CHARACTERISTICS OF THE NEUROMUSCULAR
JUNCTION IN A FLATFISH
(Citharichthys sordidus)
Roger Wobig
Bio 175H
Hopkins Marine Station
June 15,1989
ABSTRACT
The neuromuscular junction of Pacific Sanddabs was studied. Neither
fast nor slow muscle fibers in the fin muscles of sanddabs produce action
potentials. Muscle activation by indirect stimulation could be inhibited with
TTI
D-tubocurarine (curare), tetrodotoxin (11X), and GVIA omega-conotoxin.
This indicated some similarities in the neuromuscular system of sanddabs
compared to other vertebrates. Motor nerve and synaptic transmission are
sensitive to sodium and calcium channel blocks, and acetylcholine is likely to
be the neurotransmitter, as suggested by the curare sensitivity of
neuromuscular transmission. However, direct stimulation of muscle fibers
could be continued after the neuromuscular transmission had been blocked.
Although they do not produce action potentials, the muscles do produce end
plate potentials. A single end plate potential has proved adequate to
produce a muscle twitch.
INTRODUCTION
Pacific Sandabs are interesting animals because they have fast twitch
muscle fibers that do not generate action potentials, presumably, because
they lack voltage-sensitive sodium channels (Gilly and Aladjem, 1987). This
is a rare occurence in vertebrate skeletal muscle systems. Sanddabs also
possess tonic fibers which hold contractions for extended periods of time
(i.e. minutes). These fibers do not produce action potentials either. Other
vertebrates use tonic fibers to control a wide range of specialized behaviors
Instead of using action potentials, the tonic system is activated through
several axons contacting the muscle fiber at multiple sites along its length.
This produces localized depolarizations and enables graded force
development. The sanddab also has intermediate fibers which contract at
rates that are neither fast nor tonic (less than 1 second). The sanddab must
rely on multiple innervation and summation of end plate potentials in order
to control contractions of its fin muscle; this is accomplished through
depolarizations along the entire course of its fibers. This makes the sanddab
an intriguing experimental system. It would be interesting to understand
more about this system and its relations to systems in other animals.
Neuromuscular transmission has been extensively studied in amphibia
and higher vertebrates, but work on fish is limited. At the end plate a motor
axon makes contact with a muscle fiber, and a synapse exists between the
two cellular elements. When an action potential in the axon invades the
synaptic ter minals, neurotransmitter (acetylcholine) is secreted and quickly
binds to receptors in the muscle cell. These receptors are cation-selective
channels which open upon binding to ACh. Fatt and Katz (1951) first
recorded end plate potentials (e.p.p.) in frog muscle by recording
intracellularly from muscle fibers during indirect stimulation of the motor
nerve. They found that one could reduce the size of the e.p.p. by adding
curare to the preparation. This drug binds to the postsynaptic ACh receptor
molecules. It was later established by Del Castillo and Katz (1954) that the
e.p.p. was produced by a nuber of small quantal components. Further work
was done by Martin in 1955 in which he described end plate potentials as
the summation of many miniature potentials. It is now understood that
these miniature e.p.p. are all-or-none. and therefore, correspond to packets
of ACh being released either spontaneously or in response to nerve
stimulation. In a frog muscle fiber, a single e.p.p. due to one action potential
in the motor axon causes summation of many miniature e.p.p. and generally
leads to the production of an all-or-none action potential in the muscle cell.
However, sanddabs are able to produce muscle excitation without producing
action potentials.
Since sanddabs do not appear to normally have sodium channels in their
muscle fibers, it would be interesting to see if they have the capabilities to
express them under appropriate conditions. Denervation of skeletal muscles
is well known to produce this effect (Keynes and Aidley, 1981). A severed
nerve will cause the muscle it supplies to dramatically change its
physiological properties. After a few days, the muscle will develop more
sodium channels and ACh receptors. Moreover, the whole muscle cell
surface becomes sensitive to ACh, which nor mally is effective just at the end
plates. It would be interesting to see if sanddabs possess the capablities to
produce these mor phological change:
The present study focuses on three aspects of neuromuscular
transmission. The first is to look at the the summation of e.p.p. in order to
produce muscle fiber contraction. The second is to gain a better
understanding of the phar macological aspects of the neuromuscular junction,
and finally, to look at the question of functional changes due to denervation.
MATERIALS AND METHODS
Pacific Sand Dabs, Ctharicthys sordidus were caught with a hook and line
at Monterey Bay. California. They were maintained in a flow-through sea
water tank. They were killed with decapitation so that drugs or higher
motor control centers would not influence results. Dissections were carried
out to produce viable muscle and nerve preparations (see below and Figure
1). The preparations were maintained in Ringer solution and placed on a
cooling plate (see below). They were secured with pins to the Sylgard (Dow
Corning. Midland, Michigan) floor of an experimental chamber. The fin
spines were tied with thread and connected to a force transducer which was
supported by a micromanipulator.
The main nerves and trunks running along the proximal ribs branch out
less dramatically in more anterior regions of the fish. Results in this report
are all obtained from the posterior end of the fish. Smaller fish were
préferred because they possess significantly less connective tissue in the
experimental region.
FORCE MEASUREMENTS
Contractile responses to indirect stimulation (via nerve) were studied.
Motor nerves were stimulated with a polished glass suction electrode
(approximately 250 um in diameter) which was supported by a
micromanipulator. The electrode was gently placed against the nerve and a
slight suction was applied. The same technique was also used to directly
stimulate muscle fibers. Spurious stimulation of tissue away from the
pipette tip by the indifferent wire outside of the glass electrode was avoided
by placing the wire as far away from the muscle as possible, but still in the
experimental chamber. Force was then measured with a transducer made
from Pixie semiconductor elememts (Endevco, Pasedena, California; Gilly and
Hui. 1980). Recordings were made on both a chart recorder and an
oscilloscope.
INTRACELLULAR RECORDINC
In order to record voltage differences from muscle fibers, electrodes were
filled with 3M KCI. Electrodes were 10-20 x 106 Ohms in resistance. They
were carefully manipulated until they were near a muscle fiber, and then.
upon a swift tap to the micromanipulator, they punctured the cell.
Recordings were made from fin-muscle fibers only on the fin spine side of
the end plate band (see figure 1). Contractile responses appeared to be
strong and fast in this region.
SOLUTIONS
Composition of the ringer used was (mmol 1-D): 200 Nacl, 3 KCI, 4MgCI2,
and 4 CaCl. This was buffered to pH 7.2 with SmM Tris. Ringer was kept
refrigerated and the experimental chamber was maintained at
approximately 20° C. Chemicals were obtained from Sigma Chemical (St.
Louis, Missouri). Omega-conotoxin and the paralytic peptide were obtained
from Dr. Baldomero Olivera, University of Utah.
RESULTS
ANATOMY AND DEVELOPMENT OF A NEUROMUSCULAR JUNCTION
PREPARATION
The pattern of the fin spine musculature of the sanddab is shown for the
left side in Figure 1. The pattern is repeated for every dorsal and anal fin
spine along the length of the animal on both left and right sides. The figure
demonstrates that each fin spine is connected to two muscles on the left side;
two identical muscles are present on the right as well. These muscles are
connected to bone shafts with a triangular head. These bone shafts are
connected to the proximal rib and to the fin spines with extensive connective
tissue. Also connected to each fin spine is a skin muscle (not illustrated)
which is connected in the middle of the fin spine and at a junction
approximately halfway up the bone shafts. The skin muscles run between
the two bone shafts and on top of the other two muscles. They were cut
away so that the motor nerves could be exposed. Skin muscles also exist on
the both sides of the fish, and the total is thus six muscles connected to each
fin spine.
In carrying out physiological studies of fin muscle, the skin muscles were
dissected off of both sides, and the preparations were carefully cleaned from
fin spine to proximal rib. Therefore, the preparations appeared as in Figure
1. except that the antagonistic muscles were also usually connected on the
other side. Although there appeared to be no differences between the two
sides of the fish, in order to maximize consistency of results, all preparations
were taken ventrally from the right side of the fish. Previous work had
employed left dorsal muscles.
Motor nerves are indicated in Fig. I as running along the course of a
proximal rib and between two triangular-headed bone shafts.
Approximately midway down the length of the bone shafts, the nerve
branches and turns to run laterally across the muscle surface. Stimulation
applied to the nerve in this area was effective, and it appears that endplates
lie in this middle portion of the muscles (see below). A large nerve
présumably sensory in function, branches off the main nerve as shown in
Fig. I, and runs between the skin muscles and courses down the length of
the lin spine through its hollow interior. The nerves run in parallel down
every spine on both left and right sides of the fish, but are physically
seperated from one another, even the spine itself,
One difficulty in producing good preparations is the large amount of
connective tissue in the area used for stimulation (end plate band). There is
also extensive connective tissue which forms part of the proximal
termination of the skin muscles in this region. It was difficult to obtain a
clean preparation without damaging the muscle fibers or nerves in this area;
however, this procedure was necessary since a cleaner preparation was more
readily excitable and was also suitable for intracellualr recording. It was
also important to visually observe the muscle contractions and have an
unobstructed contact between the nerve and the electrode.
Several other difficulties were experienced in trying to produce viable
preparations. Results are probably dependent on the fiber type being
stimulated. Contractions were not reliably produced in the same area of the
muscle fiber every time. Differences might be representitive of stimulating
different fibers. Since the sanddab has three different muscle fiber types, it
is important to stimulate the same fiber groups every time in order to
control for variation, especially when measuring force from the whole
muscle. Different regions of contractions could be representitive of different
muscle fibers. The nerves also appeared to be extremely fragile, so that
sucking a cut end of a nerve up into the electrode appeared to damage its
conductive properties. This was allieviated by gently placing the electrode
on the nerve (en passant) and applying a gentle suction. This technique is
effective in producing indirect stimulation, but care must be exerted to avoid
direct muscle stimulation in the immediate area of the electrode tip.
Because of the difficulties associated with the fin muscle, an alternative
preparation was sought. Extraocular muscle was found to be an excellent
preparation, and it could be isolated with intact innervation. The preparation
consisted of the superior oblique extraocular muscle and its intact
innervation. The nerve could be sucked up into the electrode without
apparent damage, and produce reliable contractions with repetitive
stimulation.
CHARACTERISTICS OF INDIRECT STIMULATION
Indirect stimulation of fin muscle was carried out using single shocks, and
the resulting contractile force was measured. We routinely found that the
resting length of the muscle greatly influences contractile strength (see
Figure 2). Over a small change in length (slack length- 1 cm), the muscle
displays a large change in contractile strength. Force increases smoothly up
to a 10x stretch beyond slack length, where the maximal contractile force is
reached. A slight additional stretch (0.2 mm) causes a sharp drop in
contractile force. All experiments were carried out near the maximum of the
length-force curve which was determined for each preparation.
When a nerve is excited, its axons fire in an all or none manner. As
shown in figure 3a, extraocular muscle is indirectly excited to a maximal
contractile strength over a very small range of applied voltages. The region
of gradation in force probably reflects increasing excitation of more motor
axons in the nerve over this range of stimulating voltages. This
characteristic of a gradable response of the whole muscle is expected for
indirect stimulation. Apparently, all of the motor axons in the oculomotoi
nerve, which has been sucked up onto the stimulating electrode, can be
uniformly excited at a shock of less than 1 volt.
The fin spine muscles showed a much more gradable force response with
shock strength (Fig. 3b), at least using single shock stimuli. A major
difference between the two preparations is that the fin spine motor nerve
has an extremely large amount of connective tissue around the stimulating
region. Because the en passant configuration of nerve stimulation was used
with fin spine preparations, it may have been impossible to equally excite
the whole nerve, except with very strong shocks. Since the nerve was not
cut and sucked up into the electrode, there is also the possibility of directly
exciting muscle fibers with large shocks.
The gradation of force with indirect stimulation of fin spine muscle is
largely different from that seen with direct muscle stimulation. Direct
stimulation shows that contractile strength is even more graded with voltage
applied than the results shown in Fig. 3b. (Gilly and Aladjem. 1987.) Force
elicited by direct muscle activation is also highly responsive to changes in
the stimulus duration over a range that does not affect indirect stimulation
(e.g. changing from 0.5 to 5 ms shocks. Data not illustrated). Direct
stimulation is also not very responsive (if at all) to reverse polarity of the
stimulus (i.e. inside of pipette positive), presumably because sanddab muscle
does not have sodium channels. Nerves can be stimulated by this anode¬
break method, because the hyperpolarizing stimulus will induce Na channels
to activate upon termination of the hyperpolarization and return of the
membrane potential to its normal value. A final difference is that direct
stimulation always produces visually detectable contractions im mediately in
the region stimulated, whereas indirect stimulation often produces
contractions far away from the stimulating site, e.g. from several groups of
muscles.
PRODUCTION OF AN END PLATE POTENTIAL AT THE NEUROMUSCULAR
JUNCTION
Intracellular recording revealed that an end plate potential (e.p.p.) is
produced in response to indirect stimulation. This is shown in Figure 4a.
The rise after the e.p.p. is due to the muscle twitch dislodging the
microelectrode. As the electrode is moved closer to the end plate band
described above, (in another muscle fiber), the e.p.p. becomes larger and
faster and occurs at a shorter delay (Fig. 4b). Hence, the electrode has been
moved closer to the endplate.
Although the e.p.p. appears to be all or none, this arises from the all or
none firing of the motor axon and not from the excitability of the muscle cell.
Fig. 4c shows that the same e.p.p. is produced with shocks of 2.1. 2.2, and 2.3
V. However, a shock of 2.0 V. produced no e.p.p. at all. Nothing resembling
an action potential is ever observed. The electrical signs of muscle activation
in sanddab muscle differ markedly from more 'typical' vertebrate skeletal
muscle. As a comparison, the action potential recorded intracellularly from
tadpole myotome muscle is shown in Fig. 4d.
Figure 5 shows some additional properties of the e.p.p. in fish. Even with
repetitive stimulation, no action potential is formed. The e.p.p. depicted in
Figure 4 were all produced in response to single shocks, which also produced
brisk muscle twitches. This result differs from those in previously published
work (Gilly and Aladjem, 1987). According to this paper, trains of stimuli
were necessary to indirectly excite muscle. Also, figure 5 does not show the
same pattern of summation of junction potentials to a plateau depolarization
that was documented in the aforementioned paper (see Discussion).
PHARMACOLOGY OE NEUROMUSCULAR TRANSMISSION
Once it was clear that the contraction was accomplished through indirect
stimulation, it was possible to attempt some phar macological experiments
Figure 6 documents the block of the e.p.p. with 100 uM curare. This was
accomplished in the course of three minutes. Since curare blocks ACh
receptors, it is reasonable to assume that sanddabs use ACh as their
neurotransmitter for motor neurons. This is supported by the fact that a
strong contracture is produced when carbochol, an ACh analog, is added to
the preparation.
TTX is a toxin that blocks Na channels. Figure 7a shows the result of
adding TTX to a muscle being stimulated indirectly with single shocks. Force
is completely eliminated in 8 minutes (Fig. 7b). Presumably, conduction of
the nerve’s Na channels are blocked so that electrical excitation of the nerve
is stopped and no ACh can be released at the neuromuscular junction. TTX
was effective at concentrations of 200 nM, although its time course ranged
over à number of minutes. Because TTX blocks Na channels, it blocks direct
muscle excitation in muscles with action potentials, e.g. tadpoles (not
illustrated). However, sanddabs apparently do not have muscle Na channels
and therefore, direct stimulation is still possible after indirect stimulation
had been completely blocked by TTX. These phar macological results
reinforce what is already known about sanddab muscle.
The extraocular muscle preparation was used for further phar macological
studies. These results are summarized in Figure 8. Stimulation was not done
with à single shock, but rather with tetani of 200 Hz. for 0.5 sec. The shocks
were at 0.5 V. and lasted for 0.4 msec. It appears as if 0.6 mM of the
paralytic peptide (an uncharacterized conotoxin: obtained through personal
communication with B. Olivera) had no effect. Either higher dosages or more
time would be needed to document any effect this drug had. The next drug
applied was 0.3 mM of GVIA omega-conotoxin. This drug blocks presynaptic
calcium channels in frog neuromuscular junctions (Olivera et al. 1985), and
therefore, blocks the release of ACh from the presynaptic terminal. This
appeared to have an inhibitory effect in the present experiment. Finally, as
controls, curare and TTX were used to induce comlpete loss of response,
DENERYATION
As à final experiment, it was attempted to denervate sanddab fin muscle
A cut was made proximaf to the skin muscles. The incision was made
laterally along one half of the fish on the right side. The muscle was pulled
back to expose the main motor nerves diagrammed in Fig. 1. Several motor
nerves on the right ventral side were cut proximal to the end plate band
area nor mally used for stimulation. The fish were then allowed to recover
for at least four days. After four days, one fish was dissected. Due to the
operation, the damaged skin muscles appeared extremely white, tore easily
and had lost their elasticity. Some of the experimental muscles had begun to
take on this appearance also, but others which had clearly been denervated
still appeared healthy and responded to direct muscle stimulation with
repetitve shocks (not illustrated). Muscles on the left side of the fish
maintained their normal appearance. It was anticipated that the denervated
muscles would develop Na channels. This, along with increased sensitivity to
ACh, has been documented in other animals. Unfortunately, any increase in
muscle excitability was not detectable in this preliminary experiment.
Perhaps more time is needed for these changes to develop or finer recording
techniques may be necessary (e.g. intracellular recording).
DISCUSSION
Despite its relatively simple and highly repetitive anatomical layout, the
sanddab is a rather difficult animal to study. The muscles and nerves
appear to be rather fragile, and one must take great care during dissections
so as not to damage the preparation. The muscles might also be easily
damaged by stretching since there clearly is a maximal stretch which can be
applied to the fin muscles. The sanddab's force vs. length curve (Fig. 2) is
extremely more dramatic than other animals. Most animals show a slow loss
of force after a maximal stretch due to the loss of overlap between the actin
and myosin filaments (Keynes and Aidley. 1981). The sharp loss of force in
the sanddab is a mystery. Perhaps multiply innervated systems are much
more responsive to stretch and endplates are more easily disrupted.
It appears that work might be easier to accomplish on the extraocular
muscles. These muscles can be easily dissected out with a large intact nerve
attached. In this system, stimulation could be accomplished with a cut nerve
sucked up into the electrode. However. even in this preparation, some of the
muscles did not appear responsive
It appears as if there are some standard occurences at the neuromuscular
junction of the sanddab. Indirect stimulation can be blocked to some extent

by curare, 11A, and omega conatoxin. Presumably this is because sanddabs
use ACh as their neurotransmitter, and their nerves have Na and Ca channels
which can be blocked by appropriate toxins.
However, some of the drugs appeared to take a long time to show an
effect. Perhaps there is a problem with chemical diffusion in this fish, so
that the endplates are extremely well insulated and perfect conditions are
necessary to chemically alter behavior. Repeated studies need to be
accomplished in order to understand the time course of the drugs used.
With repeated experiments, perhaps a pattern would develop which would
lead to greater insight. It would also be advantageous if the drugs were
more responsive to washing out, so that work could be reliably reproduced
on the same muscle.
It is not clear why the preparations produced in these experiments were
responsive to a single shock, while previous studies have needed to use
repetitive stimuli to shock indirectly (Gilly and Aladjem. 1987). Perhaps this
is caused by differences in the innervation patterns of fast twitch versus
intermediate twitch muscle fibers. It would be interesting to see if there
were significant differences between the neuromuscular junctions of the
different fiber types. The previous study accomplished work on the left side
of the fish, and there is the possibility that differences exist between the left
and right sides of the sanddab. Differences were also observed in the
summation of end plate potentials for probably the same reasons. Previous
work showed a large summation of e.p.p. and then a plateau depolarization.
Figure 5 shows a lack of dramatic facilitation, so that there is not a
considerable amount of summation to a plateau. The first e.p.p. is also much
larger in comparison to the second than those seen in the earlier study. This
could explain why a single shock was a sufficient stimulus to produce
muscular contraction in our present experiments.
This paper is the first de monstration of the physiological effect of GVIA
omega-conotoxin in fish. This is rather ironic since the snails are fish killing
animals. Nevertheless, the toxin appears to block the neuromuscular
junction of fish as expected. The paralytic peptide appeared to have no
effect on peripheral nerve transmission or end plate transmisssion.
However, it has been shown to have a paralytic effect when injected into an
intact fish (Kuo, E., 1989). This implies that its effects are restricted to
higher motor centers.
The sanddab appears to be an intriguing experimental system. Two large
sensory nerves can be seen supplying each fin spine. Presumably, these
nerves are sensory in function. They might be linked to proprioception or
substrate identification. The innervation of the skin muscle may also help to
shed some light on the interesting muscles of the sanddab. The sanddab
appears to have developed a complex system in order to finely orchestrate
its movement. A more detailed characterization of this neuromuscular
system should be interesting.
ACKNOW LEDGEMENTS
My sincere thanks and gratitude go to W. F. Gilly for his guidance and
support throughout this project.
REFERENCES
Del Castillo,J. and Katz, B. (1954) Quantal components of the end plate
potential. J Physiol. 124, 560-573.
Fatt, P. and Katz. B. (1951) An analysis of the end plate potential
recorded with an intracellular electrode. 1 Physiol 115, 320-369.
Gilly, W. F. and Aladjem, E. (1987) Physiological properties of three muscle
fibre types controlling dorsal fin movements in a flatfish. 1 Musc. Res
Cell Mot. 8, 407-417.
Gilly, W.F. and Hui, C.S. (1980) Mechanical activation in slow and twitch
skeletal muscle fibers. Dependence on voltage and external calcium.
J. Physiol 301, 137-156.
Keynes and Aidley. Nerve and Muscle. Cambridge Univ. Press, Cambridge
pg. 151 (1981)
Kuo, E. (1989) Mauthner cell recording from Senorita, Oryjulis californica
Biology 175H, Hopkins Marine Station of Stanford University, Pacific
Grove, California (unpublished report).
Martin A. R. (1955) A further study of the statistical components of the end
plate potential. J Physiol 130, 114-122.
Olivera, B.M. et al (1985) Peptide neurotoxins from fish-hunting cone snails.
Science 230, 1338-1343.
FIGURE LEGENDS
FIG. 1. Schematic of fin spine preparation.
This depicts a representation of the skeletal components, muscle, and nerves
which control the fin spine. See text for details.
FIG. 2. Relationship between force generated and muscle stretch.
Single indirect shocks of 10 V. were applied for a duration of 0.4 msec each.
FIG. 3. The relationship between force generated and voltage applied.
(a) Indirect shocks lasting 0.4 ms were delivered at frequencies of 200 Hz.
for 0.5 sec. to the extraocular preparation. (b) Single shocks of 0.4 ms were
delivered to the fin spine muscles.
FIG.Aa. Properties of end plate potentials.
An end plate potential is produced by a 2.2 V. shock of 0.4 msec. (b). A
picture of the e.p.p. produced as the electrode is moved closer to the
endplate band. (c) This shows that the same end plate is produced at shocks
of 2.1, 2.2, and 2.4 V. However, there is no production of an e.p.p. at 2.0 V
(d) An action potential from a frog muscle produced by electrical
stimulation.
FIG. 5. Temporal summation of end plate potentials.
End plate potentials are produced at the indicated frequencies. Indirect
shocks consisted of 2.0 V. and were 0.4 msec. in duration.
FIG. 6. Block of the end plate potential with D-tubocurarine.
This represents the decay of the e.p.p. over 3 minutes. Block accomplished
with 100 uM of curare. Shocks were 2.2 V. and lasted for 0.4 msec.
FIG. 7. (a) Block of force generated with 100 nM TTX.
Indirectly applied shocks consisted of 40 V. and lasted 0.4 msec. (b) Time
course of 7a.
FIG. 8 Phar macology of the extraocular preparation.
There is a block of force due to 0.3 mM omega conatoxin, 200 uM curare, and
200 nM TTX. However, there is no apparent effect due to the application of
0.6 mM of paralytic peptide. Shocks were delivered indirectly at 0.6 V. for
durations of 0.4 msec. They were delivered at tetani of 200 Hz. for 0.5 sec.
FIGURE 1
MOTOR NERVE —
MUSCLE
4
BONE S
V
PROXIMAL RIB
END PLATE BAND
—
SENSORY NERVE
T
GEN SPNE
3
FORCE (mV)
FIGURE 3
BELATIONSHIP BETWEEN FORCE GENERATED AND VOLTAGE APPLIED
EXTRAOCULAR
40
20
FIN SPINE
40
20
—
-
—
40
20
VOLTAGE (V)
60
FIGURE 4
O m
-60 mV
O mV
-60 mV
END PLATE POTENTIALS
4 ms
END PLATE POTENTIAL VS ACTION POTENTIAL
4 ms
FIGURE 5
200Hz
O I
50 Hz
40 mV
REPETITIVE STIMULATION
4 ms
FIGURE 6
O mV
-60 mV
LOSS OF EPP BY CURARE
4 ms
FORCE (mV)
.. . .
FORCE (mV)