Journal of Neurocytology 20, 969-981 (1991)

Structure and function of the neuromuscular junction

in young adult mdx mice
P. R. L Y O N S * a n d C. R. S L A T E R
The Muscular Dystrophy Group Laboratories, Newcastle General Hospital, Newcastle upon Tyne NE4 6BE, UK

Received 25 April, 1991; revised 27 June 1991; accepted 8 July 1991

Summary
Dystrophin, the protein product of the gene responsible for X-linked muscular dystrophies, shares structural features with
the cytoskeletal proteins spectrin and c~-actinin. Like spectrin, it is localized at the cytoplasmic surface of the sarcolemma and
is particularly concentrated in the subsynaptic region of the neuromuscular junction. Mdx mice have a profound deficiency of
dystrophin and develop a necrotizing myopathy in the first weeks of life. Abnormalities of the neuromuscular junction,
including a redistribution of postsynaptic molecules and reduction in synaptic folding, are also observed. We have studied
these mice to see whether the lack of dystrophin has a specific effect on the structure and function of their neuromuscular
junctions.
Using a fore-limb muscle from 8 week old mdx mice we confirm the previously described postsynaptic structural changes
and in addition show that many nerve terminals are abnormally'complex. We demonstrate that these structural abnormalities
are found exclusively at neuromuscular junctions on regenerated muscle fibres. Despite these structural abnormalities,
miniature endplate potential frequency, the quanthl content of endplate potentials, the amplitude and time course of
miniature endplate currents and the number of acetylcholine receptors at the postsynaptic membrane are normal in mdx mice
of this age. We conclude that in the mdx mouse the absence of dystrophin from the postsynaptic membrane has little direct
effect on the function of the neuromuscular junction but that degeneration and regeneration of muscle fibres leads to
remodelling of both its pre- and postsynaptic components.

Introduction
The protein dystrophin is present in normal skeletal
muscle underlying the plasma membrane. While its
function is unknown, the homology of regions of its
amino acid sequence with that of spectrin and oL-ac-
tinin (Koenig et al., 1988), and its close association with
a membrane glycoprotein (Campbell & Kahl, 1989) at
the cytoplasmic surface of the sarcolemmal membrane
(Zubrzycka-Gaarn et al., 1988) suggest that it may be a
component of the membrane associated cytoskeleton.
Dystrophin is particularly concentrated at the normal
neuromuscular junction (NMJ) (Miike et al., 1989;
Shimizu et al., 1989) and it is reasonable to enquire
whether it has an influence on neuromuscular trans-
mission, perhaps by influencing the distribution of
synaptic molecules such as acetylcholine receptors
(AChRs). Such a role is consistent with the finding of a
significant amount of dystrophin in purified mem-
brane fractions rich in AChRs, derived from the
electrocyte of the Torpedo marmorata (Jasmin et al.,
1990).
The structural and functional consequences of the
absence of dystrophin from the NMJ can be examined
in the mdx mouse (Bulfield et al., 1984), which, due to a
point mutation on the X-chrontosome ,(Sicinski et at.,
1989) has a profound deficiency of dystrophin (Hoff-
man et al., 1987). Outwardly there is little evidence of
myopathic disorder in this animal but, from about the
third week of life, acute muscle fibre necrosis occurs
followed by fibre regeneration with minimal fibrosis
(Tanabe et al., 1986; Camwath & Shotton, 1987;
Coulton et aI., 1988; Karpati et aI., 19881). At the NMJ,
structural abnormalities consisting of a reduction of
secondary synaptic folding and disruption of acetyl-
cholinesterase (ACHE) staining areas develop in 4- to
6-week-old animals and become more pronounced
with increasing age (Torres & Duchen, 1987). Torres
and Duchen suggest that these structural changes are
a secondary consequence of muscle fibre necrosis and
regeneration rather than a direct effect of dystrophin
deficiency.
In this report, we describe some striking structural
abnormalities of the motor axon terminal and show
that these are associated with redistribution of the key
postsynaptic molecules, AChR and ACHE. We show

* To whom correspondenceshouldbe addressed

0300-4864/91 $03.00 +.12 9 1991 Chapman and Hall Ltd

970
that these c h a n g e s occur exclusively on r e g e n e r a t e d
muscle fibres, consistent with the h y p o t h e s i s of Torres
a n d D u c h e n . We also s h o w that in y o u n g adult mdx
mice there is no detectable defect of transmitter release
or action, n o r a n y c h a n g e in the n u m b e r of A C h R s per
NMJ. We conclude that the deficiency of d y s t r o p h i n in
mdx mice has little direct or immediate effect o n the
events in n e u r o m u s c u l a r transmission but m a y lead
indirectly to the long term reorganization of the NMJ.
Methods
All studies were performed on C57/B110 mice. Individuals of
this strain, but homozygous for the mdx mutation, were
derived from breeding pairs originally supplied by Dr G.
Bulfield from the Poultry Research Centre, Midlothian,
Scotland. All animals were within a week of two months of
age except where indicated. Animals were killed by cervical
dislocation and the epitrochleoanconeus muscle (ETA), a
thin sheet-like muscle on the inner aspect of the upper
fore-limb (Bradley et aI., 1989) was removed and immedi-
ately placed in an appropriate physiological solution (Liley,
1956) prior to morphological and physiological studies.
MORPHOLOGICAL STUDIES
Muscle histology
Histological features of the ETA muscle were examined in
6 Ixm transverse frozen sections stained with haematoxylin
and eosin, from four control and four mdx animals. Fibre
diameters and the percentage of muscle fibre profiles
containing central nuclei were determined in these prep-
arations.
The proportion of muscle fibres containing central nuclei
was also estimated in muscles which had been disrupted
into single muscle fibre fragments and labelled with a
fluorescent nuclear dye. This was done using a 2 mm wide
strip cut from the middle of ETA muscles that had been fixed
in 4% p araformaldehyde in 0.1 M phosphate buffer (pH 7.2)
at room temperature for I h. Each strip was disrupted into
single fibre fragments using an Ultraturrax homogenizer
and then labelled with bisbenzimide (Sigma) 0.05 txg ml -~
for 15 min. Following washing and centrifugation, the cell
fragments were resuspended in 0.5 ml of deionised water,
placed on a slide and viewed under incident illumination
with suitable filters.
Analysis of innervation pattern
Whole muscles from five control and five mdx mice were
fixed in 4% paraformaldehyde in 0.1M phosphate buffer
(pH 7.2) at room temperature for 2 h prior to washing
overnight in Liley's solution at 4~C. Acetylcholinesterase
(ACHE) was stained using a slightly modified Karnovsky
and Roots technique (Karnovsky & Roots, 1964), in which
maleate was used as a buffer and incubation in a solution
containing a 1:20 concentration of substrate (Acetylthio-
choline iodide) for I h was performed prior to incubation
with full strength substrate for 10 min. Axons were then
impregnated with silver (Tsuji & Tobin-Gros, 1980) and the
L Y O N S a n d SLATER
preparations were mounted in glycerol. The course and
branching of intramuscular nerves and the configuration of
nerve terminals were examined, and the functional terminal
innervation ratio (FTIR), defined as the ratio of the number
of muscle fibres innervated by a given number of terminal
axons (Co6rs & Woolf, 1959), was calculated.
AChE and AChR distribution
The total area of AChE staining at individual NMJs was
measured in control and mdx mouse muscles prepared by
the method described above and, following fixation in 5%
glutaraldehyde in phosphate buffer (0.1 M, pH 7.2) for 90
min, by the method described by Strum and Hall-Craggs
(1982). Both types of preparation were teased into single
fibres and the areas of AChE staining were measured by
tracing around the individual zones of labelling at each NMJ
visualized 'en-face' using computer-based morphometry
(Imagan; Kompira). The diameter of the associated muscle
fibre, taken to be the greatest width of the fibre perpendicu-
lar to its long axis in the region of the AChE staining, was
also measured. As a result of shrinkage during glutaralde-
hyde fixation the two methods of preparation produce
different mean measurements, but no systematic differences
were seen when measurements from control and mdx
muscles were compared. Only quantitative data from para-
formaldehyde fixed material are included.
The distribution of AChR at individual NMJs was also
examined in muscles from four control and four mdx mice.
Aceytlcholine receptors were labelled with c~-bungarotoxin
conjugated with rhodamine (Slater, 1982) during incubation
at 4~ for 2 h. Following fixation in paraformaldehyde in
0.1M phosphate buffer (pH 7.2) at 4~ for 30 min, the
muscles were teased into bundles, and mounted in glycerol.
In one set of muscles the relationship between the
distribution of AChR molecules and muscle fibre regener-
ation, ~denoted by the presence of central nuclei, was
examined. Muscles were initially labelled with rhodamine-
conjugated c,-bungarotoxin, then fixed and muscle frag-
ments containing NMJs were prepared and labelled as
described above.
AChR binding sites per NMJ
125I-o~-bungarotoxin labelling was employed to assess the
number of AChR binding site present at NMJs in ETA
muscles. Unfixed muscle from two control and two mdx
animals were incubated at room temperature with 30 ~1
12SI-c,-bungarotoxin (Amersham, specific activity 200
Ci mmol 1) in 120 ixl physiological solution, for 3 h. After
washing for a total of 1 h in four changes of Liley's solution,
the specimens were kept overnight at 4~ C. Following
fixation in 5% glutaraldehyde for i h the muscles were cut
transversely into 8 or 9 sections, each I mm wide, which
were then counted in a gamma counter for 30 min. Large
values indicating the presence of NMJs were obtained in one
or two sections from the middle of each muscle. The
remaining sections gave low values, all of similar magni-
tude, representing the nonspecific background labelling of
muscle in sections not containing NMJs. Following com-
pletion of radioactive counts, a section from the middle of
each muscle was dehydrated in ethyl alcohol and embedded
in Araldite and from these complete transverse sections,
0.5 }xm thick, were prepared and stained with Toluidine
NMJs in mdx mice
Blue. The number of muscle fibres counted in these com-
plete sections provided an accurate estimate for the number
of NMJs present in the muscle at the end-plate zone and,
following subtraction for background, the amount of label-
ling per NMJ was calculated. Results were expressed as
binding sites per NMJ, rather than the number of receptors
per NMJ, but it is assumed that each receptor has two
binding sites.
Ultrastructural studies
ETA muscles from four control and four mdx animals were
incubated with c~-bungarotoxin labelled with horseradish
peroxidase (Nakane & Kowaoi, 1974) for 12 h at 4~ C prior to
fixation in Karnovsky's fixative (Karnovsky, 1965) for I h~
They were then reacted with 0.025% diaminobenzid~ne
(Sigma) before being processed for electron microscopy.
After embedding in Araldite, ultrathin sections were cut and
fields containing NMJs were photographed at a primary
magnification of • 10 000 and then negatives were printed
at a final magnification of x 30 000 for morphometric
studies.
Electronmicrographs were analyzed morphometrically
using methods based on Engel and Santa (1971). Where
several profiles of the same nerve terminal were visualized
on a section through an individual NMJ, the mean of their
areas was determined and this value used to calculate the
'mean nerve terminal area' for each of the animal groups.
Many of the nerve terminal profiles had definable regions of
surface membrane that were closely apposed, across the
synaptic cleft, to postsynaptic membrane labelled with
c~-bungarotoxin. The sum of the lengths of these regions at
individual NMJs were used to calculate the 'mean presynap-
tic length' for the two animal groups. To express the extent
of folding, we have defined the 'folding index' as the ratio of
the total postsynaptic length, including folds, to the length
of the postsynaptic surface, measured across the top of the
folds.
Physiological recordings
All physiological recordings were made from whole ETA
muscles dissected with an intact nerve supply. Muscles were
pinned out under slight tension in bathing fluid and the
proximal end of the nerve was drawn into a suction electrode
for stimulation. All recordings were made at 22-23~ C in
solutions (see below) bubbled with 95% 02 and 5% CO2
using intracellular glass capillary micro-electrodes, filled
with acidified 3 M potassium acetate and with resistances
Table 1. Morphology and innervation pattern in 2-rnonth-old control and mdx mouse ETA
muscles
Mean fibre diameter (~m) (in
frozen sections)
Preterminal sprouting
Ultraterminal sprouting
FTIR
Mean number of boutons per NMJ
See text for definition of FTIR. Figures in parentheses: (SD,number of muscles studied).
971
ranging from 3 to 7 Kfk Recordings were stored on magnetic
tape (Racal store 4, 7.5 i.p.s.). For analysis, responses were
filtered at 5 kHz for potentials and 1.5 kHz for currents.
Analogue signals were digitized at a frequency of 17.5 kHz
using a CED 1401 interface (Cambridge electronics) con ~
trolled by a BBC B microcomputer programmed to extract
peak amplitudes and decay kinetics (Fawcett et al., 1978).
Only recordings of potentials with a rise time (10% to 90% of
peak amplitude) of less than 1.2 ms were accepted.
Passive electrical properties of muscle fibres were exam-
ined by inserting a pair of electrodes with their tips less than
50 ~m apart, one to pass current and the other to record the
induced voltage change. The membrane potential was
initially set to - 8 0 mV. Input resistance of the muscle fibre
(r~put), defined as the ratio of the amplitude of a 20ms
rectangular current pulse to the peak voltage change in-
duced by it, was calculated. The membrane time constant
(~rc) was taken to be the time to reach 84% of the peak
induced voltage change (Hodgkin & Rnshton, 1946).
Spontaneous miniature end-plate potentials (mEPPs) and
currents (mEPCs) were recorded in Liley's solution. Cur-
rents were recorded using a two electrode voltage clamp
(Axoclamp, Axon Instruments). Subthreshold endplate po-
tentials (EPPs) evoked by stimulation at i Hz were recorded
in a modified solution containing a reduced calcium concen-
tration (1.5 mM) and an increased magnesium concentration
(8 raM). The amplitudes of all evoked responses were
corrected to a standard membrane potential of - 8 0 m V
assuming a reversal potential of 0 mV. The quantal content
was estimated from the ratio of the mean amplitude of the
EPPs at an individual NMJ, corrected for non-linear sum-
mation (Martin, 1955), to the mean amplitude of the mEPPs
recorded at the same NMJ.
Results
MORPHOLOGY
Muscle
E p i t r o c h l e o a n c o n e u s muscles from both control a n d
mdx mice contain b e t w e e n 550 a n d 700 muscle fibres.
The m e a n fibre diameters in frozen sections of control
a n d mdx muscles did not differ significantly (Table 1).
In m o u s e muscle, r e g e n e r a t e d muscle fibres can be
identified by the presence of central nuclei. Such fibres
have been seen in previous studies o n mdx m o u s e
Control mdx t-test
28.6 (1.08,4) 25.8 (3.5,4) N.S.
5% (1.9,5) 3.9% (1.8,5) N.S.
0.9% (0.88,5) 1.3% (1.04,5) N.S.
1.07 (0.02,5) 1.08 (0.04,5) N.S.
0.97 (0.14,5) 5.30 (0.35,5) p < 0.001
972 LYONS and SLATER

Table 2. Regenerated muscle fibres and remodelling of NMJs in control and mdx ETA
muscles

control mdx

2-month-old
Profiles with central nuclei seen in transverse section 0% (2310,4) 48% (2665,4)
Cell fragments with central nuclei seen in disrupted
muscles 0% (50,2) 82% (148,4)
Cell fragments with central nuclei and remodelled
AChE labelling 0% (25,2) 81% (81,4)
Nerve terminals with > 2 terminal boutons 14% (405,5) 74% (375,5)
1-month-old
Cell fragments with central nuclei seen in disrupted
muscles 0% (5O,2) 24% (82,4)
Cell fragments with central nuclei and remodelled
AChE labelling 0% (25,2) 26% (57,2)
Nerve terminals with > 2 terminal boutons 14% (100,2) 29% (100,2)

Figuresin parentheses: (numberstudied, numberof musclesstudied).

muscles (Tanabe et al., 1986; Torres & Duchen, 1987;
Carnwath & Shotton, 1987) and have been quantified
in transverse sections. In transverse sections of the
ETA muscle from 2-month-old mdx mice, 50% of
muscle fibre profiles contain central nuclei (Table 2).
When fragments of single muscle fibres 2ram in
length are studied after labelling with bisbenzimide,
chains of central nuclei are easily seen, often interrup-
ted by short gaps (Fig. 1). In fragments prepared in
this manner from the ETA muscles of two mdx mice
aged two months, central nuclei were observed in 82%
(122/148) of muscle fibres, whereas none were seen in
fragments of similarly aged control muscles (Table 2).
Therefore, in comparison to the examination of trans-
verse sections, this method provides a more sensitive
way of estimating the number of regenerated muscle
fibres.
In muscles from two mdx animals aged one month
the number of fragments with central nuclei was only
24%, and again none were seen in muscles from two
control animals at this age (Table 2).
Innervation pattern
The pattern of terminal innervation in control and mdx
ETA muscles appeared similar with no significant
differences in the amount of collateral innervation,
pre- and ultraterminal neuronal sprouting or the
calculated FTIR (Table 1). However, nerve terminal
architecture was different in the mdx mouse muscles,
frequently appearing more complex with a great
increase in the number of fine terminal aborizations,
m a n y bearing bouton-like swellings (Fig. 2). The
number of these bouton-like swellings per nerve
terminal examined is shown graphically in (Fig. 3). In
muscles from control mice the majority of nerve
terminals have no boutons and only 14% have more
than two. In comparison, in muscles from mdx mice,
26% have two boutons or fewer and the remainder
form a separate population with a modal value of
approximately six.
In two ETA muscles from one-month-old mdx mice,
nerve terminals with more than two boutons were also
observed, but at a lower frequency (Table 2).
AChE and AChR distribution
At NMJs in control muscles the distributions of AChE
and AChR labelling appeared identical, both proteins
being confined to simple confluent postsynaptic re-
gions. At NMJs in mdx mouse muscles, AChRs were
frequently present in numerous small spots, corre-
sponding to the boutons on the motor axon terminals
(Fig. 2) (see below). A similar redistribution of AChE
shows that a close association between two postsyn-
aptic components is maintained in mdx mice.
Redistribution of postsynaptic molecules was found
exclusively on muscle fibres with central nuclei. This
was determined by studying fragments of single
muscle fibres in which the nuclei had been labelled
with bisbenzimide and AChRs with rhodamine-
conjugated o>bungarotoxin. Both the redistribution of
AChRs and the presence of central nuclei were un-
ambiguous and easy to score in these preparations
(Fig. 1). In a sample of 81 muscle fibre fragments from
2-month-old mdx mice, the distribution of nuclei and
AChRs were either both normal (15, 19%) or both
abnormal (66, 81%) (Table 2). In 57 fragments from
t w o mdx mouse ETA muscles studied at four weeks of
age the same invariant association of altered AChR
distribution with central nuclei was present, but in
only 26% of the fragments. At neither age did any
fragments of control muscles have central nuclei or
modified AChR distribution.
Fig. 1. Muscle fibre fragments from 2-month-old animals labelled with bisbenzimide and rhodamine-coniugated c,-bunga-
rotoxin. See text for methods. (a), (c), (e) and (g) from control mice; (b), (d), f) and (h) mdx mice. Scale bar = 20 ~m. (a), (b) In
regions distant from NMJ, central nuclei visible in regenerated mdx fibre but not in control fibre. (c), (d) At the NMJ, central
nuclei again visible in regenerated mdx fibre. (e), (f) In same fragments as (c) and (d) but at different focal plane, nuclei
associated with the NMJ are seen. (g), (h) AChR labelling of the same fragments seen in (c) and (d). (See also Fig. 2c,d).
974 LYONS and SLATER

Fig. 2. (a), (b) Nerve terminals in silver impregnated whole mount preparations of the ETA musde from (a) 2-month-old
control mouse and (b) 2-month-old mdx mouse. (c), (d) AChRs labelled with rhodamine-conjugated c~-bungarotoxin in teased
fibre preparations from (c) 2-month-old control mouse ETA muscle and (d) 2-month-old mdx mouse ETA muscle. (e), (f) AChE
activity demonstrated by Strum and Hall-Craggs method in teased fibre preparation from (e) 2-month-old control mouse ETA
muscle and (f) 2-month-old mdx mouse ETA muscle. See text for methods. Scale bar = 20 ~m.

Despite these alterations in distribution, the total
area of postsynaptic AChE labelling (Fig. 2) did not
differ significantly b e t w e e n NMJs in control and mdx
m o u s e ETA muscles (Table 3). In normal muscle a
significant positive correlation b e t w e e n the area of
postsynaptic AChE labelling and muscle fibre di-
ameter has been described (Co6rs & Woolf, 1959;
Harris, 1954; Korneliussen, 1972). Scatter diagrams
relating these two parameters for control and mdx
mouse ETA muscles (Fig. 4) reveal that this correlation
NMJs in mdx mice 975

5O 80 (a)
control r////.~
'E
- 40 rndx 1
70

60
30
F= ~50
Y,
20 ~4o
A ~ z~ a
~ 3o
~

2o A A r=0.524
v
l m l i _ _ _ _ A
0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 10
Bouton swellings per nerve terminal
0 l l I J I - I - - ' - F ~ I
Fig. 3. Number of bouton-like swellings per nerve terminal 100 200 300 400 500 600 700 800
in five control and five mdx mouse ETA muscles. Results
expressed as a percentage of the total number of terminals AChE area (pm a}
examined for each group (405 control mouse terminals; 375 80
mdx mouse nerve terminals). A (b)
A
70 A
A A
Table 3. Labelling of AChR at 2-month-old control and mdx 60 A A
mouse NMJs E A
SO a a a a A /
A /
Control mdx
40
Mean postsynaptic 349 (36.1,89,4) 361 (30.7,72,4)
| 30
AChE area (p,m2) t_

2O A ~ r=0.333
Mean muscle I 2.30 2.10
AA A
receptor muscle 2 2.40 2.20 10
sites per NMJ
(X 10 7) A
0 r T I t ~ I

0 100 200 300 400 500 600 700 800

Figures in parentheses: (SD, n u m b e r of NMJs studied, n u m b e r of
muscles studied). AChE area {Nm 2)

Fig. 4. Scatter diagram relating AChE area to fibre diameter

is m a i n t a i n e d for both, a l t h o u g h the d e g r e e of cor-
relation is less m a r k e d for mdx m o u s e muscle
(r = 0.524, p < 0.001 for control, r = 0.333, p < 0.005
for mdx).
125I-oL-bungarotoxin binding sites per NMJ
The n u m b e r of 125I-oL-bungarotoxin binding sites per
NMJ was similar in control and mdx muscles (Table 3).
Since the m e a n area of postsynaptic specialization,
based o n AChE areas, does not differ significantly
b e t w e e n the two g r o u p s of muscles, the average
density of AChRs appears to be u n a l t e r e d at the
r e m o d e l l e d NMJs of mdx mice. In b o t h cases the
density of 125I-o~-bungarotoxin binding sites is 60-
70 x 10 3 t~m-2; that for AChRs is a s s u m e d to be 30-
35 x 10 3 p~m -2.
Ultrastructure of the NMJ
The usual cellular a n d sub-cellular c o m p o n e n t s of the
NMJ w e r e p r e s e n t in all sections of NMJs from mdx
m o u s e muscle studied at the EM level. M o r p h o m e t r i c
in teased fibre preparations for (a) 2-month-old control
mouse ETA muscles (4 muscles, 89 NMJs), (b) 2-month-old
mdx mouse ETA muscles (4 muscles, 72 NMJs).
analysis indicated that the size of individual m o t o r
axon terminal b o u t o n s was n o r m a l as was the extent of
their contact with the postsynaptic surface, either in
absolute terms or, as a fraction of the postsynaptic
surface length in contact with the n e r v e ('occupancy').
O n the postsynaptic side, simplification of the
postsynaptic m e m b r a n e , with a reduction in the
n u m b e r of s e c o n d a r y synaptic folds, was seen at m a n y
mdx m o u s e NMJs (Fig. 5). The extent of simplification
was variable at different NMJs a n d at different
portions of individual NMJs.
M o r p h o m e t r i c analysis confirmed a statistically sig-
nificant reduction in the a m o u n t of s e c o n d a r y synaptic
folding at NMJs from the mdx m o u s e (Table 4). Despite
this reduction in folding AChRs r e m a i n well localized
to the region of nerve contact at mdx mice NMJso N o
other significant ultrastructural abnormalities were
identified.
NMJs in mdx mice 977

Table 4. Morphometric analysis of NMJs in control and mdx mouse ETA muscles

Control mdx t-test

Mean nerve terminal area (~Lm2) 3.39 (1.53) 4.07 (2.46) N.S.
Mean presynaptic length (~m) 9.4 (4.7) 8.8 (5.2) N.S.
Mean postsynaptic surface
length (~m) 11.8 (6.5) 10.3 (6.3) N.S.
Occupancy 79.7% 85.4% N.S.
Mean postsynaptic fold length
(~m) 66.3 (36.4) 26.4 (9.97) p > 0.001
Folding index 5.75 (1.69) 3.41 (2.26) p ,'> 0.001

See text for definitions. SDin parentheses. All values relate to 19 NMJs studied in 4 control muscles and
21 NMJs studied in 4 mdx muscles.

PHYSIOLOGY stimulation at 1 H z in a r e d u c e d calcium solution

Basic electrical properties
Resting m e m b r a n e potentials a n d the passive cable
properties of control a n d mdx ETA muscle fibres w e r e
not significantly different (Table 5). These findings are
consistent with those of H o l l i n g w o r t h and colleagues
(1990) w h o , in a detailed s t u d y of linear cable con-
stants in the extensor digitorum longus muscle of
control a n d mdx mice, f o u n d n o significant differ-
ences.
Transmitter release
Neither the f r e q u e n c y of s p o n t a n e o u s mEPPs nor the
quantal content of e v o k e d transmitter release d u r i n g
differed significantly b e t w e e n control and mdx m o u s e
NMJs (Table 5).
Transmitter action
The postsynaptic r e s p o n s e to a single q u a n t u m of
ACh, assessed b y examining mEPPs in normal
physiological solutions at control a n d mdx m o u s e
NMJs, was not significantly different (Table 5). The
amplitude of mEPPs d e p e n d on the cable properties of
the muscle fibre u n d e r study. A l t h o u g h no systematic
difference in i n p u t resistance was seen b e t w e e n the
two g r o u p s of muscles, mEPC amplitudes, which are
not influenced b y passive cable properties, w e r e also
studied. Again there was n o significant difference

Table 5. Physiological properties of the control and mdx ETA muscles and NMJs

Control mdx t-test

Passive properties
Membrane potential (mV) 78 (1.5,5) 75 (2.2,5) N.S.
rinput(k~'~) 339 (85,6) 341 (36,5) N.S.
Trc (ms) 4.15 (0.62,6) 4.23 (0.7,5) N.S.
Transmitter release
Mean frequency of mEPPs
(s -1) 1.46 (0.34,7) 1.39 (0.3,6) N.S.
Mean quantal content 5.08 (1.22,5) 5.48 (0.96,7) N.S.
Transmitter action
Mean mEPP amplitude (mV) 0.55 (0.12,9) 0.45 (0.15,7) N.S.
Mean mEPC amplitude (nA) 3.08 (0.59,6) 3.11 (0.88,4) N.S.
Mean mEPCr (ms) 1.18 (0.20,6) 1.25 (0.32,4) N.S.

Figures in parentheses (SD,number of muscles studied).

Fig. 5. Electron micrographs of ot-bungarotoxin/immunoperoxidase labelled NMJ from (a) 2-month-old control mouse ETA
muscle and (b) 2-month-old mdx mouse ETA muscle. Scale bar = 1 ~m. Note complete absence of postsynaptic folds at one of
contacts in the mdx mouse,
978 LYONS and SLATER
5o (a) no dystrophin, we conclude that this protein does not
control r////J
play a significant role in the events governing the
40 rndx I immediate effects of ACh on the postsynaptic surface.
The saturated disc model for neuromuscular trans-
mission (Salpeter, 1987) predicts that AChR density
has a major influence on the conductance change
occurring in response to a single quantum of trans-
mitter. In a study of mdx mouse diaphragm, Nagel and
12~176
lO colleagues (1990) found no significant difference in
mEPP amplitude at 2.5 and 8.5 weeks of age when
compared to control animals. Because mEPP ampli-
0 i ; ~ i i ; I~', ,'~', ,' I" tude is influenced by the passive cable properties of
1 2 3 4 5 6
the muscle fibre, we have also measured the ampli-
tude of mEPCs, which is directly related to the
mEPC amplitude (hA)
magnitude of the conductance change through the
50 (b) postsynaptic membrane. The amplitudes of mEPCs at
NMJs in ETA muscles from mdx mice are normal,
control which suggests that AChR density is also normal at
_ 40 mdx
these NMJs. The results of our studies of 1251-
e~-bungarotoxin binding are consistent with this sug-
"6 30 gestion, and are in agreement with similar findings of
Nagel and colleagues (1990) in their studies of 8.5

x2010 1 i week old animals.

o P/I, !
0.375 0.625 0.875 1.125 1.375' '1.625'
m E P C T (ms)
Fig. 6. Distribution of (a) mEPC amplitudes and (b) mEPC
exponential decay time constants (mEPCu) at 26 control and
29 mdx mouse NMJs. Results expressed as a percentage for
each animal group. In (a) the bin size is I nA with the value
given being the mid point. In (b) the bin size is 0.25 ms with
the value given being the mid point.
between the values obtained for control and mdx
mouse muscles. (Table 5 and Fig. 6).
Under normal circumstances, the time course of the
decay of mEPCs is primarily determined by, and
provides an estimate of, the mean channel open time
for the ACh gated ion channel at the NMJ. There was
no significant difference between the time constant of
the exponential decay of mEPCs (mEPCr at control
and mdx mouse NMJs (Table 5 and Fig. 6) suggesting
that the ion channels have similar properties.
Discussion
At the NMJ, the action of ACh is largely dependent on
the characteristics of the postsynaptic membrane. We
have found that both the amplitude and time course of
mEPCs at NMJs of mdx mice are indistinguishable
from those in genetically normal animals of the same
age and background. Since mdx mice have essentially
We assessed the kinetics of the AChR ion channel by
examining the exponential decay time constant of
postsynaptic currents, which has been shown to be
similar to the mean channel open time (Anderson &
Stevens, 1973; Colquhoun et al., 1977). Our values are
1.875 2.125
similar to those reported by others for mouse dia-
phragm (Quastel & Linder, 1975) and we have found
no significant differences between control and mdx
animals, which suggests that the receptor channel
kinetics at the mutant mouse NMJ are essentially
normal. Since the great majority of mdx mouse muscle
fibres do not contain detectable amounts of dys-
trophin, these results taken collectively imply that its
absence has no primary effect on the density of AChRs
at the NMJ or their function.
Although the density and function of AChRs is
normal in the muscles we studied from mdx mice, their
distribution is not. Our studies with rhodamine-
conjugated bungarotoxin show that in the mutant
mice, the AChRs tend to be present in small clusters a
few p,m in diameter. Our EM studies indicate that
these correspond closely to the regions of contact with
the numerous individual boutons that characterize the
axon terminals of the mdx mouse. This redistribution
of AChR molecules is paralleled by that of ACHE, an
enzyme closely associated with the extracellular basal
lamina. Thus, both these key molecular components
of the NMJ come to be associated with the remodelled
axon terminals of the mdx mice.
Reduced secondary synaptic folding has been de-
scribed in a number of muscles in the mdx mouse
(Torres & Duchen, 1987). In the ETA muscle, reduced
folding is often restricted to regions of individual
NMJs. The mechanisms underlying folding are un-
NMJs in mdx mice
known but the differences in amount of folding at
different boutons of the same NMJ suggest that a very
local influence of the nerve is involved.
Qualitatively similar postsynaptic abnormalities are
seen in other inherited myopathic disorders such as
dy/dy mouse dystrophy (Harris & Ribchester, 1979),
the human disease Duchenne muscular dystrophy
(Harriman, 1976; Sakakibara et al., 1977) and in the
toxin-induced necrotizing myopathies (Duchen et al.,
1974; Grubb et al., 1991). In all three conditions, when
allowance is made for varying fibre diameter and input
resistance, the amplitude of postsynaptic response
does not differ significantly from control values (Har-
ris & Ribchester, 1979; Sakakibara et al., 1977; Grubb et
al., 1991) implying little change in postsynaptic recep-
tor density.
Torres and Duchen's findings (1987) indicate that
postsynaptic abnormalities in the mdx mouse become
more obvious with increasing age, possibly reflecting
recurrent damage in the region of the NMJ due to
repeated muscle necrosis followed by regeneration.
Certainly by the age of 37 weeks in diaphragm the
abnormalities are conspicuous (Nagel et al., 1990) and
at this age they are associated with a significant
decrease in AChR density and in the postsynaptic
response to ACh. Our observations indicate that the
remodelling of postsynaptic structures is very closely
associated with degeneration and regeneration of the
muscle fibre and may well be a result of that process,
rather than a direct response to dystrophin deficiency.
However, we can not exclude the possibility that the
absence of dystrophin has some subtle effect on the
process of regeneration.
The abnormalities of nerve terminal architecture
noted in this study are particularly striking. Increasing
complexity of the nerve terminal seems to parallel
changes in the distribution of AChR and ACHE. There
is little dystrophin in normal motor neurons and
therefore these structural changes in the mdx mouse
are unlikely to reflect its absence from the nerve.
Quantal content and the frequency of spontaneous
release in 8-week-old animals do not differ signifi-
cantly, indicating that the intrinsic ability of the nerve
terminal to release transmitter remains normal. In a
study of diaphragm, using curare to block action
potentials and a 'variance' method to estimate quantal
content, Nagel and colleagues (1990) noted a small but
statistically significant increase in quantal content in
mdx mouse muscles at 8.5 weeks of age, which became
References
ANDERSON, C. R. & STEVENS, C. F. (1973) Voltage clamp
analysis of acetylcholine produced end-plate current
fluctuations at the frog neuromuscular junction. Journal of
Physiology 235, 655-91.
979
more pronounced by 37 weeks. There is evidence that
these methods overestimate quantal content due to
presynaptic effects of curare (Hubbard & Wilson, 1973;
Glavinovic, 1979; Ferry & Kelly, 1988) and marked
deviation from Poisson statistics at higher levels of
transmitter release (Cull-Candy et al., 1980). These
factors may have accentuated any increase in quantal
content in mdx mice.
In normal muscle, one of the factors determining
quantal content is the extent of synaptic contact
between the nerve terminal and the muscle fibre. In
the muscles we studied, neither the total postsynaptic
area, estimated from the area of high AChE activity
measured in the light microscope, nor the fraction of
that area in contact with nerve estimated by the ratio of
'presynaptic length' to 'postsynaptic surface length'
were significantly different in muscles from control
and mdx mice. In older mice, Nagel and colleagues
(1990) found an increase in the overall length of the
NMJ when compared with controls. This may reflect
an increase in the synaptic area which could underlie
the increased quantal content they report.
Although the function of dystrophin in muscle cells
remains unclear, our findings and those of others
indicate that dystrophin does not play any essential
role in the immediate events of neuromuscular trans-
mission or in the events responsible for localizing
some key synaptic molecules. On the basis of im-
munocytochemical studies it has been reported that
other 'dystrophin-like' proteins are concentrated at
the NMJ and, in contrast to dystrophin itself, are
present in mdx mice (Fardeau et al., 1990). It may be
that any functional deficit caused by the absence of
dystrophin from the NMJ is compensated for by the
other proteins. In any case the aspects of neuromuscu-
lar transmission we studied in the mdx mice were
substantially normal in spite of the marked structural
abnormalities of the NMJo It seems that altered form of
the NMJ is not always associated with altered func-
tion.
Acknowledgements
We thank Carol Young for expert technical assistance
and Mr B. White for maintenance of the mouse
colonies. We gratefully acknowledge the financial
support of the Muscular Dystrophy Group of Great
Britain and Northern Ireland.
BRADLEY, S., LYONS, P. R. & SLATER, C. R. (1989) The
epitrochleoanconeus muscle (ETA) of the mouse: a useful
muscle for the study of motor innervation in vitro. Journal
of Physiology 415, 3P.
980
BULFIELD, G., SILLER, W. G,, WIGHT, P. A. & MOORE, K. J.
(1984) X chromosome-linked muscular d y s t r o p h y (mdx)
in the mouse. Proceedings of the National Academy of Sciences
(USA) 81, 1189-92
CAMPBELL, K. P. & KAHL, S. D. (1989) Association of
d y s t r o p h i n 'and an integral m e m b r a n e glycoprotein.
Nature ~338, 259-62.
CARNWATH, J. W. & SHOTTON, D. M. (1987) Muscular
d y s t r o p h y in the mdx mouse: histopa thology of the soleus
and extensor digitorum longus muscles. Journal of the
Neurological Sciences 80, 39-54.
COERS, C. & WOOLF, A. L. (1959) The Innervation ofMuscle, a
Biopsy Study. Oxford: Blackwell Scientific Publications.
COLQUHOUN, D., LARGE, W. A. & RANG, H. P. (1977) A n
analysis of the action of a false transmitter at the
neuromuscular junction. Journal of Physiology 266, 4 6 4 ~ .
COULTON, G. R., MORGAN, J. E., PARTRIDGE, T. A. &
SLOPER, J. C. (1988) The mdx mouse skeletal muscle
myopathy: I. A histological, morphometric and bio-
chemical investigation. Neuropathology and Applied Neuro-
biology 14, 53-70.
CULL-CANDY, S. G., MILEDI, R., TRAUTMANN, A. &
UCHITEL, O. D. (1980) O n the release of transmitter at
normal, myasthenia gravis and myasthenic s y n d r o m e
affected h u m a n end-plates. Journal of Physiology 299,
621-38.
DUCHEN, L. W., EXCELL, B. J., PATEL, R. & SMITH, B. (1974)
Changes in motor end-plates resulting from fibre necrosis
a n d regeneration: a light and electron microscopic s t u d y
of the effects of the depolarizing fraction (cardiotoxin) of
Dendroaspis jamesoni vernon. Journal of the Neurological
Sciences 21, 391-417.
ENGEL, A. G. & SANTA, T. (1971) Histometric analysis of the
ultrastructure of the n e u r o m u s c u l a r junction in myas-
thenia gravis a n d in the myasthenic syndrome. Annals of
the New York Academy of Science 183, 46-63.
FARDEAU, M., TOME, F. M. S., COLLIN, H., AUGIER, N.,
PONS, N. & LEGER, J. (1990) Presence of dystrophin-like
protein at the neuromuscular junctions in Duchenne
muscular d y s t r o p h y a n d in the mdx m u t a n t mouse.
Comptes Rendus de l'Academie des Sciences (Paris) 311,
197-204.
FAWCETT, P. R. W., SCHOFIELD, I. S., SLATER, C. R. &
WALLS, T. J. (1978) Structure a n d function of the neuro-
muscular junction in h u m a n n e u r o m u s c u l a r diseases.
Journal of Physiology 391, 12P.
FERRY, C. B. & KELLY, S. S. (1988) The nature of the
presynaptic effects of (+)-tubocurarine at the m o u s e
n e u r o m u s c u l a r junction. Journal of Physiology 403, 425-37.
GLAVINOVIC, M. 1. (1979)Presynaptic action of curare.
Journal of Physiology 290, 499-506.
GRUBB, B. D., HARRIS, J. B. & SCHOFIELD, I. S. (1991)
N e u r o m u s c u l a r transmission at n e w l y formed neuro-
muscular junctions in the regenerating soleus muscle of
the rat. Journal of Physiology, 441, 405-21.
HARRIMAN, D. G. F. (1976) A comparison of fine structure of
motor end-plates in D u c h e n n e d y s t r o p h y and in h u m a n
neurogenic diseases. Journal of the Neurological Sciences 28,
233--47.
HARRIS, C. (1954) The m o r p h o l o g y of the m y o n e u r a l junc-
tion as influenced b y neurotoxic drugs. American Journal of
Pathology 30, 501-19.
HARRIS, J. B. & RIBCHESTER, R. R. (1979) The relationship
L Y O N S a n d SLATER
between end-plate size and transmitter release in normal
and dystrophic muscles of the mouse. Journal of Physiology
296, 245-65.
HODGKIN, A, L. & RUSHTON, W. A. H. (1946) The electrical
constants of a crustacean nerve fibre. Proceedings of the
Royal Society of London B 133, 444-79.
HOFFMAN, E. P., BROWN, R. H. JR. & KUNKEL, L. M. (1987)
Dystrophin: the protein product of the Duchenne muscu-
lar d y s t r o p h y locus. Cell 51, 919-28.
HOLLINGSWORTH, S., MARSHALL, M. W. & ROBSON, E.
(1990) Excitation contraction coupling in normal and mdx
mice. Muscle and Nerve 13, 16-20.
HUBBARD, J. I. & WILSON, D. F. (1973) Neuromuscular
transmission in m a m m a l i a n preparation in the absence of
blocking drugs and the effect of d-tubocurarine. Journal of
Physiology 228, 307-25.
JASMIN, B. J., CARTAUD, a . , LUDOSKY, M. A., CHANGEUX,
J. P. & CARTAUD, J. (1990) Asymmetric distribution of
d y s t r o p h i n in developing and adult Torpedo marmorata
electrocyte: evidence for its association with the acetyl-
choline receptor-rich membrane. Proceedings of the
National Academy of Sciences (USA) 87, 3938-41.
KARNOVSKY, M. J. (1965) A formaldehyde-glutaraldehyde
fixative of high osmolality for use in electron microscopy.
Journal of Cell Biology 27, 137A.
KARNOVSKY, M. J. & ROOTS, I. a. (1964) 'Direct colouring'
thiocholine m e t h o d for cholinesterases. Journal of Histo-
chemistry and Cytochemistry 112, 219-21.
KARPATI, G., CARPENTER, S. & PRESCOTT, S. (1988) Small-
calibre skeletal muscle fibres do not suffer necrosis in mdx
mouse dystrophy. Muscle and Nerve 11, 795--803.
KOENIG, M., MONACO, A. P. & KUNKEL, L. M. (1988) The
complete sequence of d y s t r o p h i n predicts a r o d - s h a p e d
cytoskeletal protein. Cell 53, 219-26.
KORNELIUSSEN, H. (1972) Ultrastructure of normal and
stimulated motor endplates. Zeitschrift far ZeUforschung
130, 28-57.
LILEY, A. W. (1956) A n investigation of spontaneous activity
at the neuromuscular junction of the rat. Journal of
Physiology 132, 650-66.
MARTIN, A. R. (1955) A further s t u d y of the statistical
composition of the end-plate potential. Journal of Physi-
ology 130, 114-22.
MIIKE, T., MIYATAKE, M., ZHAO, J., YOSHIOKA, K. &
UCHINO, M. (1989) Immunohistochemical dystrophin
reaction in synaptic regions. Brain and Development (Tokyo)
11, 344-6.
NAGEL, A., LENMANN-HORN, F. & ENGEL, A. G. (1990)
N e u r o m u s c u l a r transmission in the mdx mouse. Muscle
and Nerve 13, 742-9.
NAKANE, P. K. & KOWAOI, A. (1974) Peroxidase labelled
antibody, a n e w m e t h o d of conjugation. Journal of Histo-
chemistry and Cytochemistry 5, 1280-3.
QUASTEL, D. M. J. & LINDER, T. M. (1975) Pre- and
post-synaptic actions of central depressants at the mam-
malian neuromuscular junction. Progress in Anesthesiology
1, 157-68.
SAKAKIBARA, H., ENGEL, A. G. & LAMBERT, E. H. (1977)
Duchenne dystrophy: ultrastructural localization of the
acetylcholine receptor and intracellular microelectrode
studies of neuromuscular transmission. Neurology 27,
741-5.
SALPETER, M. M. (1987) Vertebrate neuromuscular junc-
NMJs in mdx mice
tions: general morphology, molecular organization, and
functional consequences. In The Vertebrate Neuromuscular
Junction (edited by SALPETER, M. M.), pp. 35-43. New
York, Alan R. Liss.
SHIMIZU, T., MATSUMURA, K., SUNADA, Y. & MANNEN, T.
(1989) Dense immunostaining of both neuromuscular
and myotendon junctions with an anti-dystrophin anti-
body. Biomedical Research 10, 405-9.
SICINSKI, P., GENG, Y., RYDER COOK, A, S., BARNARD, E.
A., DARLISON, M. G. & BARNARD, P. J. (1989) T h e
molecular basis of muscular dystrophy in the mdx mouse:
a point mutation. Science 244, 1578-80.
SLATER, C. R. (1982) Postnatal maturation of nerve-muscle
junctions in the hindlimb muscles of the mouse. Develop-
mental Biology 94, 11-22.
STRUM, J. M. & HALL-CRAGGS, E. C. B. (1982) A m e t h o d
demonstrating motor endplates for light and electron
microscopy, Journal of Neuroscience Methods 6, 305-9.
981
TANABE, Y., ESAKI, K. & NOMURA, T. (1986) Skeletal m u s c l e
pathology in X chromosome-linked muscular dystrophy
(mdx) mouse. Acta Neuropathologica (Berlin) 69, 91-5.
TORRES, L. F. & DUCHEN, L. W. (1987) T h e m u t a n t mdx:
inherited myopathy in the mouse. Morphological studies
of nerves, muscles and end-plates. Brain 110, 269-99.
TSUJI, S. & TOBIN-GROS, C. (1980) A s i m p l e silver n i t r a t e
impregnation of the nerve fibres with preservation of
acetylcholinesterase activity at the motor end-plate. Ex-
perientia 36, 1317-19.
ZUBRZYCKA-GAARN, E. E., BULMAN, D. E., KARPATI, G.,
BURGHES, A. H., BELFALL, B., KLAMUT, H. J., TALBOT,
J., HODGES, R. S., RAY, P. N. & WORTON, R. G. (1988) T h e
Duchenne muscular dystrophy gene product is localized
in sarcolemma of human skeletal muscle. Nature 333,
466-9.