- Email: [email protected]
Motoneuron morphological alterations before and after the onset of the disease in the wobbler mouse
Brain Research 930 (2002) 53–57 www.elsevier.com / locate / bres
Research report
Motoneuron morphological alterations before and after the onset of the disease in the wobbler mouse b ¨ Brigitte Blondet a , *, Gilles Carpentier a , Ali Aıt-Ikhlef , Monique Murawsky b , b Franc¸ois Rieger a
´ ´ ´ , France De Gaulle, 94010 Creteil Laboratoire CRRET, Faculte´ des Sciences, Universite´ Paris XII, Avenue du General b ˆ . G. Pincus, 80 rue du General ´ ´ ˆ , France INSERM U488, Bat Leclerc, 94276 Le Kremlin Bicetre Accepted 10 December 2001
Abstract The wobbler mutant mouse displays a recessively inherited neurological disease with degeneration of motoneurons and is considered to be an animal model for human motoneuron diseases. Mutant mice can be clinically recognised at about 3–4 weeks of age but a polymorphic marker close to the wobbler gene offers the opportunity of a preclinical diagnosis. Using this polymorphic marker we performed morphometric (cell size) analysis of spinal cord motoneurons from 10 to 40 days post natal (PN). We observed at day 16 PN a transient appearance of swollen motoneurons, probably those that present vacuolar degeneration a little later and possibly die. One week later, from 21 days onwards, we found that the subpopulation of large motoneurons was depleted in the mutant mice. The absence of large motoneurons may have important physiological consequences and the loss or absence of differentiation of this particular subpopulation of motoneurons may be a key event in the course of the disease. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neuromuscular diseases Keywords: Animal model; Motoneuron degenerative disease; Preclinical diagnosis
1. Introduction The wobbler mutant mouse, homozygous for the gene wr, displays a recessively inherited neurological disease with degeneration of motoneurons, most prominent in the cervical spinal cord [7]. It has been proposed as an animal model for human neurodegenerative disorders, including amyotrophic lateral sclerosis and infantile spinal muscular atrophy, the second most common fatal autosomal disease of childhood [11]. The corresponding wr gene, located on mouse chromosome 11 [5] is as yet unknown. The most significant abnormality found in wobbler mice is early vacuolar neuronal degeneration followed by a deficit in the number of motoneurons, particularly in the cervical ventral horns of the spinal cord [7,1,15]. Neurodegeneration is also observed in thalamus, in deep cerebellar nuclei and in brain stem [16]. The wobbler mice display an astrogliosis *Corresponding author. Tel.: 133-1-4517-1454; fax: 133-1-45171816. E-mail address: [email protected] (B. Blondet).
in the spinal cord, with the presence of reactive astrocytes increasing in number as the disease progresses [9]. Mutant mice can be clinically recognized at about 3–4 weeks of age by their smaller size, tremor and atrophy of forelimb muscles. A polymorphic marker close to the wr gene offers the opportunity of a preclinical diagnosis [5,3]. Using this polymorphic marker and in order to determine the state of the motoneurons at both pre- and postclinical stages of the disease, we performed morphometric (cell size) analysis of spinal cord motoneurons from 10 to 40 days post natal (PN), and showed a loss or absence of differentiation of large motoneurons subpopulation.
2. Materials and methods
2.1. Experimental animals All experiments were carried out in accordance with French institutional animal care guidelines.
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )03405-9
54
B. Blondet et al. / Brain Research 930 (2002) 53 – 57
The original wobbler mouse mutation (wr) arose spontaneously as an autosomal recessive mutation in the congenic C57BL / 6J stock. Linkage analysis has mapped the wr gene very near the pseudogene glutamine synthetase (glns-ps1) on the proximal segment of mouse chromosome 11 [5] and recombination frequencies between glnsps1 and the wr gene were very low. Because the allelic size of the glns-ps1 microsatellite differs between the congenic C57BL / 6J wobbler strain and the New Zealand Black (NZB) strain, the glns-ps1 microsatellite can be used as a genetic marker close to the wobbler mutation in a new hybrid, the C57BL / 6J3NZB wr /wr mouse, providing a way to investigate the presymptomatic stages in the wobbler disease [3]. Briefly, female NZB mice (possessing the large glns-ps1 microsatellite, G) were crossed with the congenic C57BL / 6J-wr /1 heterozygotes (possessing the small glns-ps1 microsatellite, g). The resulting hybrid litters (F1) were backcrossed with the congenic C57BL / 6J-wr /1 heterozygotes to identify F1 heterozygotes (wr /1 ) that would possess a small and a large glns-ps1 microsatellite (g / G). The F1 heterozygotes were then intercrossed to produce F2 littermates that were either homozygous for the wobbler mutation (wr /wr, g /g), heterozygous (wr /1, g /G) or homozygous for the wild type (1 /1, G /G). Polymerase chain reaction amplification of the glns-ps1 microsatellite region was performed on genomic DNA extracted from tail samples of the F2 littermates as previously described [3] and permitted the molecular diagnosis of the wr /wr ( g /g) mutants before the appearance of clinical symptoms. The accuracy of this molecular diagnosis technique was tested in older phenotypically identified wr /wr ( g /g) mutants or 1 /wr (G /g) and 1 /1 (G /G) clinically normal animals.
2.2. Histology and morphometric analysis Mutant (wr /wr, g /g) and control (1 /1, G /G) animals were examined at different ages between 10 and 40 days PN. Because previous data on number of surviving motoneurons in wobbler mice suggest that motoneuron loss is notable from 21 days PN, we selected for our studies two preclinical stages, i.e. 10 and 16 days old, the stage of early motoneuron loss, i.e. between 21 and 24 days PN and a later stage, mice 40 days old, when clinical symptoms are obvious. Three to five mutant animals and three to five control animals were examined for each stage. Animals were killed by an overdose of ether, perfused with Bouin’s fluid and their spinal cords were dissected out, fixed in Bouin for several hours then processed for histology. Segments C5–T3, embedded in paraffin, were serially sectioned at 12 mm thickness. Motoneuron size determination, in the lateral motor column of spinal cords sections stained with cresyl violet, was performed blind as to the genetic status of the mice. Briefly, motoneurons were identified on the basis of their location in the latero-ventral regions of the spinal cord and
the criteria for selecting included cells with a large nucleus containing a clear nucleoplasm and one or two visible nucleoli, and a large, distinctly stained cytoplasm. To determine motoneuron size, several sections were randomly selected from the C5–T3 spinal cord segments of each animal. Between 30 and 80 motoneurons were measured in each animal. Images were taken with a CCD black and white video camera (Panasonic, WV-BL200) which was mounted on a light microscope (Olympus BH-2 with a MTV-3-C mount adaptor). Image digitizing was obtained with a Biocom 200 station (Biocom, les Ulis, France). Image analysis was performed on a Macintosh 7200 / 90 computer using the public domain NIH image program (developed at the US National Institute of Health and available on the Internet at http: / / rsb.info.nih.gov / nihimage / ). Motoneuron outlining and measure of areas were performed with an automatic thresholding macro procedure, based on Sobel edge detection.
2.3. Muscle choline acetyltransferase activity determination Muscles (triceps) of 10 to 25 days PN mutant and control mice were dissected and homogenised in a glass– glass conical homogenizer, in 10% w / v medium containing 1 M NaCl, 1 mM EGTA, 1% Triton and 0.01 M Tris–HCl, pH 7.2. Homogenates were centrifuged at 20 0003g for 15 min and the supernatants were taken for analysis. Choline acetyltransferase activity was determined by the method of Fonnum [8].
3. Results
3.1. First detectable motoneuronal dysfunction and histologic alterations As an index of the functional state of motoneurons, we measured choline acetyltransferase (ChAT) activity in different muscle extracts from mutant (wr /wr, g /g) and control (1 /1, G /G) mice between 10 and 25 days PN. No differences were observed at 10 days but we showed that ChAT activity was decreased as early as 16 days PN, in triceps mutant muscles (Fig. 1), i.e. several days before the first clinical symptoms. This decrease was more marked in mutant muscles at 25 days. We showed in previous work [2] that ChAT deficit is still present in adult mutant mice. Vacuolar degeneration (Fig. 2) was present at 21 days PN, just before clinical symptoms. It is observed in about 10% of motoneurons at this stage. Some interneurons also presented vacuolar degeneration.
3.2. Motoneuron size determination at different stages before the onset of the disease and in adult mutant mice We measured the motoneuron sizes in the lateral motor column of spinal cord sections stained with cresyl violet,
B. Blondet et al. / Brain Research 930 (2002) 53 – 57
55
Fig. 3. Example of motoneuron size determination at 10 days PN. Spinal cord sections stained with cresyl violet; (a) 10-day-old control mouse; (b) 10-day-old wobbler mouse. Scale bar: 12 mm. Inset: example of motoneurons outlining by computer.
Fig. 1. Choline acetyltransferase activity in triceps muscles from 10 to 25 PN day mutant and control animals. Ordinates: choline acetyltransferase activity per muscle; arbitrary units. Data are means6S.E.M. for three or four animals for each point.
from mutant and control animals between 10 and 40 days. An example of image analysis is given at 10 days PN (Fig. 3). Fig. 4 shows average motoneuron soma size at different ages. No differences between control and mutant motoneurons were observed at 10 days PN (Fig. 4). At 16 days PN, the motoneuron size of the mutants was significantly increased, compared to controls (P,0.01). Later, mutant motoneuron sizes decreased while control sizes
Fig. 2. Motoneuronal vacuolar degeneration in 21-day-old mutant mouse. Spinal cord section stained with cresyl violet. Motoneurons (arrows) in the anterior horn which exhibit vacuolar degeneration. Scale bar: 10 mm.
increased rapidly to reach the adult values (Fig. 4) (P, 0.05 control vs. mutant for 21–24 days and adult).
3.3. Depletion of a subpopulation of large motoneurons in the wobbler mouse In order to determine motoneuronal size changes be-
Fig. 4. Average motoneuron soma size in mutant and control mice at different ages. Values are expressed as means6S.E.M.; n, number of animals in each group; black columns, control; white columns, mutant; three to five mutants and three to five controls were examined for each stage. Between 30 and 80 motoneurons were measured in each animal. We calculated the mean of neuronal size in each animal and compared between controls and mutants. P,0.01 in a paired Student’s t-test mutant versus control for 16 days PN; P,0.05 in a paired Student’s t-test mutant versus control for 21–24 days and in adult.
56
B. Blondet et al. / Brain Research 930 (2002) 53 – 57
tween 10 and 40 days, we studied the percentages of motoneurons belonging to 30 area size classes, distributed between 20 and 600 mm 2 (Fig. 5). At 10 days PN, mutant and control distributions do not differ. At 16 days, a major peak in the distribution is noted around 100 mm 2 for mutant as for controls. However, at this time, large motoneurons (.300 mm 2 ) appear in the mutant animals. At 21–24 days, the same major peak around 100 mm 2 is observed for mutants but large neurons have disappeared. At the same time, the size distribution of neurons in the control animals presents a major peak around 160 mm 2 and a second one around 350 mm 2 , corresponding to the appearance of large motoneurons. In adult animals, mutant motoneuron size distribution stays with one major peak around 100 mm 2 while the control size distribution exhibits three size classes of motoneurons around 180, 350 and 470 mm 2 corresponding to the formation of a second class of large motoneurons.
4. Discussion The present study documents two abnormal aspects in wobbler motoneuron before the onset of the disease: • a transient increase in motoneuron soma size at 16 days PN in temporal correlation with the onset of an altered muscular innervation; • an absence of a specific subpopulation of large motoneurons from 21 days PN onwards.
Fig. 5. Relative frequency distribution histograms for motoneuron areas in control and mutant mice between 10 and 40 days PN; black columns, control; white columns, mutant.
As for other neurological diseases, the disease progression in wobbler mice may be unequal from one animal to another and from one mouse strain to another. Nevertheless, data on number of surviving motoneurons in wobbler mice [15,18] suggest that motoneuron loss, negligible before the first clinical symptoms appear, is notable around 21 days PN, reaches its maximum between 4 and 6 weeks and stops after 9 weeks. Here, we demonstrate the appearance of swollen motoneurons at 16 days, that is several days before the beginning of motoneuron loss. It is possible that these swollen motoneurons are those that present vacuolar degeneration in the days after and, finally, are lost. Perikaryal swelling is also detected in the cervical spinal cord of the wobbler mouse taken at early stage of the disease by Dockery et al. [6]. Rathke-Hartlieb et al. [16] observe groups of degenerating motoneurons in the ventral horn of the spinal cord between 16 and 18 days PN but show that the first signs of neurodegeneration in wobbler mice appear in the palliothalamus, in brain stem, in the cerebellum and in interneurons of the spinal cord by day 13 PN. The deficit in muscular ChAT activity that we observe from 16 days PN is in temporal correlation with the appearance of swollen motoneurons. At the same time, another sign of altered muscle innervation is observed by
B. Blondet et al. / Brain Research 930 (2002) 53 – 57
Sedehizade et al. [17] that shows an up-regulation of the mRNA level for acetylcholine receptor subunit in forelimb muscles of wobbler mice. The increase in mutant motoneuron soma size that we observed at 16 days PN is transient and, in contrast, from 21 days PN to adulthood, we showed an absence of large motoneuron classes in mutant mice. At the same stage (18–21 days PN), we showed in recent work [3] a massive although very transient DNA fragmentation in motoneurons and glial cells of wobbler mice spinal cords that could be related to the loss or altered differentiation of the subpopulation of large motoneurons. Murakami et al. [13] have shown that protein synthesis and RNA content are reduced in all motoneurons in wobbler mice, vacuolated or not, and suggested that the genetic defect is expressed in all or in the majority of surviving neurons in the anterior horn of the spinal cord. This statement fits the widespread defect of key synaptic components of the mutant neuromuscular junctions that we have previously described [11]. In other respects, Bose and Vacca-Galloway [4] have demonstrated the sprouting of neuronal processes in the spinal ventral horn of wobbler mouse which contain substance P, methionine and leucine enkephalins as well as thyrotropin-releasing hormone. This sprouting occurs before the appearance of symptoms, around 14 days PN. These authors suggested that these early sprouts are toxic to motoneurons and may be causal to their loss. It is possible that this early sprouting plays a role in the loss or altered differentiation of large motoneurons that we observed 7 days later. Alternative hypotheses include the possibility that sprouting as well as loss or altered differentiation of large motoneurons may result from a general systemic neurotrophin disturbance. Indeed, neurotrophic factors promote neuronal development and survival [14] and, among those, ciliary neurotrophic factor and brain-derived neurotrophic factor [12] as well as insulin-like growth factor I [10] have been shown to slow the progression of motoneuron disease when injected into symptomatic wobbler mice. Moreover, glycosaminoglycans boost IGF-I-promoted neuroprotection and block motoneuron death in the wobbler mouse [18]. With combined IGF I / glycosaminoglycans treatment, the total counts of C5 and C6 motoneurons larger than 20 mm in 9-week-old wobbler was almost identical to that determined at 3 weeks of age, the time when treatment was begun. Moreover, a more complex dendritic arborization of motoneurons is also observed in IGF-I / glycosaminoglycan-treated wobbler mice, suggesting a more pronounced differentiation [18]. In conclusion, the loss or absence of differentiation of the subpopulation of large motoneurons in wobbler mice may be a key event in the course of the disease and a target for new potential treatments.
57
References [1] M. Baulac, F. Rieger, V. Meininger, The loss of motoneurons corresponding to specific muscles in the wobbler mutant mouse, Neurosci. Lett. 37 (1983) 99–104. [2] B. Blondet, G. Barlovatz-Meimon, B.W. Festoff, C. Soria, J. Soria, ¨ Plasminogen activators in the neuromuscular F. Rieger, D. Hantaı, system of the wobbler mutant mouse, Brain Res. 580 (1992) 303–310. ¨ [3] B. Blondet, A. Aıt-Ikhlef, M. Murawsky, F. Rieger, Transient massive DNA fragmentation in nervous system during the early course of a murine neurodegenerative disease, Neurosci. Lett. 305 (2001) 202–206. [4] P. Bose, L.L. Vacca-Galloway, Increase in fiber density for immunoreactive serotonin, substance P enkephalin and thyrotropin-releasing hormone occurs during the early presymptomatic period of motoneuron disease in wobbler mouse spinal cord ventral horn, Neurosci. Lett. 260 (1999) 196–200. [5] V. Des Portes, M. Coulpier, J. Melki, P. Dreyfus, Early detection of mouse wobbler mutation: A model of pathological motoneuron death, Neuroreport 5 (1994) 1861–1864. [6] P. Dockery, Y. Tang, M. Morais, L.L. Vacca-Galloway, Neuron volume in the ventral horn in Wobbler mouse motoneuron disease: a light microscope stereological study, J. Anat. 191 (1997) 89–98. [7] L.W. Duchen, S.J. Strich, An hereditary motor neuron disease with progressive denervation of muscle in the mouse: the mutant wobbler, J. Neurol. Neurosurg. Psychiatry 31 (1968) 535–542. [8] F. Fonnum, A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem. 24 (1975) 407–409. [9] D. Hantaz-Ambroise, B. Blondet, M. Murawsky, F. Rieger, Abnormal astrocyte differentiation and defective cellular interactions in the wobbler mouse spinal cord, J. Neurocytol. 23 (1994) 179–192. ¨ M. Akaaboune, C. Lagord, M. Murawsky, L. Houenou, [10] D. Hantaı, B.W. Festoff, J.L. Vaught, F. Rieger, B. Blondet, Beneficial effects of IGF I on wobbler mouse motoneuron disease, J. Neurol. Sci. Suppl. 129 (1995) 122–126. [11] J. Melki, B. Blondet, M. Pinc¸on-Raymond, P. Dreyfus, F. Rieger, Generalized molecular defects of the neuromuscular junction in skeletal muscle of the wobbler mutant mouse, Neurochem. Int. 18 (1991) 425–433. [12] H. Mitsumoto, K. Ikeda, B. Klinkosz, Arrest of motor neuron disease in wobbler mice cotreated with CNTF and BDNF, Science 265 (1994) 1107–1110. [13] T. Murakami, F. Mastaglia, D. Mann, Abnormal RNA metabolism in spinal motor neurons in the wobbler mouse, Muscle Nerve 4 (1981) 407–412. [14] R.W. Oppenheim, The neurotrophic theory and naturally occurring motoneuron death, Trends Neurosci. 12 (1989) 252–255. [15] M.M. Pollin, S. McHanwell, C.R. Slater, Loss of motor neurons from the median nerve motor nucleus of the mutant mouse wobbler, J. Neurocytol. 19 (1990) 29–38. [16] S. Rathke-Hartlieb, V. Schmidt, H. Jockusch, T. Schmitt-John, J.W. Bartsch, Spatiotemporal progression of neurodegeneration and glia activation in the wobbler neuropathy of the mouse, Neuroreport 10 (1999) 3411–3416. [17] F. Sedehizade, R. Klocke, H. Jockusch, Expression of nerve regulated genes in muscles of mouse mutants affected by spinal muscular atrophies and muscular dystrophies, Muscle Nerve 20 (1997) 186–194. [18] L. Vergani, M. Losa, E. Lesma, A.M. Di Giulo, A. Torsello, E.E. ¨ Muller, A. Gorio, Glycosaminoglycans boost insulin-like growth factor-I-promoted neuroprotection: blockade of motoneuron death in the wobbler mouse, Neuroscience 93 (1999) 565–572.
Research report
Motoneuron morphological alterations before and after the onset of the disease in the wobbler mouse b ¨ Brigitte Blondet a , *, Gilles Carpentier a , Ali Aıt-Ikhlef , Monique Murawsky b , b Franc¸ois Rieger a
´ ´ ´ , France De Gaulle, 94010 Creteil Laboratoire CRRET, Faculte´ des Sciences, Universite´ Paris XII, Avenue du General b ˆ . G. Pincus, 80 rue du General ´ ´ ˆ , France INSERM U488, Bat Leclerc, 94276 Le Kremlin Bicetre Accepted 10 December 2001
Abstract The wobbler mutant mouse displays a recessively inherited neurological disease with degeneration of motoneurons and is considered to be an animal model for human motoneuron diseases. Mutant mice can be clinically recognised at about 3–4 weeks of age but a polymorphic marker close to the wobbler gene offers the opportunity of a preclinical diagnosis. Using this polymorphic marker we performed morphometric (cell size) analysis of spinal cord motoneurons from 10 to 40 days post natal (PN). We observed at day 16 PN a transient appearance of swollen motoneurons, probably those that present vacuolar degeneration a little later and possibly die. One week later, from 21 days onwards, we found that the subpopulation of large motoneurons was depleted in the mutant mice. The absence of large motoneurons may have important physiological consequences and the loss or absence of differentiation of this particular subpopulation of motoneurons may be a key event in the course of the disease. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neuromuscular diseases Keywords: Animal model; Motoneuron degenerative disease; Preclinical diagnosis
1. Introduction The wobbler mutant mouse, homozygous for the gene wr, displays a recessively inherited neurological disease with degeneration of motoneurons, most prominent in the cervical spinal cord [7]. It has been proposed as an animal model for human neurodegenerative disorders, including amyotrophic lateral sclerosis and infantile spinal muscular atrophy, the second most common fatal autosomal disease of childhood [11]. The corresponding wr gene, located on mouse chromosome 11 [5] is as yet unknown. The most significant abnormality found in wobbler mice is early vacuolar neuronal degeneration followed by a deficit in the number of motoneurons, particularly in the cervical ventral horns of the spinal cord [7,1,15]. Neurodegeneration is also observed in thalamus, in deep cerebellar nuclei and in brain stem [16]. The wobbler mice display an astrogliosis *Corresponding author. Tel.: 133-1-4517-1454; fax: 133-1-45171816. E-mail address: [email protected] (B. Blondet).
in the spinal cord, with the presence of reactive astrocytes increasing in number as the disease progresses [9]. Mutant mice can be clinically recognized at about 3–4 weeks of age by their smaller size, tremor and atrophy of forelimb muscles. A polymorphic marker close to the wr gene offers the opportunity of a preclinical diagnosis [5,3]. Using this polymorphic marker and in order to determine the state of the motoneurons at both pre- and postclinical stages of the disease, we performed morphometric (cell size) analysis of spinal cord motoneurons from 10 to 40 days post natal (PN), and showed a loss or absence of differentiation of large motoneurons subpopulation.
2. Materials and methods
2.1. Experimental animals All experiments were carried out in accordance with French institutional animal care guidelines.
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )03405-9
54
B. Blondet et al. / Brain Research 930 (2002) 53 – 57
The original wobbler mouse mutation (wr) arose spontaneously as an autosomal recessive mutation in the congenic C57BL / 6J stock. Linkage analysis has mapped the wr gene very near the pseudogene glutamine synthetase (glns-ps1) on the proximal segment of mouse chromosome 11 [5] and recombination frequencies between glnsps1 and the wr gene were very low. Because the allelic size of the glns-ps1 microsatellite differs between the congenic C57BL / 6J wobbler strain and the New Zealand Black (NZB) strain, the glns-ps1 microsatellite can be used as a genetic marker close to the wobbler mutation in a new hybrid, the C57BL / 6J3NZB wr /wr mouse, providing a way to investigate the presymptomatic stages in the wobbler disease [3]. Briefly, female NZB mice (possessing the large glns-ps1 microsatellite, G) were crossed with the congenic C57BL / 6J-wr /1 heterozygotes (possessing the small glns-ps1 microsatellite, g). The resulting hybrid litters (F1) were backcrossed with the congenic C57BL / 6J-wr /1 heterozygotes to identify F1 heterozygotes (wr /1 ) that would possess a small and a large glns-ps1 microsatellite (g / G). The F1 heterozygotes were then intercrossed to produce F2 littermates that were either homozygous for the wobbler mutation (wr /wr, g /g), heterozygous (wr /1, g /G) or homozygous for the wild type (1 /1, G /G). Polymerase chain reaction amplification of the glns-ps1 microsatellite region was performed on genomic DNA extracted from tail samples of the F2 littermates as previously described [3] and permitted the molecular diagnosis of the wr /wr ( g /g) mutants before the appearance of clinical symptoms. The accuracy of this molecular diagnosis technique was tested in older phenotypically identified wr /wr ( g /g) mutants or 1 /wr (G /g) and 1 /1 (G /G) clinically normal animals.
2.2. Histology and morphometric analysis Mutant (wr /wr, g /g) and control (1 /1, G /G) animals were examined at different ages between 10 and 40 days PN. Because previous data on number of surviving motoneurons in wobbler mice suggest that motoneuron loss is notable from 21 days PN, we selected for our studies two preclinical stages, i.e. 10 and 16 days old, the stage of early motoneuron loss, i.e. between 21 and 24 days PN and a later stage, mice 40 days old, when clinical symptoms are obvious. Three to five mutant animals and three to five control animals were examined for each stage. Animals were killed by an overdose of ether, perfused with Bouin’s fluid and their spinal cords were dissected out, fixed in Bouin for several hours then processed for histology. Segments C5–T3, embedded in paraffin, were serially sectioned at 12 mm thickness. Motoneuron size determination, in the lateral motor column of spinal cords sections stained with cresyl violet, was performed blind as to the genetic status of the mice. Briefly, motoneurons were identified on the basis of their location in the latero-ventral regions of the spinal cord and
the criteria for selecting included cells with a large nucleus containing a clear nucleoplasm and one or two visible nucleoli, and a large, distinctly stained cytoplasm. To determine motoneuron size, several sections were randomly selected from the C5–T3 spinal cord segments of each animal. Between 30 and 80 motoneurons were measured in each animal. Images were taken with a CCD black and white video camera (Panasonic, WV-BL200) which was mounted on a light microscope (Olympus BH-2 with a MTV-3-C mount adaptor). Image digitizing was obtained with a Biocom 200 station (Biocom, les Ulis, France). Image analysis was performed on a Macintosh 7200 / 90 computer using the public domain NIH image program (developed at the US National Institute of Health and available on the Internet at http: / / rsb.info.nih.gov / nihimage / ). Motoneuron outlining and measure of areas were performed with an automatic thresholding macro procedure, based on Sobel edge detection.
2.3. Muscle choline acetyltransferase activity determination Muscles (triceps) of 10 to 25 days PN mutant and control mice were dissected and homogenised in a glass– glass conical homogenizer, in 10% w / v medium containing 1 M NaCl, 1 mM EGTA, 1% Triton and 0.01 M Tris–HCl, pH 7.2. Homogenates were centrifuged at 20 0003g for 15 min and the supernatants were taken for analysis. Choline acetyltransferase activity was determined by the method of Fonnum [8].
3. Results
3.1. First detectable motoneuronal dysfunction and histologic alterations As an index of the functional state of motoneurons, we measured choline acetyltransferase (ChAT) activity in different muscle extracts from mutant (wr /wr, g /g) and control (1 /1, G /G) mice between 10 and 25 days PN. No differences were observed at 10 days but we showed that ChAT activity was decreased as early as 16 days PN, in triceps mutant muscles (Fig. 1), i.e. several days before the first clinical symptoms. This decrease was more marked in mutant muscles at 25 days. We showed in previous work [2] that ChAT deficit is still present in adult mutant mice. Vacuolar degeneration (Fig. 2) was present at 21 days PN, just before clinical symptoms. It is observed in about 10% of motoneurons at this stage. Some interneurons also presented vacuolar degeneration.
3.2. Motoneuron size determination at different stages before the onset of the disease and in adult mutant mice We measured the motoneuron sizes in the lateral motor column of spinal cord sections stained with cresyl violet,
B. Blondet et al. / Brain Research 930 (2002) 53 – 57
55
Fig. 3. Example of motoneuron size determination at 10 days PN. Spinal cord sections stained with cresyl violet; (a) 10-day-old control mouse; (b) 10-day-old wobbler mouse. Scale bar: 12 mm. Inset: example of motoneurons outlining by computer.
Fig. 1. Choline acetyltransferase activity in triceps muscles from 10 to 25 PN day mutant and control animals. Ordinates: choline acetyltransferase activity per muscle; arbitrary units. Data are means6S.E.M. for three or four animals for each point.
from mutant and control animals between 10 and 40 days. An example of image analysis is given at 10 days PN (Fig. 3). Fig. 4 shows average motoneuron soma size at different ages. No differences between control and mutant motoneurons were observed at 10 days PN (Fig. 4). At 16 days PN, the motoneuron size of the mutants was significantly increased, compared to controls (P,0.01). Later, mutant motoneuron sizes decreased while control sizes
Fig. 2. Motoneuronal vacuolar degeneration in 21-day-old mutant mouse. Spinal cord section stained with cresyl violet. Motoneurons (arrows) in the anterior horn which exhibit vacuolar degeneration. Scale bar: 10 mm.
increased rapidly to reach the adult values (Fig. 4) (P, 0.05 control vs. mutant for 21–24 days and adult).
3.3. Depletion of a subpopulation of large motoneurons in the wobbler mouse In order to determine motoneuronal size changes be-
Fig. 4. Average motoneuron soma size in mutant and control mice at different ages. Values are expressed as means6S.E.M.; n, number of animals in each group; black columns, control; white columns, mutant; three to five mutants and three to five controls were examined for each stage. Between 30 and 80 motoneurons were measured in each animal. We calculated the mean of neuronal size in each animal and compared between controls and mutants. P,0.01 in a paired Student’s t-test mutant versus control for 16 days PN; P,0.05 in a paired Student’s t-test mutant versus control for 21–24 days and in adult.
56
B. Blondet et al. / Brain Research 930 (2002) 53 – 57
tween 10 and 40 days, we studied the percentages of motoneurons belonging to 30 area size classes, distributed between 20 and 600 mm 2 (Fig. 5). At 10 days PN, mutant and control distributions do not differ. At 16 days, a major peak in the distribution is noted around 100 mm 2 for mutant as for controls. However, at this time, large motoneurons (.300 mm 2 ) appear in the mutant animals. At 21–24 days, the same major peak around 100 mm 2 is observed for mutants but large neurons have disappeared. At the same time, the size distribution of neurons in the control animals presents a major peak around 160 mm 2 and a second one around 350 mm 2 , corresponding to the appearance of large motoneurons. In adult animals, mutant motoneuron size distribution stays with one major peak around 100 mm 2 while the control size distribution exhibits three size classes of motoneurons around 180, 350 and 470 mm 2 corresponding to the formation of a second class of large motoneurons.
4. Discussion The present study documents two abnormal aspects in wobbler motoneuron before the onset of the disease: • a transient increase in motoneuron soma size at 16 days PN in temporal correlation with the onset of an altered muscular innervation; • an absence of a specific subpopulation of large motoneurons from 21 days PN onwards.
Fig. 5. Relative frequency distribution histograms for motoneuron areas in control and mutant mice between 10 and 40 days PN; black columns, control; white columns, mutant.
As for other neurological diseases, the disease progression in wobbler mice may be unequal from one animal to another and from one mouse strain to another. Nevertheless, data on number of surviving motoneurons in wobbler mice [15,18] suggest that motoneuron loss, negligible before the first clinical symptoms appear, is notable around 21 days PN, reaches its maximum between 4 and 6 weeks and stops after 9 weeks. Here, we demonstrate the appearance of swollen motoneurons at 16 days, that is several days before the beginning of motoneuron loss. It is possible that these swollen motoneurons are those that present vacuolar degeneration in the days after and, finally, are lost. Perikaryal swelling is also detected in the cervical spinal cord of the wobbler mouse taken at early stage of the disease by Dockery et al. [6]. Rathke-Hartlieb et al. [16] observe groups of degenerating motoneurons in the ventral horn of the spinal cord between 16 and 18 days PN but show that the first signs of neurodegeneration in wobbler mice appear in the palliothalamus, in brain stem, in the cerebellum and in interneurons of the spinal cord by day 13 PN. The deficit in muscular ChAT activity that we observe from 16 days PN is in temporal correlation with the appearance of swollen motoneurons. At the same time, another sign of altered muscle innervation is observed by
B. Blondet et al. / Brain Research 930 (2002) 53 – 57
Sedehizade et al. [17] that shows an up-regulation of the mRNA level for acetylcholine receptor subunit in forelimb muscles of wobbler mice. The increase in mutant motoneuron soma size that we observed at 16 days PN is transient and, in contrast, from 21 days PN to adulthood, we showed an absence of large motoneuron classes in mutant mice. At the same stage (18–21 days PN), we showed in recent work [3] a massive although very transient DNA fragmentation in motoneurons and glial cells of wobbler mice spinal cords that could be related to the loss or altered differentiation of the subpopulation of large motoneurons. Murakami et al. [13] have shown that protein synthesis and RNA content are reduced in all motoneurons in wobbler mice, vacuolated or not, and suggested that the genetic defect is expressed in all or in the majority of surviving neurons in the anterior horn of the spinal cord. This statement fits the widespread defect of key synaptic components of the mutant neuromuscular junctions that we have previously described [11]. In other respects, Bose and Vacca-Galloway [4] have demonstrated the sprouting of neuronal processes in the spinal ventral horn of wobbler mouse which contain substance P, methionine and leucine enkephalins as well as thyrotropin-releasing hormone. This sprouting occurs before the appearance of symptoms, around 14 days PN. These authors suggested that these early sprouts are toxic to motoneurons and may be causal to their loss. It is possible that this early sprouting plays a role in the loss or altered differentiation of large motoneurons that we observed 7 days later. Alternative hypotheses include the possibility that sprouting as well as loss or altered differentiation of large motoneurons may result from a general systemic neurotrophin disturbance. Indeed, neurotrophic factors promote neuronal development and survival [14] and, among those, ciliary neurotrophic factor and brain-derived neurotrophic factor [12] as well as insulin-like growth factor I [10] have been shown to slow the progression of motoneuron disease when injected into symptomatic wobbler mice. Moreover, glycosaminoglycans boost IGF-I-promoted neuroprotection and block motoneuron death in the wobbler mouse [18]. With combined IGF I / glycosaminoglycans treatment, the total counts of C5 and C6 motoneurons larger than 20 mm in 9-week-old wobbler was almost identical to that determined at 3 weeks of age, the time when treatment was begun. Moreover, a more complex dendritic arborization of motoneurons is also observed in IGF-I / glycosaminoglycan-treated wobbler mice, suggesting a more pronounced differentiation [18]. In conclusion, the loss or absence of differentiation of the subpopulation of large motoneurons in wobbler mice may be a key event in the course of the disease and a target for new potential treatments.
57
References [1] M. Baulac, F. Rieger, V. Meininger, The loss of motoneurons corresponding to specific muscles in the wobbler mutant mouse, Neurosci. Lett. 37 (1983) 99–104. [2] B. Blondet, G. Barlovatz-Meimon, B.W. Festoff, C. Soria, J. Soria, ¨ Plasminogen activators in the neuromuscular F. Rieger, D. Hantaı, system of the wobbler mutant mouse, Brain Res. 580 (1992) 303–310. ¨ [3] B. Blondet, A. Aıt-Ikhlef, M. Murawsky, F. Rieger, Transient massive DNA fragmentation in nervous system during the early course of a murine neurodegenerative disease, Neurosci. Lett. 305 (2001) 202–206. [4] P. Bose, L.L. Vacca-Galloway, Increase in fiber density for immunoreactive serotonin, substance P enkephalin and thyrotropin-releasing hormone occurs during the early presymptomatic period of motoneuron disease in wobbler mouse spinal cord ventral horn, Neurosci. Lett. 260 (1999) 196–200. [5] V. Des Portes, M. Coulpier, J. Melki, P. Dreyfus, Early detection of mouse wobbler mutation: A model of pathological motoneuron death, Neuroreport 5 (1994) 1861–1864. [6] P. Dockery, Y. Tang, M. Morais, L.L. Vacca-Galloway, Neuron volume in the ventral horn in Wobbler mouse motoneuron disease: a light microscope stereological study, J. Anat. 191 (1997) 89–98. [7] L.W. Duchen, S.J. Strich, An hereditary motor neuron disease with progressive denervation of muscle in the mouse: the mutant wobbler, J. Neurol. Neurosurg. Psychiatry 31 (1968) 535–542. [8] F. Fonnum, A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem. 24 (1975) 407–409. [9] D. Hantaz-Ambroise, B. Blondet, M. Murawsky, F. Rieger, Abnormal astrocyte differentiation and defective cellular interactions in the wobbler mouse spinal cord, J. Neurocytol. 23 (1994) 179–192. ¨ M. Akaaboune, C. Lagord, M. Murawsky, L. Houenou, [10] D. Hantaı, B.W. Festoff, J.L. Vaught, F. Rieger, B. Blondet, Beneficial effects of IGF I on wobbler mouse motoneuron disease, J. Neurol. Sci. Suppl. 129 (1995) 122–126. [11] J. Melki, B. Blondet, M. Pinc¸on-Raymond, P. Dreyfus, F. Rieger, Generalized molecular defects of the neuromuscular junction in skeletal muscle of the wobbler mutant mouse, Neurochem. Int. 18 (1991) 425–433. [12] H. Mitsumoto, K. Ikeda, B. Klinkosz, Arrest of motor neuron disease in wobbler mice cotreated with CNTF and BDNF, Science 265 (1994) 1107–1110. [13] T. Murakami, F. Mastaglia, D. Mann, Abnormal RNA metabolism in spinal motor neurons in the wobbler mouse, Muscle Nerve 4 (1981) 407–412. [14] R.W. Oppenheim, The neurotrophic theory and naturally occurring motoneuron death, Trends Neurosci. 12 (1989) 252–255. [15] M.M. Pollin, S. McHanwell, C.R. Slater, Loss of motor neurons from the median nerve motor nucleus of the mutant mouse wobbler, J. Neurocytol. 19 (1990) 29–38. [16] S. Rathke-Hartlieb, V. Schmidt, H. Jockusch, T. Schmitt-John, J.W. Bartsch, Spatiotemporal progression of neurodegeneration and glia activation in the wobbler neuropathy of the mouse, Neuroreport 10 (1999) 3411–3416. [17] F. Sedehizade, R. Klocke, H. Jockusch, Expression of nerve regulated genes in muscles of mouse mutants affected by spinal muscular atrophies and muscular dystrophies, Muscle Nerve 20 (1997) 186–194. [18] L. Vergani, M. Losa, E. Lesma, A.M. Di Giulo, A. Torsello, E.E. ¨ Muller, A. Gorio, Glycosaminoglycans boost insulin-like growth factor-I-promoted neuroprotection: blockade of motoneuron death in the wobbler mouse, Neuroscience 93 (1999) 565–572.