Calcium Spikes in Metridium
Introduction
In preliminary experiments designed to observe isometric
contractions in the sea anemone Metridium senile it was noted
that Tetrodotoxin in low or high concentrations (0.1 mg/1 and
10 mg/1) had no effects on responses to electrical stimuli.
Tetrodotoxin has the specific effect of holding closed the sodium
gate in other nervous systems and thus blocking conduction
(Kao and Fuhrman, 1967; Takata, Moore, Kao, and Fuhrman, 1965).
Following this observation experiments were undertaken
to show that Na was not the ion used in depolarizing the axon
by influx. The ion responsible for depolarization is usually
termed the spike ion. Noting the work of D. M. Ross (1969)
it was hypothesized that calcium might be used instead, since
Ross shows that when calcium is removed from artificial sea
water the anemone no longer responds. It is concluded that the
evidence indicates that calcium is the spike ion in Metridium
senile.
Materials and Methods
Apparatus
Mechanical recordings were made with a counterbalanced
lever system (Robson and Josephson, 1969), modified in that
the transducer used was a Grass model FTOB force transducer.
Thus the contractions were measured isometrically, and were
Calcium Spikes in Metridium
recorded on a Grass model 5 polygraph. Temperature was held
to the range between 12-16°0.
Stimulus
The stimulus electrode consisted of two platinum wires,
sixteen guage, insulated to lmm from the tip, placed 2mm apart.
Electrical pulses were obtained from a Grass model S6 stimulator,
through a Grass model S.I.U. 4678 stimulus isolation unit.
Voltages listed on figures are output across the electrode tips.
Preparation of animal
Metridium senile with average diameter of 5 cm were collected
from a depth of 6 meters on Wharf Number 2 of Monterey Harbor.
Monterey, California. In all cases the animals were maintained
for one week in aquariums with fresh running sea water before
dissection. Preparations were obtained by the method of Batham.
Pantin, and Robson (1960), and allowed to equilibrate for eight
hours.
For each test run the mesentary was removed from running
sea water and placed directly onto the apparatus. The prepara¬
tion was then stimulated and responses recorded until a series
of five similar responses were evidently typical of that mesen¬
tary.
The mesentary was then removed from apparatus and placed
in the test solution. A control placed in fresh sea water and
another in artificial sea water were held under otherwise iden¬
tical conditions. For tests longer than four hours, all
Calcium Spikes in Metridium
solutions were changed every four hours. At the end of the test
period, each preparation was placed on the apparatus in fresh
test solution and a series of stimuli were applied, similar to the
series used initially. Thus, results reported refer to the
differences between the initial and final recordings relative
to the differences in the controls.
est solutions
The five solutions used were prepared as follows. Sea
water was used from the running sea water system. Its temper-
ature ranged from 12-15°0. Tetrodotoxin in.53M Nacl in dis¬
tilled water at 100 mg/ml was added to 100 ml of sea water.
Artificial sea water was prepared using formula M. B. L. No.
given in the Woods Hole Marine Biological Laboratory Manual
(Cavanaugh, 1956). Calcium-free artificial sea water was pre-
pared by formula number 6, that of Moore, given in the same
reference. Cobaltous chloride solution was prepared by
adding isotonic Cocly to sea water in the same final concen¬
tration as Caclo, 10 mM.
Electrical recording
Attempts were made to record nerve and muscle potentials
from the preparation by means of a glass suction electrode of
inner diameter 150u. A Tektronix type 122 preamplifier and
Tektronix type 502 scope were used for display. Recordings
were seen that resembled those reported by Robson and Joseph-
son (1968), but these were rare and are not further reported.
Calcium Spikes in Metridium
Results
Ideal versus non-ideal sites
1. There are two distinct types of stimulus site, and
some gradation in between these. The first, termed ideal, is
is characterized by giving full responses to two pulses of
10 msec duration, 0.2 sec apart at 5 volts. Response immediately
follows the second pulse, as shown in Figure 1.
2. At the second type, termed a non-ideal site, more
pulses in a series, or higher voltage is required to illicit
a similar responseto an ideal site, as shown in Figure 2.
3. These sites are not automatically apparent and are
found by trial and error.
Stimulus site decrement
1. Stimulation at any site decrements the ability of the site
to respond to further stimulation, as shown in Figure 3
2. The rate of decrement at an ideal site is slower than
that at a non-ideal site. (Figure 4 ys. 3)
3. The rate of decrement depends on the condition of the
preparation, since by approximately sixty hours after dissection
the preparation fails to respond and necrosis is evident.
4. The more non-ideal the site is, the faster it decre¬
ments. Also, the larger or longer the stimulus is used, the
faster decrementation occurs. (Figure 5)
5. Any site once decremented will return to its original
Calcium Spikes in Metridium
potency in less than thirty minutes, whether or not the elec¬
trodes are physically removed, as long as no electrical stimuli
are used.
Distinguishing-nerve net initiated contractions from electrically
initiated contractions.
The nerve net was considered deactivated after four hours
in MgClo may also slightly impair muscle ability while CoCl
in relatively small concentrations quickly deadened nervous
response but did not seem to impair the muscle's ability to
contract.
A comparison of differences in response to electrical
stimuli between preparations in sea water and preparations in
Cocl or MgCl is shown in Table I. Note that in all cases
a nerve net initiated contraction requires two pulses or more,
while muscle may be stimulated by one pulse.
Responses to stimuli in test solutions
Tetrodotoxin
Six preparations were tested at the concentrations and
times given below.
l at 1 mg/ for 4 hours
1 at 2.5 mg/1 for 12 hours
1 at 5 mg/ for 12 hours
2 at 10 mg/ for 24 hours
(Figure 7)
1 at 1 mg/ for24 hours
(Figure 8)
1 at 2.5 mg/ for 1 hour
(Figure 9)
l at 5 mg/ for 1 hour
Calcium Spikes in Metridium
The last preparation, held in increasing concentrations
over time, is shown in Figure 6 and is representative of all of
the mesentaries that were tested. In all cases the preparation
in Tetrodotoxin solution responded better than its control at any
point in the test period. Tetrodotoxin was found to have no
effect on responses to stimuli and no effect on specific site
decrement.
Artificial sea water, M. B..L. No. 1
Artificial sea water was used as a control for tests
in calcium-free artificial sea water. This formula does not
have the full complement of ions found in real sea water and per¬
haps for this reason tends to increase the rate of decay of the
preparation as a whole, as seen in Figure 10. This overall
decay was barely noticeable in thirty minutes but was apparent
after four hours. The preparation decays uniformly in that
each site is effected in the same way, giving smaller responses
and showing heightened rates of stimulus site decrement.
Calcium-free artificial sea water
Within ten minutes after application the nervous
response is extinguished. The preparation relaxes continually,
and immediately after application the rate of stimulus site
decrement increases markedly. After twelve hours of application
muscle responses to 100v. stimuli are still present, but are
slower and of less magnitude than produced by a mesentary in
Calcium Spikes in Metridium
in MgCl, solution. (Figure 11 and Figure 12)
Cobaltous chloride in sea water
The effect of 10 mM CoCl, is found to be very similar
to calcium-free artificial sea water, as seen in Figure 13, CoCl
is seen to work much quicker than either Mgol, or calcium-free
mediums and is seen to have less blocking effect to muscle con¬
tractions illicited by high voltage stimuli (Figurellvs.12).
Stimulus site decrement is more rapid with CoCl, than any other
solution tested. (Figure 15)
Discussion
In concentrations of 0.1 mg/1 Tetrodotoxin completely
inhibits conductance in frog sciatic nerves within ten minutes
after application (Kao and Fuhrman, 1967). Tetrodotoxin has high
rates of diffusion in frog neurons, with or without myelin
sheath, and the preparation used here is quite thin with a high
surface to volume ratio (Kao and Fuhrman, 1963, 1967). Thus it
is reasonable to conclude that Tetrodotoxin is reaching the
neurons but is having no effect.
D. M. Ross has shown that preparations in sodium-free sea
water show no change in response to electrical stimuli (Ross,
1960). Thus it is reasonable to conclude that sodium is not the
spike ion, since neither the absence of sodium nor the presence
of an inhibitor of its conduction affects the system.
It has been shown here that the absence of calcium ions or
Calcium Spikes in Metridium
the presence of any of several other divalent cations extinguishes
nervous activity. These observations agree with those of Ross.
He further notes that if both calcium and magnesium ions are
removed from sea water the preparation's activity and responses
to stimulation are heightened (Ross, 1960). This particular
experiment was only carried out for thirty minutes, which in the
absence of magnesium ions (which Ross showed to be critical)
may not be long enough to deplete calcium ion reserves around the
neuron. It is thus theorized that the spike ion is calcium and
that magnesium ion, in competing with calcium controls the activity
of the nerve net.
In MgClo or calcium-free sea water the ability of muscles
to contract is impaired and eventually extinguished. This effect
is found to be much less with CoCl. It is theorized that cobalt
ion blocks nervous conduction by diffusing through interstitial
fluid and competing with calcium ion influx around the neuron
in the same manner as magnesium ion. But because of its larger size
cobalt ion is unable to diffuse into the muscle end plate to
block the calcium ion output there, or does so at a much slower
rate. A single experiment using manganous chloride showed its
effects on electrically initiated muscle contractions to be less
than magnesium ion but greater than cobalt ion.
These results cannot be regarded as conclusive because of
difficulties in two main areas. First, kWestfall has shown that
the synapses in Gonionemus contain dense core vessicles similar
to those in the vas deferens of the rat (Westfall, 1970). Since
release of these vessicles by exocytosis is catalyzed by calcium
Calcium Spikes in Metridium
ion, it is possible that calcium-free sea water with magnesium
ion present inhibits vessicle release. Second, calcium-free
sea water or the presence of other divalent cations may in¬
hibit muscle end plate potentials, rather than the nerve net.
Considerable evidence has been presented here to contradict this
idea, but the possibility is still present.
In order that these findings be made conclusive, it will
be necessary to record electrically from the nerve net during
stimulation, a test which has been found difficult. The evidence
given here is clear in itself and it is reasonable to believe
that calcium is the spike ion in the anemone nervous system.
Summary
1. There are two distinct types of stimulus site: one
which responds to two pulses at low voltage, termed ideal, and
the other which requires more pulses at higher voltage, termed
non-ideal.
2. Stimulation at any site decrements the ability of the
site to respond to further stimulation.
3. Nerve net initiated contractions may be differentiated
from electrically initiated contractions.
4. Tetrodotoxin has no effect on the responses of Metridium
senile to electrical stimulation.
5. Calcium-free sea water extinguishes response to elec¬
trical stimulation.
10
Calcium Spikes in Metridium
11
6. The presence of other divalent cations which are believed
to compete with calcium extinguishes response.
7. It is theorized that calcium is the spike ion in the
nervous system of Metridium senile
I thank Dr. and Mrs. Frederick Fuhrman for their immense
help and guidance throughout this project, as well as Nathan
Howe for reading this manuscript and his advice on performing
dissections. I further thank Dr. D. P. Abbott and the faculty,
staff, and students of Hopkins Marine Station of Stanford Univer-
sity for their continued encouragement, especially C. J. Rogers,
without whose help this manuscript would still be untyped.
Calcium Spikes in Metridium
Literature cited
Batham, E. J., C. F. A. Pantin, and E. J. Robson, 1960. The
nerve-net of the sea anemone Metridium senile:
the mesenteries and the column. Quart. J. Microcop.
Sci. 101: 487-510.
Cavanaugh,
G. M., ed., 1956. Formulae and Methods V. of the
Marine Biological Laboratory. Chemical Room.
Marine Biological Laboratory, Woods Hole, Massachu-
setts.
Kao, C. Y., and F. A. Fuhrman, 1963. Pharmacological studies
on tachichatoxin, a potent neurotoxin. J. Pharmacl.
exp. Ther. 140: 31.
Mosher, H.
S., F. A. Fuhrman, H. S. Buchwald, and H. G. Fischer,
1964. Tarichatoxin-tetrodotoxian, a potent neuro-
toxin. Science 144: 1100.
Robson, E. A. and R. K. Josephson, 1969. Neuromuscular properties
of mesenteries from the sea anemone Metridium. J. Exp.
Bio. 50: 151-168.
Ross, D. M., 1960. The effects of ions and drugs on neuromuscular
preparations of sea anemones. I. On preparations of the
column of Calliactis and Metridium. J. Exp. Bio. 37:
732-752.
12
0
Calcium Spikes in Metridium
13
Takata, M., J. W. Moore, C. Y. Kao, F. A. Fuhrman, 1965,
Blockage by tarichatoxin of sodium conductance change
in excitation. J. gen. Physiol. 49: 977
Westfall, J. A., 1970. Ultrastructure of synapses in a prima¬
tive coelenterate. J. Ultrastructure Res. 32: 237-
246.
Calcium Spikes in Metridi
Caption to Table
Table 1. Comparison of Electrically Initiated Contractions
to Nerve Net Initiated Contractions
Calcium Spikes in Metridium
lable 1
Parameter
Voltage Applied
Response to 1
Stimulus
Response to'
Stimuli
Response to a
Series of
Stimuli
Relaxation
Time
Electrically
Initiated
Contractions
90-100v.
small
small, additive
graded,
additive
long
Nerve Net
Initiated
Contractions
5-30v.
none
immediate
complete
contraction
sustained
complete
contraction
short
Calcium Spikes in Metridium
Captions to Figures
Figure 1.
Stimulation at an ideal site showing immediate
response to the second pulse.
Figure 2. Stimulation at a non-ideal site using 30v.
Figure 3. Decrement at an ideal site.
Figure 4. Decrement at a non-ideal site using 30v.
Figure 5. Decrement at a non-ideal site showing effect
of number of stimuli.
Figure 6. Initial control for Tetrodotoxin tests.
Figure 7. Stimulus after 24 hours at 1 mg/1 TTX
Figure 8. Stimuli after 1 hour at 2.5 mg/1
Figure 9. Stimuli after 1 hour at 5.0 mg/1 TTX
Figure 10. Stimuli after 12 hours in M.B.L. 1
Figure 11. Lack of response after Calcium free sea water, 1 hr.
Figure 12. Muscle response after 24 hours in Car- free sea water.
Figure 13. Lack of response after 1 hr. in CoCl, solution.
Calcium Spike in Metridium
Captions to Figures (Continued)
Figure 14. Muscle response after 24 hrs. in Coclo solution.
Figure 15. Site Decrement after 5 min. in CoClo solution.
Calcium Spikes
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Calcium Spikes in Metridium
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Calcium Spikes in Metridium

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Calcium Spikes in Metridium
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