Abstract
Neuromuscular transmission in the squid is described in terms of the
slow and fast fibers characterizing circular mantle muscle. Various
potential transmitter substances and antagonists to synaptic
transmission were tested by electrically stimulating giant fibers and
recording twitch responses of the circular muscle in the presence of the
substances tested. Of these, bath application of L-glutamate had an
excitatory effect on circular mantle tissue. Antagonists to L- glutamaté
known to mediate synaptic transmission in other neuromuscular
systems had no reversible inhibitory effect on transmission. It is
concluded that L- glutamate or a glutamate analog may directly excité
the slow circular fiber. The apparently inhibiting action of glutamate on
fast fibers innervated by the giant axons may arise either
presynaptically or postsynaptically.
Introduction
The functions and properties of the giant axons in squid have been
well studied, but little is known about the obliquely striated mantle
muscle (Rosenbluth, 1972) innervated by the giant axon. For example,
motor ter minals of the giant axon have not been identified anatomically
(Bone and Pulsford, 1981), and the identity of neuromuscluar
transmitter is unknown. Similarly, little is known about the muscle,
e.g. whether the fibers generate action potentials. By applying potential
neurotransmitters as well as known blockers to synaptic transmission,
the synapses onto the muscle could be more easily understood.
Organization of squid mantle muscle has been deter mined by
anatomical data and physiological studies have yet to be conducted
(Gosline and DeMont, 1985). The mantle is composed primarily of
circular muscle tissue. The outer most layers are comprised of
slow-twitch fibers whereas the inner fibers are the fast-twitch muscle.
The slow muscles are used most often and are characterized by large
stores of mitochondria to provide ample amounts of ATP. The fast
muscles are only used in escape jetting when they are activated by the
giant axons. J. Z. Young (1938) reported that these giant axons act in an
"all or none" fashion, but that the muscles do not.
One unresloved issue involving these different muscle types
concerns the innervation of the muscles themselves. In another
invertebrate with obliquely striated muscle, Ascaris, innervation does
not occur via peripheral fibers, but through elongated processes that
arise from the muscle cells themselves and run to the ventral nerve
cords (Rosenbluth, 1972). In the squid, two motor systems exist and
the possibility exists of dual innervation of single muscle fibers(Wilson,
1960). The outer zone of the mantle containing the slow muscle is
probably not innervated in the same way as the inner zone. The giant
axon branches supply the circular muscle of the inner zone and there
are probably smaller axons which innervate both the outer and inner
circular muscle (Bone and Pulsford, 198 1).
Traditionally, fast muscle in vertebrates is thought to generate
actions potentials which aid in simultaneous activation of a long muscle
fiber over its entire length. However, systems have been described
where the fastest fibers in at least one vertebrate, a fish, do not show
action potentials. Instead, these muscles rely on the temporal
summation of end plate potentials (Gilly and Aladjen, 1987). Action
potentials in squid fast muscle have not been observed although Ca
action potentials have been observed in many other invertebrates.
Nothing is known about the transmitters at either slow or fast fiber
neuromuscular junctions in the squid. Glutamate is a good candidate,
though based on work on chromatophore muscle (Florey et al, 1985). It
has also been shown to be capable of both excitation and inhibition by
depolarizing and hyperpolarizing squid neurons. Glutamate may work
via a presynaptic action as it din crustacean muscle (Florey and
Woodcock, 1968) by causing an increase in miniature potentials which
summate to act postsynaptically.
Materials and Methods
Squid mantles were prepared from Loligo opalescens netted from
Monterey Bay. Preparations were obtained by decapitating the animals
and making a ventral cut along the length of the mantle. The pen as
well as all interior organs were removed and the resulting full mantle
preparation was left intact with both stellate ganglia and all the
accompanying giant fibers. A second mantle preparation involved
manually removing the inner collagen tunic to enable quicker diffusion
of bath applied solutions. An excised muscle preparation was also
obtained by cutting a strip (20mm X 3mm) of tissue in a direction
transverse to the animals long axis (ie., in parallel wtih the circular
muscle fibers).
A suction electrode with a nalgene tip was attached to a Grass S44
stimulator which was used to directly stimulate giant fibers in full
mantle preparations. Twitch responses were measured with a force
transducer and recorded on a Gould 220 dual pen chart recorder.
Muscle were stimulated using C-shaped Pt/Pt black foil electrodes
(3XSmm) mounted 1.5 cm apart and a few mm from the muscle. A
force transducer made from Pixie Se miconductor elements (Gilly and
Schuer, 1984) was used to measure twitch responses. Stimulations
were nor mally given once every 20 seconds with 70 V 3 msec pulses.
A polyethylene contact electrode (diameter 2.5 mm) with silver/silver
chloride wire was also used to record electrical activity and infor mation
was conveyed via a Nicolet 3091 oscilloscope.
Potential neurotransmitters used were L- glutamate, acetylcholine,
betaine, taurine, isotheonic acid, and octopamine all in 2mM
concentrations. Antagonistic che micals in concentrations ranging from
2-20mM include glutamate diethyl ester, glutamate A methyl ester, and
2 amino 4 phosphoro butyric acid. Also used were TTX (20-200 nM),
Na-free sea water (450 mM N-methyl glucamine-Cl. 60mM Cacl,, 10
mM KCI, 10 mM HEPES), low Ca seawater, and 10 mM Co seawater.
Preparations and solutions were all kept at 9-11° C and muscle tissure
was manually washed by pippetting solutions into and out of the glass
preparation dish with the tissue pinned down to a piece of Sylgard.
Individual muscle cells were obtained by soaking tissue in 1mg/ml
sterilized seawater solution of Type 1 A Collagenase (obtained from
Sigma) for 1- 3 hours. Tissue was then centrifuged for 5 minutes and
the collagenase was replaced by an L-15 (Gibco) based culture medium
containing 62 fetal bovine serum medium (Hyclone) and extra salts to
achieve final concentrations 10 mM CaCl,, 10 mM KCl, 50 mM MgCL,, and
434.2 mM Nacl in distilled water. The tissue was then vortexed for 3
minutes and centrifuged for one more minute then plated on 12
poly-L-lysine coated coverslips. Voltage clamp experiments were done
as described by Brismar and Gilly (1987). Glutamate was also applied
directly to isolated cells in sterile seawater via a micropippette and a
pressure injection apparatus.
Results
Full mantle preparations, even with the inner collagen tunic
remoyed, proved to be far less effective than isolated muscle strips in
washing chemicals in and out quickly. Therefore, the majority of
experiments were carried out using isolated pieces of lateral mantle
tissue containing both slow and fast twitch fibers. Figure lA shows the
electrical activity recorded through a seawater filled polyethylene tube
pressed up against the muscle in a full mantle preparation. Such
activity is followed by muscle twitch as shown in figure IB. Figure 2A
shows a portion of this electrical activity at a faster sweep speed. An
initial small, fast action potential is visible and is followed by a larger
and much slower response. Following stimualation for several seconds
at 20 Hz. the larger response is greatly reduced in amplitude (Figure
3B), while the earlier action potential is unaffected. Presumably the
small spike reflects activity in small branches of axons which send
identical signals to the neuromuscular junction even after repetitive
stimulation. The origin of the slower response is not so clear but it may
reflect electrical activities of muscle cells, because this activity précèdes
force development. The electrical activity at the muscle obviously
changes although the axon spike remains unchanged. Another type of
fatigue is seen in measuring muscle twitches. Figure 3A shows how the
contractile responses also decline after repetitve stimulation at à much
lower frequency (0.05 Hz). Following 20 min of such stimulation, twitch
amplitude fell to one-fifth its original value and a second, slow phase of
the twitch becomes obvious. Such slow muscle responses were only
observed in isolated muscle strip preparations. These responses are
most visible following fatigue of the fast component, but in reality the
slow twitch is probably always present.
In order to conclude that test solutions were diffusing through the
muscle tissue, various known antagonists to axon propagation and
synaptic transmission were applied. Blockers were considered effective
if muscle twitches declined until the antagonist was removed and then
then gradually increased back towards the original level. Tetrodotoxin
prevents action potential transmission along axons by blocking Na
channels. Even in low concentrations, TTX effectively blocked
transmission in muscle strip preparations (figure 4). Sodium-free
artificial seawater (ASW) acts similarly to TTX by re moying
extracellular sodium and preventing action potentials. Solutions can
thus gain access to axonal elements in muscle strip preparations.
Solutions were also applied which were designed to disrupt
neuromuscular transmission. Cobalt ions also block twitches.
presumably by inhibiting the amount of Ca influx at the presynaptic
motor ter minal and thereby decreasing neurotransmitter release (figure
5). Figure 6 shows how low Ca seawater acts similarly by reducing the
amount of extracellur Ca and thereby effecting the amount of
neurotransmitter released (Llinas, 1982).
Mantle strips were then exposed to bath application of various
potential excitatory solutions. Acetylcholine proved ineffective as did
betaine, taurine, isotheonic acid. and octopamine. These amino acids
known to exist in the axoplasm of axons (Prosser, 1972). Only
L-glutamate gave an excitatory response characterized by a gradual
contraction extending for about I minute and gradually decreasing to a
near-baseline level. The response appears more similar to slow muscle
contraction as glutamate does not give rapid twitch-like responses
assosciated with fast muscle. Instead, it acts antagonistically to inhibit
the fast responses which decrease following glutamate application and
increase again upon washing out with seawater (figure 7). The
excitatory effect of glutamate does not appear to be due to activation of
axons as shown in figure 8. TTX was first applied to block the axons
innervating the muscle, but the glutamate response is unaffected.
Glutamate blockers were also applied which act antagonistically to
glutamate receptors in other systems (Florey et al, 1985). Figure 9
shows typical results of three such blockers on an isolated muscle strip
None of the antagonists exhibited convincing reversal. When a
glutamate solution was applied in the presence of antagonist, the muscle
still exhibited contraction though at a lessened amplitude. It was
therefore concluded that glutamate was probably not the transmitter
involved at the neuromuscular junction of the fast muscle since the
blockers did prevent synaptic transmission.
Discussion
It appears that glutamate acts to excite certain muscle in the squid
and may possibly act to inhibit others. In similar experiments where
glutamate was hypothesized as a neurotransmitter on fast and slow
muscle in molluscs, it affected fast muscle by causing a series of rapid
contractions (Bone and Howarth. 1980, Florey et al. 1985). Slow muscle
responded by a slow and elongated contraction as observed in this
investigation. Similarly, TTX effectively blocked nerve propagation, but
failed to block a response to direct application of glutamate (see figure
6). However, this alone is not enough to conclude that glutamate acts
postsynaptically as Florey and Woodcock (1968) discovered in their
work with crab muscle. Although L-glutamate has been found to be
generally excitatory, there is also evidence for it acting as a blocker to
transmission at the giant synapse of the squid stellate ganglion (Kelly
and Gage, 1969) and as an inhibitory transmitter on cell bodies of
stellate ganglion neurons (Bevan et al, 1975).
The three glutamate antagonists all proved ineffective at being able
to reversibly block neuromuscular transmission although they were
shown to be effective blockers to glutamate in squid chromatophore
muscles (Florey et al, 1985). While the glutamate itself was excitatory
on at least the slow muscle, the blockers presumably did not diminish
twitch activity because the slow muscle was not being stimulated
significantly with single shocks. The giant axons are thought to run only
into the central circular muscle where the fast muscle fibers are found.
Therefore, if only fast muscle is being stimulated, the blockers would
have no effect if the glutamate were only affecting the slow muscle.
This is consistent with the data that shows that the blockers were
largely ineffective at blocking neuromuscular transmission.
Although only a few of the criteria for establishing the identity of a
neurotransmitter have been met (Dorsett 1975), the evidence suggests
References
Bevan S.J., Katz B., and Miledi R. 1975. Membrane potential fluctuations
produced by glutamate in nerve cells of the squid. Proc. R. Soc. Lond. B.
191. 561-565.
Bone Q., and Howarth, J. V. 1980. The role of L- glutamate in
neuromuscular transmission in some molluscs. I mar. biol. Ass. UK. 60.
619-626.
Bone Q., Pulsford A., and Chubb A.D. 1981. Squid mantle muscle. I mar
biol. Ass. UK. 61.327-342.
Brismar, T., and Gilly, W. 1987 Synthesis of sodium channels in the cell
bodies of squid giant axons. Proc. Natl. Acad. Sci. USA. 84. 1459-1463.
Dorsett, D. A. 1975. Some apects of neural organization in molluscs.
Simple Nervous Systems Crane, Russak, and Co.
Florey, E., Dubas, F., and Hanlon, R.T. 1985. Evidence for L-Glutamate as
a transmitter substance of motoneurons innervating squid
chromatophore muscles. Comp. Biochem. Physiol. 820. 259-268.
Florey, E. and Woodcock, B. 1968. Presynaptic excitatory action of
Glutamate applied to crab nerve-muscle preparations. Comp. Biochem.
Physiol. 26. 631-661.
Gilly, W. and Aladjem, E. 1987. Physiological properties of three muscle
fibre types controlling dorsal fin movements in a flatfish, Ctharichthys
sordidus. Jor. Mus. Res. Cell Mot. Vol 8 (in press).
Gosline J. and Demont, E. 1985. Jet-propelled swimming in squids. Sci
Am. 253.96-104.
Llinas, R. 1982. Calcium in synaptic transmission. Sci Am. 247. 56-65.
Kelly, J. and Gage, P. 1969. L-glutamate blockade of transmission at the
giant synapse of the squid stellate ganglion. Jor. Neurobio. 2. 209-219.
Prosser, C. 1973. Comparative Animal Physiology. 3rd Edn. 79-110.
Rosenbluth, J. 1972. Obliquely striated muscle. The Structure and
Function of the Cell. Vol 1. 2nd Edn. 389-420.
Scheuer, T. and Gilly, W. 1986. Charge movement and
depolarization-contraction coupling in arthropod vs. skeletal muscle.
Proc. Natl. Acad. Sci. USA 83. 8799-8803.
Wilson, D. Nervous control of movement in cephalopods. I exp. Biol.
37. 57-72.
Young. J. Z. The functioning of the giant nerve fibres of the squid./ exp
Biol. 15. 1938.
Figure Legend
Figure 1: Electrical and contractile recordings from full mantle
preparation. A shows electrical activity recor ded from contact electrode
in muscle and B shows fast muscle contraction following electrical
activity.
Figure 2: Electrical activity from stimulation of full mantle preparation.
A and B show electrical muscle response before and after repeated
stimulation.
Figure 3: Fast and slow contractile responses.
A demonstrates a fast contraction at high speed and B shows the slow
fiber activation after fast muscle fatique.
Figure 4: Effect of 20 nM TTX on twitch response ofmuscle strip
preparation.
Inactivation of axons is demonstrated by decreased response then
reversal with washing. Downward arrow represents washing in of TIX
and upward arrow represents washing out.
Figure 5: Effects of 10mM Co/ Na free (50 mM Ca) on twitch response.
Same muscle strip preparation shows inactivation of axon in case of Na
and presynaptic terminal in the case of Co. Upward and downward
arrows represent washing in and out of antagonists.
Figure 6: Effect of low Ca on twitch response.
Experiment on full mantle preparation shows effect of blocking
neurotransmitter release at presynaptic ter minal. Upward arrow
represents washing in and downward arrow represents washing out.
Figure 7: Application of L-glutamate and corresponding response.
Glutamate gives slow contraction but inhibits fast responses which
increase as glutamate is washed out of preparation.
Figure 8: Application of L-glutamate in presence of TTX.
Even with axons inactivated by TTX, glutamate response is still
significant.
Figure 9: Twitch responses to glutamate antagonists.
None of the three antagonists tested reversibly blocked stimulation.
Downward arrows represent washing in and upward arrows represent
washing out of antagonists.
Figure 10. Model for hypothesized transmission.
Glutamate appears to inhibit fast response but act excitatory to slow
response.
9
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Acknowledgments
I would like to express my gratitude to everyone involved in the
175H program. Thanks go to Carol Marzuola for finding whatever I
needed when nobody else knew where to look and to Bruce Hopkins for
spending long and lonely nights at the Wharf to keep us supplyed with
squid. I also appreciated the enthusiasm and constructive criticisms of
Stuart Thompson, the help from Chuck Baxter in locating several
applicabse articles, and the equipment loaned to me by Mark Denny.
am particularly indebted to my adviser W. F. Gilly. His honesty and
criticisms were an invaluable asset in completing this project but it was
his guidance and seemingly endless hours of attention which I value the
most.