Exp Brain Res (1998) 121:35±45 � Springer-Verlag 1998

R E S E A R C H A R T I C L E

C. Strazielle ´ P. KrØmarik ´ J.-F. Ghersi-EgeaR. Lalonde

Regional brain variations of cytochrome oxidase activityand motor coordination in Lurcher mutant mice

Received: 15 September 1997 / Accepted: 3 March 1998

C. Strazielle ())UniversitØ de Nancy 1,Laboratoire de Neuroanatomie Fonctionnelle,FacultØ Dentaire, BP 34, rue du Dr Heydenreich,F-54012 Nancy, Francee-mail: straziel@odonto.u-nancy.fr,Tel.: +33-3-83-39-18-00 ext 881, Fax: +33-3-83-35-41-01

P. KrØmarikLaboratoire de Biologie Cellulaire du DØveloppement,UniversitØ de Nancy I-FacultØ des Sciences,F-54506 Vandoeuvre les Nancy Cedex, France

J.-F. Ghersi-EgeaINSERM U325, Institut Pasteur, 1, rue du Pr. Calmette, BP 245,F-59019 Lille Cedex, France

R. LalondeLaboratoire de Neurobiologie de l'Apprentissage,UniversitØ de Rouen ± FacultØ des Sciences,F-76821 Mont St Aignan FranceCHUM, Campus Hôtel Dieu, MontrØal, Canada

Abstract Lurcher mutant mice are characterized by mas-sive degeneration of cerebellar Purkinje cells and granulecells and by deficits in motor coordination. Regional brainvariations of cytochrome oxidase (CO) activity were ana-lyzed to identify those brain regions with abnormal met-abolic activity as a secondary consequence of the cerebel-lar atrophy and to establish the relationship between COactivity and motor deficits. Lurcher mutants had higherCO activity in all three cerebellar deep nuclei than normallittermate controls of the same background strain. HigherCO activity was also found in Lurcher mutants in brainregions directly connected to the cerebellum, such asthe lateral vestibular nucleus, the cochlear nucleus, thered nucleus, the ventrolateral thalamus, the dorsal raphe,the interpeduncular nucleus, and the inferior colliculus.By contrast, there was a sharp decrease in CO activityin the inferior olive. As for brain regions not directly con-nected to the cerebellum, higher CO activity was ob-served in the trigeminal motor nucleus and the CA1 mo-lecular layer of the hippocampus, which highlights prob-able transsynaptic alterations as a secondary consequenceof cerebellar atrophy. A positive correlation between COactivity in the red nucleus and latencies before falling in

two motor-coordination tests indicates that a compensato-ry increase of metabolic activity in a cerebellar efferentregion is associated with improved behavior.

Key words Cytochrome oxidase ´ Motor coordination ´Cerebellum ´ Cerebral metabolism ´ Lurcher mutants

Introduction

Cytochrome oxidase (CO) is an enzyme of the inner mi-tochondrial membrane responsible for oxidative phos-phorylation (Wong-Riley 1989). CO activity is correlatedto CO amounts (Hevner and Wong-Riley 1989) and neu-ronal activity (Wong-Riley 1989). Moreover, there is acorrelation between CO activity and other indices of cere-bral metabolism, such as glycogen phosphorylase (Harleyand Bielajew 1992).

In contrast to the deoxyglucose method used to detectshort-term metabolic alterations, CO activity is an indexof long-term metabolism allowing detection of local varia-tions among brain structures (Wong-Riley 1989; Gonzalez-Lima and Garrosa 1991; Gonzalez-Lima and Cada 1994;Gonzalez-Lima and Jones 1994; Hevner et al. 1995) andchanges during development (Mjaatvedt and Wong-Riley1988; Tuor et al. 1994) and aging (Ferrandiz et al. 1994).Changes in CO activity in animals have also been reportedafter rewarding brain stimulation (Bielajew 1991), afterelectroconvulsive but not kindled seizures (Nobrega et al.1993a, b), and in a mouse mutant with copper deficiency(Kumode et al. 1993). Brain CO activity decreases in Alz-heimer's disease (Parker and Parks 1995), while it remainsunchanged in Parkinson's disease (Vila et al. 1996).

Since clinical studies have indicated the presence ofcerebello-cortical diaschisis, as assessed by single-photonemission computed tomography (Broich et al. 1987;Botez et al. 1991; Sönmezoglu et al. 1993), we deter-mined the levels of cerebral metabolism in a mutant withselective degeneration of the olivocerebellar system. Theabnormal Lurcher gene causes a Purkinje-cell intrinsicdefect during developmental stages (Soha and Herrup

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1995). Juvenile and adult Lurcher mutant mice are char-acterized by massive degeneration of cerebellar Purkinjecells and secondary degeneration of granule cells and in-ferior olive neurons (Caddy and Biscoe 1979; Heckroth etal. 1990; Heckroth and Eisenman 1991). A milder form ofdegeneration occurs in the cerebellar deep nuclei (Heck-roth 1994). As a result of degeneration of the olivocere-bellar system, the mutants have deficient motor coordina-tion and postural learning (Caston et al. 1995; Lalonde etal. 1995, 1996). A cartography of CO staining was per-formed by quantitative histochemistry in order to studythe metabolic activity of the cerebellum as well as otherregions of the brain. We wished to determine whetherchanges in metabolic activity occur in other brain regionsas a secondary consequence of the cerebellar pathology.Using the 3,3© diaminobenzidine (DAB) technique(Wong-Riley 1979), direct quantification of CO activitywas assessed by comparing optical density of the COstaining with standards whose CO activity had been deter-mined by spectrophotometry after enzymatic reaction(Gonzalez-Lima and Garrosa 1991). We also determinedwhether there is a linear correlation between regionalCO activity and motor coordination in Lurcher mutants.

Materials and methods

Animals

Male Lurcher mutant mice and normal mice (n=10 in each group) ofthe same background strain (B6CBACa-Aw-J/A) were born and bredin the ªLaboratoire de Biologie et Physiologie du ComportementURA CNRS 1293º. The mice were kept in group cages with wood-chip bedding under a light-dark cycle of 14/10 h (lights on at07.00) with food and water available at all times. At the start of be-havior testing, the mice were approximately 3±4 months old withoutsignificant differences in mean age between Lurcher and normalmice. All procedures were in strict accordance with the ªPrinciplesof laboratory animal careº and the regulations of the University ofNancy-Henri PoincarØ.

Behavioral studies

After a few days of preliminary handling, the mice were assessedduring nine consecutive days on the following motor coordinationtests: coat-hanger (Lalonde et al. 1996), rotating grid (Lalonde etal. 1995), and rotorod (Lalonde et al. 1996). The coat-hanger consist-ed of a horizontal string (length: 40 cm, diameter: 2 mm) connectedto two diagonal bars set at a 35� angle from the horizontal and placedat a height of 50 cm above a cushioned table. The mice were sus-pended upside down in the middle part of the horizontal bar, care be-ing taken to release the mouse only when all four paws were securelyplaced. After 3 days of testing on the coat-hanger, the mice wereevaluated on the rotating grid, consisting of a 10�7 cm steel gridplaced at a height of 40 cm above a cushioned table and rotated ata speed of 1 rpm. Each mouse was placed on the screen while itwas immobile in a horizontal position. On days 7±9, the mice wereassessed on the rotorod. The rod, whose outer surface was made ofrubber, measured 5 cm in diameter and 8 cm in width, the top partbeing at a height of 11.5 cm above the table. Each mouse was placedon the rotorod while it was in motion (1 rpm) with the snout facingthe opposite direction of the rotating mast. Because of its smooth sur-face, the mice were unable to rotate passively by clinging to thebeam. Instead, in order to prevent a backward fall, they had to predictthe movement of the beam by advancing forward. In each test, the

time taken before falling was recorded for 4 trials per day, with acut-off period of 60 s per trial and an intertrial interval of 10±15 min.

Tissue preparation

Three days after termination of the behavioral studies, the animalswere killed in the afternoon by decapitation and the unperfused brainrapidly removed, frozen in ±40�C N-methylbutane, and stored at±80�C. Subsequently, the brains were serially cut into 20-�m-thick co-ronal sections at ±12�C with a cryostat, and the sections mounted ongelatin-chrome alum coated slides and stored at ±80�C until processing.

CO histochemistry

CO histochemistry was performed on two series of sections fromeach brain, according to the protocol of Wong-Riley (1979) andslightly modified in the postincubation period. Slides were incubat-ed for 1 h at 37�C in the dark in a solution of 0.1 M phosphate buffer(pH 7.4) containing 50 mg DAB, 20 mg horse-heart cytochrome c,4 g sucrose, and 18 mg catalase per 90 ml, stirred continuously. Thechemical products were purchased from Sigma Aldricht. The slideswere washed in cold buffer for 5 min and immersed in a 10% buf-fered formalin solution with 10% sucrose for 30 min. The slideswere then washed a second time in buffer (2�5 min), dehydratedin successive ethanol and xylene baths, and coverslipped with eukitt.Several complete sets of CO-activity standards were stained simul-taneously with each set of brain sections. Additional sections wereincubated for the same duration, in the absence of DAB or with0.01 M potassium cyanide added to the incubation medium. No vis-ible CO reaction product was seen in these sections.

Preparation of CO standards

To prepare standards for CO activity, cylindrical microtubes werefilled with whole-brain homogenized with a manual Dounce-glasshomogenizer in the absence of any buffer and then frozen on abed of dry ice. The frozen cylinders were kept frozen at ±80�C untilcut in a cryostat at the same time as the brain sections. Sections 10,20, and 40 �m in thickness were used to cover the range of activitymeasured in the different structures by histochemistry. Thisstandardization method has been previously validated byGonzalez-Lima and Jones (1994). In our experimental conditions,the intensity of the staining was proportional to the thickness ofthe standard sections (linear function with r=0.999).

The actual specific activity of CO was determined by the method ofHess and Pope (1953) and modified as follows. Ferrocytochrome c wasprepared extemporally by mixing a 100 mM ferricytochrome c solutionin a fresh K-phosphate buffer (pH 7.1) with sodium dithionite (3 mg/ml). The excess reducing agent was then oxidized by shaking the solu-tion vigorously in air for 3 min. As an index of reduction efficiency, theratio of optical density of the ferrocytochrome c solution between 550and 565 nm was always higher than 8.3. In the second phase, 9 mg offrozen standard was homogenized in 1 ml of 5-mM K-phosphate buffer(pH 7.1) containing 0.75% sodium deoxycholate. Aliquots of the re-sulting homogenate (135±450 �m tissue) were incubated at 22�C in1 ml of the same buffer containing 40 �M ferrocytochrome c. After15 s, the enzymatic oxidation of ferrocytochrome c was followed ina Uvikon 930 (Kontron) spectrophotometer at 550 nm, and the activitymeasured using an extinction coefficient of 19000 M±1 cm±1. Datawere corrected for the low spontaneous reoxidation of ferrocyto-chrome c occurring during the assay. In these conditions, the signalrecorded was proportional to the amount of homogenate incubatedand was fully inhibited by potassium cyanide. The specific activitymeasured, 31.7�3.7 �mol min±1�g of tissue±1, remained constant in±80�C frozen standards for several months.

Densitometric analysis

The CO histochemical-staining intensity was quantified by densito-metric analysis of the sections using a BIOCOM computer-assisted

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image-analysis system (Les Ulis, France) and standards were used toconvert optical density levels into enzymatic activity in �mol min±

1�g of tissue±1. The entire brain could be observed on each slide,the sections being separated by 600-�m intervals. Staining with a1% methylene blue solution was performed on adjacent slides in or-der to identify and delimit the anatomical structures according to theatlas of Franklin and Paxinos (1997) as well as to attest the impor-tance of degenerative lesions in the mutant mice. Multiple optical-density readings were made at a magnification of 20� to sample en-tire areas of different regions. A magnification of 100� was used forsome structures to specify cellular or extracellular localization ofCO labelling. The paired structures were measured equally fromeach side of coronal sections of the brain and the values averaged.

The analysis was performed on 55 brain regions anatomicallyidentified with the atlas of Franklin and Paxinos (1997). The cere-bral cortical areas were the primary motor cortex (frontal), the sec-ondary motor cortex (eye field), the primary sensory cortex (pari-etal), the cingulate cortex (prefrontal) as well as the agranular insu-lar cortex (AIC), the entorhinal cortex, and the primary visual (vi-sual) and auditory (temporal) cortices. These cortical areas, as wellas the neighbouring olfactory tubercles and the claustrum, weremeasured globally without any distinction between their constituentcellular layers. The neostriatum (caudate-putamen) was subdividedlongitudinally into rostral (rNS) and caudal (cNS) halves using theanterior white commissure as a landmark. The rostral neostriatumwas divided into dorsal (rNSd) and ventral (rNSv) parts. No distinc-tions were made between the different nuclei composing the amyg-dala complex, the septum, or the hypothalamus, which were ana-lyzed in their totality. The thalamic areas corresponded to the dif-ferent nuclei of the thalamus, except for the midline region, includ-ing three subnuclei (the rhomboid, the reuniens, and the submedial).The different layers and subdivisions of the hippocampal formationwere measured on the total surface of the structure. All structures ofthe brainstem were well defined. The red nucleus was measured inits magnocellular part; the solitarius complex included the nucleusof the solitary tract and the area postrema. Finally, concerning thecerebellum, the different cortical layers and the deep nuclei weremeasured independently, except for the Purkinje cell layer of thecortex, which is very sharp in normal mice and totally absent inLurchers.

Statistical evaluation

In the behavioral tests, because of unequal variances, latencies be-fore falling were evaluated by means of the nonparametric Mann-Whitney U test. For the quantitative histochemical study, CO valueswere evaluated by means of the unpaired t-test (two-tailed). Becauseof the large number of comparisons, the latter method is subject totype-I errors, so that the required level of significance was set atP=0.01 instead of the usual P=0.05. Ten Lurcher mice were com-pared with ten controls in most brain regions except when sampleswere lost.

Multiple regression analyses between performance on the motorcoordination tests and regional CO activity in Lurcher mutants weremade on those brain regions showing changes in CO activity. Suchanalyses were not made in normal mice because of the maximal ornear maximal values achieved by these animals. For those analyses,the level of significance was set at P=0.05.

Results

Cartography of CO activity

The CO-density cartography of the mouse brain and thedifferences occurring in mutant mice are, in part, illustrat-ed in Figs. 1±5 and density values of the different anatom-ical structures (55 regions) measured in the mutants andcontrols are given in Tables 1±4.

Fig. 1a±c Serial coronal sections of cytochrome-oxidase (CO) la-belling in control mice at the +4.66 mm and +2.46 mm anteriorplanes and at the interaural plane of reference (0.00 mm) (scalebar 1 mm). The sections illustrate the neostriatum region (a), thethalamic region (b), and the mesencephalic region (c), respectively

Abbreviations in figures: Acb accumbens nucleus, CBctx cerebellarcortex, Cg cingulate cortex (prefrontal), CI inferior colliculus, Cochcochlear nuclei, CS superior colliculus, Dt dentate nucleus, Ent ento-rhinal cortex, Ft fastigial nucleus, GP globus pallidus, Gr granularlayer of the cerebellar cortex, Hip hippocampus, Hyp hypothalamus,ICP inferior cerebellar peduncle, Intp interpositus nucleus, IO infe-rior olive, IP interpeduncular nucleus, LVe lateral vestibular nucle-us, M1 primary motor cortex (frontal), M2 secondary motor cortex(eye field), MD mediodorsal nucleus of the thalamus, Mid midlinenuclei of the thalamus, Mol molecular layer of the cerebellar cortex,nR red nucleus, NS neostriatum with its dorsal (rNSd), its ventral(rNSv), and its caudal (cNS) subdivisions, PAG periaqueductal graymatter, S1 primary sensory cortex (parietal), Sol solitary complex,SpiV trigeminal spinal nucleus, Spt septum, SN substantia nigra,TU olfactory tubercles, V1 primary visual cortex (visual), VL ventro-lateral nucleus of the thalamus, VTA ventral tegmental area

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The CO staining pattern observed in the normal mousewas very similar to that in the rat. The gray matter wasmore highly stained than the white matter, which wasvery pale. As each brain structure showed a specific neu-ronal activity, the labelling conformed to specific anatom-ical boundaries. To be brief, relatively moderate COstaining was measured in all neocortical regions, with atypical pattern of laminated higher labelling restricted tolayers I and III-IV, except for the entorhinal cortex, wherethe darkest CO activity was found in layer II. The label-ling in the neostriatum was relatively high and appearedªpatchyº: this alternation of dark and moderate CO densi-ty was more evident in the dorsal portion of the region(Fig. 1a). The thalamus presented varying levels of label-ling, which conforms to the different subnuclei, whilestaining of the hypothalamus was uniform and moderate

with no distinction between the different nuclei(Fig. 1b). In the limbic structures, the overall labellingwas rather moderate to low, except in certain layers orcell types, such as the CA1 molecular layer of the hippo-campus or the interpeduncular nucleus (Fig. 1c). In con-trast to the forebrain, the brainstem was characterizedby relatively dark CO staining in all structures belongingto sensory and motor systems as well as components ofthe extrapyramidal motor pathways (Fig. 1c). In the cere-bellum, the levels of CO density were high in the differentlayers of the cortex as well as in the nuclei.

In the Lurcher mutants, in comparison to the controlsand in spite of severe cell loss, CO staining was main-tained in the molecular (t18=1.38, P>0.1) and the granularlayers (t18=1.0, P>0.3) of the cerebellum. By contrast, theCO activity of Lurcher mutants was increased in the threecerebellar deep nuclei (t18=7.6, P<0.001 for the fastigial;t18=9.39, P<0.001 for the interpositus; and t18=8.25,P<0.001 for the dentate). This staining seemed to be ho-mogeneous, involving the cell bodies as well as the neu-ropils (Figs. 2 and 3a, b). Higher CO activity was present

Fig. 2 Cytochrome-oxidase (CO) labelling of the cerebellum incontrol (a) and Lurcher mutant (b) mice at the ±2.08 mm posteriorplane (scale bar 1 mm). For both sections (a and b), the plane of thesection passes through the deep cerebellar nuclei localized at eachside of the fourth ventricle and through the superior olive localizedin the ventral part of the brainstem. On the Lurcher section, the COis still visible due to atrophy of the cerebellum. A darker line be-tween the granular layer (*) and the molecular layer (V) prefiguresthe Purkinje cell layer

Table 1 Cytochrome-oxidase activity (means and S.E.M. in �mol/min±1�g of tissue±1) in the cerebellum of Lurcher mutants and nor-mal mice (n=10 in each group)

Cerebellum Normal mice Lurcher mutants

Granular layer 50.70 (1.75) 47.14 (3.07)Molecular layer 52.11 (1.37) 58.40 (4.36)Fastigial 41.05 (1.38) 59.43 (1.99)**Interpositus 52.75 (1.78) 74.81 (1.53)**Dentate 55.83 (1.69) 82.27 (2.72)**

**P<0.001 (unpaired t-test, two-tailed)

Table 2 Cytochrome-oxidase activity (means and S.E.M. in �mol/min±1�g of tissue±1) in the brainstem of Lurcher mutants and normalmice (n=7±10 in each group)

Regions Normal mice Lurcher mutants

Substantia nigra 35.36 (0.84) 35.70 (1.92)Ventral tegmentum 26.58 (0.65) 26.97 (1.22)Periaqueductal grey 29.94 (1.09) 31.40 (1.12)Dorsal raphe 42.18 (1.21) 48.69 (1.41)*Red nucleus 34.58 (0.92) 41.54 (1.19)**Interpeduncular 48.45 (1.83) 58.57 (2.02)*Superior colliculus 31.35 (1.04) 34.91 (0.80)Inferior colliculus 42.01 (1.09) 49.90 (2.18)*Medial pontine 35.80 (1.02) 36.94 (1.96)Lateral pontine 47.57 (0.89) 46.04 (2.29)Trigeminal motor 44.33 (1.57) 52.23 (1.35)*Trigeminal mesencephalic 85.38 (2.05) 77.66 (2.58)Trigeminal spinal 41.28 (1.99) 44.86 (1.29)Facial motor 51.50 (2.33) 52.59 (1.12)Cochlear 45.86 (1.69) 53.79 (1.70)*Medial vestibular 54.03 (2.08) 56.63 (1.15)Lateral vestibular 40.59 (1.26) 48.63 (1.21)**Solitarius complex 45.87 (3.06) 52.08 (1.41)Inferior olive 62.90 (2.94) 44.00 (2.40)**Superior olive 44.03 (1.41) 47.40 (0.98)Locus coeruleus 36.35 (0.91) 36.76 (1.82)

*P<0.01; **P<0.001 (unpaired t-test, two-tailed)

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in the red nucleus (t18=4.62, P<0.001), more precisely inits magnocellular portion (Fig. 4a, b). Intracellular CO la-belling measured in this nuclear portion at 100� magnifi-cation confirmed the increase of CO staining in theLurcher mutant (data not shown in the tables; t18=3.27,

P<0.01). Higher CO activity was also observed in otherextracerebellar regions of Lurcher mutants, namely thelateral vestibular nucleus (t18=4.59, P<0.001) (Fig. 3a,b), the ventrolateral thalamus (t18=2.99, P<0.01), the dor-sal raphe (t18=3.94, P<0.01), the interpeduncular nucleus

Fig. 3 Cytochrome-oxidase(CO) labelling of the cerebellarregion analyzed in control (a)and Lurcher mice (b), (scale bar0.1 mm). In the mutant mice,CO-staining intensity was sig-nificantly increased in the deepcerebellar nuclei and in the lat-eral vestibular nucleus (see Ta-bles 1 and 2 for values). Despitethe same magnification in a andb, note the smaller structures inb due to the severe atrophy ofthe cerebellum

Fig. 4 Cytochrome-oxidase(CO) labelling of the red nucle-us (magnocellular part) in con-trol (a) and Lurcher mutant (b)mice (scale bar 0.1 mm). On theLurcher section, note the CO-labelling increase in a large cellpopulation of the red nucleus(arrows). The average CO val-ues of the two animals choosenfor this figure are 37.54 for thecontrol animal C8 and 44.25 forthe Lurcher mutant L2, respec-tively

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(t18=3.72, P<0.01), the cochlear nucleus (t16=3.31,P<0.01), the inferior colliculus (t18=3.24, P<0.01), thetrigeminal motor nucleus (t18=3.81, P<0.01), and the mo-lecular layer of the CA1 region of the hippocampus(t18=4.62, P<0.01). The only brain region showing a de-crease of CO activity in all regions sampled was the infe-rior olive (t12=4.98, P<0.001; Fig. 5). There was no inter-group difference in the other brain regions (P>0.01).

The quantitative optical-density study of the sectionsstained with methylene blue showed a significant increaseof coloration per surface unit of about 30% in the threecerebellar nuclei compared to the controls. In the cerebel-lar cortex, the tissue density of the granular layer present-ed a significant decrease of 22%, while the density wasconserved in the molecular layer. The Purkinje cell layerwas absent.

Correlations between CO activity and behavior

As shown in Table 5, Lurcher mutant mice fell earlierthan normal mice on the coat-hanger (z corrected for

Fig. 5 Cytochrome-oxidase (CO) labelling of the inferior olive incontrol (a) and Lurcher mutant (b) mice at the ±3.68 mm posteriorplane (scale bar 1 mm). For both sections (a and b), the plane passesthrough the caudal part of the solitarius complex (at the level of thearea postrema). In Lurcher mutants, the inferior olive is atrophiedand CO activity is decreased to such a degree that it is no longer dif-ferentiable from the adjacent structures

Fig. 6 Correlation data between CO activity in the red nucleus andperformance on the three tests; coat-hanger (A), rotating grid (B),and rotorod (C). For values of the respective regression coefficients,see result section

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ties=3.52, P<0.001), the rotating grid (z=4.04, P<0.001),and the rotorod (z=3.86, P<0.001). Multiple regressionsrevealed that the three motor coordination tests were notlinearly correlated (coat-hanger vs. the other two tests,r2=0.07, P>0.3; rotating grid vs. the other two tests,r2=0.38, P>0.05). Therefore, separate correlations weremade between each of the three motor-coordination testsand regional CO activity. Only those regions showing in-tergroup differences and a possible relation to motor coor-dination were chosen, namely the cerebellar deep nuclei,the lateral vestibular nucleus, the red nucleus, the ventro-lateral thalamus, and the inferior olive. A multiple regres-

sion analysis indicated a high degree of correlation in COactivity between the three cerebellar nuclei (r=+0.94, ad-justed r2=0.85, P<0.001). Simple regressions revealed ahigher degree of positive correlation between the fastigialnucleus and the interpositus nucleus (r=+0.93, adjustedr2=0.86, F1,8=55.11, P<0.001) than between the fastigialnucleus and the dentate nucleus (R=+0.62, adjustedR2=0.3, F1,8=0.3, P=0.057). In contrast, multiple regres-sions revealed no correlation in CO activity between eachof the deep nuclei on the one hand and the lateral vestib-ular nucleus, the red nucleus, the ventrolateral thalamus,and the inferior olive on the other (P>0.05). Since theCO values of the deep nuclei were intercorrelated, onlythe values of the fastigial nucleus were evaluated for cor-relations with behavior, since this is the nucleus most of-ten associated with postural deficits.

Multiple regression analysis indicated that the onlysignificant correlation was between CO activity in thered nucleus and performance on the rotating grid androtorod tests (r=+0.81, adjusted r2=0.56, F2,7=6.68,P<0.05), with high levels of CO activity being associatedwith better performance. Simple regressions revealed asignificant linear correlation between CO activity in thered nucleus and performance on the rotating grid(r=+0.74, adjusted r2=0.5, F1,8=9.92, P<0.05) and onthe rotorod (r=+0.69, adjusted r2=0.41, F1,8=7.37,P<0.05) (Fig. 6A±C). The best fitting curve for each as-sociation was the fourth order polynomial, yieldingr2=0.69 for the rotating grid and r2=0.84 for the rotorod.No significant correlation was discovered between CO ac-tivity in the fastigial nucleus, the lateral vestibular nucle-us, the ventrolateral thalamus, and the inferior olive onthe one hand and behavioral performances on the other(P>0.05).

Discussion

Changes in CO activity occurred in selective brain regionsof a spontaneous mutant with degeneration of the cerebel-lar cortex (Caddy and Biscoe 1979). CO activity in-creased in the cerebellar deep nuclei, but remained un-changed in the cerebellar cortex. The total loss of Purkinjecells and 90% loss of cerebellar granule cells in Lurchermutants (Caddy and Biscoe 1979) causes a 65% reductionof the cerebellar surface area (Strazielle et al. 1996). Nev-ertheless, the CO activity was preserved in the granularand molecular layers. The CO activity is reported to be

Table 3 Cytochrome-oxidase activity (means and S.E.M. in �mol/min±1�g of tissue±1) in the limbic system, thalamus, and subthalamicnuclei of Lurcher mutants and normal mice (n=10 in each group)

Regions Normal mice Lurcher mutants

Hippocampus

CA1 molecular 41.07 (1.19) 47.75 (1.46)*CA1 polymorphic 27.74 (0.79) 30.93 (1.08)CA2-CA3 polymorphic 32.61 (1.03) 35.49 (1.46)Dentate gyrus 35.49 (0.89) 39.53 (1.25)

Thalamus

Ventrolateral 30.91 (0.99) 35.24 (1.05)*Ventromedial 29.36 (1.43) 33.12 (1.64)Dorsomedial 30.94 (1.24) 32.63 (2.18)Midline 26.61 (0.86) 33.05 (2.24)Lateral geniculate 33.76 (0.97) 37.48 (1.06)Medial geniculate 34.86 (0.87) 35.95 (0.89)Subthalamus 40.25 (1.91) 43.79 (2.03)Zona incerta 32.53 (0.86) 34.59 (0.99)Hypothalamus 32.18 (0.91) 33.88 (1.05)Amygdala 31.10 (0.87) 31.63 (1.09)Septum 31.55 (0.87) 33.59 (1.13)

*P<0.01 (unpaired t-test two-tailed)

Table 4 Cytochrome-oxidase activity (means and S.E.M. in �mol/min±1�g of tissue±1) in the telencephalon of Lurcher mutant miceand normal mice (n=10 in each group)

Regions Normal mice Lurcher mutants

Neocortex

Motor 34.87 (0.57) 36.58 (1.25)Eye field 32.29 (0.82) 34.06 (1.24)Prefrontal 35.72 (0.93) 35.82 (1.43)AIC 31.54 (0.73) 29.89 (1.12)Parietal 34.96 (1.20) 35.86 (1.06)Posterior parietal 33.55 (1.07) 33.50 (1.05)Entorhinal 46.24 (0.94) 50.30 (2.32)Temporal 34.39 (1.20) 32.25 (1.20)Temporal association 33.15 (1.18) 29.70 (0.96)Visual 35.21 (0.59) 36.26 (1.95)Olfactory tubercle 38.98 (1.04) 39.71 (1.63)Nucleus accumbens 37.13 (1.14) 41.54 (1.52)

Neostriatum

Rostrodorsal 30.76 (1.10) 34.96 (2.38)Rostroventral 37.65 (1.72) 38.66 (1.64)Caudal 35.51 (0.92) 38.56 (2.20)Globus pallidus 27.79 (0.73) 29.71 (2.02)

Table 5 Latencies before falling (means in seconds and S.E.M.) ofLurcher mutant mice (n=10) and normal mice (n=10) in three motorcoordination tests

Test Normal mice Lurcher mutants

Coat-hanger 709 (6) 393 (55)*Rotating grid 720 (0) 532 (51)*Rotorod 708 (7) 117 (35)*

*P<0.001 (Mann-Whitney U Test)

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a function of tissue weight. Thus, irrespective of the pres-ence of atrophy, CO activity is a measure of oxidativephosphorylation in the remaining cells, predominantlyneurons, since the contribution of glial cells to CO activ-ity is minimal (Wong-Riley 1989). The preservation ofcerebellar cortical metabolic activity may be due to thecontinued presence of some cerebellar afferents in thismutant, such as climbing fibers (Heckroth and Eisenman1991), mossy fibers (Caddy et al. 1977), and serotoniner-gic fibers (Strazielle et al. 1996), as well as to a preserva-tion or an increase of CO activity in the remaining cells:basket, stellate, or Bergman cells. The observation of thecerebellar cortex of both groups of animals at 100� mag-nification, showed a homogeneous CO labelling in themolecular layer of normal mice as opposed to a heteroge-neous CO labelling in the same layer of the mutant mice.However, the use of CO immunocytochemical labellingwould be more appropriate to define more preciselywhich structures are involved in CO activity of this layer.Moreover, in the normal brain, there are projections fromthe deep nuclei back to the cerebellar cortex (Buisseret-Delmas and Angaut 1989), so that these projectionsmay be involved in the maintenance of CO activity inthe cerebellar cortex as well.

Under normal conditions, the deep nuclei receive in-hibitory afferents from Purkinje cells and excitatory affer-ents from climbing and mossy fibers (Ito et al. 1970;Oertel 1993). Because of the atrophied cerebellar cortexand because of the continuing presence of the various af-ferents in the Lurcher cerebellum, it is probable that theexcitatory afferents predominate, causing an increase inmetabolic activity in the deep nuclei as a consequenceof altered neuronal activity. It has been shown that theelectrophysiological activity of the interpositus nucleusis abnormal in Lurcher mutants (Martin and Caddy1977). The high degree of intercorrelation between eachof the deep nuclei found in the present study is an indica-tion that common factors are responsible for the increaseof CO activity in this region.

An increase of CO activity in Lurcher brain was alsofound in extracerebellar sites. This increase was mainlyseen in regions directly connected to the cerebellum,namely the lateral vestibular nucleus, the red nucleus,the ventrolateral thalamus, the interpeduncular nucleus,the dorsal raphe nucleus, the cochlear nucleus, and the in-ferior colliculus. Concerning the lateral vestibular nucle-us, two-way interactions exist between this nucleus andthe cerebellum (Walberg 1972; Carpenter and Batton1982). Like the cerebellar deep nuclei, the lateral vestib-ular nucleus directly receives projections from Purkinjecells. Thus, the increased activity in this nucleus is prob-ably due to the loss of inhibitory input from Purkinjecells.

The red nucleus (Asanuma et al. 1983) and the ventro-lateral thalamic nucleus (Aumann et al. 1994, 1996) aretwo cerebellar efferent regions involved in motor control.The CO activity of the red nucleus was measured in themagnocellular part, which receives cerebellar efferentsfrom the interpositus nucleus and in turn projects to the

spinal cord (Daniel et al. 1987; Padel 1993). It is possiblethat the increased CO activity in the red nucleus and theventrolateral thalamus is a direct consequence of the in-creased CO activity in the cerebellar deep nuclei. In sup-port of the hypothesis that red nucleus activation is due todeep nuclei activation, it is known that, in the normalbrain, there are tonic excitatory impulses from the inter-positus nucleus to the magnocellular cells of the red nu-cleus (Toyama et al. 1967). However, this is probablynot the only cause, as no correlation in Lurcher brainwas found between CO activity in the deep nuclei onthe one hand and CO activity in the red nucleus and inthe ventrolateral thalamus on the other. This result is anindication that the CO activity of these two regions ismodulated by the activity of other brain regions as well.

The dorsal raphe is a midbrain structure containing se-rotoninergic neurons that project to multiple brain re-gions. In particular, serotoninergic neurons of the dorsalraphe project to the cerebellum (King et al. 1993). More-over, electrophysiological studies indicate the existenceof two-way interactions between midbrain raphe neuronsand the cerebellum (Weiss and Pellet 1982a, b). A remod-elling of serotoninergic terminals has been demonstratedin the rat agranular cerebellum (Beaudet and Sotelo1981). Moreover, changes in the distribution of sero-tonin-uptake sites in different brain regions, includingthe dorsal raphe, were observed in Lurcher mutants(Strazielle et al. 1996). The increased CO activity report-ed in the present study may be due to altered activity ofserotoninergic neurons as a secondary consequence ofthe cerebellar pathology.

Another midbrain nucleus showing an increase of COactivity in the Lurcher brain is the interpeduncular nucle-us. Although the interpeduncular nucleus is known to re-ceive a cerebellar input (Snider et al. 1976), the functionalsignificance of this pathway is obscure.

The final midbrain nucleus with an increase of CO ac-tivity in Lurcher mutants was the inferior colliculus. Thecerebellum receives auditory input from the cochlear nu-cleus (Wang et al. 1991) and projects to the inferior col-liculus (Fish et al. 1979). In Lurcher brain, the cochlearnucleus was also activated. The functional role of the cer-ebellum for auditory information has not been elucidatedin great detail. However, there is evidence that the cere-bellum modulates the acquisition of auditory stimulus-re-sponse associations (Wang et al. 1991). We are unawareof any studies regarding auditory-information processingin Lurcher mutants.

In contrast to the CO activation in the brain regions de-scribed above, a decrease in CO activity was found in theinferior olive. The inferior olive is the source of climbingfibers to the cerebellar cortex and deep nuclei (Dietrichsand Walberg 1989). In turn, the deep nuclei send fibersback to the inferior olive (Brown et al. 1977; Dietrichsand Walberg 1989), in part through inhibitory GABA-ergic connections (Angaut and Sotelo 1989). Lurcher mu-tants are characterized by a cell loss of 60% in the inferiorolive (Heckroth and Eisenman 1991), probably as a sec-ondary consequence of Purkinje cell loss (Caddy and Bis-

43

coe 1979), the principal target of the abnormal Lurchergene (Soha and Herrup 1995). The hypometabolism inthe inferior olive is probably due to the Purkinje cell lossand may be a necessary phase prior to cell death. A sec-ond possible reason is excessive inhibition of the remain-ing cells caused by increased cerebellar deep nuclei activ-ity, as the nucleo-olivo projection is GABAergic (Angautand Sotelo 1989).

In contrast to the significant changes of CO activity re-ported above, other brain regions associated with the cer-ebellum were not altered, for example, the medial vestib-ular nucleus (Carpenter and Batton 1982), the basal pon-tine nuclei (Carpenter and Batton 1982; Yamada andNoda 1987; Brodal and Bjaalie 1992), the locus coeruleus(Snider 1975), the substantia nigra, the ventral tegmentalarea (Snider et al. 1976; Perciavalle et al. 1989), the supe-rior colliculus (May and Hall 1986; Gonzalez-Ruiz andLeichnetz 1987), and the hypothalamus (Haines andDietrichs 1987; Pu et al. 1995). These structures establishsingle or double-pathway connections with the cerebel-lum. However, they do not belong to well-defined closedfunctional pathways as in the cerebellar-inferior olive orcerebellar-vestibular systems described above. Thus, theyreceive many inputs arising from different parts of thebrain, interacting, as does the cerebellum, on the metabol-ic activity of these different structures. Moreover, it ispossible that the relative mildness (20±30% cell loss) ofthe deep nuclei pathology in Lurcher mutants (Heckroth1994) is insufficient to cause CO changes at these levels.

Differential results in terms of activated versus non-ac-tivated regions were also noted among the various thalam-ic subnuclei. While an enhancement of CO activity wasrevealed in the ventrolateral thalamus, there was nochange in CO activity in the other thalamic nuclei. Thecerebellar deep nuclei predominantly innervate the ven-trolateral thalamus. However, there are also less importantprojections to the ventromedial, dorsomedial, intralami-nar, and ventral lateral geniculate regions (Graybiel1974; Faull and Carman 1978; Herkenham 1979, 1986;Haroian et al. 1981; Sugimoto et al. 1981; Groenewegen1988; Aumann et al. 1994, 1996; Sakai et al. 1996). Theseresults indicate that CO activity is preferentially affectedin the thalamic nucleus receiving the densest cerebellar fi-ber projections.

The ventrolateral nucleus projects in turn to the motorcortex as well as to other neocortical regions (Herkenham1986). There was no change in CO activity in the motorcortex or in any other neocortical region of Lurcher mu-tants. Only two regions not directly connected with thecerebellum presented CO activation: the hippocampusand the trigeminal motor nucleus.

In conclusion, the present study showed that manychanges in CO activity occurred in extracerebellar brainregions in Lurcher mutant mice. They may be explainedby closed anatomical connections or by the relativestrength of the connections. It remains to be determinedwhy some brain regions with sparse or poorly defined cer-ebellar connections such as the interpeducular nucleusand the inferior colliculus, were activated as well. These

results may eventually be explained by our current studieson neurochemical changes in the same mutant. However,associated, precise anatomical and biochemical studiesare required to explain these CO alterations and to con-firm the neurochemical hypotheses concerning them.

We found positive correlations between the perfor-mance of Lurcher mutants on two motor coordinationtests on the one hand and CO activity in the magnocellu-lar part of the red nucleus on the other. A polynomialcurve of the fourth order between the two variables ex-plained 69% of the variance on the rotating grid and84% of the variance on the rotorod. The positive correla-tion between CO activity and motor performance is an in-dication that the red nucleus takes over impaired cerebel-lar functions, being an efferent cerebellar region closer tothe neuromuscular synapse. The magnocellular part of thered nucleus is situated in the caudal region and receivesprojections from the interpositus nucleus, while the parvi-cellular part is situated in the rostral region and receivesprojections from the dentate nucleus (Daniel et al.1987). The rostral part of the red nucleus is the sole ormain recipient of projections from the motor cortex andother neocortical regions in rats and primates (Gwynand Flumerfelt 1974; Kennedy et al. 1986). The caudalpart projects to the contralateral spinal cord, while the ros-tral part projects to the inferior olive (Padel 1993). Mag-nocellular neurons of the rubrospinal tract discharge inphase to the locomotor cycle, but not to parvicellular neu-rons (Orlovsky 1972). Thus, it may be hypothesized thatmagnocellular neurons are activated as a result of alteredafferent input from the cerebellum and other brain regionsand that these neurons compensate for a dysfunctionalcerebellum in tests requiring postural adjustments to themobile apparatus. This hypothesis may be tested by usingother types of cerebellar pathology.

Acknowledgements The histochemical studies were performed inthe ªLaboratoire d©Histologie, FacultØ de MØdecine de l©UniversitØNancy 1º. We would like to thank Professors G. Grignon and B. Fol-liguet for providing us with the technical facilities. This work wassupported by a grant from the ªAssociation Française de l©Ataxiede Friedreichº (AFAF).

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