Passive avoidance behaviour deficit in the mdx mouse

Ncurafnmc~l~r D•orders. Vol. I. No. 2. pp. 121-123. 1991 P,nnt~l in Gnntt Bntann

PASSIVE AVOIDANCE

0960--$966/91 $3.00 + 0.00 .~ 1991 Pergamon Press pk;

BEHAVIOUR

DEFICIT

IN THE mdx MOUSE

FRANCESCOMUNTONI,ANNAMATEDDUand GINOSERRA* lstituto di Neuropsichiatria Infantile. Via Ospedale 119. 09124 Cagliari, Italy; and *Dipartimentodi Neuroscienze B.B. Brodie.Via Porcell.09124Cagliari. Italy (Received I0 January 1991;accepted 18 April 1991)

Abstract--Thirty per cent of boys with Duchenne muscular dystrophy (DMD) suffer from various degrees of mental retardation. Since dystrophin, the protein absent in muscles of boys with DMD, is produced also in the brain, it was postulated that the deficiency of brain dystrophin might account for the mental retardation found in DMD boys. The mdx mouse, a mouse model of DMD, fails to produce dystrophin in muscle and brain. This prompted us to study the cognitive function of these animals. Learning and memory processes were studied in l0 mdx females and 9 genetically matched controls using the passive avoidance test. Statistically significant differences in the retention of the passive avoidance response was detected between mdx and control mice, indicating an impairment in passive avoidance learning in mdx mice. Our data reinforce the view that brain dystrophin deficiency is correlated with cognitive dysfunction and indicate that mdx mice might be a model for the mental retardation found in DMD boys. Key words: Duchenne muscular dystrophy, mdx mouse, cognitive impairment, passive avoidance behaviour.

INTRODUCTION

weakness, as demonstrated by their incapacity to cling as strongly to the bars of a wire grid as Since its original description in 1984 [I], the mdx normals do [9]. mutation, which causes an X-linked myopathy in Dystrophin is expressed not only in muscle the mouse, has been considered a genetic homo- cells but, to a lesser extent, in neurons [5] and Iogue candidate of human X-linked Duchenne possibly in glial cells [10]; using a very sensitive muscular dystrophy (DMD). Additional studies antibody, Lidov recently demonstrated that carried out after the discovery ofdystrophin, the dystrophin is particularly abundant in neurons protein absent in muscles of boys with DMD [2- of the cerebral cortex and in Purkinje cells in the 4], confirmed that mdx mice also produced none cerebellum [I I]. Dystrophin appears to be localof this protein [3]. This datum, coupled with the ized at postsynaptic membrane specializations of demonstration of reduced dystrophin RNA a subpopulation of neurons in the cerebral levels later noted in the mdx mouse [5], and, cortex, while in Purkinje cells immunoreaetivity finally, the discovery of the specific mutation appears to be confined to the membrane of causing the dystrophin abnormality in the mdx the soma and dendrities [ii]. A deficiency of mouse [6], supported the concept that D M D and dystrophin in a brain region most strongly the mdx mouse myopathy represent the same associated with cognitive function, like the ceregenetic disorder. As such, the mdx mouse may be bral cortex, could well be correlated with the fact considered as an animal model for human DM D, that some 30°/, of DM D patients also suffer from although more studies are needed for con- various degrees of mental retardation [12, 13]. firmation. In this respect it should be noted Interestingly, mdx mice do not produce dysthat although mdx mice have histopathological trophin in the brain [3], although no major changes similar to those seen in boys with DMD, behavioural abnormality was noted in previous they show successful muscle fibre regeneration studies [i,7-9]. and reduced endomysial fibrosis, in marked The aim of the present study was to investigate contrast to the D M D phenotype. As a result, whether md,: mice showed cognitive dysfunction. mdx do not develop obvious functional disability The acquisition and retention of a passive [I,7,8] or have only a slight degree of muscle avoidance response was investigated as a means 121

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F. MUNTONI et al.

of examining an aspect of cognitive function in mice. Passive avoidance is indeed a widely used paradigm for studying learning and memory processes in animals [I 4-16]. MATERIALS AND M E T H O D S

Animals A breeding pair of experimental mice (C57BL/ 10ScSn-mdx) were initially obtained from Drs J. Morgan and T. Partridge (London). Breeding pairs of genetically matched C57BL/10ScSn control mice were obtained from Charles River, Como, Italy. The two stocks of animals were inbred for up to three generations in our laboratory. All breeding was carried out by brothersister mating. Both strains were given conventional diet and water ad libitum. All mdx females studied were homozygous for the mutation. The homozygosity for the rod,: allele was assessed by studying dystrophin immunostaining in one female for each litter. Six micrometre cryostat sections of limb muscles were immunostained with P6 dystrophin antibodies (rabbit polyclonal antiserum raised to a fusion protein corresponding to amino acids 2814 -3028 of dystrophin, kindly donated by Tim Sherrat and Dr P. Strong). All mdx females showed a negative immunostaining pattern. The animals were aged 16-22 weeks. Passive avoidance behaviour The ability in learning to inhibit a spontaneous response (passive avoidance) was studied in a single step-through type passive avoidance situation [14-16]. The apparatus consisted of a dark box equipped with a grid floor. The box had only one opening giving access to a platform of rectangular shape (5 x 15 cm) illuminated by an incident light. The box was oriented on the bench so that the platform was in the direction of the room and suspended in the air. The physiological behaviour of mice, when placed on the platform, is to enter the dark box after a short period of time; if mice are placed in the dark box instead, they will briefly explore the platform and then they will re-enter the dark box. The experiments consisted of three parts: the adaptation trial, the learning trial and the measurement of the duration of inhibitory behaviour. Mice were first adapted (without receiving any shock) for 120 s in the dark box and then placed on the platform and allowed to enter the dark compartment (adaptation trial). Three such trials were effected the next day. Immediately after entering the dark

compartment on the third trial, the mice received a single unavoidable scrambled footshock (duration 4 s, shock intensity 0.3 mA)(learning trial). Retention of passive avoidance behaviour was tested at 24 and 120 h after learning trial by placing the mice on the platform and measuring the latency in re-entering the dark compartment up to a maximum of 300 s. Motor activi O' Motor activity was measured by individually placing the animals in motility cages (M/P40 Fc Electronic Motility Meter, Motron Products, Stockholm, Sweden). Each cage had 40 photoconductive sensors (one every 16 cm") placed under the grid floor area where the animals were left free to move. The sensors were lit uniformly by an incandescent lamp mounted 60 cm above the floor. Motor activity was defined as the total number of interruptions of a beam in an hour. RESULTS

Passive avoManee hehaviour The latcncies in entering the dark box in the learning trials (i.e. when no shocks had been given) were measured and compared in the two groups. No statistically significant difference in these values was detected between the two groups: the mean latency in controls was 33 s (range 2-120); the mean latency in mdx was 30.6 (range 2-180) (P not significant, Mann-Whitney U-test). Tablc I. Retention of one-trial learning passive-avoidanceresponse in mdx and C57BI./10 mice Animals

Number

Retention of passive avoidance (s) 24 h*

C57BL/IO mdx

9 I0

120 h*

150 s (range 26--300) 44 s (range 6--280) 25 s t (range 2-300) 22 s$.(range 4-70)

Values rcpre~nt the median (in s), * Hours after learning [riM.

t P< 0.02 (Mann-Whimcy U-test). ~P< 0.04 (Mann-Whitney U-tcsl).

The results of passive avoidance behaviour (i.e. after the shock had been given) are presented in Table I. Latcncies in re-entering the shock compartment were significantly lower in mdx females as compared with controls, not only 24 h after the learning trials (P<0.02, MannWhitney U-test) but also in the second retention test at 120 h interval (P<0.04, Mann-Whitney U-test).

Passive Avoidance Behaviour Deficit in the mdx Mouse Table 2. Spontaneous motor activity, mdx v C57BL 10 mice

C57BL. 10 mdx

Number of animals

Motility counts (60')

10 15

3284 ::1:445 2555 ± 753"

Each value represents the mean ± S.E.M. * P < 0.02 (Mann-Whitney t'-test).

Motor activity Table 2 shows that spontaneous motor activity ofmdx and control mice was statistically different (P<0.02, Mann-Whitney U-test). In fact, mdx mice showed reduced motor activity as compared with control mice of the same sex and age. DISCUSSION

123

drug_s that might be ofhelp in the treatment ofthe cognitive impairment found in DMD boys. Acknowledgements--The authors wish to thank G. L. Gessa and C. Cianchetti for their guidance and thoughtful comments on the manuscript. This work was, in part. financially supported by Telethon--Italy. REFERENCES

I. 2.

3.

Using a widely used passive avoidance test, we 4. were able to detect statistically significant differences in the retention of a passive avoidance response between mdx and control mice. The 5. differences obtained in the passive avoidance test between mdx and control mice cannot be accounted for by the observed reduced motility of 6. mdx mice: although mdx mice have a reduced spontaneous motility, they are quicker than controls in re-entering the cage. 7. This result indicates a difference in passive avoidance learning between mdx and genetically matched control mice. it should be noted that 8. various degrees of mental retardation [9,10] and specific deficits in immediate memory have been reported in DMD boys [17]. The primary bio9. chemical defect in DMD is an impaired production of dystrophin, a protein present 10. in muscle and brain; similarly, mdx mice were found to lack dystrophin in muscle and brain cortical neurons. Since the cerebral cortex II. is the brain region most strongly associated with cognitive function, it has been suggested that the defect in cerebral cortical neurons 12. secondary to dystrophin deficiency is expected to produce a cognitive deficit [11]. Our data on the presence of an impairment in learning 13. in mdx mice agree with and reinforce this view. More extensive work must be undertaken 14. before it is possible to claim that mdx mice might 15. be a model not only for the muscle impairment but also for the mental retardation found in 16. DMD boys. If this is confirmed, it should be possible not only to investigate the possible 17. biochemical abnormalities underlying the behavioural deficit, but also to test and develop

Bulfield G, Sillear W G, Wight P A L. Moore K J. X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci USA 1984: 81: 1189-1192. Koenig M, Hoffman E P. Bertelson C J. Monaco A P, Feener C. Kunkel L M. Complete cloning of the Duchenne muscular dystrophy (DMD) eDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 1987; 50: 509-517. Hoffman E P. Brown R H, Kunkel L M. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987: 51: 919-928. Hoffman E P, Fischbeck K H. Brown R H. et al. Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy. N Engl J Ah,d 1988: 3t8:1363-1368. Chamberlain J S, Pearlman J A, Muzny D M, et al. Expression of the murine Duchenne muscular dystrophy gene in muscle and brain. St'ience 1988; 239: 1416-1418. Sicinski P, Geng Y, Ryder-Cook A S, Barnard E A, Darlison M G, Barnard P J. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Scient.e 1989; 244:1578.1580. Carnwath J W. Shotton D M. Muscular dystrophy in the mdx mouse: histopathology of the soleus and extensor digitorum Iongus muscles. J Neurol Sci 1987; 1,10:39-54. Tanabe Y, Esaki K, Nomura T. Skeletal muscle pathology in X chromosome-linked muscular dystrophy (md~:) mouse. Acta Neuropathol ( Berl) 1986; 69: 91-95. Tortes L F B, Duchen L W. The mutant mdx: inherited myopathy in the mouse. Brain 1987; 110: 269-299. Chelly J, ltamard G, KoulakoffA, Kaplan J C, Kahn A, Berwald-Nener Y. Dystrophin gene transcribed from different promoters in neuronal and glial cells. Nature 1990; 344: 64-65. Lidov It G W, Byers T J, Watkins S C, Kunkel L M. Localization of dystrophin to postsynaptic regions of central nervous system cortical neurons. Nature 1990; 348: 725-727. Dubowitz V. Mental retardation in Duchenne muscular dystrophy. In: Rowland L P, ed. Pathogenesis of Human Muscular Dystrophies. Amsterdam: Excerpla Mcdica, 1977: 688-689. Karagan N. Intellectual functioning in Duchenne muscular dystrophy: a review. Psvchol Bull 1979; 86: 25O-259. Ader R, Wied D de. Effects of lysine vasopressin on passive avoidance learning. P~:vch~mSci 1972; 29: 46--48. Bertolin A, Gessa G L. Bchavioural effects of ACTH and M S! t r)cptides. J Endocrinol Invest 1981; 4:241-25 I. Wied D de, Gaffori O, Ree J M van, Jong W de. Central target for the bchavioural effects of vasopressin neuropcptides. Nature 1984; 308: 276.-278. Whelan T B. Neuropsychological performance of children with Duchenne muscular dystrophy and spinal muscular atrophy. Dev Med Child Neuro11987; 29:212220.