Stress, caffeine and ethanol trigger transient neurological dysfunction through shared mechanisms in a mouse calcium channelopathy

Abstract

Several episodic neurological disorders are caused by ion channel gene mutations. In patients, transient neurological dysfunction is often evoked by stress, caffeine and ethanol, but the mechanisms underlying these triggers are unclear because each has diverse and diffuse effects on the CNS. Attacks of motor dysfunction in the Ca(V)2.1 calcium channel mouse mutant tottering are also triggered by stress, caffeine and ethanol. Therefore, we used the tottering mouse attacks to explore the pathomechanisms of the triggers. Despite the diffuse physiological effects of these triggers, ryanodine receptor blockers prevented attacks induced by all of them. In contrast, compounds that potentiate ryanodine receptors triggered attacks suggesting a convergent biochemical pathway. Tottering mouse attacks were both induced and blocked within the cerebellum suggesting that the triggers act locally to instigate attacks. In fact, stress, caffeine and alcohol precipitated attacks in Ca(V)2.1 mutant mice in which genetic pathology was limited to cerebellar Purkinje cells, suggesting that the triggers initiate dysfunction within a specific brain region. The surprising biochemical and anatomical specificity of the triggers and the discovery that the triggers operate through shared mechanisms suggest that it is possible to develop targeted therapies aimed at blocking the induction of episodic neurological dysfunction, rather than treating the symptoms once provoked.

Figure 1. Response to adenosine receptor antagonists

A–C. Systemic injections of the nonselective adenosine receptor antagonists caffeine, theophylline or paraxanthine dose-dependently induced attacks in tottering mice (n = 10–24/dose). D–F. Systemic injections of the A1 adenosine receptor-selective antagonist DPCPX or the A2a adenosine receptor antagonist ZM 241385 did not induce attacks when administered alone or in combination (n = 10–16/dose). **p < 0.01, ***p < 0.001 as determined by logistic regression analysis.

Figure 2. Effect of dantrolene on caffeine- and stress-induced attacks

A–C. Systemic administration of the RyR antagonist dantrolene dose-dependently reduced the frequency of attacks in tottering mice triggered by 5 mg/kg caffeine, theophylline or paraxanthine (n = 10–17/dose). D. Dantrolene dose-dependently blocked attacks triggered by restraint stress (n = 11–12/dose). *p < 0.05, **p < 0.01, ***p < 0.001 as determined by logistic regression analysis.

Figure 3. Effect of intracerebellar ryanodine microinjections on tottering mouse attacks

Mice were pretreated with intracerebellar microinjections of ryanodine (100 μM) or vehicle prior to restraint stress or systemic injections of caffeine (5 mg/kg) or ethanol (1 g/kg). A. Ryanodine significantly reduced the frequency of attacks triggered by stress, caffeine or ethanol in tottering mice (n = 5–12/dose). B. Ryanodine significantly reduced the frequency of attacks triggered by stress, caffeine or ethanol in tottering^PC-haplo mice (n = 5–7/dose), which carrying an attack-causing genotype in only Purkinje cells. *p < 0.05, **p < 0.01 as determined by chi-square test.

Figure 4. Effect of intracerebellar caffeine infusion on tottering mouse attacks

Vehicle or caffeine was reverse dialyzed into cerebellum or striatum of awake-behaving mice. A. Typical location of a cerebellar microperfusion probe. Scale bar, 500 μm B. Intracerebellar infusions of caffeine dose-dependently induced attacks in tottering mice (n = 7–9/dose). *p < 0.05, ***p < 0.001 as determined by logistic regression analysis. C. Microperfusions of 2.5 mM caffeine into the tottering mouse striatum (n = 6) did not elicit any motor abnormalities. C. Systemic administration of 20 mg/kg dantrolene (+Dan), but not vehicle (+Veh), blocked attacks induced by intracerebellar microperfusions of caffeine (n = 4–5/dose). *p < 0.05, **p < 0.01 as determined by chi-square test.

Figure 5. X-gal staining as a reporter for Cre recombinase expression in mice carrying the L7-Cre transgene

The L7-Cre transgene was bred onto the Cre-reporter mouse line, Rosa26, which is permissive for ß-galactosidase expression in the presence of Cre recombinase. A–D. X-gal staining was localized to Purkinje cells. Scale bars, 100 μm

Figure 6. Attacks are mediated by Purkinje cells

The L7-Cre transgene, which expresses Cre recombinase only in Purkinje cells was bred into mice bearing one tottering allele and one floxed allele (Cacna1a^tg/flox) to generate tottering^PC-haplo mice, which carry an attack-causing genotype only in Purkinje cells. Mice bearing one tottering allele and one null allele (tottering^haplo) and tottering mice carrying the L7-Cre transgene (tottering^PC-Cre) were also tested as controls. A. Environmental challenges. Restraint induced attacks in all genotypes. Tottering^PC-haplo mice (n = 11–12/challenge) and tottering^haplo mice (n = 14/challenge) were more sensitive to the other environmental challenges than tottering mice (n = 11/challenge) and tottering^PC-Cre mice (n = 7/challenge). B. Caffeine dose-dependently induced attacks in all genotypes. Tottering^PC-haplo mice (n = 11–12 per dose) and tottering^haplo mice (n = 13–14/dose) were more sensitive to the effects of caffeine than tottering mice (n = 10–11/dose) and tottering^PC-Cre mice (n = 7/dose). C. Ethanol dose-dependently induced attacks in all genotypes. Tottering^PC-haplo mice (n = 10–16/dose) and tottering^haplo mice (n = 12–14/dose) were more sensitive to the effects of ethanol than tottering mice (n = 9–11/dose) and tottering^{PC- Cre} mice (n = 7/dose). *p < 0.05, **p < 0.01, ***p < 0.001 as determined by logistic regression analysis.