European Journal of Neuroscience, Vol. 11, pp. 233–240, 1998 © European Neuroscience Association

Cerebellar granule-cell-specific GABAA receptorsattenuate benzodiazepine-induced ataxia: evidence fromα6-subunit-deficient mice

Esa R. Korpi,1,2 Paula Koikkalainen,1 Olga Y. Vekovischeva,1 Riikka Makela,2,3 Raymonde Kleinz,4Mikko Uusi-Oukari1 and William Wisden5

1Department of Pharmacology and Clinical Pharmacology, University of Turku, FIN-20520 Turku, Finland2Department of Mental Health and Alcohol Research, National Public Health Institute, POB 719, FIN-00101 Helsinki, Finland3Tampere Brain Research Center, University of Tampere Medical School, POB 607, FIN-33101 Tampere, Finland4Clinical Research Group, Department of Psychiatry, University of Mainz, D-55101 Mainz, Germany5Medical Research Council Laboratory of Molecular Biology, Medical Research Council Centre, Cambridge CB2 2QH, UK

Keywords: alcohol sensitivity, benzodiazepines, genetic redundancy, motor coordination

Abstract

Benzodiazepine- and alcohol-induced ataxias in rodents have been proposed to be affected by the γ-aminobutyric acid type A(GABAA) receptor α6 subunit, which contributes to receptors specifically expressed in cerebellar granule cells. We have studiedan α6 –/– mouse line for motor performance and drug sensitivity. These mice, as a result of a specific genetic lesion, carry aprecise impairment at their Golgi-granule cell synapses. On motor performance tests (rotarod, horizontal wire, pole descending,staircase and swimming tests) there were no robust baseline differences in motor function or motor learning between α6 –/– andα6 1/1 mice. On the rotarod test, however, the mutant mice were significantly more impaired by diazepam (5–20 mg/kg, i.p.),when compared with α6 1/1 control and background C57BL/6J and 129/SvJ mouse lines. Ethanol (2.0–2.5 g/kg, i.p.) producedsimilar impairment in the α6 –/– and α6 1/1 mice. Diazepam-induced ataxia in α6 –/– mice could be reversed by the benzodiazepinesite antagonist flumazenil, indicating the involvement of the remaining α1β2/3γ2 GABAA receptors of the granule cells. The levelof activity in this synapse is crucial in regulating the execution of motor tasks. We conclude that GABAA receptor α6 subunit-dependent actions in the cerebellar cortex can be compensated by other receptor subtypes; but if not for the α6 subunit, patientson benzodiazepine medication would suffer considerably from ataxic side-effects.

Introduction

Neurons use multiple types ofγ-aminobutyric acid type A (GABAA)receptor, but the physiological significance of this is unknown(Stephenson, 1995; Lu¨ddens & Korpi, 1996; McKernan & Whiting,1996; Wisden & Moss, 1997). For example, these different receptorsubtypes may contribute to the selective effects of drugs such asethanol and benzodiazepines on certain types of behaviour. Thecerebellar granule cell provides a good system to study these issues.Granule cells receive only one type of inhibitory synaptic input fromGolgi interneurons, but express multiple GABAA receptor subunitgenes (mainlyα1, α6, β2, β3, γ2 andδ) (Wisdenet al., 1996; seeFig. 1). They assemble at least two subtypes of receptor at the synapticcleft and various extrasynaptic receptors on the cell body (Nusseret al., 1996, 1998; Somogyiet al., 1996). However, unlike the othersubunit genes, theα6 subunit gene is exclusively expressed incerebellar and cochlear nucleus granule cells (Kato, 1990; Lu¨ddenset al., 1990; Laurieet al., 1992a; Wisdenet al., 1992; Vareckaet al.,1994), and so may contribute specialized functional properties tocircuits regulating, for example, postural reflexes (Korpiet al., 1993a;

Correspondence: Dr Esa R. Korpi, Department of Pharmacology and ClinicalPharmacology, University of Turku, FIN-20520 Turku, Finland.E-mail: esa.korp@utu.fi

Received 12 March 1998, revised 1 July 1998, accepted 6 August 1998

Korpi, 1994). As theα6 subunit is synaptically localized with theα1, β2, β3 and γ2 subunits (Nusseret al., 1996, 1998; see Fig. 1),α6 subunit-containing receptors contribute to phasic feedback inhibi-tion of granule cells by Golgi cells. Theα6 subunit is also specificallypaired with theδ subunit in extrasynaptic locations, on the dendritesand cell body, possibly asα6β2/3δ combination (Joneset al., 1997;Jechlingeret al., 1998; Nusseret al., 1998); theseα6δ-containingreceptors may therefore be activated by GABA diffusing from thesynaptic cleft, and contribute to a tonic background inhibition neededfor optimal granule cell function (Gabbianiet al., 1994; Brickleyet al., 1996; Wall & Usowicz, 1997; Nusseret al., 1998). Slow risingand decaying inhibitory postsynaptic potentials in nonsynaptic cellshave also been suggested to result from high sensitivity ofα6 subunit-containing receptors to GABA spilling over from cerebellar corticalsynaptic glomeruli (Rossi & Hamann, 1998).

Pharmacologically, theα6 subunit-containing receptors are respons-ible for the cerebellar granule cell GABAA receptor population’srelative insensitivity to diazepam and other benzodiazepine agonists(Malminiemi & Korpi, 1989; Turneret al., 1991; Korpiet al., 1992;Wong & Skolnick, 1992; Mathewset al., 1994; Zhenget al., 1994;Joneset al., 1997). This characteristic feature is due to a single aminoacid residue (Arg-100 in theα6 subunit, replaced by His in otherbenzodiazepine-sensitiveα subunits; Wielandet al., 1992). Alcohol-and benzodiazepine-sensitive ANT (Alcohol Non-Tolerant) rats

234 E. R. Korpiet al.

FIG. 1. A simplified description of pathways and synapses in the cerebellar cortex associated with glutamatergic (Glu) granule cells. Granule cells are activatedby excitatory mossy fibre terminals and give rise to parallel fibres, which synapse onto GABAergic Purkinje cells (P) and GABAergic Golgi neurons (Go) inthe molecular layer. Purkinje cells provide the only efferent pathway from the cerebellar cortex, mainly to deep cerebellar nuclei (DCN). Golgi neurons providethe inhibitory feedback to granule cells. Known GABAA receptor subunit combinations are given for the wildtypeα6 1/1 and mutantα6 –/– mice for theGolgi-granule cell synapse (Joneset al., 1997). *, receptors containing theα6 subunit combined with theδ subunits are extrasynaptic (Nusseret al., 1998).Black arrows indicate the direction of information flow.

harbour Gln in this position of theα6 subunit polypeptide (Korpiet al., 1993a). Theα6 Arg to Gln mutation in ANT rats might explainthe abnormal sensitivity of their motor reflexes to sedative drugs,measured as adaptation of posture on a rapidly tilted, rough-surfacedplane (Hellevuoet al., 1989; Wonget al., 1996). Furthermore, drugsthat show high affinity toα6 subunit-containing receptors, such asRo 15-4513 and bretazenil, may antagonize the acute low-dose actionsof ethanol (Suzdaket al., 1988; Bonettiet al., 1989; Harriset al.,1995; see also Hellevuo & Korpi, 1988). Theα6 subunits have alsobeen implicated in the neurochemical adaptations to chronic ethanol:α6 subunit mRNA and protein levels increase following chronicethanol treatments (Mhatre & Ticku, 1992; Wuet al., 1995); quantitat-ive trait loci analysis highlights theα6 gene in the genetic backgroundof ethanol withdrawal seizures in mice (Keir & Morrow, 1994; Bucket al., 1997).

What is the α6 subunit’s specific contribution to an animal’sresponse to ethanol and benzodiazepines? Theα6 subunit-containingreceptors can be specifically inhibited by furosemide (Korpiet al.,1995; Korpi & Luddens, 1997) and antisense oligonucleotides(Zhu et al., 1996), but these reagents would be difficult to utilizein vivo (e.g. by intracerebellar injection) due to the widespreadlocalization of the granule cell layer within the cerebellar cortex.However, targeting theα6 subunit gene by homologous recombinationto makeα6 –/– mice (∆a6lacZ –/– ) provided a direct way to assessits function (Joneset al., 1997). Theα6 gene is expressed only inmature granule cells (Melloret al., 1998; Laurieet al., 1992b; Korpiet al., 1993b), and so gene ablation is unlikely to interfere withcerebellar development. The∆α6lacZ –/– mice lack functionalα6subunits, and their rough motor behaviour is indistinguishable fromwildtype littermates (Joneset al., 1997; see also Homanicset al.,1997). To further assess the significance of theα6 subunits incerebellar function, we have here used motor performance tests of

© 1999 European Neuroscience Association,European Journal of Neuroscience, 11, 233–240

varying difficulty with and without diazepam and ethanol administra-tions in ∆α6lacZ –/– mice andα6 1/1 control mouse lines.

Materials and methods

Animals

A 129/SvJXC57BL/6J mouse line,∆α6lacZ, in which the mouseα6subunit gene was disrupted at exon 8, was created by homologousrecombination (Joneset al., 1997); noα6 subunit polypeptide canbe detected in these mice by either immunoblotting or ligandautoradiography (Joneset al., 1997; Makela et al., 1997). Breedingpairs were transferred to Turku from Cambridge. After behaviouralexperiments, the genotypes of the animals were confirmed usingSouthern blotting, by probing tail-tip DNA samples with an intron8-derivedSacI-XbaI fragment, which hybridizes to 9- and 15-kbSphIfragments from theα6 –/– andα6 1/1 animals, respectively (Joneset al., 1997).

In addition to the∆α6lacZ –/– mice [strain 129/SvJXC57BL/6J,seven males and five females for the motor learning and ethanol anddiazepam experiments, 10 males for swimming experiment, 20 malesfor flumazenil (Ro 15-1788) experiment, 22 males for higher speedrotarod experiments, and six males for blood ethanol concentrations]and α6 1/1 mice (strain 129/SvJXC57BL/6J, eight males and fourfemales for the motor learning and ethanol and diazepam experiments,and 10 males for swimming experiment, 18 males for higher speedrotarod experiments, and six males for blood ethanol concentrations),also mice of the C57BL/6JBom (12 males, Bomholtgård, Denmark)and 129/SvJ (12 males, Jackson Laboratory, Bar Harbor, ME, USA,stock # 000691, 1st generation in Turku) strains were used asbackground strainα6 1/1 controls. The mice were 8–9 weeks ofage and weighed 15–29 g at the beginning of experiments. The micewere maintained at the Central Animal Laboratory (University of

Functions ofα6 subunit-containing GABAA receptors 235

Turku, Turku, Finland) in groups of 4–12 in polypropylene Macroloncages in a room artifically illuminated from 7 a.m. to 7 p.m. and air-conditioned (206 1 °C and relative humidity of 506 10%). Tapwater and pellets were availablead libitum. All experimental protocolswere approved by the Institutional Animal Care and Use Committeeof the University of Turku. Due to obvious differences in the furcolour of the mouse strains, behavioural tests could be carried out atrandomized, but not blind, fashion by several observers, each takingthe responsibility of a complete experiment.

Tests of motor skills and learning

In a staircase test(Simiandet al., 1984), which measures exploratorybehaviour and locomotion, the mice were placed individually for1–3 min in a wooden box, which consisted of five steps (2.5 cm high,10 cm wide, 7.5 cm deep) surrounded by a 12.5-cm-high wall. At thestart the mice were facing away from the steps. The number of stepsclimbed (all four paws on the next step) and the number of rearingswere counted.

In apole test(Ogawaet al., 1985), which measures motor coordina-tion, the mice were placed head upwards near the top of a rough-surfaced iron pole (1 cm in diameter and 55 cm high). The timestaken to turn completely downwards and then to descend to theplatform were recorded (with the cut-off limit of 5 min).

In a horizontal wire test, the ability of the mice to lift theirhindpaws onto a thin horizontal wire after initially hanging from theirforepaws was monitored (Hellevuo & Korpi, 1988).

The above three tests were performed one after another during onetrial and repeated five times, once a day, on succeeding days.

In a rotarod test, the ability of mice to learn to stay for 180 s onan accelerating rotating rod (Rotamex 4/8, Columbus Instruments,Columbus, OH, USA) was tested in six trials on succeeding days(Joneset al., 1997) using four animals, one of each strain at a time,with the rod positions of the strains systematically altered for eachset of animals. Before the six to eight learning trials, the mice werefirst trained to stay on a stationary rod, and then on a rod rotatingevenly at the speed of 5 r.p.m. for 90 s. In the learning trials, themice were placed on a nonmoving rod, the rotation was thenimmediately started at 5 r.p.m. and increased to 15 r.p.m. over the180-s measuring interval. The time for which the mice were able tostay on the rod was recorded. After the learning trials, the animalswere used in ethanol and diazepam experiments with at least 5 daysand at least one injection-free trial on rotarod in-between the druginjections. With some drug-naive animals, the rotarod test was mademore demanding by increasing the speed from 5 r.p.m. to either 30or 40 r.p.m. after the initial learning session with the normal speed.

In a swimming test, ∆α6lacZ –/– andα6 1/1 (129/SvJXC57BL/6J) mice were followed for 2 min in warm (25 °C) water in a 2-Lbeaker, and their immobility (all extremities nonmoving) times scoredfrom video recordings.

Ethanol-induced motor impairment

The tested doses of ethanol (Primalco, Rajama¨ki, Finland; 12%weight/volume, diluted in 0.9% NaCl) were 2.0 and 2.5 g/kg i.p. Theperformance of the mice on the accelerating rod was tested at 5, 15,30, 45 and 60 min after the injection. To test ethanol-inducedlocomotion, the same mice were observed for 2 min in the staircaseat 35 min after injection. The horizontal wire test was performed at50 min after ethanol injection.

Six male ∆α6lacZ –/– andα6 1/1 (129/SvJXC57BL/6J) micewere studied for ethanol metabolism by injecting 2 g/kg ethanol i.p.and collecting 50-µL tail-tip blood samples at 15, 30 and 90 min

© 1999 European Neuroscience Association,European Journal of Neuroscience, 11, 233–240

after the injection for head-space gas-chromatographic analysis ofwhole blood ethanol concentration (Erikssonet al., 1977).

Diazepam-induced motor impairment

The rotarod test was used to study the effects of diazepam [Stesolid(Dumex, Copenhagen, Denmark) 10 and 20 mg/kg i.p., diluted withIntralipid (Pharmacia, Stockholm, Sweden) to a final concentrationof 2 mg/mL] on motor function of the four mouse strains that hadbeen taught how to stay and move on the rod until the 180-s criterion.The time for which the animals were able to stay on the acceleratingrod was recorded at 30 min and 60 min after injections in groups offour mice, each set consisting of one mouse from each line. Horizontalwire and pole tests were also performed at 40 min and 45 min afterthe injections, respectively.

To ascertain that the effects of diazepam were mediated via thebenzodiazepine site of the GABAA receptor, a fresh group of male∆α6lacZ –/– mice (n 5 20) were trained, as described above forthe learning experiment, to stay to the criterion of 180 s on therotating rod. Then the mice were used to test whether thediazepam-induced ataxia could be abolished by flumazenil(Ro 15-1788, Hoffmann-La Roche, Basel, Switzerland), a benzo-diazepine receptor antagonist (Hunkeleret al., 1981). Diazepamwas injected at a dose of 10 mg/kg i.p., followed by the rotarodtest at 30 min, by flumazenil 10 mg/kg i.p. or vehicle [two dropsof Tween 80 (Merck, Darmstadt, Germany) per 10 mL distilledwater] injections at 40 min, and by a final rotarod trial at 60 min.After 1 week, these same animals were tested for the activity offlumazenil (2 mg/kg i.p.) in the staircase and rotarod tests 15 and20 min after the injection, respectively.

Statistics

Analysis of variance (ANOVA) with repeated measures followed byNewman–Keuls multiple comparison posthoc test (more than twogroups) and Student’st-test (two groups) were used to assess thesignificance of the differences between the mouse lines, test trials,drug treatments and interactions, using the Prism 2.0 program(GraphPad Software, San Diego, CA, USA).

Results

In the following experiments, we compared∆α6lacZ –/– mice notonly with their wildtype control hybrid strain (129/SvJXC57BL/6J),but also with two pureα6 1/1 background lines (129/SvJ andC57BL/6J), since polygenic effects on behaviour can be greater thanthose contributions made by single genes (see Gerlai, 1996; Logueet al., 1997; Owenet al., 1997), i.e. mouse strain differences cansometimes confound the results of gene ‘knockout’ experiments.

Baseline motor performance

When compared with theα6 1/1 wildtype controls from all strains,the ∆α6lacZ –/– mice showed normal motor ability in learning tostay on the rotating rod, in climbing steps and exploring a staircasebox, in learning to climb down from a rough pole, and in reflexivelylifting their hindpaws to a horizontal wire. In none of the tests couldwe find significant (F1,605 0.09–1.96,P . 0.11) differences betweenthe male and femaleα6 –/– andα6 1/1 wildtype mice; therefore,sexes were pooled for the further analyses.

Most of the animals learned to reach the rotarod performancecriterion in the course of six learning trials and could stay on a rod

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FIG. 2. Learning to walk on an accelerating rotating rod and habituation to astaircase test of theα6 –/– (∆α6lacZ –/– ),α6 1/1 (129/SvJXC57BL/6J),129/SvJ and C57BL/6J mouse strains during the first six trials. (A) Performancetimes until falling (mean6 SEM, n 5 12) on the rod accelerating from 5 to15 r.p.m. during 3 min. (B) Proportion of the mice of each strain staying onthe rod until the criterion of 180 s. (C) Number of stairs (mean6 SEM)climbed up in the staircase during the observation period of 3 min.(D) Number of rearings (mean6 SEM) in the staircase test during 3 min.(E) Performance times until falling (mean6 SEM, n 5 18–22) on the rodaccelerating from 5 to 30 or 40 r.p.m. during 3 min.

accelerating from 5 to 15 r.p.m. during 180 s (Fig. 2A,B). Two-wayANOVA on the performance times revealed significant mouse line(F3,2625 3.77,P , 0.012) and trial (F5,2625 4.71,P , 0.001) effects,but no interaction, indicating a clear learning component and a minormouse line difference, which could not be identified by apost hoctest. All animals of all strains reached the criterion before the drugexperiments started, except for one∆α6lacZ –/– mouse, whichsucceeded in doing it during the control sessions between the drugsessions. In additional experiments, we found only 1–3∆α6lacZ –/–mice in four different sets (total of 58 mice) which were slow toreach the rotarod criterion (not shown). One batch of mutant andwildtype mice was tested at higher acceleration (5–30 and5–40 r.p.m.) speeds, and the∆α6lacZ –/– mice fell off slightly earlierthan theα6 1/1 (129/SvJXC57BL/6J) mice (Fig. 2E). This differencedisappeared, however, on the next day in an identical test. Thus,drug-naı¨ve ∆α6lacZ –/– mice are not abnormally clumsy during therotarod test (see also Homanicset al., 1997; Joneset al., 1997).

The staircase test showed a clear habituation effect in all mouselines (Fig. 2C,D). The number of stairs climbed during the sixtrials was significantly affected by the mouse line (F3,2645 11.35,P , 0.0001) and decreased by the trial number (F5,2645 9.88,

© 1999 European Neuroscience Association,European Journal of Neuroscience, 11, 233–240

TABLE 1. Performance of various mouse strains in repeated trials of the pole test

α6 –/– α6 1/1 α6 1/1 α6 1 / 1129XC57BL 129/SvJ C57BL/6J

Time to turn downwards (s)trial 1 16.06 4 (11) 186 13 (5) 1016 98 (3) 9.96 2.9 (8)trial 2 3.96 1.3 (11) 7.06 3.3 (9) 556 32 (5)** 4.9 6 0.9 (11)trial 3 1.76 0.3 (11) 6.86 2.1 (10) 236 10 (7)** 4.3 6 0.8 (10)trial 4 3.26 1.2 (11) 3.46 1.1 (10) 1.66 0.4 (7) 3.46 0.6 (10)trial 5 1.96 0.3 (12) 2.06 0.3 (10) 146 11 (9) 4.56 0.9 (12)trial 6 1.66 0.3 (11) 1.96 0.3 (10) 256 23 (9) 2.66 0.4 (12)

Time to descend to platform (s)trial 1 526 17 (11) 286 12 (5) 1666 150 (2)* 186 3 (8)trial 2 116 2 (10) 146 4 (9) 626 33 (5)* 126 1 (12)trial 3 126 2 (11) 146 3 (10) 306 10 (7)** 11 6 1 (12)trial 4 206 5 (10) 146 2 (10) 146 2 (7) 9.26 0.7 (11)trial 5 326 7 (12)** 16 6 4 (10) 136 2 (7) 116 1 (12)trial 6 306 8 (11)* 146 3 (10) 126 1 (5) 9.76 1.1 (12)

Number of animals staying on top of the poletrial 1 1 7 9 3trial 2 1 3 7 0trial 3 1 2 5 0trial 4 1 2 5 1trial 5 0 2 3 0trial 6 1 2 3 0

*P , 0.05, **P , 0.01 for the statistical significance of the difference fromthe other mouse lines (Newman–Keuls test). Total number of animals was 12in each group. Number of animals meeting the criteria for turning anddescending is given in parentheses.

P , 0.0001) without interaction. The∆α6lacZ –/– mice were less(P , 0.05) active in climbing up the stairs than theα6 1/1 C57BL/6J and 129/SvJXC57BL/6J mice during the first and last trials,respectively. The number of rearings was also significantly affectedby the mouse line (F3,2645 8.30,P , 0.0001) and decreased by thetrial number (F5,2645 55.54, P , 0.0001). Theα6 1/1 129/SvJmice exhibited low rearing activity, whereas theα6 1/1 C57BL/6Jmice were active; this difference reached the statistical significance(P , 0.05) during the 2nd trial. Allα6 –/– and α6 1/1 miceregardless of the strain were similar in their rearing activities.

During the pole test, the∆α6lacZ –/– mice learned to turndownwards and descend to the ground as easily as theα6 1/1 129/SvJXC57BL/6J and C57BL/6J mice (Table 1). In the latency to turndown, ANOVA revealed a significant mouse line–trial interaction(F15,2005 1.87, P , 0.03). It took slightly longer for theα6 1/1129/SvJXC57BL/6J mice than the∆α6lacZ –/– andα6 1/1 C57BL/6J mice to learn to turn downwards, but theα6 1/1 129/SvJ micewere even slower to turn downwards (P , 0.01 for the 2nd and 3rdtrials), many of them just staying on top of the pole until the end ofthe experimental time. The time to descend from the pole was alsoaffected by mouse line–trial interaction (F15,1955 4.24,P , 0.0001),and thepost hoccomparison revealed that the 129/SvJ mice wereagain deviant during the first three trials, as they descended signific-antly slower than the other mice strains (P , 0.05). During the lasttwo trials, of the mice that did come down during the 5 min, the∆α6lacZ –/– mice were slower than the other strains (P , 0.05).

All mice could perform the horizontal wire test, although threemice of theα6 1/1 129/SvJXC57BL/6J andα6 1/1 C57BL/6Jgroups failed in several of the six trials.

During a 2-min period in room-temperature water, the∆α6lacZ –/–mice tended (P 5 0.106) to be more immobile than theα6 1/1129/SvJXC57BL/6J mice (46.06 7.4 s, mean6 SEM, n 5 10, vs.26.66 8.6 s,n 5 10, respectively).

Functions ofα6 subunit-containing GABAA receptors 237

FIG. 3. Effects of ethanol administration on the ability of theα6 –/–(∆α6lacZ –/– ),α6 1/1 (129/SvJXC57BL/6J), 129/SvJ and C57BL/6J miceto stay on the accelerating rotating rod and on the climbing and rearingactivities in the staircase test. Ethanol was injected i.p. at doses of 2.0 (A andB; E as indicated under the bars) and 2.5 g/kg (C and D; E as indicated underthe bars). (A,C) Performance times (mean6 SEM, n 5 12) until falling fromthe rod determined at 5, 15, 30, 45 and 60 min after the injections. C57BL/6J mice took significantly longer to recover from ataxia at both doses ofethanol from the other lines (*P , 0.05, **P , 0.01; Newman–Keuls test).(B,D) Proportion of the mice staying on the rod until 180 s at various timesafter the ethanol injection. (E) Number of stairs climbed up and number ofrearings in the staircase test during the 2-min observation period at 35 minafter the ethanol injection. The bars give mean6 SEM for 12 animals in eachgroup. Theα6 –/– mice were significantly less active than the other lines inclimbing up the stairs after both doses of ethanol (*P , 0.05). The C57BL/6J mice were more active than the other lines in rearing after injection of2.0 g/kg ethanol (*P , 0.05).

Ethanol-induced ataxia

Ethanol at doses of 2 and 2.5 g/kg produced a quick ataxic responsein the rotarod test in all mouse lines, with significant recoveryoccurring during the next 30–60 min (Fig. 3A–D). At both ethanoldoses, two-wayANOVA revealed significant mouse line (F3,200. 7.6,P , 0.0001) and time (F4,200. 24.5,P , 0.0001) effects, without asignificant interaction (P . 0.4). There was no difference in the acuteethanol action nor in the recovery from it between the∆α6lacZ –/–,α6 1/1 129/SvJXC57BL/6J andα6 1/1 129/SvJ mice. In apreliminary experiment, the∆α6lacZ –/– mice were slower to recoverthan theα6 1/1 129/SvJXC57BL/6J controls at the ethanol dose of2 g/kg, whereas no difference was observed at 1.5, 2.5 and 3.0 g/kg(data not shown). The C57BL/6J mice were the slowest to recoverfrom ataxia induced by ethanol at both doses. The horizontal wire

© 1999 European Neuroscience Association,European Journal of Neuroscience, 11, 233–240

FIG. 4. Effects of the benzodiazepine agonist, diazepam, on the ability ofthe the α6 –/– (∆α6lacZ –/– ), α6 1/1 (129/SvJXC57BL/6J), 129/SvJand C57BL/6J mice to stay on the accelerating rotating rod and the effectof the benzodiazepine antagonist, flumazenil, on the diazepam-inducedataxia. (A,C) Performance times (mean6 SEM, n 5 12) on the rod untilfalling at 30 and 60 min after i.p. diazepam injections of 10 mg/kg (A)and 20 mg/kg (C). Theα6 –/– mice were more impaired than all otherlines (**P , 0.01,***P , 0.001, Newman–Keuls test). (B,D) Proportion ofthe mice staying up on the rod until 180 s at 30 and 60 min after diazepaminjections at doses of 10 mg/kg (B) and 20 mg/kg (D). (E) Ataxicperformance (mean6 SEM, n 5 10 per group) of theα6 –/– mice on therod at 30 and 60 min after diazepam injection (10 mg/kg) and its reversalby flumazenil (10 mg/kg) given at 40 min after the diazepam injection.(F) Number of stairs climbed up and the number of rearings in thestaircase test at 15 min after flumazenil (2 mg/kg) or vehicle injectionduring an observation period of 3 min.

test (at 50 min after 2 g/kg ethanol) could be performed by three ofthe 129/SvJ mice and oneα6 1/1 129/SvJXC57BL/6J mouse. All∆α6lacZ –/– andα6 1/1 C57BL/6J mice failed, as did all othermice after 2.5 g/kg ethanol dose.

The α6 1/1 C57BL/6J mice were the most active in climbing upstairs and rearing in the staircase test performed at 35 min after theethanol injection (Fig. 3E), suggesting that the locomotor stimulatoryeffect of ethanol might have interfered with their motor coordinationin the rotarod test. Theα6 –/– mice were less active than the otherlines in climbing up the stairs after both doses of ethanol (P , 0.05);this was also true in a drug-naive state (Fig. 2C).

Whole blood ethanol concentrations at 15, 30 and 90 min afterintraperitoneal injection of ethanol 2 g/kg were 46.66 2.5, 48.66 1.4and 31.66 2.3 mM (mean6 SEM, n 5 6) vs. 45.56 2.2, 44.76 1.2and 29.36 0.8 mM for the∆α6lacZ –/– andα61/1 129/SvJXC57BL/6J mice (F1,295 2.46, P 5 0.1278), respectively, indicating nopharmacokinetic difference for ethanol between the mouse lines.

Diazepam-induced ataxia

Relatively high doses of diazepam were needed to produce ataxia inthe rotarod test in the mouse strains studied (Fig. 4A–D), while the

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diazepam-induced impairment in the other tests, the horizontal wireand pole tests, was so much more pronounced at these doses thatalmost all animals failed to perform (see below). Two-wayANOVA

revealed significant mouse line effects (F3,84. 17.6,P , 0.0001) atboth 10 and 20 mg/kg diazepam doses in the rotarod test, a timeeffect (F1,845 9.38,P , 0.01) at the higher dose, but no interactionfor the performance time. The∆α6lacZ –/– mice were clearly moreimpaired thanα6 1/1 129/SvJXC57BL/6J,α6 1/1 129/SvJ andα6 1/1 C57BL/6J mice, both in terms of time on the rod and thenumber of animals reaching the criterion, especially at the lowerdiazepam dose used. There was a dose-related ataxic response in allstrains except the C57BL/6J mice, which were similarly impaired byboth doses of diazepam. With another batch of∆α6lacZ –/– andα61/1 129/SvJXC57BL/6J mice, initially used to test drug-naı¨veperformance on rotarod at higher acceleration speeds (see above),5 mg/kg diazepam impaired significantly more the performance ofthe mutant than wildtype animals (data not shown).

Forty to 45 min after giving the mice 10 mg/kg diazepam, onlyone α6 1/1 129/SvJ mouse could perform the horizontal wire testand oneα6 1/1 C57BL/6J, twoα6 1/1 129/SvJ and oneα6 1/1129/SvJXC57BL/6J were able to descend from the pole. After 20 mg/kg diazepam, again oneα6 1/1 129/SvJ mouse performed the wiretest, and oneα6 1/1 C57BL/6J andα6 1/1 129/SvJXC57BL/6Jmouse succeeded in the pole test.

Another set of the∆α6lacZ –/– mice were used to confirm thatthe 10 mg/kg diazepam-induced ataxia could be reversed by thebenzodiazepine receptor antagonist, flumazenil (Ro 15-1788,10 mg/kg) (Fig. 4E). Since the∆α6lacZ –/– mice were less active inclimbing up the stairs in a drug-naive state (Fig. 2C), we also testedwhether flumazenil (2 mg/kg) would enhance this behaviour byblocking the action of putative endogenous inverse agonists. However,flumazenil failed to affect the staircase test behaviour of thesemice (Fig. 4F).

Discussion

We have now established a correlation between a genetically alteredcerebellar GABAA receptor population and an action of the benzodiaz-epine site agonist diazepam. Although GABAA receptor∆α6lacZ –/–mice show rather normal locomotion and exploration in drug-freesituations (this study; Homanicset al., 1997; Joneset al., 1997), thesemice are strongly impaired by diazepam during a learned motor taskon a rotarod when compared with wildtype controls (this study).Diazepam-induced ataxia in∆α6lacZ –/– mice could be reversed bythe benzodiazepine site antagonist flumazenil (Ro 15-1788), indicatingthe involvement of the remainingα1β2/3γ2 GABAA receptors at theGolgi neuron-granule cell synapse (Fig. 1); benzodiazepine agonistswould be expected to decrease the deactivation rate of such receptors(Mellor & Randall, 1997). In addition, the receptors located extra-synaptically on the dendrites and cell bodies would also becomemore benzodiazepine sensitive: in∆α6lacZ –/– mice, theδ subunitlevels are severely depleted, and so theα6β2/3γ2 and α6β2/3δpopulations would be gone (Joneset al., 1997; Nusseret al., 1998),leaving mainly extrasynapticα1β2/3γ2 receptors, and possibly aminority of α1β2/3δ receptors. The enhanced function of diazepamin the ∆α6lacZ –/– mice is in agreement with the emergence orexpansion of a benzodiazepine agonist-sensitive GABAA receptorpopulation with relatively low GABA and methyl-6,7-dimethoxy-4-ethyl-β-carboline sensitivities as observed by ligand autoradiographyon ∆α6lacZ –/– granule cells (Ma¨kela et al., 1997). This impliesincreased inhibition of the granule cell parallel fibre synapses bydiazepam-induced facilitation of GABAergic action of the Golgi

© 1999 European Neuroscience Association,European Journal of Neuroscience, 11, 233–240

neurons, leading to decreased glutamatergic excitation of the Purkinjeneuron dendrites in the cerebellar molecular layer. The fact thatsystemic benzodiazepines promote ataxia in∆α6lacZ –/– mice isproof that the level of activity in this Golgi-granule cell synapse and/or at the extrasynaptic receptors of the granule cells is critical for thecorrect execution of motor programs, and our study provides the firstdemonstration of this. Our results also confirm the suggestion thatthe abnormal sensitivity of the alcohol-sensitive ANT rats to benzo-diazepine agonists is due to the point mutation altering theirα6subunit-containing receptors from diazepam-insensitive to sensitiveones (Korpiet al., 1993a; see Korpi, 1994). In humans, if it were notfor the α6 subunit, patients on benzodiazepine medication wouldsuffer from considerable ataxia.

Diazepam induced some impairment in motor coordination evenin the mouse strains having wildtype cerebellar GABAA receptorpopulations, indicating that other brain regions might be also involved.In the∆α6lacZ –/– mutant mice, a major contribution can be assignedto the cerebellar receptors. A pharmacodynamic action has beendemonstrated to be responsible for differential ataxic sensitivitybetween diazepam-sensitive and diazepam-resistant mouse lines (Gal-laher et al., 1987), which indicates that pharmacokinetic differencesare not needed to produce wide sensitivity differences to diazepambetween mouse lines. The mechanism of enhanced diazepam actionin the ∆α6lacZ –/– mutant mice is also likely a pharmacodynamicone, since the mutant mice are not expected to have increaseddiazepam concentrations just due to a modification of a gene encodingfor a brain neurotransmitter receptor, especially as the backgroundlines did not differ from each other in diazepam sensitivity (Fig. 4).

Ethanol sensitivity was not altered in the∆α6lacZ –/– mutantmice, clearly indicating thatα6 subunit-containing GABAA receptorsare not responsible for the ethanol-induced motor impairment.Although disappointing, this finding is not surprising: ethanol actsalso on other GABAA receptor subtypes (e.g. Criswellet al., 1993)and on other ligand-gated ion channels, including ionotropic glutamatereceptors (Lovingeret al., 1989; Kuneret al., 1993). Thus, the R/Qpoint mutation in theα6 gene of the ANT rats is probably notresponsible for these animals’ enhanced ethanol sensitivity and,actually, other qualitative alterations in the cerebellar granule cellGABAA receptors of the ANT rats have now been found (Ma¨kelaet al., 1996). Mihic et al. (1997) have described amino acids invarious GABAA and glycine subunits critical for ethanol and volatileanaesthetic actions at the extracellular portion of transmembranedomains TM2 and TM3. Therefore, it is plausible that the ANT ratsharbour unknown mutations which are more significant for the ethanolthan benzodiazepine sensitivity. Since the ethanol sensitivity on themotor task (the present study) and anaesthetic response (Homanicset al., 1997) does not differ in the∆α6lacZ –/– mice compared withα6 1/1 mice, it remains to be established how this subunit couldbe involved adaptating the GABAergic system to chronic ethanoladministration or in the severity of ethanol withdrawal symptoms(see Introduction). Thus, our results and those of Homanicset al.(1997) indicate that GABAA receptorα6 subunit-dependent actionsin the cerebellar cortex can be compensated by other receptorsubtypes, at least in mice.

The lack of any obvious motor-related phenotype in∆α6lacZ –/–mice is surprising. Theα6 protein is abundant, contributing to about50% of benzodiazepine sites on granule cells (Malminiemi & Korpi,1989), but the knockout experiment tells us that granule cells have alarge reserve of receptors, far in excess of what they apparently needto function. Even though deleting theα6 protein leads to a 75%reduction in cerebellarδ subunit protein levels, and substantialreductions inβ2 (– 50%),β3 (– 15%) andγ2 (– 40%) polypeptide

Functions ofα6 subunit-containing GABAA receptors 239

levels (Z. Nusseret al., unpublished observation; Joneset al., 1997),the function of the cerebellum is unimpaired unless the animals aregiven drugs. Yet evolutionary considerations argue that this gene isimportant. The coding sequences (including Arg-100) and the granulecell-specific expression pattern of theα6 gene have been conservedin extreme vertebrate classes: teleost fish through to mammals (Bahnet al., 1996; Hadinghamet al., 1996). In contrast, the intron sequencesbetween pufferfish, chicken and mouse are totally nonconserved(S. Bahnet al., unpublished observation). There are several explana-tions. We cannot rule out that a complex wildtype environment, notjust simple laboratory tests, is needed to see the selective advantageof the α6 gene; alternatively, there may be suppressor genes in themixed 129/C57BL background of the mutants. Producing congenicGABAA receptorα6 –/– lines on different strain backgrounds willresolve this. Finally, theα6 gene might be genuinely redundant(Thomas, 1993; Cookeet al., 1997), since redundancy can be a stableevolutionary strategy (Nowaket al., 1997).

AcknowledgementsThis work was partly supported by the Academy of Finland (E.R.K.), TEKES(E.R.K.), the Finnish Foundation for Alcohol Studies (R.M.), the MedicalResearch Council (W.W.), the British Council (E.R.K. and W.W.), the GermanAcademic Exchange Service (R.K.) and the Center for International Mobility(E.R.K. and O.V.). We thank Alison Jones for expert help with transgenicmice, Antti Haapalinna for kind advice and Elisa Riuttala for expert help inbehavioural tests.

AbbreviationsGABA, γ-aminobutyric acid; Ro, 15-1788 flumazenil.

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