Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Feb 1.
Published in final edited form as: Neurobiol Dis. 2012 Sep 23;50:151–159. doi: 10.1016/j.nbd.2012.09.005

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

Robert S Raike a, Catherine Weisz b, Freek E Hoebeek c, Matthew C Terzi d, Chris I De Zeeuw c,e, Arn M van den Maagdenberg f, HA Jinnah d,g, Ellen J Hess a,d,†
PMCID: PMC3534906  NIHMSID: NIHMS421533  PMID: 23009754

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 CaV2.1 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 CaV2.1 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 suggests that it is possible to develop targeted therapies aimed at blocking the induction of episodic neurological dysfunction, rather than treating the symptoms once provoked.

Keywords: episodic, channelopathy, cerebellum, Purkinje, calcium channel, tottering

Introduction

Recurrent transient neurological dysfunction superimposed on a relatively normal baseline is observed in several disorders such as familial hemiplegic migraine, episodic ataxia, and periodic paralysis. In each case, transient neurological impairments are superimposed on a relatively normal baseline. Although the clinical features of these disorders are diverse, the underlying causes of the condition and the triggers that bring on attacks are strikingly similar. Many of these disorders are associated with ion channel gene mutations, and are therefore known as channelopathies (Bhatia et al., 2000; Cannon, 2006; Ptacek, 1999). Further, regardless of etiology, attacks are induced in many episodic disorders by psychological or physical stress, and the consumption of caffeine or alcohol (Bhatia, 2001; Frucht et al., 2000; Ptacek, 1999).

The mechanisms underlying the induction of neurological dysfunction in patients who are otherwise normal between attacks are unknown primarily because the triggers have a variety of effects on the nervous system. Stress activates the limbic-hypothalamic-pituitary-adrenal axis, causing the release of several neurochemicals, including corticotrophin releasing factor (CRF), adrenocorticotropic hormone, corticosteroids and catecholamines (Koob, 1999). Caffeine acts as a stimulant through adenosine receptor antagonism, inhibits phosphodiesterase and mobilizes calcium from intracellular stores (Daly, 2007; Muller and Daly, 1993). Ethanol acts through multiple signaling cascades involving GABA, glycine, adenosine and NMDA receptors (Kumar et al., 2009; Newton and Messing, 2006). Each of these biochemical mechanisms may impinge on multiple brain areas. Despite the diverse effects of these triggers, their ability to provoke symptoms across a broad range of episodic disorders suggests the possibility of a common mechanism.

Because the attacks are observed only in intact animals, the trigger mechanisms must be assessed in vivo. Mouse mutants that recapitulate both genotypic and phenotypic characteristics of episodic neurological disorders are therefore essential for this type of analysis. The tottering mouse mutant exhibits stereotyped attacks of dystonia (Shirley et al., 2008) that are caused by a mutation in the Cacna1a gene (Fletcher et al., 1996). Mutations in this gene in man and mouse are associated with aberrant function of the pore-forming α1 subunit of CaV2.1 (P/Q-type) voltage-gated calcium channels (Jen et al., 2007; Wakamori et al., 1998). In humans, CACNA1A mutations are associated with various disorders, including episodic ataxia type 2 (EA2) and familial hemiplegic migraine type 1 (Ophoff et al., 1996). Similar to humans, the attacks in tottering mice are triggered by stress, caffeine and alcohol (Fureman et al., 2002). Further, drugs that prevent episodic dysfunction in patients, such as 4-aminopyridine, also block attacks in tottering mice (Weisz et al., 2005). Therefore, we used the attacks in tottering mice as a readout to explore the pathomechanisms of the triggers.

Materials and Methods

Animals

Mice were bred and housed at Emory University or Johns Hopkins University vivaria. Tottering mice (Cacna1atg/tg) congenic with C57BL/6J were originally obtained from The Jackson Laboratory (Bar Harbor, ME). Mice carrying floxed Cacna1a alleles (Cacna1aflox/flox) were on a C57BL/6J background, as described previously (Todorov et al., 2006). Tg(Pcp2-Cre)2Mpin/J (common name, L7-Cre) on C57BL/6J were purchased from The Jackson Laboratory. Mice carrying a null allele (Cacna1a+/−) on a mixed C3H-C57BL/6J background were kindly provided by Dr. David Yue (Johns Hopkins University, Baltimore, MD). PCR was used to genotype mice with forward and reverse primers: for the Cacna1a− allele, 5'-ATAATAAGTCACCTCTCGTTCTAAAG-3' and 5'-CTGACTAGGGGAGGAGTAGAAG-3'; for the Cacna1aflox allele, 5'-ACCTACAGTCTGCCAGGAG-3' and 5'-TGAAGCCCAGACATCCTTGG-3'; for the Cacna1atg allele, 5'-GGAAACCAGAAGCTGAACCA-3'and 5'-GAAATGGAGGAATTCAGGG-3'; for L7-Cre, 5'-GCGGTCTGGCAGTAAAAACTATC-3' and 5'-TCTCTGACCAGAGTCATCCTTAGC-3'. The PCR product for the Cacna1atg/tg allele was sequenced to detect the C1802T mutation.

Male and female mice (8–20 wks of age) were used in all experiments since we have previously shown that tottering mouse attacks are comparable across age and between genders (Weisz et al., 2005). Mice had access to food and water ad libitum throughout all experiments. All procedures conformed to the NIH Guidelines for the Care and Use of Animals and were approved by the Emory University or Johns Hopkins University Animal Care and Use Committees.

Motor behavior analyses

Mice were transferred from the vivarium to the laboratory at least 4 hours prior to drug challenge or restraint stress drug. For restraint stress, mice were placed in a plastic tube with air holes for 10 min, and then scored for attacks following release. Pretreatment drugs were administered 30 min before challenge with caffeine or ethanol or 10 min before restraint challenge. After drug challenge or restraint stress, mice were observed for 40 min for the presence or absence of an attack of generalized dystonia. Each mouse was scored for 30 sec every 10 min using a disability scoring system previously described (Jinnah et al., 2000), where D0 = normal motor behavior; D1 = slightly slowed or abnormal movements; D2 = fleeting abnormal postures; D3 = moderate impairment, limited ambulation even when disturbed, frequent abnormal postures; D4 = severe impairment, sustained abnormal postures. An attack was defined as a disability score of D ≥ 3. The percent of mice exhibiting an attack during the 40 min following challenge was used as a measure of attack frequency. Observers were blinded to treatment.

To assess the central effects of adenosinergic antagonists, gross locomotor activity was quantified using photocell activity chambers (San Diego Instruments, San Diego, CA). Mice were individually placed into a clean activity chamber and habituated overnight. The following morning mice were treated systemically with drug or vehicle and the total number of beam breaks in the ensuing 40 min period was used for analysis.

To assess the effects of dantrolene on strength and coordination, the cling and pole tests were used. The cling test was performed as previously described (Devanagondi et al., 2007), where mice were habituated for 1 min on a horizontal 20 × 20 cm framed grid of wire mesh suspended above a padded surface. The frame was then rotated 90° to vertical for 1 min, and then rotated another 90° for 1 min so that the mice were inverted. The time to fall was recorded with a 180 sec cut-off. For the pole test, a vertical wooden rod measuring 50 cm in length and 1 cm in diameter affixed to a square base (12 × 12 cm) was used. The base of the pole was placed in the home cage. Mice were placed with the head facing up at the top of the pole. The time for each mouse to turn around, descend the pole and place all four limbs onto the base was recorded. Mice performed 5 trials of training each day for 2 days prior to testing. On the test day, the best time of the 5 trials was used.

Systemic drug administration

All drugs were first tested in normal C57BL/6J mice for deleterious side effects such as sedation or seizures. Each drug was tested on at least 2 separate dates and mice were pseudorandomly assigned to a drug dose or vehicle group. Observers were blinded to treatment. Caffeine, dimethylxanthine (theophylline) and 1,7-dimethylxanthine (paraxanthine) were obtained from Sigma (St. Louis, MO). ZM241385 and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) were obtained from Tocris (Ellisville, MO). Dantrolene was obtained from Proctor & Gamble (Cincinnati, OH). Caffeine, theophylline and paraxanthine were administered subcutaneously (s.c.). All other drugs were administered intraperitoneally (i.p.). Dantrolene was administered 30 min before drug challenge or 10 min before restraint challenge.

Intracerebellar microinjection

Intracerebellar microinjections were performed as previously reported (Pizoli et al., 2002; Weisz et al., 2005). Mice were anesthetized with isoflurane and injected with 0.5 μl of 100 μM ryanodine (Tocris) or saline vehicle. For cerebellum, coordinates were: −6.5 mm AP from bregma, 0 mm ML from midline, −2 mm V from skull. For lateral ventricle, coordinates were: −0.5 mm AP, 1.25 mm ML, −3 mm V. The wound was sealed with Nexaband topical skin closure (Veterinary Products Laboratories, Phoenix, AZ). Mice were administered caffeine or ethanol approximately 2 min after microinjection. Restraint began approximately 4 min after microinjection. Microinjection sites were histologically verified.

Caffeine microperfusion

Concentric microdialysis probes were used to reverse dialyze caffeine into select brain regions. Mice were anesthetized with tribromoethanol (Sigma), a small incision made and a hole drilled for implantation of the probe, which was constructed as previously described (Fan and Hess, 2007). For cerebellum the coordinates for probe implantation were the same as for microinjections. For striatum the coordinates were: −0.6 mm AP, 1.7 mm ML, −5.4 mm V. After surgery, the probe was connected to a microinjection pump (Harvard Apparatus, Holliston, MA) through a liquid swivel (Instech, Plymouth Meeting, MA) and perfused continuously with artificial cerebrospinal fluid (147 mM NaCl, 3.5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 1 mM NaH2PO4, 25 mM NaHCO3, pH 7.4) at a flow rate of 1.5 μl/min. Mice were allowed 14–16 hr recovery after surgery and then caffeine was added to the perfusate. Percent recovery of amino-acid neurotransmitters in vitro was used to determine the relative exchange rate of each probe prior to use in vivo. Perfusion sites were histologically verified.

Histology

To verify injection and infusion sites, sections were stained with cresyl violet. For X-gal staining, tissue was incubated in 0.02% Igepal (Sigma), 0.01% sodium deoxycholate, 2 mM MgCl2, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 1 mg/ml X-gal in PBS overnight and counterstained with Nuclear Fast Red (Vector Laboratories, Burlingame, CA).

Statistical analysis

Nonparametric logistic regression and chi-square techniques were used to analyze attack frequency data. Student's t test was used for cling and pole test data. Analyses were performed using Statview (SAS Institute, Cary, NC).

Results

Common biochemical mechanisms of stress, caffeine and ethanol

Although each trigger has many effects on the CNS, all increase arousal. The stimulatory effects of caffeine are attributed to its action as a nonselective A1/A2a adenosine receptor (ADR) antagonist (Williams and Jarvis, 1988). Stress and ethanol are also implicated in A1 and A2a ADR signaling (da Silva Torres et al., 2003; Minor et al., 2001; Newton and Messing, 2006), suggesting a common mechanism of action. To determine if adenosinergic signaling mediates neurological attacks, we challenged tottering mice with a battery of nonselective and selective ADR antagonists. Centrally active doses were first determined by assessing drug-induced increases in locomotor activity in normal mice. All compounds increased locomotor activity at the doses tested (Table 1). As previously reported (Fureman et al., 2002), caffeine dose dependently induced attacks (Figure 1A). Similar to caffeine, the mixed A1/A2a ADR antagonists, 1,3-dimethylxanthine (theophylline) and 1,7-dimethylxanthine (paraxanthine) dose-dependently induced attacks in tottering mice (Figure 1B & C). To determine if these effects were specific to a particular ADR subtype, tottering mice were challenged with DPCPX, an A1-selective ADR antagonist, or ZM 241385, an A2a-selective antagonist. Neither DPCPX (Figure 1D) nor ZM 241385 (Figure 1E) induced attacks at any dose tested, even at doses that significantly increased motor activity (Table 1). Although these results suggest that attacks are not instigated through either adenosine receptor subtype alone, it is possible that coincident antagonism of A1 and A2a ADRs, similar to the action of caffeine, is necessary to trigger attacks. However, co-administration of the A1-selective ADR antagonist DPCPX and the A2a-selective antagonist ZM 241385 failed to trigger attacks (Figure 1F), suggesting adenosinergic signaling is not likely involved in the instigation of attacks by caffeine.

Table 1.

Effect of adenosinergic antagonists (5 mg/kg) on the locomotor activity of normal mice

Drug (ADR subtype) Beam breaks (% vehicle)
Caffeine (mixed) 281 ± 33***
Theophylline (mixed) 223 ± 42*
Paraxanthine (mixed) 346 ± 72**
DPCPX (A1) 234 ± 54*
ZM 241385 (A2a) 281 ± 36***
DPCPX + ZM 241385 (A1+A2a) 369 ± 63**
Open in a new tab

Data are expressed as mean ± SEM and were analyzed with Student's t test (n = 6–11/drug).

*

p < 0.05,

**

p < 0.005,

***

p < 0.001 compared to vehicle treatment.

Figure 1. Response to adenosine receptor antagonists.

Figure 1

Open in a new tab

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.

At high concentrations, caffeine acts at ryanodine receptors (RyR) to facilitate the mobilization of calcium from intracellular stores (Kano et al., 1995; Womack et al., 2000). The caffeine analogs theophylline and paraxanthine also promote ryanodine-sensitive increases in the intracellular Ca2+-concentration (Muller and Daly, 1993). Further, tottering mice carry a mutation in a calcium channel gene, supporting the idea that calcium-dependent mechanisms may be involved. To test the hypothesis that RyRs mediate attacks, tottering mice were treated with systemic injections of the RyR antagonist dantrolene before challenge with caffeine, theophylline or paraxanthine. Pretreatment with dantrolene dose-dependently reduced the frequency of attacks induced by all 3 drugs (Figure 2A–C), suggesting that RyR activation is necessary to induce attacks.

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

Figure 2

Open in a new tab

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.

In addition to its action at neuronal RyRs, dantrolene blocks RyRs in skeletal muscle and is used as a muscle relaxant in humans. Because it is possible that dantrolene spuriously inhibited the expression of attacks in tottering mice by acting as a muscle relaxant rather than blocking the trigger per se, the effect of dantrolene on volitional motor function was examined. Dantrolene did not significantly affect the performance of tottering mice on either the cling test or the pole test, which both require strength and motor control (Table 2). These results do not rule out the possibility that dantrolene alters muscle function but do suggest that, at the highest dose used in these experiments, dantrolene did not likely mask tottering mouse attacks by weakening muscles.

Table 2.

Effect of dantrolene (20 mg/kg) on the motor performance of tottering mice

Time after injection
30 min 60 min
Pole Test (sec)
Vehicle 17 ± 2 13 ± 1
Dantrolene 14 ± 2 15 ± 1
Cling Test (sec)
Vehicle 150 ± 6 142 ± 2
Dantrolene 156 ± 7 144 ± 6
Open in a new tab

Data are expressed as mean ± SEM and were analyzed with Student's t test (n = 9–10/group). There were no significant differences between vehicle and treated mice..

To determine whether stress triggers attacks through a mechanism similar to caffeine, the ability of dantrolene to block stress-induced attacks was assessed. Pretreatment with dantrolene significantly reduced the frequency of attacks triggered by restraint stress (Figure 2D). Similar experiments using dantrolene to block attacks triggered by ethanol were not feasible due to the sedating effects of this drug combination in vivo. Overall, these results suggest that stress and caffeine trigger attacks through similar biochemical mechanisms.

Triggers act in the cerebellum

Localization of (dys)function is critical for understanding pathogenesis; yet stress, caffeine and ethanol act in many brain regions. It is not known whether neurological dysfunction is triggered via diffuse effects, or if attacks are initiated within discrete regions of the brain. We hypothesized that the triggers act locally because the transient motor symptoms arising from mutant CaV2.1 channels in humans and mice are associated with cerebellar dysfunction (Jen et al., 2007; Walter et al., 2006). Therefore, ryanodine, a RyR antagonist, was microinjected into tottering mouse brain prior to systemic administration of caffeine. Intracerebellar ryanodine injections significantly reduced the frequency of attacks triggered by caffeine compared to intracerebellar injection of saline vehicle (Fig 3A; p < 0.05). However, microinjection of ryanodine into the lateral ventricle had no effect on the frequency of attacks triggered by caffeine whereby four out of five tottering mice treated with vehicle exhibited attacks and four out of six tottering mice treated with ryanodine exhibited attacks (p = 0.62; chi-square test). These results suggest that ryanodine injected into the cerebellum did not diffuse to other brain regions to block attacks.

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

Figure 3

Open in a new tab

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 totteringPC-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.

To determine if the cerebellum is also the site of action for other triggers, we tested the ability of intracerebellar ryanodine injections to block attacks induced by stress or ethanol. Similar to caffeine, intracerebellar injections of ryanodine, but not vehicle, significantly reduced the frequency of attacks induced by stress or ethanol (Figure 3A; p < 0.01; chi-square test) suggesting that all three triggers work through the cerebellum.

To determine if attacks are triggered within the cerebellum, we performed localized microperfusions of caffeine in freely moving tottering mice (Figure 4A). Microperfusion of caffeine into the cerebellum dose-dependently triggered attacks in tottering mice with an average latency of 31 ± 4 min after the start of the infusion, whereas vehicle alone had no effect (Figure 4B). Microperfusion of caffeine into the normal mouse cerebellum did not cause attacks (n = 4; not shown). Microperfusion of caffeine into the tottering mouse striatum did not induce attacks, even after 80 min (Figure 4B) suggesting that the effects of microperfusion are specific to the cerebellum and that microperfused caffeine was not diffusing throughout brain. Furthermore, pretreatment of tottering mice with systemic administration of dantrolene completely blocked attacks triggered by microperfusion of caffeine into the cerebellum (Figure 4C; p < 0.01). Thus, attacks were triggered and blocked within the cerebellum.

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

Figure 4

Open in a new tab

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.

Attacks are mediated by Purkinje cells

Although stress, caffeine and ethanol act as nonselective agents throughout the CNS, the previous results suggest that attacks are triggered locally within the cerebellum. In light of this localized effect, we asked if a single cell type might mediate the response. Purkinje cells provide the sole output of cerebellar cortex and their firing patterns are exquisitely sensitive to changes in CaV2.1 currents (Hoebeek et al., 2005; Walter et al., 2006). Therefore, we tested the hypothesis that Purkinje cells mediate the response to triggers by using the conditional floxed Cacna1a allele in combination with the tottering allele to achieve cell-specific expression (Table 3). Heterozygous tottering mice (Cacna1atg/+; n = 14) did not exhibit attacks (Campbell and Hess, 1998). Likewise, Cacna1atg/flox mice, which bear one tottering allele and one normoactive floxed Cacna1a allele (Todorov et al., 2006), did not exhibit attacks (n = 8). To isolate the attack-causing genotype to Purkinje cells, the L7-Cre transgene, which drives Cre recombinase expression in Purkinje cells (Figure 5), was bred into Cacna1atg/flox mice. These mice carried one normoactive allele and one tottering allele everywhere except Purkinje cells, where the tottering allele and a null allele were present (totteringPC-haplo mice). Tottering mice carrying the L7-Cre transgene (totteringPC-Cre mice) were produced to control for possible effects of Cre recombinase expression in Purkinje cells on the response to the triggers. Mice bearing one tottering and one null allele throughout the nervous system (totteringhaplo mice) were also generated to control for Cacna1a gene dose and to determine if other cell types contribute to the response to triggers.

Table 3.

Nomenclature of experimental and control mice

Common Name Genotype Description Phenotype
Normal Cacna1a +/+ Normal C57BL/6J mice Normal
Tottering Cacna1atg/tg Two tottering alleles Attacks
Heterozygous tottering Cacnala +/tg One tottering and one normal allele Normal
Heterozygous tottering-fioxed Cacna1atg/flox One tottering and one normal floxed allele Normal
TotteringPC-Cre Cacna1atg/tg; L7-Cre +/− Tottering mice expressing Cre recombinase in Purkinje cells Attacks
Totteringhaplo Cacna1a tg/− One tottering and one null allele throughout Attacks
TotteringPC-haplo Cacna1atg/flox; L7-Cre +/− One tottering and one null allele in Purkinje cells but otherwise heterozygous throughout Attacks
Open in a new tab

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

Figure 5

Open in a new tab

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

The response to stress in tottering mice and totteringPC-Cre mice was similar (Figure 6A). Likewise, the response to caffeine in tottering mice (ED50 = 3.19 mg/kg) and totteringPC-Cre mice (ED50 = 2.96 mg/kg) was comparable (Figure 6B) and the response to ethanol in tottering mice (ED50 = 0.69 g/kg) did not differ from totteringPC-Cre mice (ED50 = 0.85 g/kg; Figure 6). These results demonstrate that the response to triggers is not modified by Purkinje cell expression of Cre recombinase per se. All three triggers also induced attacks in totteringhaplo mice. However, totteringhaplo mice were more sensitive to the triggers compared to tottering mice. Even minor stressors triggered attacks more frequently in totteringhaplo than in tottering mice (Figure 6A). The dose response curves for both caffeine and ethanol (ED50 = 0.10 g/kg) were shifted to the left for totteringhaplo mice compared to tottering mice (Figure 6B & C), although it was not possible to calculate the ED50 for caffeine in totteringhaplo due to the high vehicle baseline. These results suggest that there is an effect of gene dose whereby mice that are haploinsufficient for the tottering allele are more sensitive to the triggers than mice that are homozygous for the tottering allele. Stress, caffeine and ethanol also induced attacks in totteringPC-haplo mice. These mice were also more sensitive to stressors than tottering mice (Figure 6A). The ED50 for caffeine was shifted ~5-fold to the left in totteringPC-haplo mice (ED50 = 0.62 mg/kg) compared to tottering mice. A similar 5-fold shift in the dose-response for ethanol was observed in totteringPC-haplo mice (ED50= 0.14 g/kg) compared to tottering mice. Overall, mice with the attack-causing genotype in only Purkinje cells had preserved and even exaggerated responses to triggers.

Figure 6. Attacks are mediated by Purkinje cells.

Figure 6

Open in a new tab

The L7-Cre transgene, which expresses Cre recombinase only in Purkinje cells was bred into mice bearing one tottering allele and one floxed allele (Cacna1atg/flox) to generate totteringPC-haplo mice, which carry an attack-causing genotype only in Purkinje cells. Mice bearing one tottering allele and one null allele (totteringhaplo) and tottering mice carrying the L7-Cre transgene (totteringPC-Cre) were also tested as controls. A. Environmental challenges. Restraint induced attacks in all genotypes. TotteringPC-haplo mice (n = 11–12/challenge) and totteringhaplo mice (n = 14/challenge) were more sensitive to the other environmental challenges than tottering mice (n = 11/challenge) and totteringPC-Cre mice (n = 7/challenge). B. Caffeine dose-dependently induced attacks in all genotypes. TotteringPC-haplo mice (n = 11–12 per dose) and totteringhaplo mice (n = 13–14/dose) were more sensitive to the effects of caffeine than tottering mice (n = 10–11/dose) and totteringPC-Cre mice (n = 7/dose). C. Ethanol dose-dependently induced attacks in all genotypes. TotteringPC-haplo mice (n = 10–16/dose) and totteringhaplo mice (n = 12–14/dose) were more sensitive to the effects of ethanol than tottering mice (n = 9–11/dose) and totteringPC- Cre mice (n = 7/dose). *p < 0.05, **p < 0.01, ***p < 0.001 as determined by logistic regression analysis.

To determine if the triggers of attacks in totteringPC-haplo mice also depend on RyR activity within the cerebellum, localized microinjections of ryanodine were conducted. Similar to tottering mice, intracerebellar injections of ryanodine significantly reduced the frequency of attacks induced by stress, caffeine or ethanol (Figure 3B; p < 0.01, p < 0.05 and p < 0.01; as determined by chi-square test) indicating that the mechanisms for attacks in totteringPC-haplo mice are similar to those of tottering mice.

Discussion

Here, we described the biochemical and anatomical targets of stress, caffeine and ethanol in the induction of attacks of neurological dysfunction due to a calcium channelopathy. At the biochemical level, we found that compounds that potentiate RyR activity, including caffeine, theophylline and paraxanthine, triggered attacks. Conversely, dantrolene and ryanodine, which are structurally unrelated RyR antagonists, prevented attacks induced by stress, caffeine or ethanol. Taken together, these results suggest that the triggers act through a common biochemical pathway that includes RyR signaling. At the anatomical level, we found that the attacks are triggered locally. Intracerebellar delivery of ryanodine blocked attacks and intracerebellar infusions of caffeine induced attacks. Further, the results of the conditional genetic manipulations implicate a single cell type – Purkinje cells. Thus, the seemingly disparate triggers share biochemical and anatomical substrates. These results suggest that despite the diverse and relatively diffuse actions of the triggers throughout the CNS, the substrates of attack induction are surprisingly well circumscribed.

RyR signaling was necessary for the induction of attacks by stress, caffeine and ethanol. RyRs mediate the release of intracellular stores of calcium from the endoplasmic reticulum in response to changes in the cytosolic calcium concentration. Acute changes in cytosolic calcium concentrations can have an immediate impact on excitability through the regulation of transmitter release and by mediating the activity of other ion channels and receptors. While it is possible that stress, caffeine and ethanol exert their effects directly at RyR, it is more likely that each trigger disrupts calcium homeostasis through unique mechanisms and the requirement for RyR activity is downstream of the primary effects. Stress induces the release of CRF, which evokes ryanodine-sensitive Ca2+-release from internal Ca2+-stores (Riegel and Williams, 2008; Yamamori et al., 2004). Stress also stimulates noradrenergic transmission to promote calcium release from intracellular stores through α1-adrenergic receptors (Kirischuk et al., 1996; Paladini and Williams, 2004). In fact, α-adrenergic receptor signaling is required for stress-induced attacks in tottering mice (Fureman and Hess, 2005). At millimolar concentrations in vitro, caffeine induces rapid and sustained increases in cytoplasmic calcium concentrations through RyR activation (Kano et al., 1995; Womack et al., 2000). However, only micromolar concentrations of caffeine are typically achieved in vivo. Low caffeine concentrations evoke an increase in the rate of calcium sparks (Kong et al., 2008; Usachev and Thayer, 1997), which are short-lived local increases in intracellular calcium concentrations (Cheng and Lederer, 2008). It is possible that caffeine triggers attacks by promoting calcium sparks. It is also possible that the other diverse actions of caffeine, such as phosphodiesterase inhibition, contribute, although we have ruled out adenosinergic signaling. Ethanol indirectly mediates the disposition of calcium through the regulation of numerous calcium-permeable channels (Crews et al., 1996; Haorah et al., 2007). Thus, RyR blockade may preclude attacks by damping abnormal fluctuations in cell calcium caused by the triggers without direct interaction of the triggers at RyRs. Although additional experimentation is needed to describe the precise role of RyR receptors in the response to triggers, the experiments presented here provide a clear direction for examining shared biochemical features, despite the complex physiological effects of stress, caffeine and alcohol.

Several different lines of evidence converge to suggest that the triggers act specifically within the cerebellum but not other brain regions. Infusion of caffeine directly to the cerebellum, but not the striatum, triggered attacks. Attacks induced by stress, caffeine and ethanol were blocked by intracerebellar injection of ryanodine, but not by microinjection of ryanodine into the lateral ventricle. Finally, attacks persisted even after isolating the attack-causing genotype to Purkinje cells in totteringPC-haplo mice suggesting that the abnormal response to the triggers originates specifically within these neurons. However, it is possible that the triggers instigate abnormal presynaptic signals that then provoke abnormal Purkinje cell responses, but not likely because the abnormal activity in tottering mouse Purkinje cells is intrinsically generated and independent of synaptic input (Chen et al., 2009; Walter et al., 2006). This finding does not rule out the possibility that other cells contribute. Although restricting the attack-causing genotype to Purkinje cells in totteringPC-haplo mice caused an increase in sensitivity to stress, caffeine and ethanol compared to tottering mice, totteringhaplo mice also exhibited a similar increase in sensitivity. Indeed, totteringhaplo mice appeared even more sensitive to stress than totteringPC-haplo and a similar trend for an increase in sensitivity to caffeine and ethanol is also evident for totteringhaplo mice. The exaggerated response in both totteringPC-haplo and totteringhaplo mice is likely caused by haploinsufficiency of the Cacna1a gene in Purkinje cells. The further augmentation of the response in totteringhaplo mice suggests that other cells may also contribute but abnormal Purkinje cells alone are sufficient for the response to stress, caffeine and ethanol.

The abundant expression of CaV2.1 channels in Purkinje cells may, in part, explain the selective vulnerability of these neurons (Volsen et al., 1995; Westenbroek et al., 1995). The intrinsic pacemaking function of Purkinje cells is mediated by calcium-dependent small-conductance type (SK) potassium channels, which are exclusively activated by calcium entry through CaV2.1 channels (Womack and Khodakhah, 2004). Loss of precision in Purkinje cell firing patterns has been detected in tottering mice presumably due to inefficient SK channel activation (Hoebeek et al., 2005; Walter et al., 2006). Furthermore, potassium channel activators correct the irregular Purkinje cell firing patterns in mice (Alvina and Khodakhah, 2010) and ameliorate attacks of cerebellar dysfunction in both EA2 patients and tottering mice (Strupp et al., 2004; Weisz et al., 2005). The effects of the persistent subclinical abnormalities in Purkinje cell calcium handling caused by the calcium channel mutation plus the typically inconsequential effects of the triggers combine to precipitate overt behavioral dysfunction in the form of an attack. Thus, rather than indiscriminately acting throughout the CNS, the triggers likely exploit cell-type specific differences in gene expression and electrophysiological properties.

Understanding the mechanisms underlying the induction of transient neurological dysfunction is essential for the development of therapies that eliminate or reduce the incidence of attacks. Though dantrolene is most commonly used clinically to treat spasticity, the drug's ability to stabilize abnormal neuronal calcium handling (Chen et al., 2008; Liu et al., 2009; Nagatomo et al., 2001) and to prevent attacks in tottering mice and seizures in other mice (Nagatomo et al., 2001; Swiader et al., 2002) suggests that it could be used similarly to block episodic neuronal dysfunction in patients. Indeed, compounds that regulate calcium signaling, particularly verapamil which is an L-type calcium channel blocker, have been used as treatments for hemiplegic migraine, severe myoclonic epilepsy of infancy and paroxysmal dyskinesia (Hsu et al., 2008; Iannetti et al., 2009; Links et al., 1998; Szczaluba et al., 2009). Despite the global CNS effects of the triggers, the surprising biochemical and anatomical specificity of the triggers suggests that it is possible to develop targeted therapies aimed at blocking the induction of episodic neurological dysfunction, perhaps by damping or stabilizing calcium signaling in target neurons, rather than treating the symptoms after they arise.

Highlights

  • ■

    RyR signaling is necessary for the induction of attacks by stress, caffeine and ethanol in tottering mice

  • ■

    stress, caffeine and ethanol act specifically within the cerebellum but not other brain regions, to trigger attacks in tottering mice

  • ■

    Abnormal Purkinje cells mediate the response to stress, caffeine and ethanol to instigate neurological dysfunction in tottering mice

Acknowledgements

This work was supported by the United States National Institutes of Health (R01 NS033592, R01 NS040470, F32 NS055584), the Dystonia Medical Research Foundation, ZonMW and the NeuroBasic-PharmaPhenomics consortium.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Alvina K, Khodakhah K. The therapeutic mode of action of 4-aminopyridine in cerebellar ataxia. J Neurosci. 2010;30:7258–68. doi: 10.1523/JNEUROSCI.3582-09.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bhatia KP. Familial (idiopathic) paroxysmal dyskinesias: an update. Semin Neurol. 2001;21:69–74. doi: 10.1055/s-2001-13121. [DOI] [PubMed] [Google Scholar]
  3. Bhatia KP, Griggs RC, Ptacek LJ. Episodic movement disorders as channelopathies. Mov Disord. 2000;15:429–33. doi: 10.1002/1531-8257(200005)15:3<429::AID-MDS1001>3.0.CO;2-R. [DOI] [PubMed] [Google Scholar]
  4. Campbell DB, Hess EJ. Cerebellar circuitry is activated during convulsive episodes in the tottering (tg/tg) mutant mouse. Neuroscience. 1998;85:773–83. doi: 10.1016/s0306-4522(97)00672-6. [DOI] [PubMed] [Google Scholar]
  5. Cannon SC. Pathomechanisms in channelopathies of skeletal muscle and brain. Annu Rev Neurosci. 2006;29:387–415. doi: 10.1146/annurev.neuro.29.051605.112815. [DOI] [PubMed] [Google Scholar]
  6. Chen G, Popa LS, Wang X, Gao W, Barnes J, Hendrix CM, Hess EJ, Ebner TJ. Low-frequency oscillations in the cerebellar cortex of the tottering mouse. J Neurophysiol. 2009;101:234–45. doi: 10.1152/jn.90829.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen X, Tang TS, Tu H, Nelson O, Pook M, Hammer R, Nukina N, Bezprozvanny I. Deranged calcium signaling and neurodegeneration in spinocerebellar ataxia type 3. J Neurosci. 2008;28:12713–24. doi: 10.1523/JNEUROSCI.3909-08.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheng H, Lederer WJ. Calcium sparks. Physiol Rev. 2008;88:1491–545. doi: 10.1152/physrev.00030.2007. [DOI] [PubMed] [Google Scholar]
  9. Crews FT, Morrow AL, Criswell H, Breese G. Effects of ethanol on ion channels. Int Rev Neurobiol. 1996;39:283–367. doi: 10.1016/s0074-7742(08)60670-4. [DOI] [PubMed] [Google Scholar]
  10. da Silva Torres IL, Bonan CD, Crema L, De Leon Nunes M, Battastini AM, Sarkis JJ, Dalmaz C, Ferreira MB. Effect of drugs active at adenosine receptors upon chronic stress-induced hyperalgesia in rats. Eur J Pharmacol. 2003;481:197–201. doi: 10.1016/j.ejphar.2003.09.045. [DOI] [PubMed] [Google Scholar]
  11. Daly JW. Caffeine analogs: biomedical impact. Cell Mol Life Sci. 2007;64:2153–69. doi: 10.1007/s00018-007-7051-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Devanagondi R, Egami K, LeDoux MS, Hess EJ, Jinnah HA. Neuroanatomical substrates for paroxysmal dyskinesia in lethargic mice. Neurobiol Dis. 2007;27:249–57. doi: 10.1016/j.nbd.2007.05.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fan X, Hess EJ. D2-like dopamine receptors mediate the response to amphetamine in a mouse model of ADHD. Neurobiol Dis. 2007;26:201–211. doi: 10.1016/j.nbd.2006.12.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fletcher CF, Lutz CM, O'Sullivan TN, Shaughnessy JD, Hawkes R, Frankel WN, Copeland NG, Jenkins NA. Absence epilepsy in tottering mutant mice is associated with calcium channel defects. Cell. 1996;87:607–617. doi: 10.1016/s0092-8674(00)81381-1. [DOI] [PubMed] [Google Scholar]
  15. Frucht MM, Quigg M, Schwaner C, Fountain NB. Distribution of seizure precipitants among epilepsy syndromes. Epilepsia. 2000;41:1534–9. doi: 10.1111/j.1499-1654.2000.001534.x. [DOI] [PubMed] [Google Scholar]
  16. Fureman BE, Hess EJ. Noradrenergic blockade prevents attacks in a model of episodic dysfunction caused by a channelopathy. Neurobiol Dis. 2005;20:227–32. doi: 10.1016/j.nbd.2005.03.004. [DOI] [PubMed] [Google Scholar]
  17. Fureman BE, Jinnah HA, Hess EJ. Triggers of paroxysmal dyskinesia in the calcium channel mouse mutant tottering. Pharmacol Biochem Behav. 2002;73:631–637. doi: 10.1016/s0091-3057(02)00854-7. [DOI] [PubMed] [Google Scholar]
  18. Haorah J, Knipe B, Gorantla S, Zheng J, Persidsky Y. Alcohol-induced blood-brain barrier dysfunction is mediated via inositol 1,4,5-triphosphate receptor (IP3R)-gated intracellular calcium release. J Neurochem. 2007;100:324–36. doi: 10.1111/j.1471-4159.2006.04245.x. [DOI] [PubMed] [Google Scholar]
  19. Hoebeek FE, Stahl JS, van Alphen AM, Schonewille M, Luo C, Rutteman M, van den Maagdenberg AM, Molenaar PC, Goossens HH, Frens MA, De Zeeuw CI. Increased noise level of purkinje cell activities minimizes impact of their modulation during sensorimotor control. Neuron. 2005;45:953–65. doi: 10.1016/j.neuron.2005.02.012. [DOI] [PubMed] [Google Scholar]
  20. Hsu DA, Stafstrom CE, Rowley HA, Kiff JE, Dulli DA. Hemiplegic migraine: hyperperfusion and abortive therapy with intravenous verapamil. Brain Dev. 2008;30:86–90. doi: 10.1016/j.braindev.2007.05.013. [DOI] [PubMed] [Google Scholar]
  21. Iannetti P, Parisi P, Spalice A, Ruggieri M, Zara F. Addition of verapamil in the treatment of severe myoclonic epilepsy in infancy. Epilepsy Res. 2009;85:89–95. doi: 10.1016/j.eplepsyres.2009.02.014. [DOI] [PubMed] [Google Scholar]
  22. Jen JC, Graves TD, Hess EJ, Hanna MG, Griggs RC, Baloh RW. Primary episodic ataxias: diagnosis, pathogenesis and treatment. Brain. 2007;130:2484–93. doi: 10.1093/brain/awm126. [DOI] [PubMed] [Google Scholar]
  23. Jinnah HA, Sepkuty JP, Ho T, Yitta S, Drew T, Rothstein JD, Hess EJ. Calcium channel agonists and dystonia in the mouse. Mov Disord. 2000;15:542–551. doi: 10.1002/1531-8257(200005)15:3<542::AID-MDS1019>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  24. Kano M, Garaschuk O, Verkhatsky A, Konnerth A. Ryanodine receptor-mediated intracellular caclium release in rat cerebellar Purkinje neurons. J Physiol. 1995;487:1–16. doi: 10.1113/jphysiol.1995.sp020857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kirischuk S, Matiash V, Kulik A, Voitenko N, Kostyuk P, Verkhratsky A. Activation of P2-purino-, alpha 1-adreno and H1-histamine receptors triggers cytoplasmic calcium signalling in cerebellar Purkinje neurons. Neuroscience. 1996;73:643–7. doi: 10.1016/0306-4522(96)00205-9. [DOI] [PubMed] [Google Scholar]
  26. Kong H, Jones PP, Koop A, Zhang L, Duff HJ, Chen SR. Caffeine induces Ca2+ release by reducing the threshold for luminal Ca2+ activation of the ryanodine receptor. Biochem J. 2008;414:441–52. doi: 10.1042/BJ20080489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Koob GF. Corticotropin-releasing factor, norepinephrine, and stress. Biol Psychiatry. 1999;46:1167–1180. doi: 10.1016/s0006-3223(99)00164-x. [DOI] [PubMed] [Google Scholar]
  28. Kumar S, Porcu P, Werner DF, Matthews DB, Diaz-Granados JL, Helfand RS, Morrow AL. The role of GABA(A) receptors in the acute and chronic effects of ethanol: a decade of progress. Psychopharmacology (Berl) 2009;205:529–64. doi: 10.1007/s00213-009-1562-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Links TP, Arnoldus EP, Wintzen AR, van der Hoeven J, Gerritsen JJ, Brandenburg HC. The calcium channel blocker verapamil in hypokalemic periodic paralysis. Muscle Nerve. 1998;21:1564–5. doi: 10.1002/(sici)1097-4598(199811)21:11<1564::aid-mus38>3.0.co;2-y. [DOI] [PubMed] [Google Scholar]
  30. Liu J, Tang TS, Tu H, Nelson O, Herndon E, Huynh DP, Pulst SM, Bezprozvanny I. Deranged calcium signaling and neurodegeneration in spinocerebellar ataxia type 2. J Neurosci. 2009;29:9148–62. doi: 10.1523/JNEUROSCI.0660-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Minor TR, Rowe MK, Soames Job RF, Ferguson EC. Escape deficits induced by inescapable shock and metabolic stress are reversed by adenosine receptor antagonists. Behav Brain Res. 2001;120:203–12. doi: 10.1016/s0166-4328(00)00376-4. [DOI] [PubMed] [Google Scholar]
  32. Muller CE, Daly JW. Stimulation of calcium release by caffeine analogs in pheochromocytoma cells. Biochem Pharmacol. 1993;46:1825–9. doi: 10.1016/0006-2952(93)90589-o. [DOI] [PubMed] [Google Scholar]
  33. Nagatomo I, Hashiguchi W, Tominaga M, Akasaki Y, Uchida M, Takigawa M. Effects of MK-801, dantrolene, and FK506 on convulsive seizures and brain nitric oxide production in seizure-susceptible EL mice. Brain Res. 2001;888:306–310. doi: 10.1016/s0006-8993(00)03101-2. [DOI] [PubMed] [Google Scholar]
  34. Newton PM, Messing RO. Intracellular signaling pathways that regulate behavioral responses to ethanol. Pharmacol Ther. 2006;109:227–37. doi: 10.1016/j.pharmthera.2005.07.004. [DOI] [PubMed] [Google Scholar]
  35. Ophoff RA, Terwindt GM, Vergouwe MN, van Eijk R, Oefner PJ, Hoffman SM, Lamerdin JE, Mohrenweiser HW, Bulman DE, Ferrari M, Haan J, Lindhout D, van Ommen GJ, Hofker MH, Ferrari MD, Frants RR. Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4. Cell. 1996;87:543–652. doi: 10.1016/s0092-8674(00)81373-2. [DOI] [PubMed] [Google Scholar]
  36. Paladini CA, Williams JT. Noradrenergic inhibition of midbrain dopamine neurons. J Neurosci. 2004;24:4568–75. doi: 10.1523/JNEUROSCI.5735-03.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pizoli CE, Jinnah HA, Billingsley ML, Hess EJ. Abnormal cerebellar signaling induces dystonia in mice. J Neurosci. 2002;22:7825–7833. doi: 10.1523/JNEUROSCI.22-17-07825.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ptacek LJ. Ion channel diseases: episodic disorders of the nervous system. Semin Neurol. 1999;19:363–9. [PubMed] [Google Scholar]
  39. Riegel AC, Williams JT. CRF facilitates calcium release from intracellular stores in midbrain dopamine neurons. Neuron. 2008;57:559–70. doi: 10.1016/j.neuron.2007.12.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shirley TL, Rao LM, Hess EJ, Jinnah HA. Paroxysmal dyskinesias in mice. Mov Disord. 2008;23:259–64. doi: 10.1002/mds.21829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Strupp M, Kalla R, Dichgans M, Freilinger T, Glasauer S, Brandt T. Treatment of episodic ataxia type 2 with the potassium channel blocker 4-aminopyridine. Neurology. 2004;62:1623–5. doi: 10.1212/01.wnl.0000125691.74109.53. [DOI] [PubMed] [Google Scholar]
  42. Swiader M, Borowicz KK, Porebiak J, Kleinrok Z, Czuczwar SJ. Influence of agents affecting voltage-dependent calcium channels and dantrolene on the anticonvulsant action of the AMPA/kainate receptor antagonist LY 300164 in mice. Eur Neuropsychopharmacol. 2002;12:311–9. doi: 10.1016/s0924-977x(02)00045-7. [DOI] [PubMed] [Google Scholar]
  43. Szczaluba K, Jurek M, Szczepanik E, Friedman A, Milewski M, Bal J, Mazurczak T. A family with paroxysmal nonkinesigenic dyskinesia: genetic and treatment issues. Pediatr Neurol. 2009;41:135–8. doi: 10.1016/j.pediatrneurol.2009.02.013. [DOI] [PubMed] [Google Scholar]
  44. Todorov B, van de Ven RC, Kaja S, Broos LA, Verbeek SJ, Plomp JJ, Ferrari MD, Frants RR, van den Maagdenberg AM. Conditional inactivation of the Cacna1a gene in transgenic mice. Genesis. 2006;44:589–94. doi: 10.1002/dvg.20255. [DOI] [PubMed] [Google Scholar]
  45. Usachev YM, Thayer SA. All-or-none Ca2+ release from intracellular stores triggered by Ca2+ influx through voltage-gated Ca2+ channels in rat sensory neurons. J Neurosci. 1997;17:7404–14. doi: 10.1523/JNEUROSCI.17-19-07404.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Volsen SG, Day FC, McCormack AL, Smith W, Craig PJ, Beattie R, Ince PG, Shaw PJ, Ellis SB, Gillespie A, Harpold MM, Lodge D. The expression of neuronal voltage-dependent calcium channels in human cerebellum. Mol Brain Res. 1995;34:271–282. doi: 10.1016/0169-328x(95)00234-j. [DOI] [PubMed] [Google Scholar]
  47. Wakamori M, Yamazaki K, Matsunodaira H. Single tottering mutations responsible for the neuropathic phenotype of the P-type calcium channel. J Biol Chem. 1998;52:34857–34867. doi: 10.1074/jbc.273.52.34857. [DOI] [PubMed] [Google Scholar]
  48. Walter JT, Alvina K, Womack MD, Chevez C, Khodakhah K. Decreases in the precision of Purkinje cell pacemaking cause cerebellar dysfunction and ataxia. Nat Neurosci. 2006;9:389–97. doi: 10.1038/nn1648. [DOI] [PubMed] [Google Scholar]
  49. Weisz CJ, Raike RS, Soria-Jasso LE, Hess EJ. Potassium channel blockers inhibit the triggers of attacks in the calcium channel mouse mutant tottering. J Neurosci. 2005;25:4141–5. doi: 10.1523/JNEUROSCI.0098-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Westenbroek RE, Sakurai T, Elliott EM, Hell JW, Starr TV, Snutch TP, Catterall WA. Immunochemical identification and subcellular distribution of the alpha 1A subunits of brain calcium channels. J Neurosci. 1995;15:6403–18. doi: 10.1523/JNEUROSCI.15-10-06403.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Williams M, Jarvis MF. Adenosine antagonists as potential therapeutic agents. Pharmacol Biochem Behav. 1988;29:433–41. doi: 10.1016/0091-3057(88)90182-7. [DOI] [PubMed] [Google Scholar]
  52. Womack MD, Khodakhah K. Dendritic control of spontaneous bursting in cerebellar Purkinje cells. J Neurosci. 2004;24:3511–21. doi: 10.1523/JNEUROSCI.0290-04.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Womack MD, Walker JW, Khodakhah K. Impaired calcium release in cerebellar Purkinje neurons maintained in culture. J Gen Physiol. 2000;115:339–46. doi: 10.1085/jgp.115.3.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yamamori E, Iwasaki Y, Oki Y, Yoshida M, Asai M, Kambayashii M, Oiso Y, Nakashima N. Possible involvement of ryanodine receptor-mediated intracellular calcium release in the effect of corticotropin-releasing factor on adrenocorticotropin secretion. Endocrinology. 2004;145:36–8. doi: 10.1210/en.2003-1222. [DOI] [PubMed] [Google Scholar]

RESOURCES