Summary
The compound ascidian Botrylloides sp. was anaesthetized through
the application of two types of substances. An isotonic solution of
Tris chloride was found to be an effective anaesthetic. The addition
of various concentrations of the divalent cations magnesium and
calcium altered its action. Addition of calcium caused a slowing of
this action, while addition of magnesium accelerated the process.
Three local anaesthetics, procaine, dibucaine, and tetracaine were
testedfor anaesthetic effectiveness on Botrylloides. All three paralyzed
the animals, but only procaine was found to be useful as a reversible
anaesthetic. Full recovery times were obtained for various exposure
periods to procaine and a dosage-response curve for both anaesthesia and
reversal were constructed.
Introduction
Anaesthetizing ascidians has been a problem in working with them
because of the large time lag between application of the anaesthetic and
final complete narcotization. Classical anaesthetics include magnesium
chloride, magnesium sulfate, and menthol (Personal communication from
D. P. Abbott, Hopkins Marine Station), and all of these are subject to
the above criticism. The actual literature on ascidian anaesthesia is
severely lacking, being limited to little more than isolated, brief
observations in various studies concerned with other aspects of ascidians.
I describe here two new methods for tunicate narcotization involving
manipulation of the cationic composition of sea water and application of
local anaesthetics. Both methods are very useful in that they quickly
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immobilize the animal, allowing for dissection or other manipulation.
Further, narcotization is easily reversible upon application of fresh
sea water if a conscious animal is again required.
Materials and Methods
The ascidian Botrylloides sp. was obtained from the fouling community
in Monterey Harbor and transferred to five cm by eight om glass slides
for easier handling. This was accomplished by removing a colony of animals
from its natural substrate and after removing as much extraneous tunic as
possible, attaching the animals to the slide by tying them on with thread.
Approximately five days later, after the ascidians had attached to the
glass, the thread was cut to allow further growth and budding. These
colonies were kept in a slide tray under slowly running sea water, and would
remain healthy in this form for at least five weeks.
The tentacles in the oral siphons of these animals were found to be
very sensitive, causing a contraction of the entire pharyngeal region when
mechanically stimulated. This response, thought to be a natural ejection
mechanism (Hecht, 1918) to rid these filtering animals of oversized bits
of detritis, served as a good indication of the irritability of the animal
to tactile stimuli. Thus, the oral tentacles of the animals were stimulated
with a small, stainless steel pin, and the response was either easily
visible or absent.
While in a dissecting bowl, the colonies were examined by observation
under a dissecting microscope throughout the experiment. Before exposure to
the anaesthetic, the animals were examined to confirm that the colony was
healthy and that the normal pharyngeal contraction mechanism was present in
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every individual. At this point, the sea water was replaced by the the
appropriate anaesthetic solution. Immediately, the colony was examined for
pharyngeal contractile activity by mechanically stimulating the tentacles,
accomplished by gently circling the inner lip of the oral siphon with the pin.
This operation was performed on twenty individuals in the colony on a
regular schedule of either every five or ten minutes.
After the appropriate exposure time, the anaesthetic was removed by
pouring out the solution surrounding the colony and refilling the dissecting
bowl with fresh sea water. The bowl was filled twice and dumped out, in
order to more thoroughly wash the colony. The same data collection pro¬
cedure was used on this recovery phase of the experiment.
The Tris solutions were prepared daily by using a combination of Trizma
HCl and Trizma Base (Sigma Chemical, St. Louis, Mo.) in the correct
proportions to create a 450 mM solution with a pH of 8.0. MgCl, and Cacl,
were added to the Tris solutions from 1M stock solutions (see also Results).
Procaine, dibucaine, and tetracaine (Sigma Chemical) solutions were prepared
by adding the local anaesthetic directly to fresh sea water. These solutions
were used immediately.
Results and Discussion
Tris Studies.
450 mM Tris chloride with no addition of Caclor MgCl, was found to
have a potent anaesthetic effect on tunicates. None of the monovalent cations
normally present in sea water were present in the Tris solution. The
mechanism of Tris anaesthesia is unknown but two possibilities are likely.
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Sodium is known to be important in nervous propagation in many systems, and
its absence is an obvious possible explanation for the anaesthetic effect
observed. Tris may also have other, more subtle neuropharacological effects
of course.
Calcium is another cation known to be important in both nervous and
muscular electrical activity and impulse propagation (Hagiwara & Byerly,
1981). Figure 1 displays the relationship between the percent of the 20
stimulated individuals in the colony under study which showed pharyngeal
contraction in response to stimulation vs. time at various concentrations
of calcium in the presence of 450 mM Tris. The results of Figure 1
indicate that anaesthesia is most rapid in Tris-O Ca, showing that Ca ions
in the solution must retard or inhibit the anaesthetic action of Tris.
These results suggest that calcium is important at some point in the
reflex pathway from tentacle stimulation to mantle smooth muscle contraction.
A possible site of calcium's action might be directly on the smooth muscle
mediating contraction, as extracellular calcium is of prime importance in
activating smooth muscle contraction in the tunicate Ciona intestinalis
(personal communication from Ms. Gabrielle Nevitt, Hopkins Marine Station).
All synaptic transmission would also likely be blocked in such a O Ca
solution (Katz & Miledi, 1967).
Observations on recovery time after washing out Tris showed a similar
effect. Recovery following Tris-O Ca followed a much slower time course
than with either 25 mM or 50 mM Ca. This possibly represents the time
necessary for the displaced calcium to return to the tissues, where it
becomes important for smooth muscle contraction and/or nervous transmission.
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Figure 2 represents a similar but more detailed study on the solitary
tunicate, Ascidia ceratodes. More concentrations of calcium were studied,
but the sample sizes (i.e., number of animals) are considerably smaller,
varying from three to five in each concentration. The data in Figure 2
indicate a trend similar to that of Figure 1. The final level of recovery,
however, also appears to be a graded function of calcium concentration.
Figure 3 and all subsequent figures are again for Botrylloides.
Figure 3 represents a study similar to that in Figure 1, but for Tris-Mg
solutions rather than Tris-Ca. In this case, anaesthetization in lower
concentrations of the divalent Mg cation follows a slower time course than
in the higher concentrations. This is ascribed to be due to a competitive
inhibition-type interaction between magnesium and calcium, though conclusive
proof is not available. Mg is known to show such inhibition of calcium's
involvement in synaptic transmission, for example (Ross & Stuart, 1978).
A discrepency between the time courses for 450 Tris-O Ca in Figure 1 and
450 Tris-O Mg in Figure 3 can be seen upon close examination. Though they
were performed at different times, both the Ca study and Mg study employed
precisely the same solutions for all three concentrations, varying only in
the amount of Cacl, or MgCl, added. Further, all three data sets for each
cation were obtained simultaneously. To settle this point, a control
study was run using three colonies of Botrylloides and a 450 mM Tris-O Ca
solution. Data from the three colonies were very similar, and followed
a time course intermediate to the O Mg and 0 Ca study. Thus, while data
from Figure 1 may not be strictly comparable to that of Figure 3, the
comparisons within graphs are justifiably so.
Finally, the animals, while anaesthetized with Tris, were severely
-6-
insulted by poking a sharp steel probe through the wall of the pharynx
and lifting upward to create a large rip in the pharynx. The animals
failed to respond,and this was interpreted as a good indication that a
true stateof anaesthesia had been reached. Although animals exposed to
Tris solutions were found to recover fully, while they were anaesthetized
the colonies often appeared somewhat shrunken and compressed, and many of
the oral siphons were partially or completely closed. Also, blood flow
through the colony was substantially slowed, and the colony generally had
an unhealthy appearance.
Local Anaesthetic Studies
Three local anaesthetics were also tested for anaesthetic effectiveness.
Figure 4 shows a compilation of results for procaine, dibucaine, and
tetracaine at dosages varying by a factor of 1000. Figure 4a shows an
immediate response caused by all three anaesthetics at 1 mM concentration
(SW+A arrow). However, upon washing after thirty minutes exposure (SW
arrow), only those individuals exposed to procaine recovered fully. Figure
Ab (100 uM anaesthetic) shows a much slower anaesthetic response by those
individuals exposed to procaine compared to those exposed to tetracaine and
dibucaine. Again, only the procaine sample recovered. Figure lc (10 uM)
shows no response to procaine, but still a fairly rapid reaction to dibucaine
and tetracaine. However, dibucaine and tetracaine were irreversible at
this dosage and exposure time. Figure hd (1 uM) shows no responsiveness to
any of the three drugs. The 50% anaesthetized point at four hours for
tetracaine actually represents lethality for these individuals at that
time. Other tetracaine dosages displayed similar lethal activity. In
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all cases with tetracaine, although recovery may have been transiently
complete (e.g. Fig. 4c), the individuals were compressed and constricted and
ultimately died. Of the three local anaesthetics tested, only procaine
appears to be completely reversible.
Varying the concentration of procaine over a narrow range caused great
deviation in anaesthetic responsiveness. Figure 5 shows the time courses
for disappearance and recovery of pharyngeal contraction in Botrylloides to
three concentrations of procaine between 100 - 200 uM. Over this narrow
range, the time until anaesthesia varied by forty minutes, with the lowest
concentration showing the longest time course. The reversal time course
after a standard sixty minute exposure showed a similar trend, with the
lowest concentration recovering more rapidly than the other two.
Recovery time was also found to be a function of exposure period length.
Colonies were exposed to lmM procaine for varying periods, and recovery
time course data was gathered and plotted. Figure 6 shows the recovery
time course following washing in fresh sea water (at time zero) after the
indicated exposure periods. In the low range of exposure times, small dif¬
ferences in exposure time made large differences in recovery rates (compare
15 min., 30 min., and 1 hr.). The recovery time course for two, three, and
four hour exposures are all similar. This seems to indicate that procaine's
maximum pharmacological effect was reached at approximately two hours exposure,
and additional exposure length changes recovery time very little.
A dosage-response curve for procaine's effects was constructed by
exposing colonies of Botrylloides to various concentrations of procaine for
a standard sixty minute period. The "threshhold" dose below which all
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members of the colony did not become completely narcotized was 100 uM.
At 75 uM, the colony did not become narcotized even after three hour exposure.
However, at this dosage, certain individuals would be unresponsive at a
certain time, but responsive at a later time while still exposed to the
procaine solution. This did not represent an anaesthetized state, as defined
by this study. As procaine concentration increased above 100 uM, the time
before the entire colony was completely anaesthetized decreased sharply, as
indicated by the circles in Figure 7. Essentially, immediate action of
procaine was reached by the 1mM point.
Recovery time vs. concentration is also plotted in Figure 7 (triangles).
The recovery points of zero at 75 and 50 uM represent dosages which were
not effective. As concentration increased from these points, recovery
time also increased until a concentration of 2 mM, where the colony did not
recover after a five hour washing in sea water. All dosages at this level
and above were deemed lethal.
Individuals anaesthetized with procaine in the nonlethal concentration
range maintained ciliary pumping throughout their exposure. Upon gross
examination, heart rate did not appear to be affected. Thus, except for
the apparent absence of sensory or motor nerve activity and smooth muscle
responsiveness, the animals seemed viable and healthy. This is also
supported by the ready reversibility of procaine.
Finally, animals exposed to procaine could be preserved in 3% formalin
in sea water and maintain their living, filtering appearance, whereas those
not anaesthetized prior to preservation would undergo fairly violent
pharyngeal contraction and thus appear distorted and substantially different
-9-
than when alive.
Procaine thus appears to be the choice anaesthetic of those studied.
being easily reversible, maintaining the animals in an open and relaxed
state, and being useable in a rather wide dosage range. Tris is slightly
less useful, requiring a longer period until anaesthesia, and subjecting
the animals to a temporary unhealthy looking state. Both, however.
allow the researcher to limit muscular responses in Botrylloides, if such
a state is required.
-10-
Acknowledgements
Special thanks to William F. Gilly for advice and guidance throughout
the experiments, and to Donald P. Abbott for additional help.
References
Hagiwara, S., and L. Byerly. Calcium channel. Ann. Rev. Neurosci. 4: 69-125.
Hecht, S. 1918. The physiology of Ascidia atra Leseur II Sensory Physiology.
Am. J. Physiol. 15: 157-187.
Hille, B. 1977. The pH dependent rate of action of local anaesthetics
on the node of Ranvier. J. Gen. Physiol. 69: 475-496.
Hille, B. 1977. Local anaesthetics: Hydrophilic and hydrophobic pathways
for the drug-receptor reaction. J. Gen. Physiol. 69: 197-515.
Katz, B., and R. Meledi. 1967. The release of acetylcholine from nerve
endings by graded electrical pulses. Proc. R. Soc. London B. 167: 23-38.
Narahashi, T. Neurophysiological basis of drug action: Ionic mechanism,
site of action and active form in nerve fibers. In: Biophysics and
physiology of excitable membranes (W. J. Adelman, ed.). New York:
Van Nostrand Reinhold Co. 1971.
Ross, W.N., and A.E. Stuart. 1978. Voltage sensitive calcium channels in
the presynaptic terminals of decrementally conducting photoreceptor.
J. Physiol. London 283: 197-209.
Wagner, H. H., and W. Ulbricht. 1976. Saxitoxin and procaine act independently
on separate sites of the sodium channel. Pfluegers Arch. Eur. J.
Physiol. 364: 65-70.
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Figure Captions
Figure 1: Time course of pharyngeal contraction response in Botrylloides
exposed to 450 mM Tris, pH 8.0 with varying amounts of calcium. Decreased
calcium correlated with faster anaesthesia time.
Figure 2: Time course of pharyngeal contraction response in Ascidia
ceratodes in response to 450 mM Tris, pH 8.0 with varying amounts of
calcium. Decreased calcium correlated with faster anaesthetic time.
Figure 3: Time course of pharyngeal contraction response in Botrylloides
in response to 450 mM Tris, pH 8.0 with varying amounts of magnesium.
Decreased magnesium correlated with greater anaesthetic time, suggesting
competitive inhibition of magnesium for calcium.
Figure 4: Comparitive study of procaine, dibucaine, and tetracaine at
various concentration levels. Anaesthetics were applied at zero time
(SW4A) and washed out at various times later (SW arrow). a) ImM. Anaes-
thetic response immediate for all three substances, only procaine reversible.
b) 100 uM. Anaesthetic response less immediate for procaine. Dibucaine and
tetracaine irreversible. c) 10 uM. Procaine ineffective at this dosage.
Dibucaine and tetracaine ultimately lethal. d) 1 uM. All three
anaesthetics ineffective at this dosage, tetracaine lethal.
Figure 5: Procaine critical range dosage response. Slight decrease in
procaine dosage causes extension of time needed to full anaesthesia. Recovery
time also depends on procaine dose.
-12
Figure 6: Recovery time course following varying times of exposure to
1 mM procaine concentration. Increasing exposure causes increase in time
needed until full recovery. Further exposure after 2 hours couses little
change in recovery time.
Figure 7: Dosage response curve. Semi-log plot of Botrylloides colonies
exposed to varying concentrations of procaine for 60 minutes. Left
ordinate is drawn at threshhold dosage, right ordinate is drawn at lethal
dosage. Increasing concentration causes decreased time to anaesthesia and
increased recovery time.
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