Episodic ataxia type 2 (EA2) is a rare autosomal dominant inherited neurological disorder caused by loss-of-function mutations in the CACNA1A gene, which encodes the pore-forming α1A-subunit of the voltage-gated P/Q-type calcium channel (Ophoff et al., 1996). Dysfunctions in the channel lead to decreased Ca²⁺ influx in cerebellar Purkinje cells (PCs), the sole output neuron of the cerebellar cortex. Clinically, EA2 is characterized by recurrent episodes of ataxia, vertigo and dystonia which can last a few hours to days and can be triggered by physical or emotional stress, ethanol or caffeine (Jen and Wan, 2018).

To elucidate the pathophysiology of EA2, several mouse models have been developed (Mark et al., 2011; Maejima et al., 2013; Fureman and Hess, 2005). The tottering^tg/tg mouse, a well-established mouse model for EA2, displays a spontaneous mutation in the Cacna1a gene, resulting in ataxia and stress-induced dystonia associated with aberrant PC activity (Walter et al., 2006). Recent work from our laboratory demonstrated that selective blockade of α1D-adrenergic receptors (ARs) is sufficient to abolish stress-induced dystonia and improve ataxia in tottering^tg/tg mice by stabilizing PC firing (Bohne et al., 2025), suggesting α1-AR blockade as a potential treatment option (Snell et al., 2022). Our laboratory previously established Cacna1a^purk(-/-) mice (purky) as a complementary EA2 model. In purky mice, the P/Q-type calcium channel is selectively deleted from PCs, resulting in severe ataxia and paroxysmal stress-induced dystonia (Mark et al., 2011), providing a valuable system for dissecting PC-dependent mechanisms in EA2 pathophysiology. In this study we aimed to identify noradrenergic receptor subtypes mediating stress induced dystonia in purky mice, where the P/Q-type calcium channel is specifically ablated from PCs.

To investigate whether the beneficial effects of AR blockade observed in tottering^tg/tg mice can also be transferred to purky mice, we intraperitoneally injected different α1-, and α2-AR antagonists and exposed the animals to a new environment (cage change stress) (Figure 1). The general α1-AR prazosin (Praz, green, 10 mg/kg) increased the frequency of cage change stress-induced dystonia in Cacna1a^{purk(−/−)} mice from 60 to 100% (n = 10), but reduced dystonia duration compared to vehicle injected purky mice (Figure 1a). Interestingly, the α1D-AR subtype specific antagonist BMY-7378 (brown, 10 mg/kg) substantially alleviated stress-induced dystonia by 75%, without affecting duration or onset of attacks (n = 8). A complete absence of stress-induced dystonia was observed when we blocked α2-ARs (Figure 1a) with both the general α2-AR blocker yohimbine (Yoh, blue, n = 10), as well as the α2A adrenergic autoreceptor agonist clonidine (Clon, pink, n = 12) (Figure 1a). Clonidine primarily binds to presynaptically expressed α2-AR, thereby decreasing the release of NE and abolishing stress-induced dystonia in a dose-dependent manner (Fureman and Hess, 2005). Yohimbine, however, blocks α2-ARs, therefore likely preventing NE-induced effects postsynaptically. However, injection of Yoh had no rescuing effect on ataxia in purky mice (Table 1) and did not improve motor coordination in control mice (Supplementary Table 3). Interestingly, tottering^tg/tg mice do not show alleviation of stress-induced dystonia after yoh injection (Fureman and Hess, 2005; Bohne et al., 2025), thus our purky mice are the first EA2 model which respond to α2-, and α1D-AR blockade. Since yohimbine was effectively preventing stress-induced dystonia in purky mice, we explored the effects of α2-AR blockade using Yoh on the norepinephrine (NE)-induced inhibition of PC simple spike (SS) firing in anaesthetized Cacna1a^{purk(−/−)} mice (Figure 1b, N = 3 mice). Pressure injection of 10 μM NE inhibited PC SS firing by 87% compared to baseline recordings (Ref, grey, N = 7 cells) (Figures 1c–e). However, application of 5 μM Yoh significantly recovered the PC SS firing, suggesting a rescinding effect on the NE-induced inhibition (Figure 1e). Surprisingly, we did not find positive effects of Yoh on PC regularity or the predominant firing rate of PCs, suggesting contributions of other cerebellar neurons to alleviation of dystonia by α2-AR blockade in purky mice.

Norepinephrine containing terminals in the cerebellum originate solely from the locus coeruleus (LC) (Olson and Fuxe, 1971). The LC-NE⁺ fibres mainly form synapses with PC dendrites (Olson and Fuxe, 1971; Bloom et al., 1971; Hökfelt and Fuxe, 1969), but also synapse on PC soma, granule cell dendrites, and the deep cerebellar nuclei (Gould et al., 1997). Interestingly, several cerebellar degeneration animal models show alterations in their cerebellar monoaminergic system (Landis et al., 1975; Muramoto et al., 1982). For example, tottering^tg/tg mice display increased noradrenergic innervation (Levitt and Noebels, 1981), while the number of LC neurons is not altered (Noebels, 1984). Similarly, Purkinje cell degeneration (pcd) mice, a mouse model of human degenerative ataxia, display PC loss accompanied by cerebellar degradation and ataxia (Mullen et al., 1976), similar to what we observe in our purky mice (Mark et al., 2011). The pcd mouse exhibits increased amounts of NE transporters in the cerebellar cortex, especially in vermis and paravermis which are associated with motor functions. To investigate whether dystonia is accompanied by altered noradrenergic innervation in our Cacna1a^{purk(−/−)} mice, we performed immunohistochemical staining against dopamine-β-hydroxylase (DβH, red), the enzyme which catalyses dopamine to norepinephrine, in motor coordination involved cerebellar lobules 4 and 5, vestibular controlling lobule 9 and cognition related Crus2 (Figure 2). In accordance with the literature, we found severe loss of PCs in purky mice in all regions analysed (Mark et al., 2011; Ghetti et al., 1981; Todorov et al., 2012). Remaining PCs were identified by calbindin staining (white, Figure 2a), which revealed significantly smaller somata compared to control mice (Figure 2b). However, purky PCs showed increased DβH immunoreactivity, displayed as mean fluorescence intensities (mFl), compared to control mice (Figure 2b). This also reflects in a higher ratio of the mFl to PC area in purky meaning smaller PC soma exhibit brighter fluorescence, suggesting increased noradrenergic innervation from the LC to PCs (Figure 2c). In addition, we also found more DβH⁺ soma in the LC of purky compared to control mice, potentially suggesting that the increased DβH⁺ immunoreactivity in the cerebella of purky mice is a consequence of an increased number of neurons in the LC projecting to the PC layer (Figure 2d). To confirm this hypothesis, we additionally analysed the area comprising the LC in both control and Cacna1a^{purk(−/−)} mice. We could not identify alterations in the LC size or the amount of DβH⁺ pixels within this area, but analysis revealed that purky mice display increased neuronal density in the LC, suggesting alterations in neurogenesis (Figure 2e). Our findings strengthen the idea that EA2 and the formation of stress-induced dystonia is correlated to altered noradrenergic innervation.

Many studies correlated AR activation to stress-induced dystonia in EA2 mouse models (Fureman and Hess, 2005; Bohne et al., 2025; Snell et al., 2022; Fureman et al., 2002). A predominant role of α1-ARs is commonly accepted, and we recently reported the contribution of α1D-AR specifically expressed on PCs to dystonia formation in tottering^tg/tg mice (Bohne et al., 2025). Interestingly, both the specific α1D-AR blocker BMY-7378, but also general α2-AR yohimbine alleviated stress-induced dystonia in purky mice, suggesting a coordinated engagement of α1- and α2-ARs during dystonia formation. In mice, α1D-ARs are almost exclusively expressed in cerebellar PCs⁷ and both the pharmacological block, as well as shRNA-induced knock-down of specifically cerebellar α1D-ARs alleviated stress-induced dystonia in tottering^tg/tg mice (Bohne et al., 2025), suggesting a PC specific effect. Similarly, blocking α1D-ARs in purky mice decreased, but not abolished, stress-induced dystonia, suggesting that both α1D-ARs and PCs are required for dystonia in purky mice, but are likely not the only key players. Injection of yoh alleviated stress-induced dystonia, possibly by simultaneously blocking multiple α2-AR subtypes expressed throughout the cerebellar network (Bohne et al., 2025; Wang et al., 1996). This interpretation is based on previously published studies linking dystonia to the cerebellum (Bohne et al., 2025; Snell et al., 2022). Since yoh was administered systemically, we cannot exclude contributions from other brain regions or peripheral off targets. Our hypothesis of yoh targeting multiple subtypes simultaneously and thereby preventing dystonia, is strengthened by the selective blockade of α2B-ARs via i.p. injection of imiloxan hydrochloride which did not alleviate stress-induced dystonia in purky mice (Supplementary Figure 1). This observation suggests that under our experimental conditions, selective blockade of α2B-ARs is insufficient to modify dystonia and that the other α2 subtypes or additional mechanisms are involved.

We hypothesise that yohimbine antagonizes different subtypes of α2-ARs in the cerebellar network, thereby attenuating the NE-induced inhibition of PCs, as demonstrated in our electrophysiological recordings (Figure 1). However, our electrophysiological recordings show only a partial restoration, which may not fully explain the observed phenotypical effect of complete stress-induced dystonia abolishment. A likely explanation could be that NE already activated both α1- and α2-ARs and respective G-protein pathways, where belated injection of yoh only partially reversed these NE-induced effects. We previously made these observations for NE-induced inhibition of PCs in tottering^tg/tg mice using BMY-7378, where BMY-7378 showed better protection of PC SS firing when injected before NE (Bohne et al., 2025). Similar effects could apply to yoh, too. Additionally, we recorded from anaesthetized mice, thus the observed effects do not necessarily correlate to the observed behavioural effects. Lastly, since we only recorded from PCs, we cannot rule out the effects of other cerebellar neurons on PCs or the whole cerebellar network, which could contribute to the absence of dystonia. In accordance with this hypothesis, a recent study showed that activation of presynaptic α2A- and α2B-ARs downregulated parallel fibre (PF) to PC synaptic transmission (Wang et al., 2023), thereby reducing glutamatergic-induced SS firing. The PCs of purky mice display severe firing deficits (Mark et al., 2011), but in the presence of NE, the firing can be further decreased via presynaptic activation of α2A- and α2B-ARs on the PF synapse. Blocking the NE-mediated activation of presynaptic α2A- and α2B-ARs using yohimbine could promote normal PC excitation and help maintain physiological PC firing.

Our data suggest that stress-induced dystonia in Cacna1a^{purk(−/−)} mice involves both α1D- and α2-adrenergic receptor signalling, thereby strengthening ARs as therapeutic targets in EA2. However, given that our conclusions are based on a specific genetic mouse model, systemic pharmacological manipulations and a particular stress paradigm, further studies using more selective and region-specific interventions, different EA2 models and ideally human data will be required to fully assess the translational potential and safety of targeting adrenergic receptors in EA2.