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Architectural changes of the cortico-spinal system in the dystrophin defective mdx mouse
ELSEVIER
Neuroscience Letters 200 (1995) 53-56
NEUROSCIENC[ I[[I[IIS
Architectural changes of the cortico-spinal system in the dystrophin defective mdx mouse Alessandro Sbriccoli a, Marialaura Santarelli b, Donatella Carretta b, Francesco Pinto b, Alberto Granato c, Diego Minciacchib,* alnstitute of Neurology, Catholic University, Largo F. Vito l, 00168 Rome, Italy bDepartment of Neurological and Psychiatric Sciences, University of Florence, Viale Morgagni 85, 50134 Florence, Italy Clnstitute of Anatomy, Catholic University, Largo F. Vito 1, 00168 Rome, Italy
Received 22 July 1995; revised version received 23 September 1995; accepted 29 September 1995
Abstract
The mutant mdx mice which lack the protein dystrophin are an animal model of Duchenne muscular dystrophy. We studied the organization of the cortico-spinal (CS) system in mdx mice using the horseradish peroxidase retrograde tracing technique. Tracer injections were placed in the cervical spinal cord of mutant and control mice. The tangential and radial distribution of CS labeled neurons were similar in mdx and normal mice. Conversely, the absolute number and the cell packing density of labeled CS neurons were considerably lower in mdx than in controls. In mdx, the average size of CS cells was smaller while the perikaryal sizes displayed a normal distribution. In addition, CS neurons of mdx appeared round-shaped compared to the pyramidal cells labeled in control animals. The structural modifications described here should prompt a reconsideration of the involvement of central nervous system in the dystrophin deficient mdx mice. Keywords: Horseradish peroxidase; Muscular dystrophy; Pyramidal tract; Motor control; Sensorimotor cortex; Spinal motoneurons
The X-linked Duchenne muscular dystrophy (DMD) is characterized by degeneration of muscles; the basic molecular defect is the lack of dystrophin, a membrane cytoskeletal protein of the spectrin family [7,12]. Dystrophin is present in skeletal, cardiac, and smooth muscles [1,3]. In the central nervous system (CNS), dystrophin has been found within cortical pyramidal neurons, cerebellar Purkinje cells, hippocampus, substantia nigra, and spinal motoneurons [8,13,14]. This protein appears to influence cell motility, regulate cell shape, contribute to intracellular transport [17], and interfere with developmental events [21]. The mdx mouse shares with D M D a similar X-linked genetic defect and represents an acknowledged animal model for the study of this human pathology [ 1,2,10,19]. Although various degrees of mental retardation and modest changes in some CNS regions of D M D patients are described [5,9,11,20], the evidence of peripheral in* Corresponding author. Tel.: +39 55 4277788; fax.: +39 55 290662; e-mail: diego@cesit 1.unifi.it.
volvement led to the opinion that central structures are not affected by dystrophin deficiency. We hypothesized, however, that the localization of dystrophin in the CNS is important both for the development and maintenance of the structural and functional properties of neuronal networks. We have thus investigated the neurons of origin of the major motor pathway, the cortico-spinal (CS) system. To this purpose, CS neurons of normal and mdx mice were labeled by injecting into the spinal cord the retrograde tracer wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Surgery was performed under deep barbiturate anesthesia (Nembutal, 40 mg/kg i.p.). W G A - H R P (Sigma, 0.1/~1, 7.5% aqueous solution) was injected into the left spinal cord (2nd cervical segment, Fig. IA) to involve both the spinal gray and white matter. Animals were perfused after 48 h with buffered saline and 2.5% glutaraldehyde. Brains and spinal cords were dissected out, soaked in 30% buffered sucrose, cut into 40/~m thick coronal sections, and incubated with tetramethylbenzidine [15]. Adjacent sections were counterstained with thionin. Spi-
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(95) 12079-G
54
A. Sbriccoli et al. / Neuroscience Letters 200 (1995) 53-56
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Fig. 1. Data obtained in representative normal (B,D) and mdx (C,E) mice. Injections into the left cervical cord (A) labeled neurons in the cortex of the right hemisphere (gray area). (B,C) Dorsal views of right hemispheres; dark gray areas are regions containing packed CS cells in the lateral agranular and medial granular cortex, and light gray areas indicate regions with sparse cells. (D,E) Neurons in layer 5 of the motor cortex; arrows indicate the border between lateral agranular and medial granular cortex. Borders have been evaluated by comparing tetramethylbenzidine-incubated and adjacent thionin-stained sections. Numbers in (D,E) refer to levels in (B,C).
nal sections were incubated with diaminobenzidine for evaluation of injection sites. Cells retrogradely labeled in the sensorimotor cortex of three m d x (AFRS, Physiology and Genetics Research Station, Roslin, Midlothian, UK) and three C57BL/6 normal mice were analyzed. Experiments were performed on 3 months old male animals. Microphotographs were captured by digital scanner (Umax UC630) and morphometric analysis was performed on the public domain NIH Image program (US National Institutes of Health). Labeled cells were counted and their number corrected using the formula by Floderus [6]. Differences were always evaluated by ANOVA for nested designs. The cell packing density (CPD) of CS neurons was evaluated as follows: CPD = Nc/(AI × 40), where N c is the corrected number of cells per section, A1 is the area containing labeled neurons, and 40 is the section thickness. To estimate the CPD of all layer 5 neurons, counts into square samples of 19600/~m 2 were performed on thioninstained material and then corrected [6]. Further experiments were carried out on two m d x and two C57BL/6 mice to evaluate the number and morphology of spinal motoneurons. Experiments were performed on 3 months old male animals. WGA-HRP (0.1/d, 7.5% aqueous solution) was injected into the left sciatic nerve.
Animals were perfused after 48 h; spinal cords were dissected out, and processed as described above for visualization of WGA-HRP reaction products. The distribution of CS cells in normal mice was analogous to that in other rodents [4,16]. Stripes of CS neurons extended along the cortex through most of the primary motor field lateral agranular cortex, and the medial part of the granular cortex (Fig. 1B). CS neurons were also found in other cortical fields: rostral part of the medial agranular cortex, second somatosensory cortex, and areas 18a, 18b, 14, 39 and 40, not analyzed in the present study. The same pattern of tangential distribution was observed in m d x mice (Fig. 1C). The radial distribution was also matching in normal and m d x animals: labeled neurons were located exclusively within layer 5. Interestingly, CS neurons of normal mice were pyramidal in shape (Fig. 2A,C), whereas those of m d x were round-shaped (Fig. 2B,D). CS cells appeared always less numerous in mdx than in normal animals (Fig. 1D,E). Stereological counts revealed that CS neurons were reduced of about one half in m d x mice (7362.7 _+439.4 and 3258.4 _+ 1104.3 cells per animal in normal and m d x mice, respectively; F],4 = 35.78; P < 0.01). The CPD of layer 5 neurons was higher in m d x than in normal animals whereas the CPD of CS neurons showed a reverse tendency (Fig. 3A). CS cells represented about 50% of layer 5 neurons in normal animals and only about 35% in m d x (Fig. 3A). The CPD of CS cells, considered taking as covariate the CPD of all layer 5 cells, was significantly different in the two groups (F1,3 = 10.96; P < 0.05). The reduction of labeled neurons in m d x is due to selective damage of the origin of the CS tract rather than to a diffuse shrinkage of all layer 5 cell populations. To evaluate differences on soma size, we measured the cross-section areas of labeled neurons. Values in m d x were significantly lower than those in normal animals (124.71 _+ 12.94pm 2 in normal and 96.15 _+6.31/tm 2 in m d x ; FI, 4 = 11.80; P < 0.05); the two populations of CS neurons showed similar modes of areal distribution but a peak shift of about 30/tm 2 (Fig. 3B). To substantiate the primary impairment of CS cells we counted the motoneurons labeled in the lumbar ventral horn after injections of WGA-HRP into the sciatic nerve of m d x and normal animals. The average number of cells per section was closely similar in both groups (24.57 _+ 8.15 in normal and 24.23 _+9.84 in mdx; F1, 4 = 0.43; P > 0.05). The modifications of the CS tract in mdx mice could be interpreted by considering the role of dystrophin in the cerebral cortex and its complete lack of expression in the brain of m d x [7,13,14,23]. In muscles, the functional significance of dystrophin is related to the evidence that this protein belongs to the cytoskeletal system [7,12]. Should dystrophin play an analogous role in cortical cells, its lack would yield defects in ~ uronal maturation and migration.
55
A. Sbriccoli et al. / Neuroscience Letters 200 (1995) 53-56
Fig. 2. Microphotographs of layer 5 neurons labeled in the lateral agranular cortex. (A) CS neurons in normal animals are pyramidal shaped. (B) In mdx animals neurons are round-shaped. (C) Inset of A. (D) Inset of B. Bar = 50/.tm (A,B), 20 ~ m (C,D).
Indeed, dystrophin is expressed at very early stages of development [10,21]. In addition, cortical neurons of D M D patients display an alteration of the dendritic tree [9] which could be ascribed to anomalies in the cytoskeletal system. Similarly to what is proposed to explain muscle fiber damage in D M D [ 18], it is plausible to assume that a normal number of CS neurons are generated in mdx
mice but bear a reduced ability to survive. Alternatively, the decrease of CS neurons could be secondary to a loss of spinal motoneurons. This is unlikely since we observed that the number of spinal motoneurons is not reduced in mdx. In addition, a normal amount of nerve fibers has been found in lumbar motor roots of mdx [22]. What remains to be clarified is why structural changes
B
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Fig. 3. Quantitative analyses on CS populations in normal and mdx mice. (A) CPD of thionin-stained (white and black columns) versus WGA-HRP labeled neurons (gray columns) in normal and mdx animals. (B) Frequency distribution of cross-section areas of labeled neurons in mdx and normal mice.
56
A. Sbriccoli et al. / Neuroscience Letters 200 (1995) 53-56
o f the CS s y s t e m are c o m p a t i b l e with the apparent normal m o t o r b e h a v i o r o f m d x [2,19]. W e can only speculate that the e f f i c a c y o f the CS s y s t e m m u s t be, in mdx, s o m e w h a t i n d e p e n d e n t o f the a m o u n t o f C S neurons. Our data d e m o n s t r a t e the p r e s e n c e o f m o t o r cortical alterations in mdx m i c e and should p r o m p t a reconsideration o f the C N S i n v o l v e m e n t in D M D . M o d i f i c a t i o n s o f the CS tract, if c o n f i r m e d in D M D patients, w o u l d prov i d e a better u n d e r s t a n d i n g o f the clinical profile and specific insights on individual aspects, such as the difficulty o f rehabilitation. W e thank Dr. A. A n t o n i n i , Institute o f P h y s i o l o g y , U n i v e r s i t y o f C a l i f o r n i a San Francisco, U S A , and Dr. F. Rossi, D e p a r t m e n t o f A n a t o m y and P h y s i o l o g y , U n i v e r sity o f Turin, Turin, Italy, for helpful reading o f the manuscript. This w o r k has been supported by a T e l e t h o n grant. [1] Arahata, K., Ishihara, T., Nonaka, I., Ozawa, E. and Sugita, H., Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide, Nature, 333 (1988) 861-868. [2] Bulfield, G., Siller, W.G., Wight, P.A. and Moore, K.J., X chromosome-linked muscular dystrophy (mdx) in the mouse, Proc. Natl. Acad. Sci. USA, 81 (1984) 1189-1192. [3] Chelly, J., Kaplan, J.C., Maire, P., Gautron, S. and Kahn, A., Transcription of the dystrophin gene in human muscle and nonmuscle tissues, Nature, 333 (1988) 858-860. [4l Donoghue, J.P. and Wise, S.P., The motor cortex of the rat: cytoarchitecture and microstimulation mapping, J. Comp. Neurol., 212 (1982) 76-88. 15] Emery, A.E.H., Involvement of tissues other than skeletal muscle. In A.E.H. Emery (Ed.), Duchenne Muscular Dystrophy, Oxford Medical Publications, Oxford, 1988. [6] Floderus, S., Untersuchungen iiber den Bau der menschlichen Hypophyse mit besonderer Beriicksichtigung der qualitativen mikromorphologischen Verh~iltnisse, Acta Pathol. Microbiol. Scand., 53 (1944) 1-276. [7] Hoffman, E.P., Brown, Jr., R.H. and Kunkel, L.M., Dystrophin: the protein product of the Duchenne muscular dystrophy locus, Cell, 51 (1987) 919-1128. [8] Huard, J., Cttt, P.-Y., Parent, A., Bouchard, J.-P. and Tremblay, J.P., Dystrophin-like immunoreactivity in monkey and human brain areas involved in learning and motor functions, Neurosci. Lett., 141 (1992) 181-186.
[9] Jagadha, V. and Becker, L.E., Dendritic pathology: an overview of Golgi studies in man, Can. J. Neurol. Sci., 16 (1989) 41-50. [10] Jung, D., Pons, F., l.,tger, J.J., Aunis, D. and Rendon, A., Dystrophin in central nervous system: a developmental, regional distribution and subcellular localization study, Neurosci. Lett., 124 (1991) 87-91. [11] Karagan, N.J., Intellectual functioning in Duchenne muscular dystrophy: a review, Psychol. Bull., 86 (1979) 250-259. [12] Koenig, M., Monaco, A.P. and Kundel, L.M., The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein, Cell, 53 (1988) 219-228. [13] Lidov, H.J.W., Byers, T.J. and Kunkei, L.M., The distribution of dystrophin in the murine central nervous system: an immunocytochemical study, Neuroscience, 54 (1993) 167-I 87. [14] Lidov, H.J.W., Byers, T.J., Watkins, S.C. and Kunkel, L.M., Localization of dystrophin to postsynaptic regions of central nervous system cortical neurons, Nature, 348 (1990) 725-728. [15] Mesulam, M.M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 25 (1978) 106-117. [16] Miller, M.W., The origin of corticospinal projection neurons in rat, Exp. Brain Res., 67 (1987) 339-351. [17] Miyatake, M., Miike, T., Zhao, J.-E., Yoshioka, K., Uchino, M. and Usuku, G., Dystrophin: localization and presumed function, Muscle Nerve, 14 (1991) 113-119. [18] Petrol, B.J., Shrager, J.B., Stedman, H.H., Kelly, A.M. and Sweeney, H.L., Dystrophin protects file sarcolemma from stresses developed during muscle contraction, Proc. Natl. Acad. Sci. USA, 90 (1993) 3710-3714. [19] Sicinski, P., Geng, Y., Ryder-Cook, A.S., Barnard, E.A., Darlison, M.G. and Bamard, P.J., The molecular basis of muscular dystrophy in the mdx mouse: a point mutation, Science, 244 (1989) 1578-1580. [20] Sugimoto, S., Tsuruta, K., Kurihara, T., Ono, S., Morotomi, Y., lnoue, K. and Matsukura, S., Posterior tibial somatosensory evoked potentials in Duchenne-Type progressive muscular dystrophy, Electroencephalogr. Clin. Neurophysiol., 61 (1986) 525527. [21] Torelli, S., Sogos, V., Ennas, M.G., Muntoni, F., Clerk, A., Strong, P.N. and Gremo, F., Dystrophin immunoreactivity in normal and Duchenne human fetal neurons in culture, J. Neurosci. Res., 32 (1992) 116-125. [22] Torres, L.F.B. and Duchen, L.W., The mutant mdx: inherited myopathy in the mouse, Brain, 110 (1987) 269-299. [23] Uchino, M., Yoshioka, K., Miike, T., Tokunaga, M., Uyama, E., Teramoto, H., Naoe, H. and Ando, M., Dystrophin and dystrophin-related protein in the brains of normal and mdx mice, Muscle Nerve, 17 (1994) 533-538.
Neuroscience Letters 200 (1995) 53-56
NEUROSCIENC[ I[[I[IIS
Architectural changes of the cortico-spinal system in the dystrophin defective mdx mouse Alessandro Sbriccoli a, Marialaura Santarelli b, Donatella Carretta b, Francesco Pinto b, Alberto Granato c, Diego Minciacchib,* alnstitute of Neurology, Catholic University, Largo F. Vito l, 00168 Rome, Italy bDepartment of Neurological and Psychiatric Sciences, University of Florence, Viale Morgagni 85, 50134 Florence, Italy Clnstitute of Anatomy, Catholic University, Largo F. Vito 1, 00168 Rome, Italy
Received 22 July 1995; revised version received 23 September 1995; accepted 29 September 1995
Abstract
The mutant mdx mice which lack the protein dystrophin are an animal model of Duchenne muscular dystrophy. We studied the organization of the cortico-spinal (CS) system in mdx mice using the horseradish peroxidase retrograde tracing technique. Tracer injections were placed in the cervical spinal cord of mutant and control mice. The tangential and radial distribution of CS labeled neurons were similar in mdx and normal mice. Conversely, the absolute number and the cell packing density of labeled CS neurons were considerably lower in mdx than in controls. In mdx, the average size of CS cells was smaller while the perikaryal sizes displayed a normal distribution. In addition, CS neurons of mdx appeared round-shaped compared to the pyramidal cells labeled in control animals. The structural modifications described here should prompt a reconsideration of the involvement of central nervous system in the dystrophin deficient mdx mice. Keywords: Horseradish peroxidase; Muscular dystrophy; Pyramidal tract; Motor control; Sensorimotor cortex; Spinal motoneurons
The X-linked Duchenne muscular dystrophy (DMD) is characterized by degeneration of muscles; the basic molecular defect is the lack of dystrophin, a membrane cytoskeletal protein of the spectrin family [7,12]. Dystrophin is present in skeletal, cardiac, and smooth muscles [1,3]. In the central nervous system (CNS), dystrophin has been found within cortical pyramidal neurons, cerebellar Purkinje cells, hippocampus, substantia nigra, and spinal motoneurons [8,13,14]. This protein appears to influence cell motility, regulate cell shape, contribute to intracellular transport [17], and interfere with developmental events [21]. The mdx mouse shares with D M D a similar X-linked genetic defect and represents an acknowledged animal model for the study of this human pathology [ 1,2,10,19]. Although various degrees of mental retardation and modest changes in some CNS regions of D M D patients are described [5,9,11,20], the evidence of peripheral in* Corresponding author. Tel.: +39 55 4277788; fax.: +39 55 290662; e-mail: diego@cesit 1.unifi.it.
volvement led to the opinion that central structures are not affected by dystrophin deficiency. We hypothesized, however, that the localization of dystrophin in the CNS is important both for the development and maintenance of the structural and functional properties of neuronal networks. We have thus investigated the neurons of origin of the major motor pathway, the cortico-spinal (CS) system. To this purpose, CS neurons of normal and mdx mice were labeled by injecting into the spinal cord the retrograde tracer wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Surgery was performed under deep barbiturate anesthesia (Nembutal, 40 mg/kg i.p.). W G A - H R P (Sigma, 0.1/~1, 7.5% aqueous solution) was injected into the left spinal cord (2nd cervical segment, Fig. IA) to involve both the spinal gray and white matter. Animals were perfused after 48 h with buffered saline and 2.5% glutaraldehyde. Brains and spinal cords were dissected out, soaked in 30% buffered sucrose, cut into 40/~m thick coronal sections, and incubated with tetramethylbenzidine [15]. Adjacent sections were counterstained with thionin. Spi-
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(95) 12079-G
54
A. Sbriccoli et al. / Neuroscience Letters 200 (1995) 53-56
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Fig. 1. Data obtained in representative normal (B,D) and mdx (C,E) mice. Injections into the left cervical cord (A) labeled neurons in the cortex of the right hemisphere (gray area). (B,C) Dorsal views of right hemispheres; dark gray areas are regions containing packed CS cells in the lateral agranular and medial granular cortex, and light gray areas indicate regions with sparse cells. (D,E) Neurons in layer 5 of the motor cortex; arrows indicate the border between lateral agranular and medial granular cortex. Borders have been evaluated by comparing tetramethylbenzidine-incubated and adjacent thionin-stained sections. Numbers in (D,E) refer to levels in (B,C).
nal sections were incubated with diaminobenzidine for evaluation of injection sites. Cells retrogradely labeled in the sensorimotor cortex of three m d x (AFRS, Physiology and Genetics Research Station, Roslin, Midlothian, UK) and three C57BL/6 normal mice were analyzed. Experiments were performed on 3 months old male animals. Microphotographs were captured by digital scanner (Umax UC630) and morphometric analysis was performed on the public domain NIH Image program (US National Institutes of Health). Labeled cells were counted and their number corrected using the formula by Floderus [6]. Differences were always evaluated by ANOVA for nested designs. The cell packing density (CPD) of CS neurons was evaluated as follows: CPD = Nc/(AI × 40), where N c is the corrected number of cells per section, A1 is the area containing labeled neurons, and 40 is the section thickness. To estimate the CPD of all layer 5 neurons, counts into square samples of 19600/~m 2 were performed on thioninstained material and then corrected [6]. Further experiments were carried out on two m d x and two C57BL/6 mice to evaluate the number and morphology of spinal motoneurons. Experiments were performed on 3 months old male animals. WGA-HRP (0.1/d, 7.5% aqueous solution) was injected into the left sciatic nerve.
Animals were perfused after 48 h; spinal cords were dissected out, and processed as described above for visualization of WGA-HRP reaction products. The distribution of CS cells in normal mice was analogous to that in other rodents [4,16]. Stripes of CS neurons extended along the cortex through most of the primary motor field lateral agranular cortex, and the medial part of the granular cortex (Fig. 1B). CS neurons were also found in other cortical fields: rostral part of the medial agranular cortex, second somatosensory cortex, and areas 18a, 18b, 14, 39 and 40, not analyzed in the present study. The same pattern of tangential distribution was observed in m d x mice (Fig. 1C). The radial distribution was also matching in normal and m d x animals: labeled neurons were located exclusively within layer 5. Interestingly, CS neurons of normal mice were pyramidal in shape (Fig. 2A,C), whereas those of m d x were round-shaped (Fig. 2B,D). CS cells appeared always less numerous in mdx than in normal animals (Fig. 1D,E). Stereological counts revealed that CS neurons were reduced of about one half in m d x mice (7362.7 _+439.4 and 3258.4 _+ 1104.3 cells per animal in normal and m d x mice, respectively; F],4 = 35.78; P < 0.01). The CPD of layer 5 neurons was higher in m d x than in normal animals whereas the CPD of CS neurons showed a reverse tendency (Fig. 3A). CS cells represented about 50% of layer 5 neurons in normal animals and only about 35% in m d x (Fig. 3A). The CPD of CS cells, considered taking as covariate the CPD of all layer 5 cells, was significantly different in the two groups (F1,3 = 10.96; P < 0.05). The reduction of labeled neurons in m d x is due to selective damage of the origin of the CS tract rather than to a diffuse shrinkage of all layer 5 cell populations. To evaluate differences on soma size, we measured the cross-section areas of labeled neurons. Values in m d x were significantly lower than those in normal animals (124.71 _+ 12.94pm 2 in normal and 96.15 _+6.31/tm 2 in m d x ; FI, 4 = 11.80; P < 0.05); the two populations of CS neurons showed similar modes of areal distribution but a peak shift of about 30/tm 2 (Fig. 3B). To substantiate the primary impairment of CS cells we counted the motoneurons labeled in the lumbar ventral horn after injections of WGA-HRP into the sciatic nerve of m d x and normal animals. The average number of cells per section was closely similar in both groups (24.57 _+ 8.15 in normal and 24.23 _+9.84 in mdx; F1, 4 = 0.43; P > 0.05). The modifications of the CS tract in mdx mice could be interpreted by considering the role of dystrophin in the cerebral cortex and its complete lack of expression in the brain of m d x [7,13,14,23]. In muscles, the functional significance of dystrophin is related to the evidence that this protein belongs to the cytoskeletal system [7,12]. Should dystrophin play an analogous role in cortical cells, its lack would yield defects in ~ uronal maturation and migration.
55
A. Sbriccoli et al. / Neuroscience Letters 200 (1995) 53-56
Fig. 2. Microphotographs of layer 5 neurons labeled in the lateral agranular cortex. (A) CS neurons in normal animals are pyramidal shaped. (B) In mdx animals neurons are round-shaped. (C) Inset of A. (D) Inset of B. Bar = 50/.tm (A,B), 20 ~ m (C,D).
Indeed, dystrophin is expressed at very early stages of development [10,21]. In addition, cortical neurons of D M D patients display an alteration of the dendritic tree [9] which could be ascribed to anomalies in the cytoskeletal system. Similarly to what is proposed to explain muscle fiber damage in D M D [ 18], it is plausible to assume that a normal number of CS neurons are generated in mdx
mice but bear a reduced ability to survive. Alternatively, the decrease of CS neurons could be secondary to a loss of spinal motoneurons. This is unlikely since we observed that the number of spinal motoneurons is not reduced in mdx. In addition, a normal amount of nerve fibers has been found in lumbar motor roots of mdx [22]. What remains to be clarified is why structural changes
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150
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Fig. 3. Quantitative analyses on CS populations in normal and mdx mice. (A) CPD of thionin-stained (white and black columns) versus WGA-HRP labeled neurons (gray columns) in normal and mdx animals. (B) Frequency distribution of cross-section areas of labeled neurons in mdx and normal mice.
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A. Sbriccoli et al. / Neuroscience Letters 200 (1995) 53-56
o f the CS s y s t e m are c o m p a t i b l e with the apparent normal m o t o r b e h a v i o r o f m d x [2,19]. W e can only speculate that the e f f i c a c y o f the CS s y s t e m m u s t be, in mdx, s o m e w h a t i n d e p e n d e n t o f the a m o u n t o f C S neurons. Our data d e m o n s t r a t e the p r e s e n c e o f m o t o r cortical alterations in mdx m i c e and should p r o m p t a reconsideration o f the C N S i n v o l v e m e n t in D M D . M o d i f i c a t i o n s o f the CS tract, if c o n f i r m e d in D M D patients, w o u l d prov i d e a better u n d e r s t a n d i n g o f the clinical profile and specific insights on individual aspects, such as the difficulty o f rehabilitation. W e thank Dr. A. A n t o n i n i , Institute o f P h y s i o l o g y , U n i v e r s i t y o f C a l i f o r n i a San Francisco, U S A , and Dr. F. Rossi, D e p a r t m e n t o f A n a t o m y and P h y s i o l o g y , U n i v e r sity o f Turin, Turin, Italy, for helpful reading o f the manuscript. This w o r k has been supported by a T e l e t h o n grant. [1] Arahata, K., Ishihara, T., Nonaka, I., Ozawa, E. and Sugita, H., Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide, Nature, 333 (1988) 861-868. [2] Bulfield, G., Siller, W.G., Wight, P.A. and Moore, K.J., X chromosome-linked muscular dystrophy (mdx) in the mouse, Proc. Natl. Acad. Sci. USA, 81 (1984) 1189-1192. [3] Chelly, J., Kaplan, J.C., Maire, P., Gautron, S. and Kahn, A., Transcription of the dystrophin gene in human muscle and nonmuscle tissues, Nature, 333 (1988) 858-860. [4l Donoghue, J.P. and Wise, S.P., The motor cortex of the rat: cytoarchitecture and microstimulation mapping, J. Comp. Neurol., 212 (1982) 76-88. 15] Emery, A.E.H., Involvement of tissues other than skeletal muscle. In A.E.H. Emery (Ed.), Duchenne Muscular Dystrophy, Oxford Medical Publications, Oxford, 1988. [6] Floderus, S., Untersuchungen iiber den Bau der menschlichen Hypophyse mit besonderer Beriicksichtigung der qualitativen mikromorphologischen Verh~iltnisse, Acta Pathol. Microbiol. Scand., 53 (1944) 1-276. [7] Hoffman, E.P., Brown, Jr., R.H. and Kunkel, L.M., Dystrophin: the protein product of the Duchenne muscular dystrophy locus, Cell, 51 (1987) 919-1128. [8] Huard, J., Cttt, P.-Y., Parent, A., Bouchard, J.-P. and Tremblay, J.P., Dystrophin-like immunoreactivity in monkey and human brain areas involved in learning and motor functions, Neurosci. Lett., 141 (1992) 181-186.
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