Brain Research, 463 (1988) 78-89 Elsevier
78 BRE 14004
Abnormal neuromuscular junctions in the lateral rectus muscle of wobbler mice J e n n i f e r H. LaVail 2 and K a t h e r i n e P. Irons 1 Department of 1Anatomy and 2Neuroscience Program, The Universitv of California, San Francisco, CA 94143 (U.S.A.) (Accepted 11 May 1988)
Key words: Mutant; Extraocular: Motor neuron; Muscle; Synapse
We have studied the lateral rectus muscles and neuromuscular junctions (NMJs) of abducens motoneurons in wobbler (wr/wr) mutant mice from 26 to 58 days of age. The muscles of wr/wr weighed about 70% of the weight of linermate controls and were composed of fiber types comparable to those of controls, as assayed by succinate dehydrogenase activity. The most obvious difference between wr/wr and control NMJs was a reduction in the length of the postjunctional membrane of wr/wr mice. The mutant muscle endplate membrane was only about 70% (6.58 jxm) the length of control muscle regions (9.44 #m). There were no obvious differences at the light microscopic level in the distribution of acetylcholine (ACh) receptors at junctional regions or staining of acetylcholinesterase, as assayed with ct-bungarotoxin binding or enzyme histochemistry. Indirect immunocytochemical studies using antibodies directed against the subunits of the ACh receptor failed to indicate an abnormal presence of immature receptors clustered at the NMJs of wr/wr mice. Our findings suggest that the formation or maintenance of normal postjunctional folds and the differentiation of receptors at the junctions are under independent control during development. Furthermore, the wobbler mutation may affect muscle cell differentiation as well as neuronal differentiation. This mutant mouse should prove a useful model for study of postjunctional fold formation and function. INTRODUCTION In 1968 Duchen and Strich 11 first described the behavioral manifestations and cytopathology of mice affected by the autosomal recessive mutation, wobbler (wr/wr). They reported motoneuron loss throughout the brainstem and spinal cord and concluded that the mutation resulted in degeneration of motoneurons that innervated skeletal muscle. Subsequently, most investigators have focused on the loss of neurons in the cervical spinal cord, although reports of neuron loss in the dorsal root ganglia and elsewhere have also appeared 2'7. Based in part on the fact that extraocular function is often spared in h u m a n neurodegenerative diseases, we examined an extraocular nucleus in wobbler Is. We chose to study the abducens (VIth) nerve nucleus, due to its discrete boundaries and small size. We found the cell bodies and axons of the VIth nerve were relatively spared, i.e. no degenerating (foamy)
cell bodies were found in the nucleus, and the nerve was intact. There were, however, about 30% fewer large m o t o n e u r o n s in the nucleus than found in agematched control mice. Since there were no previous studies of wr/wr neuromuscular junctions (NMJs), we also made preliminary ultrastructural observations of motor endplates in the lateral rectus muscles that are innervated by the VIth motoneurons. The postjunctional folds were shallower and less numerous than those of age-matched control mice. The changes in endplate morphology could be interpreted as the result of simple denervation atrophy or as a result of arrested maturation of muscle cell differentiation. Reductions in postjunctional folding have been reported as characteristic of the chronic phase of experimental a u t o i m m u n e myasthenia gravis 12. It is also a feature of several other mutations in mice, e.g. dystrophy or motor endplate disease 9'3°, as well as for several experimentally induced diseases, such as tetanus toxin poisoning t°. In these cases,
Correspondence: J. LaVail, Department of Anatomy, University of California, San Francisco, CA 94143, U.S.A. 0006-8993/88/$03.5(l© 1988 Elsevier Science Publishers B.V. (Biomedical Division)
79 however, the reduction in muscle cell folding is a long-term response of the muscle to functional denervation, requiring weeks to evolve (for review, see refs. 8 and 32). To test whether the changes we found in wr/wr lateral rectus were the result of denervation and to provide the first detailed ultrastructural description of the wobbler NMJ, we have carried out a series of descriptive and experimental studies of lateral rectus muscle, a site in which the complications of denervation and muscle cell atrophy might be reduced. MATERIALSAND METHODS
Animals The mice used in the study were the offspring from two pairs of clinically normal heterozygous (+/wr) mice of the C57BL/6J mouse strain obtained from Dr. S. Averill and Mr. H. Syms (Childrens' Hospital Medical Center, Boston, MA). The mutant animals were identified behaviorally and histologically as previously described ts. The control littermate animals were either + / + or +/wr at the wr locus and therefore are designated +/-.
cluding folds adjacent to the nerve terminal membrane) were measured with a digitizer interfaced with a microcomputer. The ratio of the postjunctional membrane to the length of the primary cleft was used as an index of the amount of folding of the postjunctional membrane 22. All values are given as mean and standard deviation.
Histochemistry To estimate the number of muscle cells in the lateral rectus of mutants and controls and to determine whether muscle fibers were atrophied, muscle samples from 4 animals, 2 wr/wr and 2 + / - , were quickly frozen in freon 12 and sectioned in a cryostat. Preliminary examination of sections treated for ATPase activity at different pH confirmed the failure of this histochemical procedure to distinguish fiber types in rodent extraocular muscle 2s. Cross-sections of the muscle were treated with substrates to demonstrate succinic dehydrogenase activity 5. The outline of each muscle fiber was traced with a camera lucida and classified as having high, medium or low succinate dehydrogenase activity.
Electron microscopy
Acetylcholine receptor autoradiography Four control and 4 wr/wr muscles from 41-day-old
For the study of muscle fiber types and the ultrastructural analysis of endplates, the animals were anesthetized with diethyl ether and perfused through the heart with saline followed by cacodylate-buffered fixative containing 1% paraformaldehyde and 1.25% glutaraldehyde 19. The lateral rectus muscles were excised and embedded in an Epon-araldite mixture. Each lateral rectus muscle from at least two mutant and two control animals was examined at each age. Three regions were sampled along the long axis of the muscle at intervals of 10-15 ¢tm. Two 1 pm thick sections were cut and stained with Toluidine blue to determine the orientation of the muscle fibers. For electron microscopy 60-80 nm thick longitudinal sections of the midbelly of the muscle were also cut, and every NMJ for which both axon and postsynaptic folds could be recognized was photographed and printed at a final magnification of 25,000 or 30,000 x. On the micrographs the area and perimeter of the presynaptic element, the length of the nerve terminal membrane adjacent to muscle cell surface (length of primary cleft), and length of muscle sarcolemma (in-
mice were used to determine the distribution of abungarotoxin binding (a-BT) and thereby indicate the location of ACh receptors on the muscle cell surface. The mice were anesthetized with ether and decapitated. The lateral rectus muscles were dissected, weighed and rinsed in 0.1 M phosphate buffer (pH 7.4) containing 0.2% bovine serum albumin (BSA). The muscles were incubated for 2.5 h at room temperature in 0.4 ktM L251-ct-BT(spec. act. 0.5 mCi/ml and 240 Ci/mM, Amersham), according to the method of Bevan and Steinbach 6. The muscles were then washed overnight in 3 changes of phosphate buffer with 0.2% BSA and the next morning incubated at room temperature for 30 min to demonstrate sites of acetylcholinesterase (ACHE) activity 16. They were subsequently fixed in 4% paraformaldehyde in phosphate buffer with 5% sucrose for 2 h. The muscle fibers were teased apart on microscope slides and dried overnight. The following day the slides were coated with photographic emulsion for light microscopic autoradiography and developed after 5 days of exposure at 4 °C. Some of the muscle fiber prepara-
80 tions were subsequently counterstaind with 0.1% Neutral red. The number of silver grains was counted to determine the magnitude of extrajunctional binding of a-BT. Six regions each 49/~m 2 in area and spaced about 60/~m apart beginning at the edge of the NMJ (as defined by the absence of A C h E staining), were examined at 400x. Background levels of silver grains over the slide were subtracted from these values. To demonstrate the specificity of binding, 4 muscles of each genotype, incubated with radioactive toxin, were compared with 4 muscles of each genotype that had been incubated in 0.4 mM unlabeled toxin before incubation in radioactive toxin. The radioactivity in muscles was counted in a gamma counter. Comparison of results from labeled and preincubated and labeled muscles indicated that > 9 0 % of the binding was competitively inhibited by pretreatment with unlabeled toxin.
Indirect immunofluorescence Frozen unfixed sections of wr/wr and + / - lateral rectus muscles were incubated with antibodies specific for the fl, Y and 6 subunits of the rat and mouse A C h receptors. The Y subunit is only present at immature endplates of normal rat and mouse muscle, while the fl and 6 subunits are present at both immature and mature endplates 9. In each case rabbit antisera were made to synthetic peptides derived from the deduced amino acid sequence, and the subunitspecific antibodies were purified from the serum by
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5 0 25
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35
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40
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45
,
50
i
55
i
60
Age (Days) Fig. 1. B. wt. as a function of age. The control male (C)) mice
were slightly heavier than the corresponding females (0) at all ages. Both the wobbler male (D) and female (m) mice were generally smaller and failed to gain weight as did the control animals. All weights were determined at the time of sacrifice.
affinity chromatography 13. Sections were treated for 2 rain in 50 nM ethylamine HC1, pH 11.0 before incubation in the anti-6 antibody. The slides with sections were incubated for 2 h at room temperature followed by multiple rinses with 1% BSA in phosphate-buffered (pH 7.4) saline and then in a fluorescein-coupied goat anti-rat antibody in 1% B S A for 1 h at room temperature. Tetramethyl rhodamine coupled to aBT was also used on adjacent sections to mark the synaptic sites. The sections were viewed and photographed with filters sensitive to wavelengths emitted by rhodamine or fluorescein. RESULTS
Wobbler phenotype The homozygous wobbler mutant mice were phenotypically indistinguishable from normal littermates until about 18 days of age. The course of the disease varied from animal to animal. In general, animals with earlier and more severe signs did not survive as long as animals with less severe tremor. A typical sequence of signs characteristic of severely affected animals was seen in Animal 440, a male born in a litter of 7 mice. Beginning at 3 weeks of age the mutant developed a slight tremor that became progressively severe over the next few days. The digits of the forelimb contracted, the first and fourth toes were about 1.1 mm closer together than the normal distance of 6.1 mm in controls, and the animal was no longer able to grip. At the same time, balance was affected and the animal was unable to stand against the cage wall without falling. The following week the forelimb digits became tightly flexed and balance was further impaired. By 4 weeks of age, the eyelids drooped and the mutant appeared to have lost control of the hind limbs. The animal moved by a swimming motion of the forelimbs and could still reach and eat moistened food on the cage floor. The animal died at 36 days of age. All mutant animals lagged behind controls in weight beginning at about 3 weeks. The controls showed a steady increase from about 17 g at 26 days to an average of 24.6 g at 45 days (Fig. 1); none of the wobbler mice weighed more than 11 g, The dissected lateral rectus muscles from mutant and control animals also differed in wet weight. Control muscles weighed an average of 1.04 _+ 0.4 mg (n = 21) and
81
A
VI
B
Fig. 2. Light micrographs of cross-sections of the lateral rectus muscle of a + / - (A) and wr/wr (B) mouse. A: the control muscle contains at least 3 different types of fibers (e.g. 1-3), based on the intensity of histochemical staining for succinic dehydrogenase. VI, axons of the abducens nerve. B: the wr/wr muscle is smaller in volume than the control and contains 3 distinct fiber types (1-3), based on the intensity of staining for substrate of succinic dehydrogenase. Bar = 100 j~m.
82
Fig. 3. Electron micrographs of neuromuscular junctions in + / - (A) and wr/wr (B) lateral rectus muscles. A: the axon terminal (T) is apposed to the junctional folds (arrows) of the muscle cell. Note the m e m b r a n e staining of the muscle cell at the crest of the folds and the presence of an electron dense extracellular matrix in the gap. Bar = 1 g m . B: the n u m b e r and depth of postjunctional folds (arrow) are reduced at the wr/wr endplate. The muscle surface includes patches of densely stained m e m b r a n e and an extracellular matrix in the gap between the axon (T) and muscle cells. G, Golgi apparatus; m, mitochondria; S, Schwann cell. Bar = 1 Bm.
83 wobbler muscles weighed only 0.74 + 0.3 mg (n = 11) (t = 2.18; df = 30; P < 0.04).
TABLE I
Muscle histology and ultrastructure
Percentages of total fibers are indicated in parentheses. Crosssections of muscle fibers classified according to levels of succinic dehydrogenase activity2~.
The total number of muscle cells in the lateral rectus muscle of wobbler mice was reduced to about 60% of the number in controls of comparable ages (Fig. 2A,B and Table 1). The average diameter of the individual muscle fibers was also slightly less in mutant animals. When the widths of individual fibers were measured at the thickest region of the midbelly in 1 ~m epoxy-embedded sections, we found the mean fiber diameter in control muscles was 12.34 + 2.85/~m (n = 54) and the diameter of fibers in wobbler mice was 11.37 + 2.27 um (n = 56) (t = 1.97; P = 0.05). Despite these changes, we found no evidence of on-going degeneration in the muscle, e.g. central nuclei, increased endomysial connective tissue or lymphocyte infiltration. The extraocular muscles of the wild-type mouse have been studied in some detail 28"29"33. Three classes of fibers found within the midbelly of the muscle can be distinguished histochemically on the basis of succinic dehydrogenase activity: (1) a multiply innervated class with low enzyme activity, (2) a class of dark red fibers with intermediate levels of enzyme activity, and (3) a class of off-white fibers with high enzyme activity. Furthermore, each muscle fiber type is characteristic of a particular type of NMJ with a specific estimate of the ratio of postjunctional/prejunctional membrane lengths 33. Because each fiber type has a different degree of postjunctional folding, a loss of one fiber type might account for a change in the frequency of junctions with increased postjunctional folds. We therefore examined the frequency of different fiber types based on succinic dehydrogenase activity. The results of these analyses are summarized in Table I and illustrated in Fig. 2. Both the mutant and control muscles had fibers with low, medium and high levels of enzyme activity. Based on a one-way analysis of variance, there was no significant difference statistically in the distribution of fiber types in the wobbler and control muscles. Additional 1 /~m thick epoxy-embedded sections of the mutant and control muscle were also examined to determine the proportions of each fiber type (data not shown). The results confirmed the findings based on succinic dehydrogenase
Summary of the comparison of fiber composition of lateral rectus muscle of mutant and control animals
Fiber type
Low
Interrnediate
High
Total
+/-
58 (9) 18 (5)
457 (71) 278 (73)
131 (20) 81 (21)
646 377
wr/wr
histochemistry. The abducens nerve enters the lateral rectus muscle at the orbital surface and penetrates the epimysium at the midbelly (VI in Fig. 2). The majority of the endplates lie in a band about 0.2 mm wide in the midregion of the muscle. The NMJs in the lateral rectus muscle of wobbler mice have most of the cytological features of those of normal mice (Fig. 3A,B). In brief, most of the endplates were formed by single axons that branched into multiple small endbulbs. In some cases, the plane of section permitted us to measure 5 or more endbulbs in the same section. Both wobbler and control muscles were covered with a dense basal lamina in the junctional cleft. The muscle cells were also similar in that thickened membranes were found in the region of the neuromuscular contact. Sole plate nuclei and Golgi apparatus were frequently found associated with the junction. There was no difference in the distribution of organelles or vesicles in the two muscle types. Although the mitochondria in the mutant axons appear larger and more spherical (Fig. 3B) than mitochondria in the axon in the control endplate (Fig. 3A), the difference was not a consistent finding and may be related to fixation artifact. The cross-sectional areas of axons of control animals were marginally larger (Table II) than those of wobbler endplates, but the difference was not statistically significant. Axons of wr/wr and + / - muscles had average aspect ratios of 3.5 and 3.3, respectively (Table II). Assuming that the average axon is a cylinder, we infer from these values that the axons were cut in similar planes, on average about 74 ° from cross-section. The length of axonal membrane that was adjacent to muscle cell surface per endplate was about 5.28/~m in control animals and about 5.06/~m in wobbler; this
84 d i f f e r e n c e was n o t s i g n i f i c a n t l y d i f f e r e n t . H o w e v e r ,
hal m e m b r a n e
t h e l e n g t h of p o s t j u n c t i o n a l p l a s m a m e m b r a n e
was
for t h e w o b b l e r m i c e was a b o u t 3 0 % less t h a n t h a t of
s i g n i f i c a n t l y d i f f e r e n t in t h e t w o g r o u p s o f a n i m a l s .
c o n t r o l s . T h e r e d u c t i o n in p o s t j u n c t i o n a l l e n g t h was
T h e s a r c o l e m m a of w o b b l e r m u s c l e s h a d f e w e r a n d
n o t c o r r e l a t e d w i t h age d u r i n g t h e p e r i o d t h a t we ex-
s h a l l o w e r folds. T h i s d i f f e r e n c e was e v i d e n c e d by t h e
a m i n e d ( T a b l e lI1).
l e n g t h s ( T a b l e III). T h e m e a n r a t i o
r a t i o s of t h e p o s t j u n c t i o n a l m e m b r a n e to p r e j u n c t i o -
A c e t v l c h o l i n e receptors TABLE II
We next determined
Summary of animals, areas of presynaptic profiles and aspect ratios n,
whether the reduced post-
j u n c t i o n a l f o l d i n g c o r r e l a t e d w i t h a loss of A C h rec e p t o r s in w o b b l e r m u s c l e . B o t h p o p u l a t i o n s of m u s cle f i b e r s d e m o n s t r a t e d n o n - u n i f o r m d i s t r i b u t i o n s of
number of axons measured.
leSl-a-BT b i n d i n g a l o n g t h e s u r f a c e o f t h e fibers. F o r
Genotype
Age (days)
n
Area (urn-')
Aspect ratio
t h e a u t o r a d i o g r a p h i c a n a l y s i s , we e x a m i n e d at least 7
+/+/+/+/+/+/Mean and S.D.
26 30 36 40 50 58
14 30 30 48 74 51
5.67 _+ 4.36 5.8(I -+ 3.95 5.48 _+ 4.40 6.10 + 5.30 8.84 _+ 7.94 4.48 _+ 2.82
3.66 4.19 3.81 3.36 3.68 3.38
b o t h wr/wr a n d + / - cells, we f o u n d t h e silver g r a i n s
wr/wr wr/wr wr/wr wr/wr wr/wr wr/wr Mean and S.D.
+ 2.07 _+ 2.53 + 2.11 _+ 1.91 _+ 1.74 _+ 1.78
slides e a c h o f w o b b l e r a n d c o n t r o l m u s c l e fibers. I n w e r e d e n s e l y p a c k e d in a r e g i o n t h a t also s t a i n e d for A C h E a n d in s o m e cases s u r r o u n d e d t h e m u s c l e f i b e r (Fig. 4). T h e r e g i o n was 31.7 + 12.gktm l o n g (n = 59) in + / - m u s c l e s a n d was 32.4 _+ l l . l # m
6 . 0 0 _+ 1.33 26 30 36 40 50 58
6 18 32 42 47 57
4.11 5.38 5.77 7.94 6.68 5.53
_+ 1.80 +- 4.26 _+ 4.82 _+ 7.02 _+ 7.46 _+ 6.19
3.56_+0.18 3.66 4.30 3.42 3.86 2.70 3.30
+ 2.07 _+ 2.27 _+ 1.69 _+ 1.89 _+ 1.49 _+ 1.41
l o n g (n -- 58)
in wr/wr m u s c l e fibers. B a s e d o n this e v i d e n c e we concluded that the control and wobbler muscles cont a i n e d A C h r e c e p t o r s at t h e N M J a n d t h a t t h e r e was n o e l o n g a t i o n o f t h e j u n c t i o n a l r e g i o n . T h e r a t e of d e c r e a s e in a m o u n t of l a b e l w i t h i n 3 0 0 / ~ m of t h e e n d p l a t e s was i n d i s t i n g u i s h a b l e in wr/wr a n d + / - fibers. T h e r e was, h o w e v e r , a n a v e r a g e i n c r e a s e in e x t r a -
5.90 + 1.30
3.30 + 0.55
j u n c t i o n a l l a b e l i n g o f w o b b l e r f i b e r s ; t h e n u m b e r of
TABLE III
Summary of animals, ages and membrane measurements Values for mean presynaptic length, mean postsynaptic length and ratio are mean and standard deviations, n, number of muscles/ number of animals.
Genotype
Age (days)
n
n of junctions
Mean presynaptic length (j~m) (a)
Mean postsynaptic length (t*m) (b)
Ratio (b/a)
+/+/+/+/+/+/+/Total
26 32 33 36 40 50 58
2/2 2/2 4/3 2/2 2/2 2/2 3/2
14 168 50 31 43 145 48 529
4.73 4.13 4.93 4.53 5.30 4.96 7.86 5.28
+_ 3.06 -+ 2.75 + 2.20 -+ 2.04 + 2.63 + 3.78 _+ 7.56 + 1.16
9.29 6.64 11.02 7.15 7.75 11.40 12.53 9.44
+ 6.33 - 4.92 _+ 5.18 -+ 3.18 _+ 3.91 _+ 10.18 _+ 11.31 _+ 2.14
2.03 1.61 2.28 1.58 1.48 2.68 1.59 1.86
+_ 0.88 _+ 0.40 _+ 0.51 +_ 0.32 -+ 0.39 + 2.79 + 0.57 + 0.42
wr/wr wr/wr wr/wr wr/wr wr/wr wr/wr wr/wr Total
26 32 33 36 40 50 58
2/2 1/2 2/2 2/2 2/2 2/2 2/2
22 158 46 33 40 89 48 471
3.49 4.64 5.34 4.57 4.85 4.42 8.13 5.06
+ 2.02 + 2.91 _+ 2.46 _+ 2.19 -+ 2.49 +_ 2.25 + 7.29 -+ 1.35
5.06 6.38 6.47 6.14 6.09 6.94 10.14 6.58
_+ 3.60 + 4.34 _+ 3.49 + 3.49 + 3.20 + 5.00 +_ 7.44 -+ 1.54
1.43 1.36 1.22 1.30 1.25 1.43 1.25 1.30
+ 0.39 _+ 0.26 _+ 0.26 _+ 0.29 _+ 0.22 _+ 0.48 +_ 0.47 +_ 0.09
85 silver grains ranged from 56 to 96% higher than corresponding values for control fibers. This increase in the vicinity of the e n d p l a t e was found in all of the 43 w o b b l e r fibers e x a m i n e d in detail and was not found in any of the 30 control fibers. Beyond 300 # m of the junction both wr/wr and control muscle fibers had equivalent background levels of labeling. The wr/wr mutant mouse had two features characteristic of NMJs in early postnatal rodents 22 and junctions that have been denervated early in postnatal life 24, i.e. reduced postjunctional folds and an increase in the distribution of extrajunctional receptors. A third characteristic of early postnatal endplates is that A C h receptors contain 7 rather than e subunits; e subunits are characteristic of adult endplates 9"23. To test whether the A C h receptors in wobbler mice resemble those of neonatal mice, we stained endplates in the lateral rectus muscle with antibodies specific for the y subunit 13. As a control, we also stained sections with antibodies to/3 and 6 subunits which are present at both neonatal and adult endplates. In sections of control adult muscle, antibodies raised against the/3 and 6 subunit bound to junctions of mature control muscle in a pattern similar to the aBT binding pattern (Fig. 5a,e,g,k). H o w e v e r , the antibody directed against the 7 subunit failed to bind to
Fig. 4. Autoradiographs of muscle fibers labeled with laSI-abungarotoxin, demonstrating the distribution of cholinergic receptors on the surface of +/- (A) and wr/wr (B) fibers. Note the concentrated label over both fibers and the higher extrajunctional labeling (arrow) of the wr/wr fiber. Bar = 50~m.
the junction in a pattern similar to that of r h o d a m i n e labeled a - B T (Fig. 5c,i). W h e n sections of wr/wr muscle were tested with the same 3 antibodies, the sections also bound the antibodies against the/3 and 6 subunits (Fig. 5b,f,h,l). However, the sections failed to bind the antibody for the y subunit of the r e c e p t o r (Fig. 5d,j). We interpret these results as evidence of the clustering of mature receptor at the NMJ of the wr/wr muscle.
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d
i
i
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: ....
ii!!i~~
/ii:
:
Fig. 5. Dark-field micrographs of frozen sections of +/- (a,c,e,g,i,k) and wr/wr (b,d,f,h,j,1) endplates. The adjacent sections were stained with either rhodamine-labeled a-bungarotoxin (a-f) or with fluorescein-labeledantibody to the/3 (g,h), y (i,]) or d (k,l) subunit of the ACh receptor. Both mutant and control endplates label with the snake toxin (a-f), indicating the presence of receptor, and the control and mutant endplates are labeled with the antibodies to the fl and 6 subunits (g,h,k,l). Neither (i and j) binds antibody to the y subunit of the ACh receptor. Bar = 10~um.
86 DISCUSSION Wobbler lateral rectus muscle Several general conclusions may be drawn from this study. First, the wobbler NMJs resemble littermate controls in many features. The axons that innervate NMJs in the lateral rectus muscles of the wobbler mouse appear relatively normal, despite the absence of about 30% of the large motoneurons in the abducens nucleus 18. The axons and terminals show none of the typical degenerative features of neurofilament bundles, cytoplasmic densities or increased numbers of dense bodies, and there was no evidence of an enlargement or shrinkage in axon cross-sectional area at any of the ages we examined. These features have been described in degenerating axons in-
nervating cervical axial musculature in wobbler mice 25, and are characteristic of the few extensor digitorum longus synapses that we examined from these wr/wr mice (unpublished results). Furthermore, we failed to find any evidence of sprouting of the nerves either in the mutant or control muscles, based on findings of similar axon cross-sectional area and aspect ratio in wobbler and control, and on preliminary experiments in which we stained cryostat sections of muscles for nerve endings and A C h E 31 (unpublished results). Sprouting has been reported • 74 by M~tsumoto-, who identified apparent attempts at regeneration in nerves of the forelimbs of mice in which the wobbler mutation was maintained on a different genetic background than used in our study. Our failure to find sprouting in lateral rectus muscle may be related to the successful match of nerve to muscle cells at least at the ages we examined (see below). Alternatively, it may be due to a difference in genetic expression between the two backgrounds or different muscles. Second, the composition of fiber types in the mutant lateral rectus is also comparable to that of controls. The fact that the same 3 classes of muscle fibers are represented in wobbler in approximately the same proportions as in normal muscle argues against any compensatory increase in one type. Furthermore, there was an overall reduction in the number of fibers in wobbler muscles which correlated with the number of motoneurons that persisted in the abducens nucleus. These findings suggest that regulation of motor unit size is normal in mutants and oc-
curs prior to 26 days, the earliest age we examined. The most significant difference between wr/wr and control synapses was the reduction in muscle plasma membrane at NMJs in the mutant. The NMJs in extraocular muscles are known to have fewer postjunctional folds than other striated muscle cells 33. Salpeter and her colleagues 33 characterized the ratio of postjunctional fold to primary cleft in the 3 main classes of fiber types and found that the dark red muscle fibers have a large, diffuse endplate with a ratio of 1.8, while the off-white fiber is characterized by a ratio of about 3. In the wobbler mice, the percentage of dark red, off-white and intermediate fibers was unchanged from that of control, yet the average ratio of postjunctional fold to primary cleft was only 1.32 compared to 1.9 in the control animals. This reduction was consistent with our other findings that the change in average fold length was not due to an altered representation of one fiber type in the muscle. Moreover, the reduced folds do not become more pronounced with age. The ratio is reduced at all ages examined, but not significantly greater in older animals. One possible explanation for these results is that the normal maturation of the muscle is arrested at an early stage 26. However, the absence of the 7 subunit of the A C h receptor at wobbler endplates suggests that a maturational defect, if present, is not a general one and is expressed after mature receptors are inserted in the NMJ. Unfortunately, a developmental study of the differentiation of NMJs in wr/wr lateral rectus muscle is impossible because the mutant behavioral phenotype is not expressed until after the junctions have differentiated. Although we have no quantitative estimates, our histochemical evidence supports the conclusion that in wr/wr lateral rectus muscle A C h E is present in the cleft normally, and the light microscopic labeling pattern of a-BT suggests that the location of A C h receptors is similar to that seen in normal mice. These resuits are consistent with other preliminary observations of normal ratios of thickened membranes at the postjunctional surface of the wobbler muscle fibers. Our conclusions about the junctional integrity are limited by the resolution and lack of quantitation available with light microscopic autoradiography. Further studies using electron microscopic autoradiography will be necessary to resolve these points. However, there was a 1.8-2-fold increase in total
87 extrajunctional binding of labeled toxin within 300 ,urn of the endplate in wobbler, and such an increase in extrajunctional labeling can be resolved satisfactorily by light microscopic inspection of autoradiograms of the mutant fibers. The increase resembles the increased binding of toxin to extensor digitorum and sternomastoid muscles of Swiss albino mice as compared to strain 129/ReJ as seen by MatthewsBellinger and Salpeter 22 and the increase in binding near the endplate seen in normal muscle immediately after denervation zl. Whatever the basis for the difference (e.g. altered insertion of receptors, metabolic stability), it suggests that at least indirectly the difference is under genetic control.
Comparison with wobbler axial musculature The pathological features of the lateral rectus endplates in the wobbler mice, specifically the reduced number of fibers and the reduced postjunctional folds, with intact and relatively normal synaptic terminals, differ from features reported for axial musculature in wr/wr innervated by cervical motoneurons in several conspicuous ways. In the latter muscles, fibers show frank atrophy 25, and some axons that innervate the muscles have been described as degenerating, based on the presence of dense accumulations of neurofilaments I and collapse of myelin sheaths 25. Furthermore, cervical spinal neurons are characterized by 'foamy cell bodies', but the abducens motoneurons fail to show any vesicular changes in the cell somas. Thus, the involvement of neurons controlling extraocular muscle of the mutant contrasts with the involvement of other motoneurons in the mutant that also innervate skeletal muscle. In this way, the wobbler mutation causes changes that resemble human motor neuron diseases, although less marked and apparently non-progressive in nature.
Other examples of NMJ abnormalities Functional denervation of normal muscle results in general muscle atrophy and, among other changes, the development of embryonic-like ACh receptors along the muscle fiber 4. To test for some subtle functional denervation effect that might not have been identifiable ultrastructurally, we applied t2SI-ct-BT and used immunocytochemistry to determine the gross distribution and maturity of ACh receptors in wr/wr lateral rectus muscle. Based on the clustering
of ACh receptors and the absence of the V subunit at the NMJ of wr/wr muscle, we conclude that there was no abnormal spread of immature receptors along the entire muscle cell surface that would be characteristic of an effect of denervation. In its lack of elaborate postjunctional folds, the wobbler model is similar to two other mutations, dystrophic and motor endplate disease, in which there is also an abnormal infolding of the postsynaptic membrane 91°'3°. In the dystrophic mouse, however, the mutation affects not only neuronal and muscle cell viability but also early developing Schwann cells 15. In motor endplate disease of mice the muscle becomes atrophied, despite profuse sprouting of axons and the absence of any degenerative changes in motor neuron cell somas. A similar reduction in muscle folds is characteristic of the chronic phase of experimental autoimmune myasthenia gravis t2. In all of these diseases, the interdependence of motoneuron and muscle cells is conspicuous. The transplantation of mutant muscle cells into control host animals provided important insight into the primary locus of the mutant gene action in the dystrophy model14'21; a similar approach may be amenable for future studies of the wobbler mutation.
CONCLUSIONS This is the first report of a study of the ultrastructure of the NMJ of the wobbler mutant mouse. We have found the most distinctive feature in lateral rectus muscle of these animals is a reduction in the length of the postjunctional folds. Since the ACh receptors at the NMJ are not dispersed along the surface of the muscle and are apparently composed of mature subunits, we speculate that the formation or maintenance of normal postjunctional folds and the differentiation of the receptor are independent events in the development of the normal junction. Our results are consistent with those of Schuetze and Vicini 34, who found that the change to mature mean channel open time of ACh receptors at developing endplates was not correlated with the elaboration of postjunctional folds. How the lack of normal folds affects neuromuscular transmission or whether the structure reflects some abnormality in nerve-muscle interdependence and neuron survival remain to be
88 a n s w e r e d . O t h e r n o n - n e u r o n a l cell types a f f e c t e d by
ACKNOWLEDGEMENTS
the m u t a t i o n in wr/wr mice include: s p e r m a t o z o a 17"2°, l y m p h o c y t e s 3 and h e p a t o c y t e s 2. W e suggest that the
W e are grateful to Ms. Nusi P. D e k k e r and Mr.
w o b b l e r m u t a t i o n m a y also directly involve muscle
R a m a R a n g a n a t h y for their technical assistance, to
cells. F u r t h e r m o r e , the w o b b l e r m u t a n t m o u s e m a y
Mr. Y o n g G u for his g e n e r o u s gift of purified anti-
p r o v i d e a n o v e l and useful animal m o d e l for studying
bodies, to Mr. G u and Dr. Z a c h H a l l for v a l u a b l e ad-
p o s t j u n c t i o n a l fold f o r m a t i o n and the role this m e m -
vice and to Mr. V o j t e c h Licko for helpful discussions.
b r a n e specialization plays in n o r m a l n e u r o m u s c u l a r
T h e w o r k was s u p p o r t e d in part by N . I . H . G r a n t
physiology.
P H S R01 N S 13533.
REFERENCES
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