was analyzed by NeuroExplorer (Nex Technologies, Littleton, MA).
Similar to the recording of cerebellar PCs, tetrodes were implanted to monitor neuronal activity in the BG (caudate putamen and globus pallidus, 0.5 mm posterior to the Bregma, 2.0-3.0 mm lateral to the midline, 2.0-4.0 mm depth from the surface) during dystonic movements. Multi-unit neuronal activity data were sampled with a minimal interval of 200 μm by slowly advancing the tetrodes. For behavior analyses, we defined rigid posture as the duration in which mice hunched their backs and extended their paws to maintain the posture. In addition, we defined opisthotonus as an abnormal posture in which the mouse's neck was completely held at the bridging position (i.e., bent fully toward the upper back). We judged the beginning of opisthotonus when the neck was held at the maximal bridging position, and defined the ending as its complete return to the normal (horizontal/unbent) position.

Footprint analysis
The hindpaws of 8-week-old Emx1-Cre;Itpr1 flox/flox and Gpr88-Cre;Itpr1 flox/flox mice, or of 19-day-old mice for Lurcher experiments, were dipped in non-toxic water-based black paint, and allowed to walk down an enclosed runway lined with white paper, to determine their gait characteristics.

STATISTICAL ANALYSES
The significance of differences between groups was analyzed using Student's t-test, paired Student's t-test, Mann-Whitney U-test, Dunnett's test, or ANOVA followed by the Bonferroni's test as appropriate. A value of P < 0.05 is reported as significant.
Unlike the premature death in Itpr1 −/− mice, Wnt1-Cre;Itpr1 flox/flox mice grew to adulthood by hand-feeding. Body weight of Wnt1-Cre;Itpr1 flox/flox mice was about 45% of Itpr1 flox/flox mice at 5 weeks. Cerebellar size of Wnt1-Cre;Itpr1 flox/flox mice at 8 weeks was significantly smaller than that of Itpr1 flox/flox mice, whereas cerebral cortex size was comparable (Figures 1C,D). The apparent morphological constituents of the cerebellum, such as granular layer, PC layer, and molecular layer seemed normal and no apparent cell death occurred as judged by DAPI staining for nuclear condensation. The expression level of IP 3 R1 in the cerebellum of 8 week-old Wnt1-Cre;Itpr1 flox/flox mice was significantly lower than that of Itpr1 flox/flox mice, whereas expression in the hippocampus, striatum, and cerebral cortex of Wnt1-Cre;Itpr1 flox/flox mice was equivalent to that of Itpr1 flox/flox mice (Figures 1E-G). Residual IP 3 R1 expression in the cerebellum of the 8-week-old Wnt1-Cre;Itpr1 flox/flox mice was attributed at least partly to PCs still expressing IP 3 R1 protein after  incomplete Cre/flox recombination in the PCs of the Wnt1-Cre Tg mice (Figures 1F, 2G).
To further delineate the neurons responsible for the expression of dystonia in Itpr1 −/− and Wnt1-Cre;Itpr1 flox/flox mice, we investigated the expression of cfos mRNA, a neural activity marker (Morgan et al., 1987). Interestingly, we found strong c-fos mRNA expression in PCs localized to the caudal parts of the cerebellum in both Itpr1 −/− and Wnt1-Cre;Itpr1 flox/flox mice, but not in that of Itpr1 +/+ or Itpr1 flox/flox mice (Figures 2A,B, upper panels). No apparent elevation of cfos mRNA was observed in the hippocampus and cortex in either Itpr1 −/− or Wnt1-Cre;Itpr1 flox/flox mice (Figures 2A,B, lower panels). We also examined CaM kinase II-mediated phosphorylation levels of Homer 3 as a marker of PC depolarization (Mizutani et al., 2008). Only weak Homer 3 phosphorylation (S120) signals were observed in the soma and proximal dendrites of PCs in 8-week-old Itpr1 flox/flox mice (Figure 2C), as reported previously (Mizutani et al., 2008). In 8-week-old Wnt1-Cre;Itpr1 flox/flox mice, however, intense Homer 3 phosphorylation was observed at the soma and proximal dendrites, including the apical dendrites of PCs in caudal lobules 9 and 10 ( Figure 2C, right).
In addition, we found aberrant tyrosine hydroxylase (TH) expression which is induced by cFos (Nagamoto-Combs et al., 1997) in the Itpr1 −/− ( Figure 2D) and Wnt1-Cre;Itpr1 flox/flox PCs ( Figure 2E). The TH-positive PCs were mainly observed in the vermis and flocculus of the Wnt1-Cre;Itpr1 flox/flox and Itpr1 −/− cerebellum ( Figure 2E) and were localized in a banded pattern ( Figure 2F). In the wild-type cerebellum, we detected some THpositive PCs as reported previously (Hess and Wilson, 1991). However, TH expression levels were relatively weak, and the regions expressing TH in a banded manner were both fewer and smaller in size in the Itpr1 +/+ and Itpr1 flox/flox mice than in the Itpr1 mutant mice ( Figure S1). Although PCs normally express a large amount of IP 3 R1 in the brain, the abnormal TH expression in the Wnt1-Cre;Itpr1 flox/flox cerebellum was observed in PCs regardless of IP 3 R1 expression (Figure 2G), suggesting that altered neural inputs onto PCs may trigger the aberrant TH expression.
Since climbing fibers (CF) innervate PCs localized within banded patterns (Oscarsson, 1979), olivocerebellar inputs may be the cause of abnormal involuntary movements in the Wnt1-Cre;Itpr1 flox/flox mice. Harmaline evokes synchronous firing across large populations of PCs via the olivocellebelar pathway by electrical coupling of IO neurons, resulting in the expression of tremor in mice (Llinas and Sasaki, 1989). We intraperitoneally injected harmaline into wild-type mice and examined Homer 3 phosphorylation and TH expression in PCs. We found that IO activation rapidly increased the phosphorylation levels of Homer 3 in the soma and dendrites of PCs in a time dependent manner ( Figure 2H). Intense phosphorylation signals were first observed in the soma of PCs within 15 min after injection ( Figure 2H, arrow), then progressed into proximal and apical dendrites as time passed (Figure 2H, arrowhead). Furthermore, we observed elevation of TH signals in PCs at the caudal region of the cerebellum, especially lobules XI and X at 24 h after injection ( Figure 2I). These results closely resembled those observed in the Wnt1-Cre;Itpr1 flox/flox mice. Therefore, we hypothesized that abnormal PC firing caused by excessive olivocerebellar input is the cause of involuntary movements in the Itpr1 −/− and Wnt1-Cre;Itpr1 flox/flox mice.

PC ACTIVITY CORRELATES WITH DYSTONIC MOVEMENTS OF MICE
To reveal the nature of abnormal PC firing underlying the expression of dystonia in the Itpr1 −/− mice, we first measured spontaneous PC activities by loose cell-attached recording using acute cerebellar slices, in which neuronal inputs from climbing and mossy fibers were severed. Because the PCs highly expressing TH were mainly observed in caudal parts of the cerebellar vermis of Itpr1 −/− mice (Figure 2E), we measured spontaneous activities of PCs mainly from those areas. However, we found no apparent difference in spike frequency and coefficient of variation (CV) between PCs from wild-type and total Itpr1 −/− mice under these conditions (Frequency: Itpr1 +/+ : 25.89 ± 2.89 (Means ± sem), n = 22 cells from 3 mice; Itpr1 −/− : 28.86 ± 3.41, n = 26 from 3 mice, Student's t-test P = 0.51. CV: Itpr1 +/+ : 0.23 ± 0.03, n = 22; Itpr1 −/− : 0.22 ± 0.02, n = 26, Student's t-test P = 0.72).
Since the cerebellar slice is devoid of neuronal inputs arising from other brain structures, we next asked whether spontaneous PC activities are altered in anesthetized mice. We performed extracellular recording of PC activities from caudal lobules in the cerebellar vermis (Figure 3A), which is responsible for the coordination of body trunk movement. We observed high amplitude PC spiking in both total Itpr1 −/− and Wnt1-Cre;Itpr1 flox/flox mice as well as in wild-type mice ( Figure 3B). Typical SSs and CSs were seen in both total Itpr1 −/− and Wnt1-Cre;Itpr1 flox/flox mice and were used as an indication of PC activity in vivo ( Figure 3B). Spontaneous firing rates of Wnt1-Cre;Itpr1 flox/flox mice were decreased compared to Itpr1 flox/flox mice (Itpr1 flox/flox : 17.12 ± 1.61 (N = 5 mice, n = 37 cells); Wnt1-Cre;Itpr1 flox/flox : 12.84 ± 1.59 (N = 4, n = 55). Mann-Whitney U-test: * P = 0.015.), and this tendency was also observed in total Itpr1 −/− mice, although this was not significant [Itpr1 +/+ : 26.01 We also found that averaged auto-correlograms of PC activity in Wnt1-Cre;Itpr1 flox/flox mice lacked a peak around 0-100 ms compared to Itpr1 flox/flox mice ( Figure 3C). The peak height (25-35 ms) was significantly lower than that of Itpr1 flox/flox (Itpr1 flox/flox : n = 37 from 5 animals; Wnt1-Cre;Itpr1 flox/flox mice: n = 55 from 4 animals, Student's t-test P < 0.01, Figure 3C), suggesting an alteration of PC activity patterns caused by IP 3 R1 deletion. Given that the spontaneous activity pattern was little affected in isolated cerebellar slices, these results suggest that neural inputs to PCs, such as parallel fiber or CF inputs, were changed by IP 3 R1 deletion in vivo. Anesthetics influence synaptic neurotransmission or cellular communication (Keane and Biziere, 1987) and suppress animal behavior and movement. To gain more insight into the possible link between PC firing and the expression of dystonia, we recorded multiple unit activity of PCs from freely moving mice (Figure 4A). High amplitude putative PC spiking and low amplitude background activity were alternately recorded as the electrode advanced (400-800 μm). Multiple spiking and increased background activity of awake animals sometimes made discrimination between SSs and CSs difficult. In Itpr1 flox/flox mice, putative PC activity showed regular tonic firing similar to the firing pattern of anesthetized mice (Figure 4A, upper panel). We did not see a drastic change of firing rate associated with particular movements or behaviors in the caudal part of the vermis. Likewise, sleep-wake state associated changes of PC firing rate were not evident.
In contrast, a sharp increase of firing rates was observed intermittently (about once/10 s) in Wnt1-Cre;Itpr1 flox/flox mice and was tightly coupled to body movement related to paroxysmal dyskinesia ( Figure 4A). Typically, Wnt1-Cre;Itpr1 flox/flox mice gradually increased rigidity during the low firing period (indicated by the orange bar in Figure 4A, lower right panel). Then, they abruptly extended their trunk and limbs simultaneously ( Figure 4A, lower left panel) during the high frequency period (indicated by the blue bar). This sequence of high and low frequency firing recurred while the animal was awake, but not during stiff ambulation. Though clear isolation of CSs was difficult, CS activity prevailed during the rigid posture (the orange bar), while SS activity became dominant with high frequency firing during the body-extension (the blue bar) (Figure 4A, lower panels).
Interestingly, the intraperitoneal injection of harmaline (30 mg/kg) in awake wild-type mice reduced SS, and caused CSdominant spike patterns in PCs ( Figure 4B) reminiscent of their firing patterns in the Wnt1-Cre;Itpr1 flox/flox mice during rigid posture. When Wnt1-Cre;Itpr1 flox/flox mice fell asleep, these particular patterns of PC activity diminished. The frequency of CSs was significantly increased in behaving Wnt1-Cre;Itpr1 flox/flox mice as compared to Itpr1 flox/flox mice, whereas the difference was not evident under anesthetized condition ( Figure 4C). An increase of multiunit activity corresponded to the body-extension phase (high firing rate period, blue arrowhead) as judged by an independent observer (Figure 4D). Multiunit firing peaks in several recordings from lobules in caudal portions of the vermis were averaged (N = 4, 20 MUA recordings), and we confirmed distinct cerebellar activity changes associated with rigid and extended postures ( Figure 4E).

TEMPORAL INACTIVATION OF THE CEREBELLUM AMELIORATES DYSTONIA
To confirm the involvement of cerebellar activity in the dystonic movements of Wnt1-Cre;Itpr1 flox/flox mice, we inhibited cerebellar activity by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist (CNQX) infusion. Inactivation of the cerebellum was confirmed by ataxia of CNQX-infused wild-type mice (Figure 5A), including abnormal footprints with shorter step length and wider gait after 2 h of infusion that recovered by 5 h (Figure 5A). Cerebellum specific infusion of the drug was confirmed by Fluo Ruby (Figure 5B). No ataxic gait was observed in wild-type mice infused with saline. Strikingly, CNQX infusion into the cerebellum of Wnt1-Cre;Itpr1 flox/flox mice improved their voluntary movement significantly: dyskinesia such as opisthotonus, rigid posture, and tremor was abolished (Figures 5C,D). Although the mutant mice still showed ataxia, they exhibited partially restored gait within 2 h (Figures 5E,F; Movie S2). After 5 h, dystonic movements of Wnt1-Cre;Itpr1 flox/flox mice appeared.
To examine whether altered IO activity was associated with dystonia in the Wnt1-Cre;Itpr1 flox/flox mice, we pharmacologically inhibited IO activities. We found that lidocaine injection into IO decreased opisthotonus of Wnt1-Cre;Itpr1 flox/flox mice (Figure 7F), although tremor of limbs and ataxia were still observed (Movie S4). In contrast, pharmacological inhibition of bilateral BG (entopeduncular nucleus) activity by lidocaine injection did not significantly affect frequency of opisthotonus in Wnt1-Cre;Itpr1 flox/flox mice ( Figure 7G; Movie S5). These results suggested that altered activities of olivocerebellar tracts cause dystonia in Wnt1-Cre;Itpr1 flox/flox mice in a BG-independent manner.

DISCUSSION
In this study, we demonstrated that genetic deletion of IP 3 R1 within cerebellum and brainstem is sufficient to cause dystonia in mice, and that further pharmacological inactivation of the cerebellum or the IO and deletion of PCs ameliorate the dyskinesia. Thus, our data suggested that dystonia is a gain of function rather than loss of function of olivocerebellar pathways, which is in line with the previous findings (Campbell et al., 1999;Pizoli et al., 2002). Moreover, using electrophysiological recordings of PC activity from freely behaving dystonic mice, we have also demonstrated the relationship between temporal changes of PC spike activity possibly triggered by altered IO activation and the expression of dystonia. Although altered PC activity was found in the movement-restricted dystonic rat (Ledoux and Lorden, 2002), how the temporal changes of PC firing patterns are related to ongoing dystonic movements were unknown. We revealed a distinct pattern of PC firing in freely moving Wnt1-Cre;Itpr1 flox/flox mice during distinct dystonic postures, which could not be observed in neither the anesthetized preparation nor the cerebellar slices. During dystonic movements, PC activities exhibiting repetitive CS patterns were predominant. Since CSs are thought to be important for voluntary movements (Welsh et al., 1995;Kitazawa et al., 1998;Welsh, 2002), the repetitive abnormal synchronized CSs with high frequency during rigid posture may in part underlie dystonia.
Chen et al. recently reported the low-frequency oscillations of flavoprotein autofluorescence in the cerebellar cortex of tottering mice (Chen et al., 2009), and showed that the oscillation was accentuated during dystonia. However, the cellular types and mechanisms that contribute to the enhancement of the oscillation in the mutant mice were unknown. By measuring the PC activities from behaving Wnt1-Cre;Itpr1 flox/flox mice, here we found a precise temporal association between CS-dominant PC firings and distinct dystonic movements. Thus, increase of CF frequency through IO activation may underlie the expression of dystonia in Wnt1-Cre;Itpr1 flox/flox mice. This hypothesis is in line with our finding that infusion of AMPAR blocker in the cerebellum ameliorates dystonia in Wnt1-Cre;Itpr1 flox/flox mice, because AMPA receptor blocker inhibits CF-PC synapse transmission. Although we don't know the relationship between the CS-dominant PC firings in the present study and the low-frequency oscillations in cerebellar cortex shown in the Chen's paper, the CS-dominant PC firings is most likely to be independent of the cerebellar oscillation, since the oscillation was reported to be intrinsic to the cerebellar cortex and the cerebellar blockade by AMPA receptor or by electrical stimulation of PFs did not affect the oscillation (Chen et al., 2009). Our results also suggest a previously unknown pathogenesis of dystonia induced by abnormal cerebellar activity in mice, namely BG-independent dystonia, based on the following facts; no apparent motor abnormality of BG-specific IP 3 R1 conditional mice, no difference in CO staining intensity in Itpr1 flox/flox and Wnt1-Cre;Itpr1 flox/flox mouse's BG, little correlations of BG activity and dystonic movement, and the ineffectiveness of pharmaceutical BG inactivation on dystonia of Wnt1-Cre;Itpr1 flox/flox mice. Thus, we propose that altered cerebellar activity causes dystonia by a mechanism, which does not involve BG activity in Wnt1-Cre;Itpr1 flox/flox mice. It is possible that the abnormal cerebellar outputs generated by IO might be directly sent to spinal cords via red nucleus or reticular formation. It is also worth mentioning that the distinctive CSs appeared only in awake Wnt1-Cre;Itpr1 flox/flox mice, and that altered activation of IO itself was not sufficient for generation of dystonia, since harmaline, which evokes similar CS dominant spike patterns of PCs, does not cause dystonia. Therefore, uncoordinated timings between voluntary corticospinal signals and the involuntary cerebellarreticulospinal signals generated by IO activation with spinocerebellar (somatosensory) inputs may cause simultaneous activation of agonist-and antagonist muscles, leading to dystonia in the Wnt1-Cre;Itpr1 flox/flox mice.
In sum, our study suggests that BG-independent dystonia is triggered by abnormal cerebellar outputs in mice. Wnt1-Cre;Itpr1 flox/flox mice may provide a therapeutic dystonia model solely dependent upon abnormal neural activities within the cerebellum and brainstem. Recently, it was reported that a deletion of the Itpr1 gene is associated with involuntary movements in patients of spinocerebellar ataxia type 15 (Di Gregorio et al., 2010;Marelli et al., 2011), which has been thought to be pure cerebellar ataxia (Hara et al., 2008). The above involuntary movements may be dystonia-related, given that dystonia can be a prominent symptom in SCAs, including some cases with exclusively cerebellar pathology (Manto, 2005). However, since spinocerebellar ataxia type 15 is a slow progressive autosomal dominant disease exhibiting cerebellar atrophy with PC death (Knight et al., 2003;Gardner et al., 2005), severe dystonia would not happen in human. Nevertheless, IP 3 R is known to interact with Na-K ATPase, a causal gene for DYT12 dystonia, and a Na-K ATPase inhibitor, ouabain, causes aberrant Ca 2+ release from the IP 3 Rs (Zhang et al., 2006). Thus, it is possible that dysfunction of IP 3 R1 could be associated with dystonia in human. Further studies on the mechanism by which disturbed Ca 2+ signals from IP 3 R1 lead to the repetitive synchronized CSs in our mutant mice, such as potential Ca 2+ -dependent regulation of gap junction among IO neurons, may contribute to the understanding of pathogenesis and the development of new therapies for dystonia.

AUTHOR CONTRIBUTIONS
Chihiro Hisatsune designed the project, performed experiments, and wrote the manuscript. Katsuhiko Mikoshiba wrote the manuscript. Hiroyuki Miyamoto, Moritoshi Hirono, Takao K. Hensch, and Masahisa Yamada. performed the electrophysiological experiments and wrote the manuscript. Naoko Ogawa, Etsuko Ebisui, and Takeyuki Sugawara performed the experiments. Naohide Yamaguchi, and Mitsuharu Hattori generated the Itpr1 flox/+ mice. Toshio Ohshima helped to establish mutant mice.