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Research Report

Modified behavioral characteristics following ablation of thevoltage-dependent calcium channel β3 subunit

Manabu Murakamia,⁎, Osamu Nakagawasaib, Kazuhiko Yanaic, Kazuo Nunokid,Koichi Tan-Nob, Takeshi Tadanob, Toshihiko Iijimaa

aDepartment of Pharmacology, Akita University School of Medicine, Akita, 1-1-1 Hondoh, Akita 010-8543, JapanbDepartment of Pharmacology, Tohoku Pharmaceutical University, Sendai, JapancDepartment of Cellular Pharmacology, Tohoku University School of Medicine, Sendai, JapandDepartment of Human Health and Nutrition, Syoukeigakuinn Colledge, Natori 918-1295, Japan

A R T I C L E I N F O

⁎ Corresponding author. Fax: +81 18 8348930.E-mail address: mmura0123@hotmail.co.j

0006-8993/$ − see front matter © 2007 Elsevidoi:10.1016/j.brainres.2007.05.041

A B S T R A C T

Article history:Accepted 27 May 2007Available online 2 June 2007

Voltage-dependent calcium channels are important for calcium influx and the ensuingintracellular calcium signal in various excitable membranes. The β subunits of thesechannels modify calcium currents through pore-forming α1 subunits of the high-voltage-activated calcium channels. In the present study, β3 subunit-null mice were used toinvestigate the importance of the β3 subunit of the voltage-dependent calcium channel,which couples with the CaV2.2 (α1B) subunit to form the major component of neuronal N-type calcium channels in the brain. Western blot analysis revealed a significant decrease inN-type calcium channels in β3 subunit-null mice, while protein levels of other high-voltage-activated calcium channel α1 subunits were unchanged. Immunoprecipitation analysiswith an anti-CaV2.2 antibody showed that reshuffling of the assembly of N-type channelshad occurred in the β3 subunit-null mice. Ablation of this subunit resulted in modifiednociception, decreased anxiety, and increased aggression. The β3 subunit-null mice alsoshowed impaired learning ability. These results suggest the importance of voltage-dependent calcium channels and the key role of the β3 subunit in memory formation,nociceptive sensory transduction, and various neurological signal transduction pathways.

© 2007 Elsevier B.V. All rights reserved.

Keywords:Calcium channelβ subunitMouseAggressionMemory

1. Introduction

Voltage-dependent calcium channels play pivotal roles in thecontrol of calcium-linked cellular functions, such as neuro-transmitter release. They have been subdivided on the basis oftheir electrophysiological and pharmacological properties intoT-, L-, N-, P/Q-, and R-types. Serial behavioral studies havedemonstrated extensive roles for these channels in the ner-vous system. N-type calcium channel antagonists have anti-

p (M. Murakami).

er B.V. All rights reserved

nociceptive effects (Murakami et al., 2000), while the L-type-specific DHP antagonist reduces ethanol intake and prefer-ence (De Beun et al., 1996), modifies locomotor activity (Wata-nabe et al., 1998), controls defensive behavior (Schenberg et al.,2000), and shows antidepressant-like activity (Cohen et al.,1997). Intracerebral injection of ω-conotoxin GVIA, a specificblocker of N-type calcium channels, induces tremors (Oliveraet al., 1984), and this toxin also disturbs circadian rhythm(Masutani et al., 1995).

.

Fig. 1 – (A)Western blot analysis of calcium channel subunitsin the brain. Each sample ofmembrane proteins (100μg/lane)was probedwith polyclonal antibodies: CaV2.1 (α1A), CaV2.2(α1B), CaV1.2 (α1C), CaV1.3 (α1D), or CaV2.3 (α1E), orβ1, 2, 3,or 4, as indicated. Each experiment was repeated at leastthree times. β3+/+ (+/+) and β3−/− (−/−) (B) Proteinsimmunoprecipitated with the anti-CaV2.2 antibody fromβ3+/+ (+/+) and β3−/− (−/−) brain membrane proteinpreparations (anti-CaV2.2 (α1B), β1, 2, 3, or 4 antibody, asindicated). (C) Saturation binding of DHP. Representativebinding curves for (+)[3H]PN 200-110 are shown for threeindependent experiments. Large open circle, total binding ofβ3+/+; small open circle, nonspecific binding of β3+/+; largeclosed circle, total binding of β3−/−; small closed circle,nonspecific binding of β3−/−.

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N-type channel null-mutant mice have disturbed circadianrhythms, resulting in hyperactivity during the dark phase(Beuckmann et al., 2003), whereas L-type channel-formingCaV1.3 (α1D) null-mutant mice showed relatively normalneurological function, with the exception of reduced audi-tory-evoked behavioral responses (Clark et al., 2003). In Cae-norhabditis elegans, mutations in the α1 subunit (unc-2 locus)disrupt physiological adaptation to dopamine and serotonin(Schafer and Kenyon, 1995). A locus associatedwith behavioraland visual mutations maps to the α1 subunit in Drosophila(Smith et al., 1996).

Voltage-dependent calcium channels are composed of α1,α2/δ, β, and γ subunits (Catterall et al., 2005). The β subunits,located on the intracellular side of the membrane, increasethe channel population, and modify channel properties. Fourβ subunit types (β1, β2, β3, and β4) have been identified. The βsubunits also play a role in the transformation of Cav2calcium channel function in immature neurons and maturesynapses (Spafford et al., 2004). The β4 subunit is abundantlyexpressed in the cerebellum; the mutant lethargic mousecarries a four-nucleotide insertion, resulting in a translationalframeshift, and is a model of absence epilepsy, chronic ata-xia, hypoactivity, and dyskinetic motor behavior (Khan andJinnah, 2002).

The β3 subunit is also expressed abundantly in the brain. Ithas been co-purified with the N-type calcium channel(CaV2.2, α1B), which is expressed in the presynaptic activezones of neurotransmitter junctions, and participates in therelease of various neurotransmitters, including γ-aminobu-tyric acid, acetylcholine, dopamine, and norepinephrine, incentral neurons (Herdon and Nahorski, 1989; Sher and Cle-mentini, 1991; Torri Tarelli et al., 1991; Komuro and Rakic,1992; Luebke et al., 1993; Turner et al., 1993; Witcher et al.,1993). It is believed that the β3 subunit participates in severalneuronal functions, because it is expressed throughout thecentral nervous system (CNS), although its exact role isunclear. As most (∼60%) N-type calcium channels areassociated with the β3 subunit, mice in which this subunitis ablated are expected to have disturbed circadian rhythms,as do N-type-deficient mice (Beuckmann et al., 2003). The β3-deficient mice show apparently decreased responses tonoxious stimuli, whereas CaV2.2-deficient mice show onlylimited change (Murakami et al., 2002; Saegusa et al., 2001),suggesting that the β3 subunit has additional roles. Thus,although little is known about its role(s) in neuronal behavior,the β3 subunit may participate in various types of neuro-transmission and may be a future pharmacological target fortreating clinical symptoms such as chronic pain. However, todate, only its role in the nociception pathway has beenclarified (Murakami et al., 2002).

In this study, we examined the physiological role of the β3subunit in the CNS by analyzing the behavior of β3-deficientmice, especially behaviors related to N-type calcium channels.We analyzed the effects of β3 gene ablation on various calciumchannel populations and channel assemblies, spontaneousactivity, pain perception with different noxious stimuli,emotion-based behaviors, and working memory. Our findingsindicate several roles for the β3 subunit in neurologicalprocesses, including nociception, anxiety, aggression, andmemory.

2. Results

2.1. Biochemical analysis

2.1.1. Western blot analysisTo analyze the protein levels of modified calcium channels inβ3-deficient (β3−/−) mice, we performedWestern blot analysesusing polyclonal antibodies specific for the Ca2+ channelsubunits CaV1.2, CaV1.3, CaV2.1, CaV2.2, and CaV2.3, and theβ1, β2, β3, and β4 subunits. Fig. 1A shows an immunoblotanalysis in wild-type (β3+/+) and β3−/− mice with an anti-CaV2.1 (α1A) antibody. The anti-CaV2.1 antibody reacted witha single band of 190 kDa. No significant difference wasobserved in the levels of CaV2.1 protein between β3+/+ andβ3−/− (98±2% compared to wild-type (wt) controls; n=4). Forthe N-type channel, we found a single 230 kDa band and a

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significant decrease in the CaV2.2 (α1B) level in the brain of β3-deficient mice (40±6% reduction by densitometry; n=4, Fig.1A, CaV2.2). No significant differences were detected in theprotein levels of other high-voltage-activated calcium channelα1 subunits: CaV1.2 (α1C, 205 kDa) (93±2% compared to wtcontrol; n=3), CaV1.3 (α1D, 190 kDa) (103±5% compared to wtcontrol; n=4), and CaV2.3 (α1E, 250 kDa) (96±4% compared towt control; n=3).

For the β subunits, Western blot analysis revealed nosignificant change in β1 (66 kDa), β2 (68 kDa), or β4 (55 kDa)subunit protein levels. The anti-β3 antibody showed a single57-kDa band in the brain of wt mice; no recognizable bandoccurred in the mutant mice, confirming that these mice hadno β3 subunit gene products (Fig. 1, β3). Western blot analysiswith an anti-GAPDH antibody revealed about the sameamount of protein (data not shown).

2.1.2. Reshuffling of N-type channelsIn the brain, N-type calcium channels consist primarily of theCaV2.2 and β3 subunits (Witcher et al., 1993; Scott et al., 1996).Our data indicate that the protein level of the CaV2.2 subunitdecreased in β3−/− mice, likely because of the null mutation inthe β3 subunit. As another possibility of channel propertymodification, reshuffling of the Ca2+ channel subunits mayhave occurred, as previously observed in a β4-deficient mouse(Burgess et al., 1999).

To detect reshuffling of N-type calcium channels as aresult of β3 gene ablation, we immunoprecipitated CaV2.2-associated proteins using an anti-CaV2.2 antibody andidentified any β subunit(s) interacting with the CaV2.2subunit (Fig. 1B). Using the anti-CaV2.2 antibody, we con-firmed that the amounts of CaV2.2 loaded were comparable(Fig. 1B, CaV2.2). Interaction between CaV2.2 and β1 increasedsignificantly (43±8% increase by densitometry; n=4, Fig. 1B,β1), while no change was observed between CaV2.2 and β2 inthe mutant mice (95±3% compared to wt control; n=4, Fig. 1B,β2). As the mutant has no β3 subunit, no interaction betweenCaV2.2 and β3 occurred in the mutant (Fig. 1B, β3). For β4, asignificantly increased interaction was seen between CaV2.2and β4 (36±9% increase by densitometry; n=4, Fig. 1B, β4).Our immunoprecipitation results indicate that the nullmutation in the β3 subunit resulted in reshuffling of the α1Bsubunit with other β subunits, specifically the β1 and β4subunits.

2.1.3. Calcium channel antagonism reveals unchanged L-typechannel populations in the brainThe L-type calcium channel consists of four different geneproducts (CaV1.1, 1.2, 1.3, and 1.4), making it difficult to esti-mate the entire population of L-type channels byWestern blotanalysis. To examine the effect of the β3 gene ablation on L-type calcium channel populations, we analyzed saturationbinding of DHP, corresponding to the L-type channel popula-tion. No significant difference was observed in DHP bindingbetween the β3+/+ and β3−/− mice (Fig. 1C), suggesting that thepopulations of L-type channels in the brains were unchanged.The maximum density (Bmax) and Kd values of (+)[3H]PN 200-110 binding were 29.2±2.7 and 26.8±5.4 fmol/mg protein, and0.153±0.010 and 0.150±0.014 nM (n=3 per group) for the β3+/+

and β3−/− mice, respectively.

2.2. Behavioral analysis (spontaneous)

2.2.1. Decreased shaking activityIn the biochemical analyses, we found a decreased amount ofCaV2.2, which means a decreased number of N-type calciumchannel pores. N-type channels exhibit preferable and irre-versible sensitivity to ω-conotoxin GVIA (Olivera et al., 1984).The bioactivity of this toxin was first identified by the shakingtremor after intracerebral injection. Thus, we examined thelevel of shaking induced by ω-conotoxin GVIA, which is aspecific bioassay for this toxin (Olivera et al., 1984). Because60% of the N-type channels are associated with the β3 subunit(Scott et al., 1996), we anticipated a significant reduction in theresponse to this toxin. Indeed,mutantmicewere less sensitiveto ω-conotoxin GVIA (Fig. 2A). The latency period of the res-ponses in β3−/− mice (n=10) to a low dose (1.72 nmol/kg) of thetoxin was approximately two-fold that in β3+/+ mice (n=10,⁎p<0.05). Importantly, β3−/− mice showed no tremors, shakes,head nods, unusual sudden bursts of activity, or any other sei-zure activities in their basal status, while other voltage-depen-dent calcium channel subunit deficiencies have often resultedin ataxic behaviors (Zhuchenkoet al., 1997; Burgess et al., 1999).

2.2.2. Impaired spontaneous locomotor activitySpontaneous activities and exploratory behaviors were exam-ined using Animex (Muromachi Kikai Co., Tokyo, Japan).Although β3−/− mice showed slightly impaired spontaneouslocomotor activities (Fig. 2B: β3+/+ and β3−/−, n=10 and n=10,respectively, ⁎p<0.05), they gave the same low-frequencyresult (7–10 rpm; data not shown) in a Rotarod test as theβ3+/+ mice, suggesting that impairment of the motor systemwas minor or negligible.

2.2.3. Alterations in circadian rhythmN-type channels are involved in circadian rhythms, and N-typechannel-deficient mice show hyperactivity during the darkphase (Beuckmannet al., 2003). Thus, β3 subunit-deficientmicewere expected to exhibit disturbed circadian rhythms. Loco-motor activities were further measured on the activity wheelfor 24 h over 2 days, to examine circadian rhythms. Thewtmicewere more active during the dark phase than during the lightphase (Fig. 2C, open circle, n=10). The mutant mice (closedcircle, n=10) showed significantly higher activity during thedark phase, especially between 03:00 and 06:00, compared withthe wt mice (⁎p<0.05). This suggests that the mutant mice hadmodified circadian rhythms, at least in terms of locomotion.

2.2.4. Impairment of motor coordinationThe β3 subunit is highly expressed in the cerebellum (Tanakaet al., 1995; Ludwig et al., 1997). Thus, we used the Rotarodtest to examine deficiencies in balance. No difference wasobserved in the retention times on the Rotarod running atlow speed (7–10 rpm, data not shown). However, at 20 rpm theβ3−/− mice showed a twofold reduction in retention time (Fig.2D: β3+/+ and β3−/−, n=10 and n=10, respectively, ⁎p<0.05).

2.3. Altered nociception

N-type calcium channel antagonists have anti-nociceptiveeffects (Murakami et al., 2000). However, N-type channel null-

Fig. 2 – (A) Shaker assay ofω-conotoxin GVIA, given by intracerebral injection, inβ3+/+ (open bar, n=10) andβ3−/− (hatched bar,n=10) mice. The mutant mice showed significantly longer latency periods than the wt mice. (B) Locomotor activities ofβ3+/+ (open circles, n=10) and β3−/− (closed circles, n=10) mice. Exploratory behavior in a new environment was evaluated withthe Animex-auto system. (C) Chronological distribution of wheel revolutions for β3+/+ (open circles, n=10) and β3−/− (closedcircles, n=10) mice. Closed bar (22:00–08:00, dark condition) and open bar (08:00–22:00, light condition). (D) Rotarod test. Theelapsed times for β3+/+ (open bar, n=10) and β3−/− (hatched bar, n=10) mice on the rod at a speed of 20 rpm were measured.

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mutant mice revealed only limited changes in nociception,such as decreased response only in the late phase of theformalin test (Saegusa et al., 2001). We previously reportedthat several types of pain perception, such as that ofthermal and mechanical stimuli, were modified by β3 geneablation (Murakami et al., 2002). Therefore, we examinedthe response to different types of stimuli in the presentstudy.

Capsaicin, which acts on vanilloid receptors, was injectedsubcutaneously into the dorsal surface of the hind paw, andlicking behavior was recorded. As we reported previously,capsaicin induces licking behavior for 10 min after injection inwt mice (Sakurada et al., 1992). No significant difference wasobserved in licking duration between the β3+/+ and β3−/− mice(Fig. 3A: β3+/+ and β3−/−, n=10 and n=10, respectively).

The tail-flick test, which is useful for evaluating anti-nociceptive effects at the spinal level, was carried out to inves-tigate the function of β3 in nociceptive transmission. The β3−/−

mice showed a significantly elevated nociceptive threshold inthe tail-flick test (Fig. 3B, β3+/+ and β3−/−, n=10 and n=10,respectively, ⁎p<0.05). We then injected intrathecally ω-cono-toxinGVIA (0.132nmol/kg), a specific blocker ofN-type calciumchannels, to examine the contribution of the remainingN-typechannels in the β3−/− mice. In both groups, ω-conotoxin GVIAincreased the nociceptive threshold significantly, while theβ3−/− mice retained significantly longer latency periods (β3+/+

and β3−/−, n=10 and n=10, respectively, ⁎p<0.05).We also examined licking pain behavior evoked by injec-

tion of formalin into the right hind paw. We found significantchanges in the early phase of the formalin test, providing a

Fig. 3 – Nociceptive behavior. The values on the y-axis represent the licking (A and C) and latency (B) periods (in seconds) of thedifferent nociceptive thresholds, expressed as the means±S.E.M. *p<0.05, **p<0.01. The genotypes are written as +/+ for β3+/+

mice, and −/− for β3−/− mice. (A) Capsaicin test. Periods of licking for β3+/+ (open bar, n=10) and β3−/− (hatched bar, n=10) miceare shown. (B) Tail-flick latency. The tail-flick latencies of β3+/+ (n=10) and β3−/− (n=10) mice, and the effect of intrathecalpretreatment with ω-conotoxin GVIA (right panel, ω-CT) in β3+/+ (n=10) and β3−/− (n=10) mice are shown. In both groups,ω-conotoxin GVIA significantly increased the periods of latency. ω-CT indicates the ω-conotoxin GVIA-treated group. (C)Formalin test. Duration of licking by β3+/+ (open bar, n=12) and β3−/− (hatched bar, n=10) mice during (i) early (first 10 min), and(ii) late (10–30 min after injection) phases, following injection with formalin. In both phases, the mutant mice showedsignificant reductions in the duration of licking. (D) Visceral pain test. Themutantmice showed significantly decreased visceralpain responses to abdominal stretching, which was produced by intraperitoneally injecting dilute acetic acid (n=10 for bothβ3+/+ and β3−/− mice).

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different measure of acute pain produced by direct chemicalactivation of C-fibers; a more profound effect on pain-relatedbehavior was noted in the late phase, probably as a result ofcentral sensitization during the early phase (Fig. 3C: β3+/+ andβ3−/−, n=12 and n=10, respectively, ⁎p<0.05, ⁎⁎p<0.01). We alsotested the mice for responses to acute visceral pain caused by

acetic acid, which induces a delayed inflammatory response,showing that the β3−/− mice had significantly elevated anti-nociceptive thresholds (Fig. 3D: β3+/+ and β3−/−, n=10 andn=10, respectively, ⁎⁎p<0.01).

Overall, β3 gene ablation had varying effects on nociceptiveperception. In the late phase of the formalin test and in the

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writhing test, the consequences of the defective gene weremore profound than in the other tests. We found nosignificant difference in the capsaicin test and a relativelyminor effect in the early phase of the formalin test, suggestingthat β3 gene deletion affects the various nociceptive signaltransduction pathways to different extents.

2.4. Behavioral analysis related to emotional status

As we found various behavioral phenotypes in the β3−/− mice,we further analyzed behavioral reactions related to emotionalstatus.

2.4.1. Forced swim testThe norepinephrine pathway in the brain is associated withdepression, and N-type calcium channels are strongly linked

Fig. 4 – (A) Behavioral models of depression in the forced swim(open bar, n=14) and β3−/− (hatched bar, n=16) mice were recordobserved forβ3−/−mice. *p<0.05. (B) Anxiety-related behavior in tharm was measured (n=13 for both β3+/+ and β3−/− mice). The muAggressive behavior of β3−/− mice in the resident-intruder assay(open bar, n=18) and β3−/− (hatched bar, n=18) mice are shown frepresents the trial number. The β3−/− mice showed significantlyaggression. The latency periods in the first trial did not differ sig

to sympathetic nerves, which also use norepinephrine as aneurotransmitter (Ino et al., 2001). TheN-type channels and β3subunit are thought to be related to norepinephrine release inthe brain, and thus we examined the responses of the β3−/−

mice in behavioral models of depression.The depressive state was induced in the mice by forcing

them to swim in a cylinder from which they could not escape.After a brief period of vigorous activity, the mice adopted acharacteristic immobile posture, which was readily identifi-able. Mutantmice showedmore prolonged periods of vigorousactivity and shorter periods of immobility than β3+/+ mice (Fig.4A: β3+/+ and β3−/−, n=14 and n=16, respectively, ⁎p<0.05). Thismay have been due to either a manic status induced by the β3genemanipulationor impaired cerebellar functionbecause themutant mice often showed an unbalanced posture whileswimming.

test. The total periods of immobility (in minutes) for β3+/+

ed. Significant decreases in the time of immobility weree elevated plus-maze test. The timemice spent on the closedtant mice spent less time in the closed arm. **p<0.01. (C). The attack latencies (i) and total fighting scores (ii) for β3+/+

or the three 5-min test trials. The number under each barshorter latency periods for attacking and increased

nificantly between the groups. *p<0.05.

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2.4.2. Elevated plus-maze testWe used the elevated-plus maze test to measure anxiety. Theelevated plus-maze test is based on the natural aversion ofrodents for high and open spaces. The β3−/− mice spentsignificantly shorter times than β3+/+ mice in the closed arm(Fig. 3B: β3+/+ and β3−/−, n=13 and n=13, respectively, ⁎⁎p<0.01),suggesting that the mutant mice had lower anxiety levels.

2.4.3. Resident–intruder testMany of the β3−/− male mice suffered hair loss when kept inthe same cage as other male mice, especially from 5 weeksafter birth. These mice had a tendency to bite their handlersand emotional disturbance was suspected. Mice with the β3−/−

genotype were often aggressive, especially when housedindividually. After a month of isolation, and later in the pre-sence of an intruder, the mutant mice were significantlymore aggressive than wt mice, as indicated by reduced attacklatencies (Fig. 4C, i) and higher fighting scores (Fig. 4C, ii: β3+/+

and β3−/−, n=18 and n=18, respectively, ⁎p<0.05).

2.5. Impairment of working memory

The step-through passive avoidance response was examinedto study learning and memory. The β3−/− mice showed sig-nificantly shorter latency periods for entry into the dark com-

Fig. 5 – Step-through passive avoidance test. The results ofthe step-through passive avoidance test for β3+/+ (opencircles, n=10) andβ3−/− (closed circles, n=10)mice. (i) Latencyof entry into the dark compartment; (ii) success rate (ii).*p<0.05.

partment starting on day 3 (Fig. 5A, i) and had lower successrates in passing through the door starting on day 2 (Fig. 5A, ii),suggesting that β3−/− mice had impaired learning andmemoryfunctions (β3+/+ and β3−/−, n=10 and n=10, respectively, ⁎p<0.05). Furthermore, after day 3, both latency and success rateof β3−/− mice worsened, suggesting that they became forgetfulor experienced memory decline.

3. Discussion

In this study, we demonstrated that disruption of the voltage-dependent calcium channel β3 subunit gene resulted in adecreased population and reshuffling of N-type calciumchannels, decreased sensitivity to ω-conotoxin GVIA in theshaker assay, altered nociception, decreased anxiety,increased aggression, and impaired memory. The N-typechannel-specific inhibitor ω-conotoxin GVIA is a 27-amino-acid peptide, purified from the fish-eating marine molluskConus geographus. This toxin was originally employed in theshaker assay and is currently used to characterize channelprofiles in electrophysiology experiments (Catterall et al.,2005). We found extended latency of tremors in the shakerassay of mutant mice, indicating decreased toxin sensitivityand suggesting a decrease or deterioration in functional N-type channels in the mutants. The decreased toxin sensitivityencouraged us to further characterize the β3 mutant mouse.

It has been demonstrated that L- and P/Q-type channels areinvolved in certain motor neuron functions (Zhuchenko et al.,1997; Mori et al., 2000; Urbano et al., 2001). Furthermore,expression of various types of channels in the brain has beenreported (Tanaka et al., 1995; Ludwig et al., 1997). Duringneurotransmitter release, highly increased local intracellularconcentrations of calcium, known as “local microdomains”and originating from voltage-dependent calcium channels,play a key role in calcium-triggered cellular events (Llinas andMoreno, 1998).

In the present study, we found impaired spontaneouslocomotor activity and motor coordination in β3-deficientmice. Because we found a decreased N-type channel popula-tion and modified channel assemblies, while other channelswere unchanged, we believe that the behavioral phenotypeswere primarily related to the modified N-type channels.Although a high level of expression of the β3 gene is knownto occur in the brain, its distribution is not completely docu-mented due to the lack of suitable antibodies for immunohis-tochemistry. Furthermore, gaps in our knowledge of therelationships between calcium channel distribution andcertain neurologically important pathways, such as theGABAergic and DOPAminergic pathways, prevent furtheranalysis of the mechanisms underlying the behavioral phe-notypes of the β3-deficient mice. Further studies are requiredto address this issue.

We have reported previously that ablation of β3 had amarked analgesic effect on mice in the tail-flick test(Murakami et al., 2002). Pretreatment with the N-typecalcium channel blocker ω-conotoxin GVIA is known to beanti-nociceptive (Diaz and Dickenson, 1997) because it inhibitsthe release of acetylcholine, substance P, and the calcitoningene-related peptide from peripheral nerve terminals (Lundy

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and Frew, 1988; Seabrook and Adams, 1989; Maggi et al., 1990).In addition, peptide analogs of ω-conotoxin GVIA were highlyeffective in the formalin and hot-plate tests (Malmberg andYaksh, 1995). The β3 subunit probably decreased calciuminflux through N-type channels and impaired neurotrans-mitter release from the presynapses, thereby increasing theanti-nociceptive effect. Note that following intrathecal injec-tion of ω-conotoxin GVIA, the β3−/− mice still showed lowernociceptive recognition than β3+/+ mice in the tail-flick test,suggesting that other types of channels that take part innociceptive neurotransmission may also be affected by β3ablation.

Anxiolytic activities have been reported for some DHPcalcium antagonists (Matsumoto et al., 1994; Balon andRamesh, 1996). Although evidence for the role of calciumremains weak compared to other potential mechanisms, suchas serotoninergic or GABAergic processes, calcium channelantagonists are thought to have antidepressant effects. In thepresent study, β3−/− mice showed moderate changes in theelevated plus-maze test. They remained in the open armslonger than β3+/+ mice, suggesting that β3−/− mice had loweranxiety levels. Thus, our present results suggest that thevoltage-dependent calcium channels are, to some extent, in-volved in the development of anxiety. In this respect, itshould be noted that Schenberg and colleagues reported theinvolvement of L-type calcium channels in defensive beha-viors, such as anxiety and aggression, due to electricalstimulation of the midbrain dorsal periqueductal gray matter(Schenberg et al., 2000).

It has been demonstrated that β4 null mutants do not havesignificant changes in the calcium channel currents in theirPurkinje neurons, resulting in the reshuffling of the CaV2.1and CaV2.2 with other β subunits (Burgess et al., 1999). Be-cause these studies found no change in the expression ofother β subunits using in situ hybridization, this reshuffling inthe β4 null mutant probably involved a posttranslationalmechanism. However, β1 null mutant mice showed a markeddecrease in L-type currents, indicating the absence of a com-pensatorymechanism (Gregg et al., 1996). In the present study,Western blot analysis showed no change in the expressionlevels of the other known β subunits (β1, β2, or β4) in the brain(Fig. 1A). Thus, it appears that no compensatory increaseoccurs in the transcription of the other β subunits. Westernblot analysis, however, revealed a decreased N-type channelpopulation (Fig. 1A). Additionally, immunoprecipitation ana-lysis showed reshuffling of N-type channels, with the β1 andβ4 subunits (Fig. 1B). Taken together, our present studyrevealed that β3 subunit ablation influenced N-type channelsin the both ways: reshuffling of channel assemblies and adecreased channel population.

In the present study, we found hyperactivity in β3-deficientmice during the dark phase. This is consistent with N-typechannel-deficient mice (Beuckmann et al., 2003). It has beenreported that the circadian rhythm in rats was disturbed by ω-conotoxin GVIA (Masutani et al., 1995). The N-type channelshave attracted particular attention with regard to neurotrans-mitter release (Catterall et al., 2005), but their widespread andcomplex expression has prevented the clarification of theirrole in detail. Dense expression of N-type channels in thelocus coeruleus (LC) and dorsal raphe nucleus (DR) regions,

associated with the ascending monoaminergic system, is pro-bably an important factor in regulating the baseline dischargerate of monoaminergic neurons. For example, N-type chan-nels relate local feedback inhibition of raphe neurons byserotonin (Bayliss et al., 1997). Thus, decreased numbers of N-type channels may reduce baseline regulation by axon colla-terals. Nevertheless, further study is needed to clarify thephysiological role of N-type channel-forming subunits, espe-cially Cav2.2 and β3.

4. Conclusion

Ablation of the β3 subunit resulted in a decreased protein levelof CaV2.2 in the brain and caused reshuffling of N-typecalcium channel assembly. Ablation of the β3 subunit resultedin various neurological phenotypes, including decreased sen-sitivity to ω-conotoxin GVIA in the shaker assay, decreasedresponsiveness to several noxious stimuli, decreased anxiety,increased aggression, and impairment of memory. These phe-notypes suggest important roles for voltage-dependent cal-cium channels and indicate an important role for the β3 sub-unit in the nervous system.

5. Experimental procedures

5.1. Animal model

The behavioral experiments were performed with theapproval of the ethics committee for animal experiments ofTohoku Pharmaceutical University. The β3-deficient mousestrain (β3−/−) was constructed from genomic DNA clones, asreported previously (Murakami et al., 2002). The wild-type (wt)and β3 mutant mice were experimentally naive and 12 to 15weeks old. They were maintained at 22±0.5°C in a 12:12 hlight–dark cycle. Each mouse was acclimatized in the testapparatus for 30 to 60min. All experiments were conducted byresearchers blinded to the genotypes of the mice.

5.2. Biochemical analysis

5.2.1. Western blot analysisPartially purified brain membranes from wt and β3 mutantmice were prepared and suspended in 50 mM Tris–HCl buffer(pH 7.4) containing protease inhibitors (Murakami et al., 2002).For each protein preparation, brains from 8 wt or β3 mutantmice were used. Aliquots of homogenate (100 μg) from eachmouse were resolved by 6% SDS-polyacrylamide gel electro-phoresis. Commercially available polyclonal antibodies spe-cific for CaV2.1 (α1A), CaV2.2 (α1B), CaV1.2 (α1C), CaV1.3 (α1D),CaV2.3 (α1E) (Alomone, Jerusalem, Israel), β1, β3, β4 (Abcamplc, Cambridge, UK), β2 (Sigma-Aldrich, St. Louis, MO, USA),and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA,USA) were used for immunodetection.

5.2.2. Immunoprecipitation analysisImmunoprecipitation (IP) analysis with anti-CaV2.2 (α1B)antibody (Abcam plc) was performed using a protein G IP kit(Sigma-Aldrich).

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5.2.3. Binding assaysPartially purified membranes were prepared from brain tissueand suspended in 50 mM Tris–HCl (pH 7.4) buffer, as describedpreviously (Striessnig et al., 1986). Themembranes (0.2 mg/ml)were incubated in the dark for 90 min at 25°C in the presenceof (+)[3H]PN 200-110 (84 Ci/mmol; Amersham, Piscataway, NJ,USA) in a final volume of 0.5 ml. Nonspecific binding wasdetermined using nicardipine (1.0 μM). The incubation wasterminated by rapid filtration through Whatman GF/B filters(Whatman, Tokyo, Japan).

5.3. Behavioral analysis (spontaneous)

5.3.1. Assay for shaker activityThe ω-conotoxin GVIA (Alomone) was injected intracerebrallyinto 6-week-old mice, which were then observed for shakingactivity for up to 15min. The doseswere administered in serialtwofold dilutions. The onset and persistence of the tremorswere dose-dependent at high doses (2–7 nmol/kg). Symptomswere observed for 2 days, and some mice died at the highestdose (7.0 nmol/kg). At low doses (1.72 nmol/kg), the shakingcould be accentuated by briefly lifting the mice by the tails, asdescribed previously (Olivera et al., 1984). The latency periodbefore shaking started was measured after intracerebralinjection of a low dose (1.72 nmol/kg) of ω-conotoxin GVIA.

5.3.2. Locomotor activity studies (spontaneous locomotoractivities)The locomotor activities of the wt and mutant mice wereanalyzed over 90min using a 12-channel Animex-auto system(Muromachi Kikai Co., Tokyo, Japan). The activity measure-ments were conducted during the light phase between 10:00and 17:00.

5.3.3. Circadian rhythmicity of locomotor activitiesLocomotor activity was measured on the activity wheel everyhour for 2 consecutive days. During the experimental period,the mice had free access to food and water. Individual,inexperienced mice were placed on the activity wheels, andmaintained in a continuous light–dark cycle (lights on at 09:00;lights off at 21:00). Age-matched (10-week-old) inexperiencedcontrol mice were also used.

5.3.4. Rotarod testMice (12-week-old) were placed on a roller at 20 rpm for 3 min.The retention times were recorded at 180 s.

5.4. Responses to noxious stimuli

5.4.1. Capsaicin testCapsaicin (1.0 μg) was injected subcutaneously into the dorsalsurface of the hind paw. Licking behavior was recorded for10 min, as described previously (Sakurada et al., 1992).

5.4.2. Tail-flick testThe tail-flick test was carried out with a tail-flick apparatus.Unanesthetized mice received intrathecal injections in the L5and L6 intervertebral spaces of either ω-conotoxin GVIA(0.132 nmol/kg) or artificial cerebrospinal fluid (aCSF), 10 minprior to the formalin test. A volume of 5 μl was injected intra-thecally using a 28 gauge needle connected to a 50 μL Hamilton

microsyringe. The test compounds were dissolved in sterileaCSF containing126.6mMNaCl, 2.5mMKCl, 2.0mMMgCl2, and1.3 mM CaCl2.

5.4.3. Formalin testThe formalin test was performed by subcutaneous injection offormaldehyde (20 μL of 1.5%) solution under the dorsal surfaceof the right hind paw. After the injection, the behavior of themousewas observed and nociceptive responseswere recordedduring the early (0–10 min after injection) and late (10–30 minafter injection) phases. The total time (in seconds) spent lickingthe injected paw was taken as an indicator of nociception.

5.4.4. Writhing testWe counted the number of abdominal stretches occurringwithin 20 min of intraperitoneal injection of 5.0 ml/kg 0.6%acetic acid. The counting of abdominal stretches started10 min after injecting the stimulus.

5.5. Behavioral analysis based on emotional status

5.5.1. Forced swimming testsOnday 1,malemicewere placed individually in a vertical glasscylinder (height 20°cm; diameter 10 cm), containing a 10 cmdepth of water at 25°C for 15 min, as a practice session. After abrief period of vigorous activity, the mice adopted the charac-teristic immobile posture. Onday 2, themicewere again placedin the glass cylinder for 5 min. A mouse was judged to beimmobile if it remained floating in the water while makingonly those movements that were necessary to keep its headabove water. The total duration of immobility was recorded.These experiments were carried out between 11:00 and 17:00.

5.5.2. Elevated plus-maze testThe elevated plus-maze, which consists of two open arms andtwo closed arms that cross a neutral 5 cm×5-cm centralsquare, was originally developed to measure anxiety levels inrodents. The entire apparatus was elevated to a height of40 cm above floor level. The mice were placed individually atthe end of one open arm, facing away from the central plat-form. The cumulative time spent in the closed arms wasobserved on amonitor using a video camera system. Themicewere allowed to explore the plus-maze for 240 s.

5.5.3. Resident male intruder assayFor the resident-intruder assay, resident male mice werehoused individually for more than 1 month before the pro-cedure. The intrudermicewere housed in cages at five to eightmice per cage. The mice underwent one 5-min session on day1. Equivalent test sessions were performed on days 2 and 3.Each test session was divided into 10-s blocks. Every block inwhich an attack took place scored 1; blocks without an attackscored 0. All scores were added to give the final fighting score.In this test, the latency periods prior to attack and the scoreswere used as indices of aggression.

5.6. Test for working memory

5.6.1. Passive-avoidance testThe step-through passive-avoidance responses were exam-ined daily between 10:00 and 13:00. The apparatus consisted of

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two compartments: one was illuminated at 60 W (150×100mm, with a distance of 150mm to the top of the chamber),and the other was in darkness. The compartments were sepa-rated by a small (20×20 mm) door. On day 1, the mice wereplaced in the illuminated safe compartment. They tended togo into the dark compartment through the door. When all fourpaws were on the grid of the dark compartment, the animalreceived a single foot shock (100 V, 0.1 mA). The latent periodsfor entry into the dark compartmentweremeasured on days 2,3, 4, 5, 10, and 16, in the same manner as in the first trial. Theperiod of latency for mice that did not move into the darkcompartment for more than 5 min was deemed to be 300 s.The success rate was calculated based on whether a mousestayed in the light cage until the end of the experiment(success, remembered) or not (failure, forgot).

5.7. Statistics

Data are expressed as means±S.E.M. and were statisticallyanalyzed using Student's t-test. In the passive-avoidance test,the χ2 test was used to compare success rates. Probability (p)values of 0.05 or less were considered statistically significant.Significant differences are designated by asterisks in thefigures. The genotypes are shown as +/+ (for β3+/+) and −/− (forβ3−/−).

Acknowledgments

We thank Drs. Soichi Hozumi, Veit Flockerzi, Kensuke Kisara,Takehiko Watanabe, and Teruyuki Yanagisawa for theirinterest in this work. This research was sponsored by grants-in-aid from the Ministry of Education, Culture, Sports, Scienceand Technology, Japan.

R E F E R E N C E S

Balon, R., Ramesh, C., 1996. Calcium channel blockers for anxietydisorders? Ann. Clin. Psychiatry 8, 215–220.

Bayliss, D.A., Li, Y.W., Talley, E.M., 1997. Effects of serotonin oncaudal raphe neurons: inhibition of N- and P/Q-type calciumchannels and the after-hyperpolarization. J. Neurophysiol. 77,1362–1374.

Beuckmann, C.T., Sinton, C.M., Miyamoto, N., Ino, M., Yanagisawa,M., 2003. N-type calcium channel α1B subunit (CaV2.2)knock-out mice display hyperactivity and vigilance statedifferences. J. Neurosci. 23, 6793–6797.

Burgess, D.L., Biddlecome, G.H., McDonough, S.I., Diaz, M.E.,Zilinski, C.A., Bean, B.P., Campbell, K.P., Noebels, J.L., 1999. βsubunit reshuffling modifies N- and P/Q-type calcium channelsubunit compositions in lethargic mouse brain. Mol. Cell.Neurosci. 13, 293–311.

Catterall, W.A., Perez-Reyes, E., Snutch, T.P., Striessnig, J., 2005.Nomenclature and structure–function relationships ofvoltage-gated calcium channels. Pharmacol. Rev. 57, 411–425.

Clark, N.C., Nagano, N., Kuenzi, F.M., Jarolimek, W., Huber, I.,Walter, D.,Wietzorrek, G., Boyce, S., Kullmann, D.M., Striessnig,J., Seabrook, G.R., 2003. Neurological phenotype and synapticfunction in mice lacking the CaV1.3 alpha subunit of neuronalL-type voltage-dependent Ca2+ channels. Neuroscience 120,435–442.

Cohen, C., Perrault, G., Sanger, D.J., 1997. Assessment of theantidepressant-like effects of L-type voltage-dependentchannel modulators. Behav. Pharmacol. 8, 629–638.

De Beun, R., Schneider, R., Klein, A., Lohmann, A., De Vry, J., 1996.Effects of nimodipine and other calcium channel antagonistsin alcohol-preferring AA rats. Alchohol 13, 263–271.

Diaz, A., Dickenson, A.H., 1997. Blockade of spinal N- and P-type,but not L-type, calcium channels inhibits the excitability of ratdorsal horn neurons produced by subcutaneous formalininflammation. Pain 69, 93–100.

Gregg, R.G., Messing, A., Strube, C., Beurg, M., Moss, R., Behan, M.,Sukhareva, M., Haynes, S., Powell, J.A., Coronado, R., Powers,P.A., 1996. Absence of the β subunit (ccb1) of the skeletalmuscle dihydropyridine receptor alters expression of the α1subunit and eliminates excitation-contraction coupling. Proc.Natl. Acad. Sci. U. S. A. 93, 13961–13966.

Herdon, H., Nahorski, S.R., 1989. Investigations of the roles ofdihydropyridine and omega-conotoxin-sensitive calciumchannels in mediating depolarization-evoked endogenousdopamine release from striatal slices. Naunyn-Schmiedeberg'sArch. Pharmacol. 340, 36–40.

Ino, M., Yoshinaga, T., Wakamori, M., Miyamoto, N., Takahashi, E.,Sonoda, J., Kagaya, T., Oki, T., Nagasu, T., Nishizawa, Y.,Tanaka, I., Imoto, K., Aizawa, S., Koch, S., Schwartz, A.,Niidome, T.*, Sawada, K., Mori, Y., 2001. Functional disorders ofthe sympathetic nervous system in mice lacking the α1Bsubunit (Cav2.2) of N-type calcium channels. Proc. Natl. Acad.Sci. 98, 5323–5328.

Khan, Z., Jinnah, H.A., 2002. Paroxysmal dyskinesias in thelethargic mouse mutant. J. Neurosci. 22, 8193–8200.

Komuro, H., Rakic, P., 1992. Selective role of N-type calciumchannels in neuronal migration. Science 257, 806–809.

Llinas, R., Moreno, H., 1998. Local Ca2+ signaling in neurons. CellCalcium 24, 359–366.

Ludwig, A., Flockerzi, V., Hofmann, F., 1997. Regional expressionand cellular localization of the alpha1 and beta subunit of highvoltage-activated calcium channels in rat brain. J. Neurosci. 15,1339–1349.

Luebke, J.L., Dunlap, K., Turner, T.J., 1993. Multiple calciumchannel types control glutamatergic synaptic transmission inthe hippocampus. Neuron 11, 895–902.

Lundy, P.M., Frew, R., 1988. Evidence of ω-conotoxinGVIA-sensitive Ca2+ channels in mammalian peripheral nerveterminals. Eur. J. Pharmacol. 156, 325–330.

Maggi, C.A., Tramontana, M., Cecconi, R., Santicioli, P., 1990.Neurochemical evidence for the involvement of N-typecalcium channels in transmitter secretion from peripheralendings of sensory nerves in guinea pigs. Neurosci. Lett. 114,203–206.

Malmberg, A.B., Yaksh, T.L., 1995. Effect of continuous intrathecalinfusion of ω-conopeptides, N-type calcium-channel blockers,on behavior and antinociception in the formalin and hot-platetests in rats. Pain 60, 83–90.

Masutani, H., Matsuda, Y., Nagai, K., Nakagawa, H., 1995. Effect ofω-conotoxin, a calcium channel blocker, on the circadianrhythm in rats. Biol. Rhythm. Res. 26, 573–581.

Matsumoto, Y., Kataoka, Y., Watanabe, Y., Miyazaki, A., Taniyama,K., 1994. Antianxiety actions of Ca2+ channel antagonists withVogel-type conflict test in rats. Eur. J. Pharmacol. 264, 107–110.

Mori, Y., Wakamori, M., Oda, S., Fletcher, C.F., Sekiguchi, N., Mori,E., Copeland, N.G., Jenkins, N.A., Matsushita, K., Matsuyama, Z.,Imoto, K., 2000. Reduced voltage sensitivity of activation ofP/Q-type Ca2+ channels is associated with the ataxic mousemutation Rolling Nagoya. J. Neurosci. 20, 5654–5662.

Murakami, M., Nakagawasai, O., Fujii, S., Hosono, M., Hozumi, S.,Esashi, A., Taniguchi, R., Okamura, T., Suzuki, T., Sasano, H.,Yanagisawa, T., Tan-No, K., Tadano, T., Kitamura, K., Kisara, K.,2000. Antinociceptive effect of cilnidipine, a novel N-typecalcium channel antagonist. Brain Res. 868, 123–127.

112 B R A I N R E S E A R C H 1 1 6 0 ( 2 0 0 7 ) 1 0 2 – 1 1 2

Murakami, M., Fleischmann, B., De Felipe, C., Freichel, M., Trost, C.,Ludwig, A., Wissenbach, U., Schwegler, H., Hofmann, F.,Hescheler, J., Flockerzi, V., Cavalié, A., 2002. Pain perception inmice lacking the β3 subunit of voltage-activated calciumchannels. J. Biol. Chem. 277, 40342–40351.

Olivera, B.M., McIntosh, J.M., Cruz, L.J., Luque, F.A., Gray, W.R.,1984. Purification and sequence of a presynaptic peptidetoxin from Conus geographus venom. Biochemistry 23,5087–5090.

Saegusa, H., Kurihara, T., Zong, S., Kazuno, A., Matsuda, Y.,Nonaka, T., Han, W., Toriyama, H., Tanabe, T., 2001.Suppression of inflammatory and neuropathic pain symptomsinmice lacking the N-type Ca2+ channel. EMBO J. 20, 2349–2356.

Sakurada, T., Katsumata, K., Tan-No, K., Sakurada, S., Kisara, K.,1992. The capsaicin test in mice for evaluating tachykininantagonists in the spinal cord. Neuropharmacology 31,1279–1285.

Schafer, W.R., Kenyon, C.J., 1995. A calcium channel homologuerequired for adaptation to dopamine and serotonin inCaenorhabditis elegans. Nature 375, 73–78.

Schenberg, L.C., de Almada Marcal, L.P., Seeberger, F., de Barros,M.R., Sudre, E.C.M., 2000. L-type calcium channels selectivelycontrol the defensive behaviors induced by electricalstimulation of dorsal periaqueductal gray and overlyingcollicular layers. Behav. Brain Res. 111, 175–185.

Scott, V.E.S., De Waard, M., Hongyan, L., Gurnett, C.A., Venzke,D.V., Lennon, V.A., Campbell, K.P., 1996. β subunitheterogeneity in N-type calcium channels. J. Biol. Chem. 271,3207–3212.

Seabrook, G.R., Adams, D.J., 1989. Inhibition of neurally evokedtransmitter release by calcium channel antagonists in ratparasympathetic ganglia. Br. J. Pharmacol. 97, 1125–1136.

Sher, E., Clementini, F., 1991. ω-conotoxin-sensitivevoltage-operated calcium channels in vertebrate cells.Neuroscience 42, 301–307.

Smith, L.A., Wang, X.-J., Peixoto, A.A., Neumann, E.K., Hall, L.M.,

Hall, J.C., 1996. A Drosophila calcium channel a1 subunit genemaps to a genetic locus associated with behavioral and visualdefects. J. Neurosci. 16, 7868–7879.

Spafford, J.D., Van Minnen, J., Larsen, P., Smit, A.B., Syed, N.I.,Zamponi, G.W., 2004. Uncoupling of calcium channel alpha1and beta subunits in developing neurons. J. Biol. Chem. 279,41157–41167.

Striessnig, J., Goll, A., Moosburger, K., Glossmann, H., 1986. Purifiedcalcium channels have three allosterically coupled drugreceptors. FEBS Lett. 197, 204–210.

Tanaka, O., Sakagami, H., Kondo, H., 1995. Localization of mRNAsof voltage-dependent Ca2+-channels: four subtypes of α1 and βsubunits in developing andmature rat brain. Mol. Brain Res. 30,1–16.

Torri Tarelli, F., Passafaro, M., Clementi, F., Sher, E., 1991.Presynaptic localization of ω-conotoxin-sensitive calciumchannels at the frog neuromuscular junction. Brain Res. 547,331–334.

Turner, T.J., Adams, M.E., Dunlap, K., 1993. Multiple Ca2+ channeltypes coexist to regulate synaptosomal neurotransmitterrelease. Proc. Natl. Acad. Sci. U. S. A. 90, 9518–9522.

Urbano, F.J., Depetris, R.S., Uchitel, O.D., 2001. Coupling of L-typecalcium channels to neurotransmitter release at mouse motornerve terminals. Pflugers Arch. 441, 824–831.

Watanabe, Y., Lawlor, G.F., Fujiwara, M., 1998. Role of nerveterminal L-type Ca2+ channel in the brain. Life Sci. 62,1671–1675.

Witcher, D.R., DeWaard, M., Sakamoto, J., Franzini-Armstrong, C.,Pragnell, M., Kahl, S.D., Campbell, K.P., 1993. Subunitidentification and reconstitution of the N-type Ca2+ channelcomplex purified from brain. Science 261, 486–489.

Zhuchenko, O., Bailey, J., Bonnen, P., Ashizawa, T., Stockton, D.W.,Amos, C., Dobyns, W.B., Subramon, S.H., Zoghbi, H.Y., Lee, C.C.,1997. Autosomal dominant cerebellar ataxia (SCA6)associated with small polyglutamine expansions in the(1A-voltage-dependent calcium channel. Nat. Genet. 15, 62–69.