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Paranodal dysmyelination and increase in tetrodotoxin binding sites in the sciatic nerve of the motor end-plate disease (medmed ) mouse during postnatal development
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Paranodal Dysmyelination and Increase in Tetrodotoxin Binding Sites in the Sciatic Nerve of the Motor End-Plate Disease (mecI/med) Mouse during Postnatal Development FRANF)OIS RIEGER, *J MARTINE PINCON-RAYMOND,* MICHEL
LAZDUNSKI,t
ALAIN LOMBET,~ GILLES PONZIO,~
AND RICHARD
L. SIDMAN*
*Groupe de Biobgie et Path&&e Neuromuaculaires, Physiopat~ des Mywhies, INSERM U 155-17, rug du FeGMoulin, 75005 Paris, France, TInatitut de Biochimie du CNRS, Univemiti de Nice, Pare Valrose, 06000 Nice, France, and Qlepartment of Neuroaci~, Children’s Hospital Medical Center, $00 Langwood Avenue, Boston, Massachusetts 0.2115 Received December 80, 1982; accepted in revised form September 6, 1983 Motor end-plate disease (mod), in the mouse, is a hereditary neuromuscular defect, caused by a single gene mutation and characterized by a progressive muscle weakness. +Med/+med mice die 21-23 days after birth and the neurobiologlcal abnormalities already reported are nerve terminal sprouting and swelling and neurotransmission failures. We studied +meoY+med mice at preclinical (9-11 days after birth) as well as at clinically recognized stages of the disease. The nonmyelinated gaps of the nodes of Ranvier in the +med/+m.ed sciatic nerve are found to be significantly widened in +med/+med animals compared to control littermates, even in the preclinical stage, although the nodes of Ranvier are not yet ultrastructurally mature. The maximal binding capacity for PHIethylene-diamine tetrodotoxin, expressed in femtomoles per milligram of protein, is significantly increased in +m.ed/+med sciatic nerves. Thus, Na+ channels, which are known to be located mainly at the nodes of Ranvier in normal myelinated axons, are increased in number in +med/+mod mice even before the disease becomes clinically established. Both the ultrastructural and biochemical developmental abnormalities of the node of Ranvier rapidly approach their maximal expression as the behavioral signs develop. Such newe abnormalities may be closely related to the physiological impairment of newe impulse conduction which leads to the pathophysiological expression of motor end-plate disease. INTRODUCTION
ical or biochemical alterations (Rieger and PinCon-Raymond, 1980). In a recent electrophysiological and ultrastructural study of triangularis sterni motor axons, we observed altered nerve conduction and widening of the nodes of Ranvier (Angaut-Petit et al, 1982). The present paper reports the ultrastructural and biochemical study of the sciatic nerves of mice homozygous for the med allele before and after the disease becomes clinically expressed; the biochemical analysis concerns the fast Na+ channel, and the Na+, K+-ATPase, which is an essential enzyme for the regulation of the resting potential. Both Na+ transport systems are essential elements of nerve conduction and localized in the nodal regions of the myelinated motor axons. We find that the nodes of Ranvier of the sciatic nerves of +med/+med mice are significantly widened at an early postnatal stage of the disease, and that these abnormalities are accompanied by a marked increase in Na+ channel number.
Motor end-plate disease (med)2 is a severe hereditary neuromuscular disorder of the mouse (Searle, 1962; Duchen et a& 1967; Duchen 1970). Three allelic forms of the mutation are known: med, medJ, and medj”, in decreasing order of severity (Sidman et aL, 1979). Mice with med/m.ed or medJ/medJ genotypes die during the fourth week after birth, while medj”/medj” mice survive for several months. Nerve terminal swelling and sprouting have been observed in both med/m.ed and medJ/ medJ mice (Duchen, 1970; Pincon-Raymond and Rieger, 1981), in all muscles studied. The nerve to the soleus muscle is characterized by a much more extensive swelling and sprouting (Pingon-Raymond et al, 1983). These pathological changes of the nerves are thought to occur as a consequence of a state of partial or total functional denervation (Duchen and Stefani, 1971; Duchen, 1979). The nerve abnormalities in motor end-plate disease seem to occur at an early period of the disease (Rieger and PinCon-Raymond, 1980; Bournaud et a& 1980) when the muscle fiber does not yet show any marked morpholog-
MATERIAL
AND METHODS
Mice. Cad is the gene symbol for directional caracul, which originally arose at the Oak Ridge National Laboratory, and med is motor end-plate disease: these genes are closely linked on chromosome 15 (Duchen et al, 1967).
1To whom reprint requests and correspondence should be addressed. * Abbreviations: en-TTX, ethylenediaminetetrodotoxin; med, motor end-plate disease. 401
0012-1606/34 $3.00 Copyright All rights
0 1984 by Academic Press. Inc. of reproduction in any form reserved.
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Breeding pairs of heterozygous Cad+/+med mice were kindly provided by Dr. A. G. Searle (Medical Research Council, Radiobiology Unit, Harwell, U. K.) through the courtesy of Dr. L. W. Duchen (The National Hospital, London, U. K.) in 1979. The med strain is kept as a closed stock, which has strains C3H and 101 in its ancestry. It is not congenic with C3H. A colony is now maintained in our laboratory by brother-sister matings and progeny testing. Mutant +med/+med mice are characterized by their smooth hair and whiskers. Doubly heterozygous Cad+/+med and normal Cad+/Cad+ (homozygous wildtype at the me& locus) have curly hair and whiskers. Cad+/Cad+ mice show a slight epilation, although only progeny testing can unambiguously differentiate them from the heterozygotes. The disease is first noticed in lo- to 12-day-old +med/+med mice and is clinically characterized by a rapid, progressive weakness. We studied mice from before the onset of behavioral signs (9-10 days after birth) until death (21-23 days after birth), discarding from our study any animal too weak to be able to feed itself. The mice were sacrificed at different ages by cervical dislocation. Light and electron microscopll. The sciatic nerves were immediately exposed after sacrifice and fixed in situ for 15 min, then dissected out for an additional fixation of 90 min and further processed (1) for nerve fiber teasing and light microscopy, by immersion into a 2% osmium tetroxide solution in a 0.14 1Mveronal acetate buffer (pH 7.4) for 24 hr, followed by 66% glycerol for 48 hr and then pure glycerol; nerve fiber teasing was performed under binocular observation with sharp insect pins and internodes measured with a micrometer (Olympus microscope), or (2) for electron microscopy, by conventional techniques (Fardeau et aL, 1978), on longitudinal ultrathin sections. Direct perfusion of fixative in anaesthetized animals gave very similar results. Binding studies. The sciatic nerves were dissected out immediately after sacrifice, and rinsed in ice-cold 20 m&f Tris-HCl (pH 7.4) containing 0.25 M sucrose and 1 mM EDTA. The two sciatic nerves from each animal were homogenized in 0.7 ml of the same medium, using a Polytron PT 10s apparatus (Brinkman Inst.-Setting 5-10). The crude homogenate obtained was directly used for binding assays, carried out in parallel for rH]enTTX and for rH]ouabain. Protein content was determined using the method of Hartree (1972) with bovine serum albumin as a standard. The amount of Na+ channels present in the tissues was determined by binding assays using the derivative rH]en-TTX, carried out in a choline chloride medium as previously described (Lombet et al, 1981). The amount of Na+, K+-ATPase was determined using C3H]ouabain as described by Desnuelle et al. (1982). In both cases the specific binding was calculated from the difference between the bound
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radioactivity measured after rapid filtration through a GF/B glass fiber filter in the absence and the presence of a large excess of unlabeled ligand (5 PM TTX or 1 mM ouabain, respectively). Chemical and drugs. rH]en-TTX was synthesized according to Chicheportiche et al. (1980). The preparation reached a specific radioactivity of 25 CVmmole and a radiochemical purity of 90%. pH]Ouabain was obtained from New England Nuclear with a specific radioactivity of 11.6 CVmmole. Citrate-free tetrodotoxin was purchased from Sankyo Chemical Co. (Tokyo, Japan) and ouabain and ATP were from Sigma Chemical Co. RESULTS
Enlargement of the Nonmyelinated Gap at the Node of Ranvier in +med/+med Sciatic Nerve The most marked and frequent abnormality observed in longitudinal ultrathin sections of +med/+m-ed sciatic nerves is a widening (elongation) of the nodes of Ranvier. A typical example of such widening is shown in Fig. 1, for an l&day-old mouse myelinated axon, in the midthigh portion of the sciatic nerve. At this stage of the disease, this enlargement is found in the majority of the nodes of Ranvier (see below), in proximal, mid, or distal portions of the nerve: only occasional nodes are of normal size and morphology, compared to nodes of Ranvier of their control, identified, Cad+/Cud+ littermates. Control l&day-old Cad-f-/Cad+ animals presented no node of Ranvier with gap widths greater than 2-2.5 pm, whereas, in +med/+med animals, about 20% of the nodes exceeded 4 pm in width. We performed a morphometric quantitative study of the size of the nodes of Ranvier at different developmental stages in +med/ +med and Cad+/Cad+ mice. All measurements were made between the extreme paranodal myelin loops of nodes of Ranvier, sectioned with an optimal longitudinal incidence. The histograms-giving the frequency distribution of axons for different classes of nodal gap widths (Fig. 2)-show that a larger size of the nonmyelinated gap is already observed at a preclinical stage in mutant relative to control mice (9 days after birth), and more clearly at the onset of the behavioral signs of the disease (12 days after birth), in an appreciable proportion of the nodes of Ranvier analyzed. The controls show a progressive shortening in nodal size between 9 and 18 days after birth corresponding to normal maturation; the mutants show a lesser trend in the same direction. The internodal distances were measured, after nerve fiber teasing, on isolated nerve fibers. The nerve fibers show a bimodal distribution in diameter (Fig. 3). We considered two groups of nerve fibers: large diameter (>4 pm) and small diameter (G3.9 pm). The frequency distributions of either large or small diameter fibers in
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normalities, which progressively became more severe during the course of the disease. At late stages, the myelin thickness was frequently asymmetric on the two sides of the widened nodes. Sometimes, when the myelin sheath on one side was very thin, the other side might be devoid of myelin for long distances (8% of the abnormal nodes). Moreover, the myelin sheath, in the par-
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FIG. 1. Widened nonmyelinated gap at the node of Ranvler of a myelinated nerve fiber in the sciatic nerve of an l&day-old +med/ +med
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function of the internodal lengths observed (Fig. 4) are not significantly different in Cad+/Cad+ or in +med/ +med nerves (nonparametric statistical test). We observed that both myelin and axoplasm in the paranodal and nodal regions presented structural ab-
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FIG. 2. Size distribution of the nonmyelinated gaps of the nodes of Ranvier of myelinated fibers during development in normal (Cad+/ Co’+) and mutant (+med/+med) mouse sciatic nerve. PN, postnatal stage (Days 9, 12, 18 after birth); abscissae, size classes of nodal gap widths, ordinates, percentage of nodes of Ranvier. Total numbers of nodes of Ranvier which were measured: PN 9: Cod+/Cod+; n = 26; +med/+med; n = 26; PN 12: Cad+/Cad+; n = 55, +med/+mad; n = 55, PN 18: Cad+/Cad+; n = 29, +rned/+me& n = 37. Cad+/Cad+; hatched columns; +med/+medq black columne.
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[3H]ouabain to their respective receptor sites are shown in Fig. 6. Sixteen nerves from 1’7-day-old Cad+/Cad+ mice and eight nerves of the same stage +med/+med mice were pooled and homogenized. The titration of PHIen-TTX binding sites was demonstrated by Scatchard plots of the data obtained with increasing cona
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FIG. 3. Similar bimodal frequency distributions of Cad+/Cud and +m.ed/+m.ed nerve fiber population in function of fiber diameter. Eighteen-day-old mice, sciatic nerve. Open columns, Cad+/Cad+; hatched columns, +med/+med Class size, 1 pm.
anodal region, often did not totally enwrap the axon, thereby producing a dyssymmetry with reference to the long axis of the axon. Other myelin abnormalities frequently encountered in the paranodal region included increased interlamellar myelin splits, aberrant myelinated bodies, or long Schwann cell processes, without lamellae, extending along the bare axolemmal regions of the very widened +med/+med nodes (Fig. 5a). However, neither generalized depletion of myelin nor significant variations in myelination in internodal regions were observed in transverse sections of the nerves of mutant compared to wild-type mice. We did not observe any discontinuity in the basal lamina of the Schwann cells surrounding myelinated fibers in the +meuY+med mutants. The axoplasm sometimes showed short protrusions or excrescences in some enlarged nodes (Fig. 5b).
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Develqnnenti Changes of the Ionic Na+ Channel and Na+,K+-ATPase in Normal Cad+/Cad+ and +med/+med Sciatic Nerves 60
The abnormality of the nodes of Ranvier of the sciatic nerves of +m.ed/+nwd mice was further investigated biochemically via binding studies. Two Na+ transport systems present in nodes of Ranvier, i.e., the ionic Na+ channel and the Na+,K+-ATPase, were quantitatively studied in sciatic nerves of mutant (+med/+med) mice and a comparison was made with normal (Cad+/Cad+) mice. The binding properties of PHIen-TTX and
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FIG. 4. Frequency distributions of small or large Cad+/Cad+ and +med/+med nerve fibers in function of internodal distances. (a) Large fibers (~4 gm diameter). The distribution appears unimodal. Mutant fibers seem to have slightly longer internodal lengths although statistical analysis (nonparametric) does not indicate a significant difference. (b) Small fibers (~4 gm diameter). The distribution is bimodal. No significant difference between control and mutant fibers. Open columns, Cad+/Cad+; hatched columns, +med/+med
FIG. 5. Myelin abnormalities in +med/+m.ed sciatic nerve. (a) Aberrant myelin in the vicinity of a widened node of Ranvier in the sciatic nerve of an 18 day-old +med/+med mouse. (b) Protrusion of axoplasm at a node of Ranvier in an B-day-old +m.ed/+med mouse. 405
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centrations of the tritiated ligand (Fig. 6A). The plots gave single straight lines indicating in each case a single population of high affinity tetrodotoxin binding sites. The equilibrium dissociation constants of the rH]enTTX-receptor complex (Kn) were 0.96 -t 0.15 and 0.80 + 0.10 nM for normal and mutant sciatic nerves, respectively. The corresponding maximal binding capacities (B,,,) were 265 and 535 fmol/mg of protein. A similar representation is given in Fig. 6B for [3H]ouabain binding. The Kn values for rH]ouabain binding were 8 + 2 and 11 +- 2 nM and B,,, values were 680 and 800 fmol/ mg of protein for the normal and mutant sciatic nerves, respectively. The binding of rH]en-TTX to normal nerve homogenates was competitively inhibited by increasing amounts of unlabeled TTX or saxitoxin with a J&,6 of 2.0 and 2.5 nM, respectively (data not shown). The maximal binding capacities for rH]en-TTX and for [3H]ouabain were obtained for different samples (38) of normal and mutant sciatic nerves at various stages of development; these data are reported in Fig. ‘7. In Fig. 7A, comparison of the number of Na+ channels in normal and mutant sciatic nerves shows that +med/ +med sciatic nerves have a twofold higher level of Na+ channels at each stage of development, from 2 to 3 days before the disease is clinically expressed, until death of the mutant mice at 22-23 days. A plateau level was observed at approximately 3 weeks which corresponds to the maturation level of the Na+ channels. The comparative evolution of rH]ouabain-binding sites is shown in Fig. 7B. At all stages of development only a slight increase in the amount of Na+,K+-ATPase was observed in the +med/+med compared to the Cad+/Cad+ sciatic nerves. At 17 days, both normal and mutant brains (without cerebellum) have the same levels of Na+ channels and Na+,K+)-ATPase: 1200 f 230 fmole of tetrodotoxin binding sites per mg of protein in the +med/+med brain against 1360 f 120 fmole per mg of protein in the control, and 15.8 + 2.5 pmol of ouabain binding sites per mg of protein in the +med/+med brain against 16.8 -t 3.0 pmol per mg of protein in the control.
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FIG. 6. Specific binding of [8Hlen-‘ITX and [sH]ouabain. Homogenates (see Materials and Methods) of sciatic nerves of normal (Cu’+/Cad+) and mutant (+med/+mcd) mice. (A) Scatchard plots of @Ien-‘ITX specific binding (A) 1%day-old Cadt/Codt mice; 16 sciatic nerves were pooled. (A) l’l-day-old td/+d mice; 8 sciatic nerves. Inset: total (0) and nonspecific (X) binding as a function of increasing concentrations of [8HJen-‘M’X. (B) Scatchard plots of [‘H]ouabain binding. Same homogenates as for the ‘I’TX experiments on A. Same symbols. Inset: total (0) and nonspecific (X) binding as a function of increasing concentration of [8H]ouabain. Determinations were performed in duplicate, by filtrating a 0.4-m] aliquot of a l-ml incubation mixture, containing 0.1-1.2 mg of protein per ml of homogenate, on GF/B filters. Nonspecific binding was measured in the presence of a large excess of unlabeled ligand (5 fl TTX or 1 PM ouabain, respectively). Specific binding presented in the Scatchard plots is the difference between total and nonspecific bindings.
DISCUSSION
Inherited motor end-plate disease in the mouse is characterized not only by abnormal modifications of the terminal motor innervation, e.g., ultraterminal sprouting (Duchen, 1970; Rieger and Pincon-Raymond, 1980; Pincon-Raymond and Rieger, 1981), and nerve terminal swelling, but also by prominent morphological abnormalities of the nodes of Ranvier. In a recent electrophysiological and morphological study of a thoracic motor nerve (Bournaud et aL, 1980; Rieger and PinconRaymond, 1980; Angaut-Petit et al, 1982), we observed
that nerve conduction alterations, namely slow nerve conduction and increased refractory period, were accompanied by ultrastructural abnormalities of the nodes of Ranvier, which are significantly widened when the disease is well established. Our present results extend these observations to a more distal nerve and bring evidence (1) for a general defect of paranodal myelination in +med/+med nerves, (2) for its occurrence at early postnatal ages, even before the onset of the behavioral signs of the disease, and (3) for a parallel in-
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DAY’S FIG. 7. Na+ channels and Na+,K+-ATPase during a period of postnatal development in normal (Cod+/Cod+) and mutant (+med/+med) mouse sciatic nerves. (A) Development of [‘HI en-TTX binding sites number in Cad+/Cad+ (A) and +medJ+med (A) sciatic nerves between Day 9 and Day 22 after birth, corresponding to a preclinical stage and death, respectively, of affected mice. (B) Development of [‘HI ouabain binding sites number in Cad+/Cad+ (A) and +mad/+med (A) sciatic nerves. For each developmental stage chosen, the determinations were performed on three to eight pairs of sciatic nerves, independently homogenized and assayed. The bars represent the standard deviation of the mean.
crease in number of Na+ channels (TTX-binding sites). We found that the nonmyelinated gap of the nodes of Ranvier in myelinated fibers was significantly widened in +med/+med sciatic nerves in regions proximal or in more distal regions to its spinal cord roots, and in small as well as larger myelinated axons. Apart from the paranodal regions, myelin does not seem to be affected: transverse sections rarely show any marked alterations, as already observed in thoracic nerves (Angaut-Petit et al, 1982). Motor end-plate disease is thus characterized by a marked and frequent paranodal dysmyelination. Our data do not discriminate whether the med mutation causes a true demyelinating process or a delayed or
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arrested development, expressed most clearly at the paranodal specializations of the myelin sheath. In any case, these paranodal abnormalities are probably related to the electrophysiological modifications previously described in a thoracic nerve (Angaut-Petit et d, 1982): the slow nerve conduction and increased refractory period of the nerve impulses. Similar abnormalities of nerve conduction have also been observed in the sciatic nerve trunk and its branch to the soleus muscle (D. Augaut-Petit; J. J. McArdle, A. Mallart, R. Bournaud, M. Pincon-Raymond, and F. Rieger, unpublished results). It seems that paranodal dysmyelination in +mecl/+med nerves cannot alone account for the important decrease in nerve conduction: it has been recently reported by Rasminsky et al. (1978) that computer simulations of conduction in internodal regions of demyelinated nerves decreased by only 18% when quadrupling nodal widths, a widening rarely observed in +med/+m.ed nerve fibers. The nerve conduction abnormalities probably explain the blocks of neurotransmission and/or failures of nerve action potentials to invade the nerve terminals, reported by other investigators (Duchen and Stefani, 1971; Harris and Ward, 1974; Weinstein, 1980). Ultrastructural alterations of the nodes of Ranvier appear early in the disease process, even before the behavioral signs are noticed. This observation strongly supports the view that the structural abnormalities are closely linked to the nerve conduction abnormalities in this neuromuscular pathological process. However, we do not know whether myelin or axon, if either, is primarily responsible for the structural and functional defects. In other nerve disorders which appear to affect the myelin sheath, usually more extensively than in motor end-plate disease, the axon has been reported to be abnormal as well. This is found, for example, in experimental diphtheric neuropathy (Waxman and Quick, 1977; Quick and Waxman, 1978), and in the dominantly inherited human hypertrophic neuropathy (CharcotMarie-Tooth disease). In the human hypertrophic neuropathy, the paucity of myelinated fibers, the segmental demyelination (Thomas, 1971; Dyck et ah, 1974) and the existence of widened nodes of Ranvier are accompanied by abnormalities of structures of the axolemma. As in Charcot-Marie-Tooth disease (Waxman and Ouellette, 1979), excrescences and protusions of the axoplasm at the nodes are sometimes observed in +med/+med sciatic nerves. Such abnormalities may be related to changes of the +med/+med axolemmal membrane and to its regenerative capacities. Na+ channel number and distribution are very important to measure, if we are to understand functional changes related to demyelination and remyelination. The biochemical data on Na+ channels quantification, with the significant increase of the TTX binding sites
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in +med/+med sciatic nerves at an early stage of the disease, show that the axolemma itself is modified at about the same stage that paranodal dysmyelination becomes effective. Demyelination of peripheral axons initially causes failure of action potential conduction probably because the internodal membrane lacks Na+ channels (Ritchie and Rogart, 19’7’7; Wood et al, 197’7; Chiu and Ritchie, 1980). However, Bostock and Sears (1978) showed that 3-14 days after demyelination of rat nerve with diphtheria toxin, some axons develop regenerative inward currents permitting continuous conduction along the internodal membrane (Ritchie and Rogart, 1977; Chiu and Ritchie, 1980). The appearance of continuous conduction in the demyelinated nerve requires either the synthesis of new Na+ channels or a redistribution of Na+ channels normally concentrated in the nodes. Ritchie et al. (1981) have examined the changes in the total number of Na+ channels, measured by saxitoxin binding capacity, in rabbit sciatic nerves which have been demyelinated in viva with lysolecithin and then allowed to remyelinate. They found no evidence for the formation of new Na+ channels during the early stage, when continuous conduction may develop, but they have shown clearly that new channels are formed during remyelination. The channel density of nerves examined about 2 months after lysolecithin injection, when remyelination is well under way, is about twice that of the control. This finding mostly resulted from the increase in nodal population associated with the formation of short internodes. The measure of internodal distances in the teased fiber preparations gives an indirect estimate of the population of nodes and Ranvier and the extension of Schwann cell territories. No significant difference has been observed between +med/+med and control nerve fibers, which eliminates the possibility that the twofold increase in TTX binding only corresponds to an increase in the number of nodes of Ranvier with short Schwann cell territories. The developmental increase in density of Na+ channels seen in the +med/+med mouse sciatic nerve as compared to the control has the following properties: (1) it does not provide a normal conduction since the course of motor end-plate disease is progressive paralysis and early death and (2) it appears very early, at the preclinical stage, which suggests that the axonal membrane is abnormal, at least with respect to Na+ channels, close to the time of onset of the disease process. Increase in Na+ channel density seems to be a general property of demyelinated, unmyelinated, or dysmyelinated peripheral nerves in mutant mice. Experiments performed on sciatic nerves of Trembler mutant mice, where Schwann cells form little or no myelin in the peripheral nervous system (Bray et &, 1981) have shown a three- to fivefold higher level of Na+ channels (A.
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Lombet, unpublished data) as compared to +med/+med mice. It is of interest to observe that increases of Na+ channels in +med/+med sciatic nerves are accompanied by only very moderate increases, if any, of Na+,K+ATPase; this may mean that there is no coordination in the regulation of the synthesis and/or of the membrane incorporation of these two macromolecular Na+ transport systems. We have not detected such modifications in the central nervous system, where the wted mutation produces no modification of the level of these two Na+ transport components. With the biochemical study of the Na+ transport systerns-Na+ channel and Na+,K+-ATPase-we further explored not only the developmental aspects of the disease process compared to normal maturation, but also the molecular properties of important components of the axolemmal membrane. Binding studies and freezeetching studies (Ritchie and Rogart, 1977; Wood et aL, 1977) strongly suggest that these Naf transport proteins are essentially located at the node of Ranvier. The widening of a majority of the nodes of Ranvier in affected +med/+med mouse sciatic nerve is accompanied by a twofold increase in the number of Na+ channels (Fig. 7A). No modification in the properties of the TTX receptor itself seems to occur, as indicated by the affinity of the toxin for its binding sites, which is about 1 nM both for +med/+med and control axons. The Na+,K+ATPase, which presents a slight increase in the number of ouabain binding sites, does not show either a change in ligand affinity. The intimacy of the developmental interactions between axon, Schwann cells, endoneurial fibroblasts, and extracellular components (Sidman and O’Gorman, 1981; Bunge et al, 1981) precludes a decision at present as to what cell is the primary target of the med genetic locus, or even whether the disease is intrinsic to the peripheral nervous system. Detailed developmental ultrastructural studies, nerve transplantation experiments, and primary cultures of muscle cells, neurons, and Schwann cells, which are underway in our laboratory, may help to define the cellular (and molecular) targets of the mutation. We thank Profs. R. Couteaux and B. Droz and Drs. M. Fardeau, A. Mallart, and R. Bournaud for fruitful discussions. This work was partially supported by research grants from Muscular Dystrophy Association of America, CNRS (ATP 3927) and MRT (Action “Dynamique du Neurone et des Ensembles Neuronaux”). REFERENCES ANGAUT-PETIT, D., MCARDLE, J. J., MALLART, A., BOURNAUD, R., PINCON-RAYMOND,M., and RIEGER, F. (1982). Electrophysiological and morphological studies of a motor nerve in motor end-plate disease of the mouse. Proc. R. Sot. London B Ser. 215,117-l%. BOSTOCK,H., and SEARS, T. A. (1978). The internodal axon membrane: Electrical excitability and continuous conduction in segmental demyelination. J. Physid (Landon) 280, 273-301.
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DUCHEN, L. W. (1979). Hereditary disorders of motor and sensory neurons in the mouse. Ann N.E AccuL Sci 317, 506-511. DUCHEN, L. W., SEARLE, A. G., and STRICH, S. J. (1967). An hereditary motor end-plate disease in the mouse. J. Physid (London) 189,4P6P. DUCHEN, L. W., and STEFANI, I. (1971). Electrophysiological studies of neuromuscular transmission in hereditary “motor end-plate” disease of the mouse. J. Phgsiol 212, 535-548. DYCK, P. J., LAIS, A. C., and OFFORD, K. P. (1974). The nature of myelinated nerve fiber degeneration in dominantly inherited hypertrophic neuropathy. Mayo Clin Proe. 49,34-39. FARDEAU, M., TOME, F., GUIMARAES, A., and CABELLO, A. (1978). Role de la microscopic Qlectronique en pathologie nerveuse p&iph&ique. Arch Anat Cytol Path& 26,283-291. HARRIS, J. B., and WARD, M. R. (1974). A comparative study of “denervation” in muscle from mice with inherited progressive muscular disorders. Exp. Ne-uroL 42,169-180. HARTREE, E. F. (1972). Determination of protein: A modification of the Lowry method that gives a linear photometric response. Anal Biochem
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LOMBET, A., RENAUD, J. F., CHICHEPORTICHE,R., and LAZDUNSKI, M. (1981). Cardiac tetrodotoxin binding components. Biochemical identification, characterization and properties. Bic&e-mtitry 20, 12791285.
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PINCON-RAYMOND, M., and RIEGER, F. (1981). The motor innervation of skeletal muscles in the motor end-plate disease mutant mouse (med and me& alleles). Bill Cell 40, 189-194. PINCON-RAYMOND,M., LUDOSKY,M. A., CARTAUD,and RIEGER, F. (1983). Intense ultraterminal sprouting from motor nerves and non-junctional sarcolemma of the soleus muscle (slow-twitch) in motor endplate disease in the mouse. Tissue CeU 15, 205-216. QUICK, D. C., and WAXMAN, S. G. (1978). Demyelination of Sternachua electrocyte fibers by injection of diphteria toxin. J. NeuroL Sci 36, 235-241. RASMINSKY, M., KEARNEY, R. E., AGUAYO, A. J., and BRAY, G. M. (1978). Conduction of nervous impulses in spinal roots and peripheral nerves of dystrophic mice. Brain Res 143, 71-85. RIEGER, F., and PINCON-RAYMOND, M. (1980). The motor innervation in motor end-plate disease (med) in the mouse. Evidence for nerve abnormalities, prior to muscle damage. In “Neurological Mutations Affecting Myelination: Research Tools in Neurobiology” (N. Baumann, ed.), pp. 163-170. INSERM Symp. No. 14. Elsevier/North Holland, Amsterdam. RITCHIE, J. M., and PELLEGRINO, R. (1981). Sodium and potassium channels in demyelinated and remyelinated mammalian nerve. Nature
(Landon)
294, 257-259.
RITCHIE, J. M., and ROGART, R. B. (1977). Density of sodium channels in mammalian myelinated nerve fibers and nature of the axonal membrane under the myelin sheath. Proc Nat. Acud Sci USA 74, 211-215. SEARLE, A. G. (1962). Personal communication. Mowe NewsLett. 27, 34.
SIDMAN, R. L., COWEN,J. S., and EICHER, E. M. (1979). Inherited muscle and nerve diseases in mice: a tabulation with commentary. Ann NY Acad Sci, 317,497-505. SIDMAN, R. L., and O’GORMAN, S. V. (1981). Cellular interactions in Schwann cell development. Adv. Neurd 29,213-235. THOMAS, P. K. (1971). The morphological basis for alterations in nerve conduction in peripheral neuropathy. Proc R. Sot Med 64, 295298.
WAXMAN, S. G., and OUELLETTE, E. M. (1979). Ultrastructural and cytochemical observations in a case of dominantly inherited hypertrophic (Charcot-Marie-Tooth) neuropathy. J. NeuropathoL Exp NewoL 38,586-595. WAXMAN, S. G., and QUICK, D. C. (1977). Cytochemical differentiation of the axon membrane in A and C fibres. J. Neural Neurosurg. Psychiatr.
40,379-385.
WEINSTEIN, S. P. (1980). A comparative electrophysiological study of motor end-plate diseased skeletal muscle in the mouse. J. PhysioL 307,453-464. WOOD, J. G., JEAN, D. H., WHITTAKER, J. N., MCLAUGHLIN, B. J., and ALBERS, R. N. (1977). Immunocytochemical localization of the sodium-potassium activated ATPase knifefish brain. J. NeurocgtoL 6, 571-581.
BIOLOGY
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(1984)
Paranodal Dysmyelination and Increase in Tetrodotoxin Binding Sites in the Sciatic Nerve of the Motor End-Plate Disease (mecI/med) Mouse during Postnatal Development FRANF)OIS RIEGER, *J MARTINE PINCON-RAYMOND,* MICHEL
LAZDUNSKI,t
ALAIN LOMBET,~ GILLES PONZIO,~
AND RICHARD
L. SIDMAN*
*Groupe de Biobgie et Path&&e Neuromuaculaires, Physiopat~ des Mywhies, INSERM U 155-17, rug du FeGMoulin, 75005 Paris, France, TInatitut de Biochimie du CNRS, Univemiti de Nice, Pare Valrose, 06000 Nice, France, and Qlepartment of Neuroaci~, Children’s Hospital Medical Center, $00 Langwood Avenue, Boston, Massachusetts 0.2115 Received December 80, 1982; accepted in revised form September 6, 1983 Motor end-plate disease (mod), in the mouse, is a hereditary neuromuscular defect, caused by a single gene mutation and characterized by a progressive muscle weakness. +Med/+med mice die 21-23 days after birth and the neurobiologlcal abnormalities already reported are nerve terminal sprouting and swelling and neurotransmission failures. We studied +meoY+med mice at preclinical (9-11 days after birth) as well as at clinically recognized stages of the disease. The nonmyelinated gaps of the nodes of Ranvier in the +med/+m.ed sciatic nerve are found to be significantly widened in +med/+med animals compared to control littermates, even in the preclinical stage, although the nodes of Ranvier are not yet ultrastructurally mature. The maximal binding capacity for PHIethylene-diamine tetrodotoxin, expressed in femtomoles per milligram of protein, is significantly increased in +m.ed/+med sciatic nerves. Thus, Na+ channels, which are known to be located mainly at the nodes of Ranvier in normal myelinated axons, are increased in number in +med/+mod mice even before the disease becomes clinically established. Both the ultrastructural and biochemical developmental abnormalities of the node of Ranvier rapidly approach their maximal expression as the behavioral signs develop. Such newe abnormalities may be closely related to the physiological impairment of newe impulse conduction which leads to the pathophysiological expression of motor end-plate disease. INTRODUCTION
ical or biochemical alterations (Rieger and PinCon-Raymond, 1980). In a recent electrophysiological and ultrastructural study of triangularis sterni motor axons, we observed altered nerve conduction and widening of the nodes of Ranvier (Angaut-Petit et al, 1982). The present paper reports the ultrastructural and biochemical study of the sciatic nerves of mice homozygous for the med allele before and after the disease becomes clinically expressed; the biochemical analysis concerns the fast Na+ channel, and the Na+, K+-ATPase, which is an essential enzyme for the regulation of the resting potential. Both Na+ transport systems are essential elements of nerve conduction and localized in the nodal regions of the myelinated motor axons. We find that the nodes of Ranvier of the sciatic nerves of +med/+med mice are significantly widened at an early postnatal stage of the disease, and that these abnormalities are accompanied by a marked increase in Na+ channel number.
Motor end-plate disease (med)2 is a severe hereditary neuromuscular disorder of the mouse (Searle, 1962; Duchen et a& 1967; Duchen 1970). Three allelic forms of the mutation are known: med, medJ, and medj”, in decreasing order of severity (Sidman et aL, 1979). Mice with med/m.ed or medJ/medJ genotypes die during the fourth week after birth, while medj”/medj” mice survive for several months. Nerve terminal swelling and sprouting have been observed in both med/m.ed and medJ/ medJ mice (Duchen, 1970; Pincon-Raymond and Rieger, 1981), in all muscles studied. The nerve to the soleus muscle is characterized by a much more extensive swelling and sprouting (Pingon-Raymond et al, 1983). These pathological changes of the nerves are thought to occur as a consequence of a state of partial or total functional denervation (Duchen and Stefani, 1971; Duchen, 1979). The nerve abnormalities in motor end-plate disease seem to occur at an early period of the disease (Rieger and PinCon-Raymond, 1980; Bournaud et a& 1980) when the muscle fiber does not yet show any marked morpholog-
MATERIAL
AND METHODS
Mice. Cad is the gene symbol for directional caracul, which originally arose at the Oak Ridge National Laboratory, and med is motor end-plate disease: these genes are closely linked on chromosome 15 (Duchen et al, 1967).
1To whom reprint requests and correspondence should be addressed. * Abbreviations: en-TTX, ethylenediaminetetrodotoxin; med, motor end-plate disease. 401
0012-1606/34 $3.00 Copyright All rights
0 1984 by Academic Press. Inc. of reproduction in any form reserved.
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Breeding pairs of heterozygous Cad+/+med mice were kindly provided by Dr. A. G. Searle (Medical Research Council, Radiobiology Unit, Harwell, U. K.) through the courtesy of Dr. L. W. Duchen (The National Hospital, London, U. K.) in 1979. The med strain is kept as a closed stock, which has strains C3H and 101 in its ancestry. It is not congenic with C3H. A colony is now maintained in our laboratory by brother-sister matings and progeny testing. Mutant +med/+med mice are characterized by their smooth hair and whiskers. Doubly heterozygous Cad+/+med and normal Cad+/Cad+ (homozygous wildtype at the me& locus) have curly hair and whiskers. Cad+/Cad+ mice show a slight epilation, although only progeny testing can unambiguously differentiate them from the heterozygotes. The disease is first noticed in lo- to 12-day-old +med/+med mice and is clinically characterized by a rapid, progressive weakness. We studied mice from before the onset of behavioral signs (9-10 days after birth) until death (21-23 days after birth), discarding from our study any animal too weak to be able to feed itself. The mice were sacrificed at different ages by cervical dislocation. Light and electron microscopll. The sciatic nerves were immediately exposed after sacrifice and fixed in situ for 15 min, then dissected out for an additional fixation of 90 min and further processed (1) for nerve fiber teasing and light microscopy, by immersion into a 2% osmium tetroxide solution in a 0.14 1Mveronal acetate buffer (pH 7.4) for 24 hr, followed by 66% glycerol for 48 hr and then pure glycerol; nerve fiber teasing was performed under binocular observation with sharp insect pins and internodes measured with a micrometer (Olympus microscope), or (2) for electron microscopy, by conventional techniques (Fardeau et aL, 1978), on longitudinal ultrathin sections. Direct perfusion of fixative in anaesthetized animals gave very similar results. Binding studies. The sciatic nerves were dissected out immediately after sacrifice, and rinsed in ice-cold 20 m&f Tris-HCl (pH 7.4) containing 0.25 M sucrose and 1 mM EDTA. The two sciatic nerves from each animal were homogenized in 0.7 ml of the same medium, using a Polytron PT 10s apparatus (Brinkman Inst.-Setting 5-10). The crude homogenate obtained was directly used for binding assays, carried out in parallel for rH]enTTX and for rH]ouabain. Protein content was determined using the method of Hartree (1972) with bovine serum albumin as a standard. The amount of Na+ channels present in the tissues was determined by binding assays using the derivative rH]en-TTX, carried out in a choline chloride medium as previously described (Lombet et al, 1981). The amount of Na+, K+-ATPase was determined using C3H]ouabain as described by Desnuelle et al. (1982). In both cases the specific binding was calculated from the difference between the bound
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radioactivity measured after rapid filtration through a GF/B glass fiber filter in the absence and the presence of a large excess of unlabeled ligand (5 PM TTX or 1 mM ouabain, respectively). Chemical and drugs. rH]en-TTX was synthesized according to Chicheportiche et al. (1980). The preparation reached a specific radioactivity of 25 CVmmole and a radiochemical purity of 90%. pH]Ouabain was obtained from New England Nuclear with a specific radioactivity of 11.6 CVmmole. Citrate-free tetrodotoxin was purchased from Sankyo Chemical Co. (Tokyo, Japan) and ouabain and ATP were from Sigma Chemical Co. RESULTS
Enlargement of the Nonmyelinated Gap at the Node of Ranvier in +med/+med Sciatic Nerve The most marked and frequent abnormality observed in longitudinal ultrathin sections of +med/+m-ed sciatic nerves is a widening (elongation) of the nodes of Ranvier. A typical example of such widening is shown in Fig. 1, for an l&day-old mouse myelinated axon, in the midthigh portion of the sciatic nerve. At this stage of the disease, this enlargement is found in the majority of the nodes of Ranvier (see below), in proximal, mid, or distal portions of the nerve: only occasional nodes are of normal size and morphology, compared to nodes of Ranvier of their control, identified, Cad+/Cud+ littermates. Control l&day-old Cad-f-/Cad+ animals presented no node of Ranvier with gap widths greater than 2-2.5 pm, whereas, in +med/+med animals, about 20% of the nodes exceeded 4 pm in width. We performed a morphometric quantitative study of the size of the nodes of Ranvier at different developmental stages in +med/ +med and Cad+/Cad+ mice. All measurements were made between the extreme paranodal myelin loops of nodes of Ranvier, sectioned with an optimal longitudinal incidence. The histograms-giving the frequency distribution of axons for different classes of nodal gap widths (Fig. 2)-show that a larger size of the nonmyelinated gap is already observed at a preclinical stage in mutant relative to control mice (9 days after birth), and more clearly at the onset of the behavioral signs of the disease (12 days after birth), in an appreciable proportion of the nodes of Ranvier analyzed. The controls show a progressive shortening in nodal size between 9 and 18 days after birth corresponding to normal maturation; the mutants show a lesser trend in the same direction. The internodal distances were measured, after nerve fiber teasing, on isolated nerve fibers. The nerve fibers show a bimodal distribution in diameter (Fig. 3). We considered two groups of nerve fibers: large diameter (>4 pm) and small diameter (G3.9 pm). The frequency distributions of either large or small diameter fibers in
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normalities, which progressively became more severe during the course of the disease. At late stages, the myelin thickness was frequently asymmetric on the two sides of the widened nodes. Sometimes, when the myelin sheath on one side was very thin, the other side might be devoid of myelin for long distances (8% of the abnormal nodes). Moreover, the myelin sheath, in the par-
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NERVE PN.9
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FIG. 1. Widened nonmyelinated gap at the node of Ranvler of a myelinated nerve fiber in the sciatic nerve of an l&day-old +med/ +med
mouse.
function of the internodal lengths observed (Fig. 4) are not significantly different in Cad+/Cad+ or in +med/ +med nerves (nonparametric statistical test). We observed that both myelin and axoplasm in the paranodal and nodal regions presented structural ab-
NODAL
GAP[jm)
FIG. 2. Size distribution of the nonmyelinated gaps of the nodes of Ranvier of myelinated fibers during development in normal (Cad+/ Co’+) and mutant (+med/+med) mouse sciatic nerve. PN, postnatal stage (Days 9, 12, 18 after birth); abscissae, size classes of nodal gap widths, ordinates, percentage of nodes of Ranvier. Total numbers of nodes of Ranvier which were measured: PN 9: Cod+/Cod+; n = 26; +med/+med; n = 26; PN 12: Cad+/Cad+; n = 55, +med/+mad; n = 55, PN 18: Cad+/Cad+; n = 29, +rned/+me& n = 37. Cad+/Cad+; hatched columns; +med/+medq black columne.
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[3H]ouabain to their respective receptor sites are shown in Fig. 6. Sixteen nerves from 1’7-day-old Cad+/Cad+ mice and eight nerves of the same stage +med/+med mice were pooled and homogenized. The titration of PHIen-TTX binding sites was demonstrated by Scatchard plots of the data obtained with increasing cona
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FIG. 3. Similar bimodal frequency distributions of Cad+/Cud and +m.ed/+m.ed nerve fiber population in function of fiber diameter. Eighteen-day-old mice, sciatic nerve. Open columns, Cad+/Cad+; hatched columns, +med/+med Class size, 1 pm.
anodal region, often did not totally enwrap the axon, thereby producing a dyssymmetry with reference to the long axis of the axon. Other myelin abnormalities frequently encountered in the paranodal region included increased interlamellar myelin splits, aberrant myelinated bodies, or long Schwann cell processes, without lamellae, extending along the bare axolemmal regions of the very widened +med/+med nodes (Fig. 5a). However, neither generalized depletion of myelin nor significant variations in myelination in internodal regions were observed in transverse sections of the nerves of mutant compared to wild-type mice. We did not observe any discontinuity in the basal lamina of the Schwann cells surrounding myelinated fibers in the +meuY+med mutants. The axoplasm sometimes showed short protrusions or excrescences in some enlarged nodes (Fig. 5b).
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Develqnnenti Changes of the Ionic Na+ Channel and Na+,K+-ATPase in Normal Cad+/Cad+ and +med/+med Sciatic Nerves 60
The abnormality of the nodes of Ranvier of the sciatic nerves of +m.ed/+nwd mice was further investigated biochemically via binding studies. Two Na+ transport systems present in nodes of Ranvier, i.e., the ionic Na+ channel and the Na+,K+-ATPase, were quantitatively studied in sciatic nerves of mutant (+med/+med) mice and a comparison was made with normal (Cad+/Cad+) mice. The binding properties of PHIen-TTX and
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FIG. 4. Frequency distributions of small or large Cad+/Cad+ and +med/+med nerve fibers in function of internodal distances. (a) Large fibers (~4 gm diameter). The distribution appears unimodal. Mutant fibers seem to have slightly longer internodal lengths although statistical analysis (nonparametric) does not indicate a significant difference. (b) Small fibers (~4 gm diameter). The distribution is bimodal. No significant difference between control and mutant fibers. Open columns, Cad+/Cad+; hatched columns, +med/+med
FIG. 5. Myelin abnormalities in +med/+m.ed sciatic nerve. (a) Aberrant myelin in the vicinity of a widened node of Ranvier in the sciatic nerve of an 18 day-old +med/+med mouse. (b) Protrusion of axoplasm at a node of Ranvier in an B-day-old +m.ed/+med mouse. 405
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centrations of the tritiated ligand (Fig. 6A). The plots gave single straight lines indicating in each case a single population of high affinity tetrodotoxin binding sites. The equilibrium dissociation constants of the rH]enTTX-receptor complex (Kn) were 0.96 -t 0.15 and 0.80 + 0.10 nM for normal and mutant sciatic nerves, respectively. The corresponding maximal binding capacities (B,,,) were 265 and 535 fmol/mg of protein. A similar representation is given in Fig. 6B for [3H]ouabain binding. The Kn values for rH]ouabain binding were 8 + 2 and 11 +- 2 nM and B,,, values were 680 and 800 fmol/ mg of protein for the normal and mutant sciatic nerves, respectively. The binding of rH]en-TTX to normal nerve homogenates was competitively inhibited by increasing amounts of unlabeled TTX or saxitoxin with a J&,6 of 2.0 and 2.5 nM, respectively (data not shown). The maximal binding capacities for rH]en-TTX and for [3H]ouabain were obtained for different samples (38) of normal and mutant sciatic nerves at various stages of development; these data are reported in Fig. ‘7. In Fig. 7A, comparison of the number of Na+ channels in normal and mutant sciatic nerves shows that +med/ +med sciatic nerves have a twofold higher level of Na+ channels at each stage of development, from 2 to 3 days before the disease is clinically expressed, until death of the mutant mice at 22-23 days. A plateau level was observed at approximately 3 weeks which corresponds to the maturation level of the Na+ channels. The comparative evolution of rH]ouabain-binding sites is shown in Fig. 7B. At all stages of development only a slight increase in the amount of Na+,K+-ATPase was observed in the +med/+med compared to the Cad+/Cad+ sciatic nerves. At 17 days, both normal and mutant brains (without cerebellum) have the same levels of Na+ channels and Na+,K+)-ATPase: 1200 f 230 fmole of tetrodotoxin binding sites per mg of protein in the +med/+med brain against 1360 f 120 fmole per mg of protein in the control, and 15.8 + 2.5 pmol of ouabain binding sites per mg of protein in the +med/+med brain against 16.8 -t 3.0 pmol per mg of protein in the control.
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FIG. 6. Specific binding of [8Hlen-‘ITX and [sH]ouabain. Homogenates (see Materials and Methods) of sciatic nerves of normal (Cu’+/Cad+) and mutant (+med/+mcd) mice. (A) Scatchard plots of @Ien-‘ITX specific binding (A) 1%day-old Cadt/Codt mice; 16 sciatic nerves were pooled. (A) l’l-day-old td/+d mice; 8 sciatic nerves. Inset: total (0) and nonspecific (X) binding as a function of increasing concentrations of [8HJen-‘M’X. (B) Scatchard plots of [‘H]ouabain binding. Same homogenates as for the ‘I’TX experiments on A. Same symbols. Inset: total (0) and nonspecific (X) binding as a function of increasing concentration of [8H]ouabain. Determinations were performed in duplicate, by filtrating a 0.4-m] aliquot of a l-ml incubation mixture, containing 0.1-1.2 mg of protein per ml of homogenate, on GF/B filters. Nonspecific binding was measured in the presence of a large excess of unlabeled ligand (5 fl TTX or 1 PM ouabain, respectively). Specific binding presented in the Scatchard plots is the difference between total and nonspecific bindings.
DISCUSSION
Inherited motor end-plate disease in the mouse is characterized not only by abnormal modifications of the terminal motor innervation, e.g., ultraterminal sprouting (Duchen, 1970; Rieger and Pincon-Raymond, 1980; Pincon-Raymond and Rieger, 1981), and nerve terminal swelling, but also by prominent morphological abnormalities of the nodes of Ranvier. In a recent electrophysiological and morphological study of a thoracic motor nerve (Bournaud et aL, 1980; Rieger and PinconRaymond, 1980; Angaut-Petit et al, 1982), we observed
that nerve conduction alterations, namely slow nerve conduction and increased refractory period, were accompanied by ultrastructural abnormalities of the nodes of Ranvier, which are significantly widened when the disease is well established. Our present results extend these observations to a more distal nerve and bring evidence (1) for a general defect of paranodal myelination in +med/+med nerves, (2) for its occurrence at early postnatal ages, even before the onset of the behavioral signs of the disease, and (3) for a parallel in-
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DAY’S FIG. 7. Na+ channels and Na+,K+-ATPase during a period of postnatal development in normal (Cod+/Cod+) and mutant (+med/+med) mouse sciatic nerves. (A) Development of [‘HI en-TTX binding sites number in Cad+/Cad+ (A) and +medJ+med (A) sciatic nerves between Day 9 and Day 22 after birth, corresponding to a preclinical stage and death, respectively, of affected mice. (B) Development of [‘HI ouabain binding sites number in Cad+/Cad+ (A) and +mad/+med (A) sciatic nerves. For each developmental stage chosen, the determinations were performed on three to eight pairs of sciatic nerves, independently homogenized and assayed. The bars represent the standard deviation of the mean.
crease in number of Na+ channels (TTX-binding sites). We found that the nonmyelinated gap of the nodes of Ranvier in myelinated fibers was significantly widened in +med/+med sciatic nerves in regions proximal or in more distal regions to its spinal cord roots, and in small as well as larger myelinated axons. Apart from the paranodal regions, myelin does not seem to be affected: transverse sections rarely show any marked alterations, as already observed in thoracic nerves (Angaut-Petit et al, 1982). Motor end-plate disease is thus characterized by a marked and frequent paranodal dysmyelination. Our data do not discriminate whether the med mutation causes a true demyelinating process or a delayed or
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arrested development, expressed most clearly at the paranodal specializations of the myelin sheath. In any case, these paranodal abnormalities are probably related to the electrophysiological modifications previously described in a thoracic nerve (Angaut-Petit et d, 1982): the slow nerve conduction and increased refractory period of the nerve impulses. Similar abnormalities of nerve conduction have also been observed in the sciatic nerve trunk and its branch to the soleus muscle (D. Augaut-Petit; J. J. McArdle, A. Mallart, R. Bournaud, M. Pincon-Raymond, and F. Rieger, unpublished results). It seems that paranodal dysmyelination in +mecl/+med nerves cannot alone account for the important decrease in nerve conduction: it has been recently reported by Rasminsky et al. (1978) that computer simulations of conduction in internodal regions of demyelinated nerves decreased by only 18% when quadrupling nodal widths, a widening rarely observed in +med/+m.ed nerve fibers. The nerve conduction abnormalities probably explain the blocks of neurotransmission and/or failures of nerve action potentials to invade the nerve terminals, reported by other investigators (Duchen and Stefani, 1971; Harris and Ward, 1974; Weinstein, 1980). Ultrastructural alterations of the nodes of Ranvier appear early in the disease process, even before the behavioral signs are noticed. This observation strongly supports the view that the structural abnormalities are closely linked to the nerve conduction abnormalities in this neuromuscular pathological process. However, we do not know whether myelin or axon, if either, is primarily responsible for the structural and functional defects. In other nerve disorders which appear to affect the myelin sheath, usually more extensively than in motor end-plate disease, the axon has been reported to be abnormal as well. This is found, for example, in experimental diphtheric neuropathy (Waxman and Quick, 1977; Quick and Waxman, 1978), and in the dominantly inherited human hypertrophic neuropathy (CharcotMarie-Tooth disease). In the human hypertrophic neuropathy, the paucity of myelinated fibers, the segmental demyelination (Thomas, 1971; Dyck et ah, 1974) and the existence of widened nodes of Ranvier are accompanied by abnormalities of structures of the axolemma. As in Charcot-Marie-Tooth disease (Waxman and Ouellette, 1979), excrescences and protusions of the axoplasm at the nodes are sometimes observed in +med/+med sciatic nerves. Such abnormalities may be related to changes of the +med/+med axolemmal membrane and to its regenerative capacities. Na+ channel number and distribution are very important to measure, if we are to understand functional changes related to demyelination and remyelination. The biochemical data on Na+ channels quantification, with the significant increase of the TTX binding sites
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in +med/+med sciatic nerves at an early stage of the disease, show that the axolemma itself is modified at about the same stage that paranodal dysmyelination becomes effective. Demyelination of peripheral axons initially causes failure of action potential conduction probably because the internodal membrane lacks Na+ channels (Ritchie and Rogart, 19’7’7; Wood et al, 197’7; Chiu and Ritchie, 1980). However, Bostock and Sears (1978) showed that 3-14 days after demyelination of rat nerve with diphtheria toxin, some axons develop regenerative inward currents permitting continuous conduction along the internodal membrane (Ritchie and Rogart, 1977; Chiu and Ritchie, 1980). The appearance of continuous conduction in the demyelinated nerve requires either the synthesis of new Na+ channels or a redistribution of Na+ channels normally concentrated in the nodes. Ritchie et al. (1981) have examined the changes in the total number of Na+ channels, measured by saxitoxin binding capacity, in rabbit sciatic nerves which have been demyelinated in viva with lysolecithin and then allowed to remyelinate. They found no evidence for the formation of new Na+ channels during the early stage, when continuous conduction may develop, but they have shown clearly that new channels are formed during remyelination. The channel density of nerves examined about 2 months after lysolecithin injection, when remyelination is well under way, is about twice that of the control. This finding mostly resulted from the increase in nodal population associated with the formation of short internodes. The measure of internodal distances in the teased fiber preparations gives an indirect estimate of the population of nodes and Ranvier and the extension of Schwann cell territories. No significant difference has been observed between +med/+med and control nerve fibers, which eliminates the possibility that the twofold increase in TTX binding only corresponds to an increase in the number of nodes of Ranvier with short Schwann cell territories. The developmental increase in density of Na+ channels seen in the +med/+med mouse sciatic nerve as compared to the control has the following properties: (1) it does not provide a normal conduction since the course of motor end-plate disease is progressive paralysis and early death and (2) it appears very early, at the preclinical stage, which suggests that the axonal membrane is abnormal, at least with respect to Na+ channels, close to the time of onset of the disease process. Increase in Na+ channel density seems to be a general property of demyelinated, unmyelinated, or dysmyelinated peripheral nerves in mutant mice. Experiments performed on sciatic nerves of Trembler mutant mice, where Schwann cells form little or no myelin in the peripheral nervous system (Bray et &, 1981) have shown a three- to fivefold higher level of Na+ channels (A.
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Lombet, unpublished data) as compared to +med/+med mice. It is of interest to observe that increases of Na+ channels in +med/+med sciatic nerves are accompanied by only very moderate increases, if any, of Na+,K+ATPase; this may mean that there is no coordination in the regulation of the synthesis and/or of the membrane incorporation of these two macromolecular Na+ transport systems. We have not detected such modifications in the central nervous system, where the wted mutation produces no modification of the level of these two Na+ transport components. With the biochemical study of the Na+ transport systerns-Na+ channel and Na+,K+-ATPase-we further explored not only the developmental aspects of the disease process compared to normal maturation, but also the molecular properties of important components of the axolemmal membrane. Binding studies and freezeetching studies (Ritchie and Rogart, 1977; Wood et aL, 1977) strongly suggest that these Naf transport proteins are essentially located at the node of Ranvier. The widening of a majority of the nodes of Ranvier in affected +med/+med mouse sciatic nerve is accompanied by a twofold increase in the number of Na+ channels (Fig. 7A). No modification in the properties of the TTX receptor itself seems to occur, as indicated by the affinity of the toxin for its binding sites, which is about 1 nM both for +med/+med and control axons. The Na+,K+ATPase, which presents a slight increase in the number of ouabain binding sites, does not show either a change in ligand affinity. The intimacy of the developmental interactions between axon, Schwann cells, endoneurial fibroblasts, and extracellular components (Sidman and O’Gorman, 1981; Bunge et al, 1981) precludes a decision at present as to what cell is the primary target of the med genetic locus, or even whether the disease is intrinsic to the peripheral nervous system. Detailed developmental ultrastructural studies, nerve transplantation experiments, and primary cultures of muscle cells, neurons, and Schwann cells, which are underway in our laboratory, may help to define the cellular (and molecular) targets of the mutation. We thank Profs. R. Couteaux and B. Droz and Drs. M. Fardeau, A. Mallart, and R. Bournaud for fruitful discussions. This work was partially supported by research grants from Muscular Dystrophy Association of America, CNRS (ATP 3927) and MRT (Action “Dynamique du Neurone et des Ensembles Neuronaux”). REFERENCES ANGAUT-PETIT, D., MCARDLE, J. J., MALLART, A., BOURNAUD, R., PINCON-RAYMOND,M., and RIEGER, F. (1982). Electrophysiological and morphological studies of a motor nerve in motor end-plate disease of the mouse. Proc. R. Sot. London B Ser. 215,117-l%. BOSTOCK,H., and SEARS, T. A. (1978). The internodal axon membrane: Electrical excitability and continuous conduction in segmental demyelination. J. Physid (Landon) 280, 273-301.
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