ABSTRACT
In a sand dab fin musc le poeparation, the electnical and mechanical
poperties of a fast twitch muscle have been studied. The "long"
(presumably multiply innervated) fast fibers have resting potentials
of -75 to -80 mU and are taken to threshold for contraction (-40 mU)
by depolarizing junction potenttals. All-on-nothing action potenttals
are not seen, even when suprathreshold shocks are applied. Twitches
ape very fast; contraction reaches a peak within 10 ms and pelaxation
is complete within 20 ms at 18'c. Direct stimulation produces unfused
tetant up to at least 200 Hz.
INTRODUCTION
Uhile certain types of vertebrate muscle have been intensely
studied, relatively little work has been conducted on fish muscle.
Work done show several distinct pecullarities of fish muscle compared
to typical vertebrate musele (e.g., amphibtan, mammalian). Highen
teleost muscle (including the sand dab's family, Acanthopterggi)
utilizes an unusual type of fast fiber innervation. Each fast fiben
is multiply innervated (Barets, 1961; Hudson, 1969), not focally like
most other vertebrate fast muscle. This type of fast muscle innervation
has been previously reported onlg in lower vertebrates. However,
multiple innervation is usual for vertebrate non-action potential
producing slou fibers. In lower ventebrates, multiply innervated
fibers are slou and are used primarily for posture (Peacheg, 1961),
while in higher vertebrates such muscles have very spectalized function
(e.q., extraocular and middle ear muscles) (Bach-g-Rita and Ito, 1966).
Fish musele is claimed further atupical for vertebrates because fish
muscles studied thus fan lack sensong ongans knoun as muscle spindles.
Only a handful of electrophysiologic studies have examined fish
fast fibers. Tost studtes have found that muscle action potentials
are characterized by ovenshoots close to 0 mU on by failure to overshoot.
Spikes tupically appear as graded regenerative responses and are not
all-og-none (Hagtwara 8 Takahashi, 1967; Hidaka 2 Toida, 1969 Hidaka 4 Kuriggam
and Barets, 1961). Other vertebrate fast fibers do show overshooting,
all-or-nothing action potentials. Additionally, few studtes have
investigated propenties of isometoie contraction in these fast fibers.
Of a limited sampling of museles, seahoose fin muscle is reponted as
one of the fastest, with an isometric contraction time as shoot as
10 ms to peak, and shous a fusion frequeneg of 120 Hz (Bone, 1978).
This project furthers our understanding of fast muscle diversitu
by charactenizing the propenties of one set of presumably multiplu
innervated fast fibers, those in sand dab fin muscle. In oueryiew,
the propenties to be examined include: properties of twitch and tetant,
the nature of neuromuscular transmission, and the absence of action
potenttals. In addition, the role of extracellular caleium in the
will 6
activation of contraction considered.
TATERIALS X TETHODS
Preparation
Specimens of Citharichthus sordidus were obtatned fishing off
Lover's Point, Tontereg Bag. Fish were maintained prior to use in
lange outdoor tanks with circulating, fresh sea water.
Phusiologg
Measurement of isometric tenston:
Experiments were performed on isolated single musele units (one
muscle, one bone, and one spine) mounted near their in vivo resting
length in a 10 ml plexiglass chamber. The ends of the muscle close
to the myotomes was pinned to the sylgard (Dow Corning) floor and the
cut spine was tied to a force transducer with silk thread (Gilly 2
Hut, 1980). Contraction was elicited by application of longitudinally
ortentad shocks generated by a Grass 5-ó stimulaton via platinum/
platinum black electrodes. Pulse duration varied between 0.5-2.0 ms
and field strength from 3-35 volts/em. Electrodes were mounted on a
micromanipulator and could be positioned close to the muscle ends.
Contractions were also elicited by local stimulation of a small
number of fibers with a small, pambling bipolar electoode. This
technique was useful in qualitativelg exploring excitability in different
egions of the muscles.
Tension pecordings were taken on a Brush 220 pen recorder and a
Textronix storage oscilloscope.
Simultaneous pecording of junction potential and isometoic tension:
A suction electrode on a motor nerve and an intracellular
microelectrode were added to the apparatus described above to record
neuromuscular transmission. The suction electrode had a 200u, diameten
tip made of polgethylene tubing mounted on a glass capillarg. Fibers
which were seen to contract in response to nerve stimulation were
impaled with the microeleetrode close to the spine end of the muscle.
Tembrane potenttal was measured as the voltage difference between the
intracellular microelectrode (30-30 megohms, filled with 3 T KC1) and
a Ag:AgCl bath electrode, taken through a UPI electmeter. Stimulating
pulses were applied to the nerve using a UPl digital pulse generator.
Experiments ended with between 0-5 mU electrode deift.
Expepiments utilizing tuo intoacellular micpoelectoodes:
Fibers were impaled with two microelectrodes (20-30 To). One was
used for injecting current (filled with 2 f KCitrate) while membrane
potential was measured as described above. Injected curpent was
collected by a Ag:Agcl bath electrode and an operational amplifien.
current-voltage converter (vintual ground) circuit. Pulses were
generated, and reconded data were sampled by a digital computer-based
system described in Gillg X Scheuer (1984). The voltage clamp circuit
used is also described in that pape.
All experiments wera conducted at room temperature, about 19c.
The composition of solutions employed is given in Table 1. Solutions
were changed by draining the chamber and subsequently adding one on
two volumes of new solution, followed by sloshing to disrupt ang
boundary layer around the muscle. For solution changes in which the
chamber was not inittally drained, four times volume was washed
through the chamber.
Experiments described in this report were conducted between
22 Tag 1984 and 1 June 1984.
RESULTS
Anatong:
The fast fin muscle fibers used for study are located along the
posterior end of the body, foom the pegions of doosal fins that ace
elevated during the pheotaxtic response (Ueland, 1984) (see fig. 1).
Two groups of fin museles exist with different onigins: 1) from bones
between the ocular and non-ocular myotome masses (see fig. 2a, arrow)
and 2) from the skin (see fig. 2a). The fin spine is the common
insertion of both types. Only the first type of muscle was studied
in the present report. As shown in fig. 2b, one fin spine is moved
by a total of four myotome-insenting museles. Of these four muscles,
an individual bundle from the ocular side was isolated for experimentation.
The accangement of fibers within a single bundle is diagrammed
in fig. 2b (fiber arrangement sketch). The fast fibers ace long and
run from a spine tendon to a tendon underneath the myotome mass (see
accou in fig. 2b). The superfictal long fibers are spindle -shaped
and have diametens of approximately 100at the centers of the fibers,
tapering doun to approximately 30at the spine end. These fibers ace
difficult to follou along their full lengths because theg weave and
insert among each other. Such weaving makes casual observation of
multiple innervation difficult, and a rigorous attempt to quantifg
innervation is get to be made.
tong fiber sarcomepes ape distinct, and measure between 7.7 and
1.9 in length at rest. This is somewhat shopter than resting
sarcomere length of most vertebrate skeletal muscle fibers.
Fibers of a shorten length relative to the long fibens ape also
depicted in fig. 2b. These oun between the long fast fibers and the
adjacent bone at about a 45° angle. They extend onlg along the
spine half of the muscle bundle. These fibers ace very slou (see
also belou) and were not used for the present studg. However, this
pegion of the muscle deserves histological examination, because
stouctures closely resembling muscle spindles in higher veotebrates
were observed at the spine end of the slow fiber pegion. This is
noteworthy, because muscle spindles in teleosts have been descriged
only in one case (Taeda, et al, 1983), and that structure was verg
primitive, containing only one intrafusal musele fiber.
Properties of tuitches and tetant:
Fig. 3a illustrates that the long fibers respond to a single
shock (between 3-20 volt/cm field strength) applied along the length
of the entire muscle with a fast twitch, 10-15 ms to peak, and a
capid relaxation, 20-30 ms, at 17-20'c. (The term twitch, as used in
this papen is defined as a capid contraction.) This speed of
contraction and relaxation is comparable to one of the fastest
vectebcate muscles described, cat extraocular muscles at 3' c
(Bach-u-Rita 8 Ito, 1966). Long fibeos are much faster than frog
(Rana) twitch fibers which follou a time course of 30-50 ms to peak
contcaction and 100 ms to relax at coom tempecature (Katz, 1966).
Fig. 3b-c indicate that shoot fibers are activated to contract
with yeru strong shocks (25-30 v/cm). The short fibers are exceedingly
slou. Peak contraction occurs in 150-200 ms (not illustrated) and
celaxation verg slow (fig. 3c). Differenttation between the fibees
involved in the slow us. fast contraction can be done by watching
contraction through the microscope. Theg mag be fuother differentiated
bu excising the slou fibers from a whole muscle preparation. Figs 3b
and 3e shou the copresponding oscilloscope polaroids (3b) and chaot
recordings (3c) of tension recorded from a muscle before and aften
cemoyal of the slow fibeos. After cutting awag the short fibers foom
the long, the slow contraction is not seen, only the fast component
remains.
The fast twitch fibers respond to direct repetitive stimulation
of up to 200 Hz (3-20 y/cm, 0.75 ms duration) with tetani of increasing
amplitude (see fig. 4a). High gain oscilloscope traces of these
tetant (not illustrated) show that the response is still not completelg
fused at 200 Hz. Thus, the chart records in fig da appean inaccurately
fused due to the lou frequency response of the chant reconder.
Indirect stimulation via nerve shocks produced fused tetani even
at quite low frquencies, as depicted in fig. 4b. At 600 Hz, maintained
contraction fails. The reason for this is unknoun.
The fast fibeos display typical, strong, extended contractures
in response to K-glutamate (134 mm) (see table 1), caffeine (20 mm)
and acetylcholine (3.5 mm). Records ane not included in this paper.
A high potassium solution depolarizes the membrane, thus causing
contraction. Caffeine induces release of intracellular calcium reserves.
ACh is the presumed neupomuscular transmitter.
A saturated solution of dantrolene sodium and 12 x 10 *  D-600
showed no effect on long fiber tension output in cesponse to direct
electrical shocks. Dantrolene sodium blocks contraction in vertebrate
muscle (e.g., frog mammal) without altering electrical activitg.
D-600 is an organte calcium-channel blocker. Both drugs show vartable
effects on various muscle and nerve preparations.
Neuromuscular Transmission:
At most of the muoneural junctions in fast vertebrate muscle,
each nerve impulse is followed by a similar impulse in the muscle
fibers which propogates rapidlg in both directions toward the tendons,
thus ensuring a suffictently synchronous contraction along the length
of the fiber. Uhile a sunchronous contraction cleanly can occur
in sand dab fast fibers, muscle impulses similar to nerve impulses
(oresumably action potentials) are not seen. Onlg depolarizing
end-plate potenttals can be doiven. The junction potentials that
ace elicited in response to repetitive nerue shocks summate temporally
to depolarize the muscle cell to about -40 mU and hold voltage at
that steady level, failing to depolarize further. Action potentials
are not seen even when high frequency stimuli are applied.
End-plate potenttals ape characterized by long decags (fig. 5a),
taking 63 ms to decag to one-half amplitude of maximum depolarization.
One unexplained phenomenon observed is that the decay accompanging
direct stimulation (fig. 5c) is much quicker than that after indirect
(fig. 5a).
stimulation. Passive depolanization following intracellulae current
injection decays to one-half amplitude in coughlg 10-15 ms (figs. 5c).
Uariation in the rise time of junction potentials with successive
cepetitive shocks is also characteristic of the fast fibers. As
illustcated in figs. 5b X 6a, the first end-plate potential in a
series rises much more slouly than subsequent end-plate potentials.
This observation is not get fully understood, but probably involves
a change in muscle cell membrane resistance with membrane potential.
That contraction will not be produced by a single or a few
junction potentials is demonstrated in figs. 5a, 5b 5c, and óa.
Multiple (at least 5-6) junction potentials must sum in time to doive
membrane potential begond threshold to cause contraction (fig. ób, 6c).
Uhile end-plate potentials fail to doive membrane potential more
positive than about -40 mU,  muscle cell can be depolapized fuothen
using a two microeleetrode protocol (see Tethods). Current pulses
are injected to obtain suprathreshold (for contraction) shocks, and
the presence or absence of action potentials can be observed. As
demonstrated in fig. 7, action potentials are not produced in the
fast fibers. This constitutes fatoly conclusive evidence that fast
fibees do not contract via an action potential propogating mechanism.
A rough estimate of input resistance can be calculated from
figure 7 if the entire fiber is treated as a behemoth resistor. A
value of about 50 Kohms is determined: this is considerably lowen than
for a frog tuitch fiben of approximately 100 Kohms (Katz, 1966).
Input resistance is directly proportional to junction potential
amplitude, and is an indicator of membrane cesistance. A lou R..
value implies a leaky resting membrane.
Involvement of extracellulap calctum in contraction:
The role of extracellular calcium in vertebrate contraction is
controversial and the mechanism of excitation-contraction coupling
is unknoun. As indicated in figure 8, a Ca-free/5 mf EGTA solution
drastically decreases tension, and a Na-free/TIX (a verg specific
sodtum-channel blocker) solution has little effect on contraction.
These results can be explained if these fast twitch fibers are unlike
most vertebrates and are akin to inventebrates in requiring extra¬
cellular calcium for contraction (see Gillg 8 Scheuer, 1984). Voltage
clamp experiments were carpied out to further clanifu the cole of
extracellular calcium in contraction activation, of fast sand dab
muscle. Tembrane potential is held at a centain holding potential
and 20 ms depolarizations are delivered with increasing amplitude
until contractile responses are seen through the microscope. In this
fashion, contractile threshold can be determined. Results of this
procedure at several holding potentials in both Ca-containing and Ca¬
free media are plotted in figure 9. Contraction occurs in O-Ca
medium at a faioly normal threshold voltage until holding potential
is ceduced to quite depolantzed values. At -20 mU holding potential,
contraction fails in the Ca-free medium. Failure is probably due to
inactivation of E-C coupling in a voltage-dependent manner, and not
pelated directly to caleium.
The absence of contraction noted in figure 8 in 0 Ca/5 mf EGTA
medium is probably due to depolarization of the membrane due to low
calcium concentration. Resting potentials measured in the Ca-free
medium tended to be substantially less negative than the noomal
value of -70 to -80 mU. Failure of 0 Na/TTX-containing solutionsto
abolish contraetion is consistent with the view that action potentials
are not involved in response to direct stimulation (fig.3) in the
activation of sand dab muscle.
DISCUSSION
The electrical and mechanical propenties of the fast twitch fibers
desepibed in this study mag seem inappropotate or imcompatible when
considered individually, but when viewed in relation to one anothee,
compose a system well-designed to produce fast, finely-controlled
movements. Threshold for contraction is reached via graded membrane
depolanizations. An all-on-none mechanism is not in operation in
these fibens and action potentials do not propogate. Thus this
system of local, graded depolarizations is capable of producing
sunchronous contraction along the length of the fibers (just like
usual vertebrate twitch muscles) if they indeed are multiplu
innervated as Bone (1978) suggests. Under this mechanism, usual
all-on-none action potentials would be unnecessarg.
At least five junction potentials in fairly rapid succession
appear to be necessany to activate contraction, though the exact
number and frequency remain unresolued at completion of this project.
As seen in figure ó membrane potential reaches threshold fon
contraction (figure 9 shous -44 mU as mean e.p.p. threshold) several
junction potentials before an increase in tension begins. Similanlg,
it is not known precisely how e.p.p.s sum to grade tension output.
Very little if ang difference exists in the depolarizations produced
by 200 Hz us. 400 Hz nenve stimulation (figure 6), get tension due to
400 Hz stimulationis much greater. It should be remembered that the
steadu-state relation between tension and membrane voltage is
exceedingly steep at threshold in frog musele (Hodgkin X Horowicz,
1960). This makes comparision of electrical activity measured in a
single musele fiber with mechantcal activity reconded from the whole
muscle very difficult under the conditions of experiments like those
in figures 5 and 6.
Several other questions posed by the data also must remain
unanswered. Uhg, as indicated in figure 4, are tetani that ace
elicited by direct shocks of the musdle unfused, while tetani
that are elicited by indirect shocks of the moton nerye fused?
Is this pechaps due to some type of sunaptic event or is it related
to the slowen decag of the junction potential; compared to that
following current injection from a microelectrode? Uhy also, does
depolapization to threshold of contraction hold at -40 mU and never
depolanize funther?
Uhat are the functional roles of these fibers in medtating
behavior of the fish? The fast fibers ace likely to be used in anu
fast fin movement, such as the characteristie burging, extension/
retraction of fins, and the initial elevation of fins in the cheotaxtic
response. In ooder to understand the functioning of the fast fibers
in celation to the other muscles that control fin movement,
knouledge of the basic properties of the other fin museles is
necessang. Speculation as to how the sand dab uses its museles for
different movements then would be more profitable.
Sand dab fast twitch muscles nepresent a highly unique solution
to the problem of fast, yet finely controlled movement with a
relatively simple motor neuron system. The mechanies of this sustem
compared to the mechanies of the typical vertebrate, action potential
propogating, singlg or dually innervated system will be of special
compacative interest. Such an analysis will lead to a more
comprehensive understanding of muscle diversitg.
C
ACKNOU LEDGETENTS
Tanu thanks are extended to my instouctors and classmates ton
making this spring a truelg "loco-" one. I further thank Gillg
foc incrediblg devoted instruction and for the rod with whichI caught
mu sand dabs. Special thanks are extended to Todd Scheuer fon
fielding numerous questions with extreme patience and enthusiasm.
And most gratefully, I thank all the sand dabs (and squid!!) who made
this project possible.
LITERATURE CITED
Barets, A. (1961). Contribution à l'étude des systèmes moteurs
lent et capide du muscle latéral des téléostéens. Arch. Anat.
Morphol. Exg. 50. Suppl., 91-187, in Eish Physiologg, vol. 7,
Locomotion. Ed. U.S. Hoar  D.J. Randall. New Vork: Academic
Press, 1978.
Bach-q-Rita, P. X F. Ito (1966). In vivo studies on fast and slow
muscle fibers in cat extraocular muscles. J. Gen. Physiol.
49:1177-1198.
Bone, 0. Locomotor muscle. In Eish Phystologg, vol 7, Locomotion.
Ed. U.5. Hoar and D.J. Randall. New Jork: Academic Poess,
1978, pp. 361-424.
Franzini-Armstrong, C. and L.D. Peacheg. Striated muscle-contractile
and control mechanisms. In Discoverg in Cell Biol., the J. of
Cell Biologg. (1981). vol. 91, no. 3, part 2, pp. 166-188.
Gillg, U.F. and C.S. Hui (1980). Techantcal activation in slow and
twitch skeletal muscle fibres of the frog. J. Physiol. 301:
137-156
U.F. and T. Scheuer (1984). Contractile activation in scorpion
Giutg
striated musele fibers: dependence on voltage and external calcium.
J. Gen. Phystol. In press.
Hagiwara, 5. and K. Takahashi (1967). Resting and spike potentials
of skeletal muscle fibens of salt-water elasmobpanch and teleost
fish. J. Phystol. 190:499-518.
Hidaka, T. and H. Kurigama (1969). Effects of catecholamines on the
cholinergie neuromuscular transmission in fish red muscle.
J. Phustol. 201:61-71.
Hidaka, T. and N. Toida (1969). Biophysical and mechanical properties
of ced and uhite muscle fibres in fish. J. Physiol. 201:49-59.
Hodgkin, A.L. and P. Horowécz (1960). Potasstum contractures in
single muscle fibres. J. Physiol. 153:386-403.
Hudson, R.C.L. (1969). Polyneuronal innervation of the fast muscles
of the marine teleost Cottus scoppius. J. Exg. Biol. 50,47-67,
in Eish Phystologg, vol 7, Locomotion. Ed. U.5. Hoae and D.J.
Randall. New Vork: Academic Press, 1978.
Hudson, R.C.L. (1973). On the function of the white muscles in
teleosts at intermedtate swimming speeds. J. Exp. Btol. 58,509-522.
Johnston 1.A. Dynamic properties of fish muscle. in Fish Biomechanics.
Ed. P.U. Uebb and D. Weihs. New Vork: Praeger, 1983, pp.36-67.
Katz, B. Nerve, muscle, and sunapse. Neu Vork: Nc Grau-Hill. 1966.
Kao, C.U. (1966). Tetrodotoxin, saxitoxin and thein significance
in the studg of excitation phenomena. Phacmacologtcal Revieus.
18:998-1049.
Lüttgau, H.C. and H.G. Glitsch. Dembeane phystologg of necve and
muscle fibres. Stuttgart: Gustay Fischen Verlag. 1976.
Luttgau, H.C. and G.D. Toisescu. Ion movements in skeletal muscle
in celation to the activation of contraction, ch. 26 in
Phystologg of membcane disonders. Ed. T.E. Andceolt, et al.
Plenum Publishing Corp., 1978.
Maeda, V., 5. Migoshi and H.Toh (1983). First obseovation of a
musele spindle in fish. Nature. 302:61-62.
Narahashi, T. (1974). Chemicals as tools in the study of excitable
membranes. Physiol. Revieug. 54:813-889.
Peacheg, L.D. Stoucture and function of slow striated muscle. In
Btophystes of physiological and pharmacological actions (1961)
by the Ameo. Assoc. foo the Advancement of Science, Uash. D.C.
Prosser, C.L. Muscles. In Compacative animal phystologg. Ed.
U.L. Prosser. Philadelphia: Saunders College, 1973, pp.719-788.
Ueland, F. (1984). Rheotaxis in Citharichthug soodidus - a dab into
hydrodgnamics. Spoing course, 175H, Hopkins Tarine Station.
C
TABLE 7
constituent concentration (mm)
solution
Iris
other
Naci
Koi Noca,
Caci
200
Normal Fish
0 Ca Fish
200
5 EGTA
Normal Gilat
diluted for fish
200
6.2 0.8
0 Na Gilat
4
diluted for fish
6.2 0.3
200 NNG
K-Glutamate, Gilai
4
diluted for fish
134
6.2 0.8
Table 1. Solutions emploged.
pH's of all solutions emploged ranged between 7.0-7.3, osmolaritu
between 400-420 mosm.
30 mm glucose was added to each solution poion to experiments.
Uantrolene sodium was obtained from U. Gillg, D-600 from T, Scheuer.
All other chemicals were obtained from Sigma Chemical.
All experiments were conducted in fish ringen except those necessitating
0 Na. and K-Glutamate. Uhen gilai eingers wene used, fish cingee was
used initially as a control. Fish and gilai oingers did not give
differing control twitches and tetani.
FIGURE LEGENDS
Figure 1. Fins up posture. Uhen ortented with its anterior end into
a waten curnent of 30 cm/s and fasteo, a sand dab elevates its posterion
(both dorsal and ventral) fins in what is termed the pheotaxtic response,
Fin elevation allous a fish to maintain a steady position in a cucrent,
due to the elatonship:P
See Ueland, 1984
aboue above belou belou'
for a biomechantcal analusis.
Eiguce 2. Fin musculature. 2a) The in situ ortentation of the
myotome-insenting muscles is depicted (see arrow). 2b) Tyotome¬
insenting museles (skin-insenting museles cemoved). The arrangements
of the bones and muscles are illustrated. Note the orientation of the
long fibers.
Figure 3. Fast contoactions. 3a) Traces of isometoic tension
elicited by shocks of increasing strength as recorded on the oscilto¬
scope. The top (horizontal) trace depicts baseline of shock strength
and the blips on the left indicate stimulus stoength measured in volts.
The bottom shous isometric tension measured in mg. Note that peak
contraction occurs in 10 ms.
3b)Isometoie tension reeoded from
a muscle before and after the shont fibens ane excised. 3e) The
chaot peconds that correspond to the oscilloscope traces of 3b. The
top cecord monitors the stimulus, while the bottom record monitors
tension. Single shocks elicited the twitches shown. Note that the
muscle was stretched after cutting, and this mag have resulted in
recordings of falsely greater tension in the "long fibers onlq" record.
Eigure 4. Contraction amplitude varies with stimulus frequency.
Aa) Uinect stimulation of the muscle. Tetani ape not completelu
fused at 200 Hz though chant recondings indicate fusion. This
discrepancy is due to the chart recorder pesponding at too lou a
frequency to accurately record unfused tetani. High gain oscilloscope
traces (not illustrated) show the unfused nature of the tetant.
4b) Indirect stimulation of nerve. Nerve shocks produce fused
tetani at lou frequeney stimuli.
Figures 5 and 6.
The top trace monitons the curnent that is passed
to shock the muscle or nerve, and also indicates the 0 mU level. The
middle trace monitor membrane potential. The lowest trace nepresents
isometrie tension.
Figuge 5. End-plate potenttals. 5a) Stimulation thoough nerve.
Junetion potentials in pesponse to shocks are illustrated.
5b) Several junctionppotentials ane reconded. Note the absence of
contraction even though threshold is reached, and note the discrepancies
in rise time of the various e.p.p.s. 5c) Stimulation via injecting
current through the intracellular microelectrode. Note the difference
in decay time with different modes of stimulation (Sa ys. 5c).
Figure 6. E.P.P.s and contraction: Contraction amplitude varies
with the number and frequency of stimuli. Tension output is sharply
dependent on the number (four in óa vs. twelve in ób) and frequencu
(200 Hz in ób us. 400 Hz in 6c) of administered shocks. Note again
that e.p.p.s hold at a steady level, failing to depolarize further.
Eiguce 7. Fast fibers do not produce action potentials. This
recording of membrane potential was taken using two intracellulae
microelectrodes. The top trace in the record indicates the injected
current in nA and the current pulses ane seen on the left. This
trace also notes the 0 mU level. The bottom trace monitors membcane
potential. Shocks which brought membrane potential to or beuond
thoeshold for contraction (about -44 mU) caused twitches. Tension
is not meconded in this experiment, but contraction was seen theough
the microscope. It'is important to abserve that while indirect
nerve shocks were unable to dive membrane potential more oositive
than - 40 mU, direct injection of current can take membrane
potential at least to a suprathreshold level of -20 mU. Undec
such suprathreshold conditions, action potentials are still not
produced.
Eiguce 8. Effects of 0 Na, TIX 2 0 Ca on contraction. Isometoic
tension recordings illustrate that Na-free/ TIX media show little
ettect on tetanic contractions while Ca-free/EGTA medta dcastically
decrease tension outout.
Eigure 9. Independence of contraction from extracellular calcium.
A voltage clamp analysis demonstrates that extracellular calcium is
not required for fast fiber contraction. The points graphed are
numbered in order of successive determinations at the various holding
potentials. The average value for contraction threshold foom e.o.o.
is -44 mU and is draun on the graph (dotted line).
—â
r



FiQuge 2
FIN MUSCULATURE, Citharichthys sordidus
myotome-inserting muscles
Transverse section
skin-inserting muscles
ocular
D



non-ocular
myotome-inserting muscles
bones
fiber arrangement
muscles & nerves
-spines


—short fibers
—bones
—iong fiers
— 4 musces

4
M4
t
myotome mass
s0q
2
—
118
EPPS & CONTRACTION: Contraction amplitude varies with
number & frequency of stimuli
—OmV
200Hz

-80mV
200 Hz
1004A 4004z
56 mg
50m
0
L
O
Z
D
L
0.
2


2
—
8
PIGURE 9
INDEPENDENCE of CONTRACTION from EXTERNAL Cat
• Normal Fish Ringer
Ca free, 5mM EGTA Ringer
X Faiure to contract
+0
o
O7
-10
06
O5
-20
9
-30
82
•2
-40
—- -- -- --- — ——— mean EPP
— —— —
S=3 mV, n-7
-50
-60
-70
-80
-90
00
-00 -90 -80 -70 -60 -50 -40 -30 -20
HOLDNG POTENTIAL (nV)