PONEC
1a
ABSTRACT: 1. The bidirectional hearts of the
tunicates Ascidia ceratodes, Ciona intestinalis,
and Clavelina huntsmani showed preferred dir-
ectionality in number, rate, and period of
beating.
2. Each species tested demonstrated a charact¬
eristic reversal pattern, but there were large
individual variations from the species averages.
3. Temperature affected both rate and period
of beating, but it did not affect number of
beats per period.
4. Salinity changes and injury had significant
effects on the cardiac function of Clavelina
huntsmani. Changes in Ph had no effect.
5. Heart rate slowed just before reversal in
all six species tested.
6. There are at least five different types of
heartbeat reversal.
7. An endogenous counting mechanism seems to be
a major means of regulating reversal.
INTRODUCTION
The tubular hearts of ascidians have fascinated research-
ers since the early 1800's. (Kuhl and v. Hasselt, 1822) The
heartbeat consists of peristaltic contractions propagating
along the length of the thin muscular tube. Periodically,
the direction of the contraction reverses so that the pro¬
pagations travel alternately in the "visceral" direction
(toward the base of the animal) and in the "pharyngeal" dir¬
ection (toward the pharynx).
Many studies have been reported concerning the causes
of this heartheat reversal and the possible regulation of it.
There are at least three reasonable theories which attempt
explaining this phenomenon. The first is the pacemaker fatique
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theory which proposes that a pacemaker region at each end of
the heart periodically fatigues. Thus, uhen one end fatiques,
the "rested" end begins beating. This idea is strongly suo¬
ported by the experiment in which the heart was removed from
the body of a Ciona and cut in half. Millar (1953), Ebara (1954).
Krijgsman (1956), and others showed that each half of the
heart had alternate periods of beating and resting, sug-
gestive of fatigue. On the other hand, proponents of the
second theory, the back pressure theory, contend that while
the heart beats in one direction, pressure increases on its
arterial side and decreases on its venous side. The decreas¬
ing venous pressure is thought to deactivate the dominant pace¬
maker so that its rate decreases and reversal can occur. (Haywood
and Moon, 1953) A third theory suggests that reversal is caused
by an active competition between the two pacemaker regions to
control beat direction. Periodically, one end subdues the
other end by iniating beats at a higher frequency.
Although the research on the tunicate heart is exten¬
sive, there are certain areas in which it is deficient. For
instance, much of the work done so far has concentrated on
Ciona intestinalis and a few other species. Few inter-
species comparative studies on rates of beating and reversal
of the heart have been done. Also, much of the analysis has
been done on excised, cannulated hearts so that the natural.
in vivo heart patterns are not clearly defined for some species.
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These deficiencies led me to perform in vivo heart rate and
reversal pattern recordings for six species of ascidians.
hese six (Ascidia ceratodes, Ciona intestinalis, Perophora
annectens, Clavelina huntsmani, Botrylloides sp., and
Botryllus sp.) were chosen to provide a variety of sizes and
morphological characteristics as well as a broad taxonomic
representation. Also, more detailed experiments testing the
reaction of the heart in the tunicate (Clavelina huntsmani)
to changes in temperature, Ph, salinity, and injury were per¬
formed. Such variations were selected as representative of
a few of the environmental challenges that a Clavelina, living
on intertidal rocks, might encounter.
MATERIALS AND METHODS
tach animal was allowed to relax in a clear finger bowl
filled with seawater at 12.5°C, which is ambient temperature
for the Monterey Bay, Ca. Then, in each living tunicate,
the beating heart was viewed via transmitted light with a dis-
secting microscope. The tunics of all species studied exceot
Ascidia ceratodes were transparent enough that the heart could
be thus viewed. Since only a few of the Ascidia were clear
enough for such observation, the rest had a small window cut
above the heart for easier viewing. The cut animals were
allowed to stabilize for 24 hours before the recordings the
recordings were made.
A polygraph chart recorder moving at Imm per second was
Used to record heartbeat. A manual switch induced a spike on
either side of a baseline (upward indicating a pharyngeal beat;
dounward for a visceral beat) every time a propagation passed
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a preselected landmark just above the heart. The hearts were
recorded for a time encompassing about twenty reversals.
The selected environmental manipulations were performed
exclusively on Clavelina huntsmani. A Clavelina often shares
its circulatory system with one of two asexually budded indi¬
viduals. This complication was avoided with the selection of
animals which lacked fully developed buds. Temperature mani¬
ulations were performed by setting a coiled copper tube coated
with nail polish in the finger bowl with the animal. A circul¬
ating cooling unit pumped antifreeze solution at various reg-
ulated temperatures through the coil. This system was also
used to keep the seawater at ambient ocean temperature (12.5°C)
during other environmental manipulations. NaDH and HEl were
used to adjust the Ph of the seawater. The low salinity sol¬
ution consisted of 20% distilled water and 80% seawater. For
high salinity, 2.69 of Nacl was added to 100ml seawater. For
injury studies, animals were stabbed with a sharp, pointed
scalpel through the branchial basket Imm below the oral
siphon. The injury test was repeated on animals anesthetized
with procaine. Procaine was obtained from Sigma Chemical Co.
and was used at a concentration of 0.125 mm.
RESULTS
Comparative Heart Study
Table 1 shows the average figures for both the visceral
and the pharyngeal directions' number of beats, period of
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beating, and rate of beating for the six species of tunicates
observed. The species means were calculated from the indi¬
cated number of animals for heartbeat in each direction over
the entire period of observation. These calculations showed
that Ascidia ceratodes beats longer, faster, and preferentially
in the pharyngeal direction. Ciona intestinalis and Clavelina
huntsmani also showed directionality in number, period, and
rate, but these two species were biased toward the visceral
direction. Unlike the first three species, Perophora annectens,
Botrylloides, and Botryllus did not show significant direc¬
tional preferences.
Figure 1 illustrates bar diagrams of average rate vs. time
of beating. This gives an overall view of the reversal pattern
of an individual selected from each species whose direction¬
ality pattern roughly matches the species average. The diagram
for Ascidia ceratodes in Figure 1 demonstrated some of the
fluctuations in rate and reversal pattern that could be observed
in the individualssamples. Of the six species, Ascidia ceratodes
showed the greatest variability in number of beats and periods
of beating. Ciona intestinalis, on the other hand, had the
had the most regular pattern with almost no fluctuations in
any of the animals recorded. The other four species had
characteristic reversal patterns, but there were sporadic
deviations from them.
Spontaneous body contractions were observed during the
recordings in all six species. Although these contractions
usually involved a dramatic compression of the animal's form,
it was never seen to affect the heart rate or reversal pattern
in any way
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Environmental Effects on Heartbeat of Clavelina huntsmani
Ihe observation of sudden changes in the heart reversal
patterns in all six species brought out the question of whether
such variations were random or induced by some unseen change
in the environment. Clavelina huntsmani was chosen for tests
on potential environmental influences because, although some
of these factors have been studied before, little work has
been applied to Clavelina huntsmani or to living samples..
Temperature had a clear effect on the rate of heartbeat
and the rate of reversal. The animal whose rate and period
of beating as a function of temperature is shown in Figure 24
demonstrated that as temperature increased, the heart rate also
increased dramatically. At the same time, the reversal freg¬
uency proportionally increased so that the number of beats in
each direction remained relatively constant over the entire
range of temperatures, as shown in Figure 28. Both ends of
the heart were similarly affected because there was no sig¬
nificant change in relative directionality. The fact that
number remained constant while period fluctuated demonstrated
that the number of beats may be the factor controlling the
occurrence of reversal.
Ph, on the other hand, had no significant on the number,
rate, or period of beating in either direction. (Figure 3)
Figure 4 illustrates the reversal pattern of another animal
in three different salinities. In low salinity, the frequency
of reversal increased, but the heart rate was not dramatically
altered. (Figure 48) The average ratio of the number of beats
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in the visceral vs. the pharyngeal direction did not signif¬
icantly change in low salinity. In high salinity, both the
ratepof beating and the reversal frequency were more sig¬
nificantly altered. Figure 40 shows that the heart beat for
an unusually long period in the visceral direction before
reversing. Its rate then slowed, and the beat became irregular
and spasmodic.
Uhen a Clavelina was injured with a scalpel, the zooid
contracted violently down inside the tunic and remained there
for over an hour. During this time, the heart rate was the
same as in the animal before injury, but the reversal frequency
increased. The pattern of reversal could become very irreg¬
ular, but in general, a regular but shorter reversal pattern
was the dominant feature as shown in Figure 5A. In order to
test whether mechanical agitation of the heart due to branchial
contraction following stabbing might be directly responsible
for the rapid heart reversals, and to question whether ner¬
vous pathways might be involved, the stabbing experiment was
repeated on animals paralyzed by 0.125mM procaine. (See Parker,
1981) Comparisons of "normal" heart recordings(ie. without
stabbing) before and after procaine anesthesia revealed that
procaine did not affect the heart's rate or reversal pat¬
tern for at least the first two hours of exposure. Exposure
to procaine for over three hours, however, caused serious
arhythmia in the heart. This effect was not studied in detail.
Figure 5B shows the reaction of a procaine anesthetized
animal to the stab insult. The animals used for the insult
experiments were exposed to procaine for one hour, after
which their pral siphons were no longer responsive to probing
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Ihen, the unresponsive animals were injured with a scalpel,
again in the branchial basket. There was no body contraction
upon injury, but again, there was severe bleeding. Like
the unanesthetized animals, the heart rates were unaltered,
but the frequency of reversal increased.
Detailed Study of Reversal Types.
In all six species studied, there was usually a noticable
slowing of the heart rate just before reversal. Figure 6
illustrates this phenomenon in a sample Clavelina huntsmani,
where the reciprocal of the inter-beat interval (ie. the
instantaneous frequency) is plotted vs. beat number for a
period of unidirectional beating. There is a clear slowing
of heart rate between beats 300 and 500 after which a reversal
occurred.
This slowing, however, was not the only pattern of beat¬
ing associated with reversal. Figure 7 illustrates five
different types of reversals that were observed. The draw¬
ings are actual tracings of the actual polygraph recordings
made on sample Clavelina. Classification of the different
categories depended on whether the heart slowed before rev¬
ersal, whether there was a reversal pause (when the heart
stopped for about 5 seconds before reversal), and whether bi¬
directional beating (beats that started at both ends and col¬
lided in the middle) was associated with reversal. As shown
in Figure 7, the most common pattern consisted of heartbeat
slowing, a reversal pause, and then takeover by the other
pacemaker. There was no noticable difference in the way this
occurred at either end of the heart. As indicated in Figurer?,
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the patterns of reversal (B-E) occurred progressively less
frequently.
DISCUSSION
The overall species averages of visceral vs. pharyngeal
humber, period, and rate of beating showed that the most sig¬
nificant differences between the six tested species were in
rate of beating and period between reversals. The differ-
ences between the average number of beats per period for each
species were much less pronounced. For instance, Clavelina
huntsmani and Botrylloides beat, on the average, the same
number of beats in the visceral direction, but their visceral
heart rates differed by more than a factor of two.
Ihe differences between the rate and perind between rever¬
sal averages for these six species can be partially ex¬
plained in terms of morphological differences. For instance,
an inverse correlation exists between the average size of a
species and the average heart rate. The three smaller species,
(Perophora annectens, Botrylloides, and Botryllus) had heart
rates almost double those of the larger species, (Ascidia
ceratodes, Ciona intestinalis, and Clavelina huntsmani).
Also, the three smaller species were colonial tunicates with
common circulatory systems. The maintenance of the high blood
flow rates observed in common blood vessels in the test probably
demanded faster beating from the individual hearts.
The colonial blood systems hight also have been responsible
for the lack of directionality preference of heartbeat in
Perophora annectens, Botrylloides, and Botryllus. If even dis-
tribution of blood throughout the colony is to be maintained,
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10
hearts beating with directionality preferences would be coun¬
terproductive. Also, the back-pressure theory (Haywood and
Moon, 1953) would suggest that this evenness is synchron¬
ized through the colony by means of pressure induced reversals.
Ubservations of natural fluctuations in the three recorded
parameters (number per period, reversal frequency, and rate
of heartbeat in each direction) as well as the environmental
effects on these living animals indicate that the regulation
of heartbeat reversal in tunicates is much more complexxthan
past investigators have suggested.
Pacemaker "fatigue", as mentioned in the introduction,
is a leading theory for cause of reversal because both ends
of an isolated heart showed alternate periods of beating and
resting even when the heart was bisected. As mentioned in the
results, the most common type of reversal in the living animal
involved slowing, a pause, and then reversal. The consistency
of this observation supports pacemaker fatique as a dominant
cause of reversal. However, "fatique", if it is defined in
the classical sense, (a decreased ability to function due to
repetative action) cannot directly cause heartbeat reversal.
The reason for this is that, over a wide range of temperatures,
the beat and reversal rate of Clavelina huntsmani changed dra¬
matically, but the numbers of beats per period remained nearly
constant. Thus, a period seems to be defined by a particular
number of beats regardless of how rapidly the heart is beating,
(See Figures 24 and 28) This is contrary to the "fatigue"
theory which would have predicted thetheart to beat for larger
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11
numbers per period at lower temperatures because a heart beating
more slowly would be less prone to fatique. Also, the occas¬
ional absence of slowing before a reversal and the variable
nature of the beating pattern before reversals strongly sug¬
gest that other factors must be involved in heart regulation.
The fact remains, however, that a regular pattern of heart
reversal can be observed in living specimens from all six
species.studied. This regularity seems primarily dependent
on the absolute number of beats, rather than on time or heart
rate. Thus, it is logical to conclude that an endogenous,
number-dependent regulatory mechanism exists in the tunicate
for the control of heart reversal. However, fluctuations from
regular patterns of reversal, such as those caused by injury,
suggest that this "heartbeat counter" is also succeptible
to influence from other regulatory mechanisms.
Une type of heartbeat counter mechanism that has been
suggested is the back-pressure theory (Haywood and Moon, 1953).
Une can see how buildup of pressure on one side of the body
during a period of unidirectional beating might depend on the
number of beats that have occurred. The mechanism of pressure's
effect on reversal is postulated to be an inhibition of the
active pacemaker on the venous end of the heart because blood
pressure decreases there. This lower rate then facilitates
a reversal. However, the idea that low pressure induced in¬
hibition causes a lower heart rate is contradicted by the
observation that severe bleeding in the animal caused neither
a directional preference nor slowing of the heart rate. (Figure 5
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12
The only change observed was an increase in reversal freg¬
uency to establish a new, regular pattern, where the back-
pressure theory predicts that a decrease in blood pressure
should have slowed at least the pharyngeal pacemaker (nearest
the cut) if not both pacemakers.
Manipulations of salinity (Figure 4) were observed to have
significant effects on both the heart rate and reversal pat¬
tern, but, again, the effects on reversal pattern were far more
dramatic. It is possible that the salinity fluctuations effec¬
tively caused blood pressure changes (due to osmotic stresses)
which might affect heartbeat. However, the time course of such
an effect as well as the osmotic gradients would be too com¬
plicated to determine. Also, possible effects of salinity
changes on muscle and nerve action potentials or on the heart's
contractile machinery might further complicate the situation.
The most interpretable of the salinity results was the fact
that high salinity consistently had a much more dramatic
effect on heart function than did low salinity. This seems
ecologically significant in that a Clavelina huntsmani living
on an intertidal rock is more likely to encounter salinities
much lower than seawater's (eg. rain) than much higher salin¬
ities. Thus, the ability of the heart to cope better with low
salinities can be interpreted as a possible ecological adapt-
ation.
Two observations discounted any important influence of
the force of body contraction on heart reversal. First,
spontaneous body contractions occurred in all six species
without any noticable effect on the heart. Second, there was
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no significant difference in the way procaine paralyzed animals'
hearts reacted to injury (even though there was no body con¬
traction) when compared with the reaction of unanesthetized
animals which had violent body contractions. (Figure 5)
Evidence for the pacemaker competition theory (Loeb, 1900)
came from observations of occasional reversals without the usually
associated decrease in heart rate. This suggests a more forcible
taking over of the heartbeat by the inactive pacemaker. A
more dramatic example of this is that, occasionally, bi¬
directional beats (beats that started at both ends of the heart
and collided in the middle) preceded reversals even in the
absence of beat slowing or any indication of "fatique". (Figure
bD and 6E) However, this type of reversal occurred so seldom
in comparison with the regular pattern that pacemaker compet-
ition must play a relatively limited role in regulating heart
reversals.
In summary, the most logical explanation for periodic
heartbeat reversal in Clavelina huntsmani is the presence
of an endogenous, number dependent regulatory mechanism or
counter, succeptible to modification by other influences such
as pacemaker competition, pressure changes, and possible ner¬
vous control. The existence of a number dependent reversal
regulator for more species of tunicates is also consistent with
the data in Table 1, where the number of heartbeats per period
has smaller inter-species differences than do reversal fre¬
quency or rate of heartbeat.
ACKNOULEDGEMENTS
I wish to express my sincere thanks to the following
people who made this project possible:
The entire faculty and staff of Hopkins Marine Station,
especially Dr. William F. Gilly whose patient support and
instruction, along with his technical assistance, helped
me immeasurably to properly apply my research efforts.
Al Vitale, with whom much of the comparative heart study was
performed.
14
REFERENCES
ANDERSUN, M. (1965). Reversal mechanism in the heart of the
tunicate Ciona intestinalis. Am. Zoologist, 5, 104.
ANDERSUN, M. (1968). Electrophysiological studies on initiation
of the heartbeat in Ciona intestinalis. J. Exp. Biol., 49,
363-385.
physio¬
BANCROFT, F. W., and C. O. ESTERLY, (1903).A case of
logical polarization in the ascidian heart. Univ. Calif.
Publ. Zool., 1, 105-114.
DAY, E. C.,(1921). The physiology of the nervous system of the
tunicate. J. Exp. 2001., 34,45-65.
EBARA, A.. (
52).
Physiological studies on the heart of an
ascidian, Polycitor mutabilis Oka iii. Observations of
heartbeats in relation to budding. Zool. Mag. 61(5), 140-144.
EBARA, A., (1952). Physiological studies on the heart of an
ascidian, Polycitor mutabilis Oka, iv. Changes of heartbeats
affected by respiration or ligation of the body. Zool.
Mag.,161(6): 159-163.
EBARA, A., (1955). The reversal of heartbeat caused by various
treatments. Zool. Mag. 64(2): 39-43
HAYUOOD, C. A. and MOON, H. P.
(1953). Reversal of heartbeat
in tunicates. Nature Lond. 172: 40..
HERUN, A. C., (1973). A new type of heart mechanism in the
invertebrates. Journal of the Marine Biological Association
of the United Kingdom, 53: 425-428.
HUNTER, G. W., Jr., (1903a) . Further notes on the heart of
Molgula manhattensis. Amer.J. Science, N. S., 17: 251
KRIEBEL, M. E., (1963).
Effect of blood pressure on the
isolated tunicate heart. Biol. Bull., 125: 358.
Studies on the cardiovascular phy¬
KRIEBEL, M. E., (1954).
siology of the tunicate, Ciona intestinalis. Master of
Science Thesis. University of Washington, Seattle,
Washington.
KRIEBEL, M. E., (1966). The role of cell nexuses in the spread
of excitation in the tunicate myocardium. Am. Zool. 6: 537.
/5
KRIEBEL, M. E., (1967). Conduction velocity and intracellular
J. gen. Physiol..
action potentials of the tunicate heart.
50: 2097-2107.
KRIEBEL, M. E., (1967c). Impulse propagation of the tunicate
heart. J. Sen. Physiol., 50: 2940.
KRIEBEL, M. E., (1968). Electrical coupling between tunicate
heart cells. Life Sciences, 7: 181-186.
KRIEBEL, M. E. (1968). Studies on cardiovascular physiology
of tunicates. Biol. Bull., 134: 434-455.
KRIEBEL, M. E. (1973). Action potentials occur only on lumen
surface of tunicate mypendothelial cells. Comp. Biochem.
Physiol.,46A; 463-468.
KRIEBEL, M. E. (1973). Cholinoceptive and adrenoceptive prop¬
erties of the tunicate heart pacemaker. Comp. Biochem.
Physiol., 48a: 745.
KRIJGSMAN, B. J. and KRIJGSMAN, N. E. (1957). Some features
of the physiology of the tunicate heart. Recent Advances
in Invertebrate Physiology, 590-606.
KRIJGSMAN, B. J. and KRIJGSMAN, N. E. (1959). Investigations
into the heart function of Ciona intestinalis, the action
of acetylcholine and eserine. Arch. int. Physiol. Biochim.
67: 567-585.
KUHL, H. and HASSELT, J. C. van, (1822). Uitrecksels uit
brieven van de Heren Kuhl en van Hasselt. Buitenzorg den
12 den Aug. In: Algemene Konst-en Letterbode, 1: 115
MILLAR, R. H., (1952). Reversal of heartbeat in tunicates.
Nature, Lond., 170: 851-852.
MILLAR, R. H., (1953b). Reply to Haywood and Moon's paper
"Beversal of Heartbeat in Tunicates."
Nature, 172:41.
WOLFE, E., (1932). Pulsation frequency of the advisceral and
abvisceral heartbeat of Ciona intestinalis in relation to
temperature. J. Sen. Physiol., 16: 89-98
6
lable 1. Dverall heart data averages for the six species tested.
Ihe numbers in the left column represent sample size for each
species. Each mean is listed with its standard error meas¬
urement and represents about twenty reversal periods per
organism tested..
10
—
2

0

Z
6

+1
+1
+1
—

—
+1
Figure 1. Bar graph plots of mean rate in each direction
(visceral and pharyngeal) for individuals whose rate, period,
and number averages were representative of their respective
species means listad in Table 1.
9
T

- I
II
—
—
es/sa
— —
I
SE

20

8
Figure 2. A) The effects of temperature on visceral and pharyn¬
geal period and rate of beating in Clavelina huntsmani. The
rate of beating in both directions increased significantly with
temperature while the periods of beating decreased.
B) The effects of temperature on visceral and pharyngeal numbers
of beats per perind. No significant change is shown for either
direction.
2
AVE
EATS/SE
—
30
AVE
4BEATS
A
2
2
A
A

A
TEMP %0
2
TEMF
sec
rate
A pharyngeal A
viscera!
0
A
O
16
pharyngeal A
viscera!
0
A
A
A
A
24
F 1400
o00
AVE
TIME
(SEC)
600
200

24
22
Figure 3. A) The effects of Ph on visceral and pharyngeal period.
and rate of beating in Clavelina huntsmani. No significant
change in either parameter was observed over the range of Ph's.
B) Ihe effects of Ph on visceral and pharyngeal numbers of beats
per period. No significant change was shown for either direction.
.8
A
A
A
rate
A
0
9
600
-4
TIN
(SEC.
200
AVE
EATS
300-
AVE
4BEATS

A
2
8
PH
sec
pharyngeal A
visceral
2
10
pharyngea! A
visceral
8
2
2

2
85
Figure 4. The effects of high and low salinity on heart rate
and reversal pattern in Clavelina huntsmani.
A represents the normal pattern in seawater of regular salinity.
B shous the plot of rate vs. time for the some organism in low
salinity seawater. Heart rate was not significantly altered,
but reversal frequency increased.
E shous the rate vs. time plot for the same organism in high
salinity seawater. After a long visceral period of beating at
the normal rate, both the rate and frequency of reversal
changed dramatically.
U
i

L
D
O



26
Figure 5. A) The effects of injury on rate and reversal
frequency of the heart in Clavelina huntsmani. Both bar
diagrams from A are from the same animal. The second was
recorded after the animal was stabbed in the branchial basket
with a scalpel. The heart rate did not change, but the reversal
frequency increased with two irregular reversals.
B) The effect of injury on rate and reversal frequency in a
Clavelina huntsmani anesthetized with procaine. An increase
of reversal frequency, similar to that of 5A can be sean.
5
1


O 1
a
o

26
Figure 6. Fluctuations of instantaneous heart rate before
reversal in Clavelina huntsmani. The rate (which is the
reciprocal of the inter-beat interval for each beat) is
plotted for every fifth beat as a function of the number of
beats which have occurred. A dramatic drog in heart rate from
beats 300 to 500 can be seen. A reversal occurred after the
last recorded beat.
2
BEATS/SECOND

6

0
O
O
Figure 7. Illustrations of five different types of reversals
of the heart of Clavelina huntsmani. A shows the most common
type of reversal in which a slowing and a pause of the active
pacemaker occurs first. B shows a reversal pause, but no slowing
of the active pacemaker before reversal. In C, there is slowing
before reversal, but no reversal pause. D shows slowing and
bidirectional beats before reversal. E shows only bidirec¬
tional beats before reversal.
most
pharyngea!
viscera!
least
EL
100
H
200
seconds