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The contractile blood vessels of the earthworm, Lumbricus terrestris
Cow@. B&hem.
Physiol., 1972, Vol. 42A, pp. 627 to 638. Pergamon Press. Printed in Great Britain
THE CONTRACTILE BLOOD VESSELS OF THE EARTHWORM, LUMBRICUS TERRESTRIS CHARLES
R. FOURTNER
and RALPH
A. PAX
Department of Zoology, Michigan State University, East Lansing, Michigan 48823 (Received 11 October 1971)
Abstract-l.
In intact resting earthworms the rate of contraction of the dorsal vessel is 11 beats/mm; in “exercised” animals, 17 beats/min. 2. In dissected animals the rate averages 8 beats/min, each contraction consisting of a peristaltic wave passing anteriorly. 3. The contraction wave has a uniform velocity of 2.4 cm/set posterior to the lateral vessels but the velocity becomes progressively less in the region of the lateral vessels. 4. Each lateral vessel possesses an endogenous rhythmicity but can be driven by activity in the dorsal vessel. 5. Increased intralumenal pressure increases the rate of beating of the dorsal vessel; decreased intralumenal pressure decreases the rate. 6. Electrical stimulation of the dorsal vessel elicits propagated contraction waves but there is a long refractory period following each contraction so that the dorsal vessel cannot be driven in a one-to-one manner at a rate greater than once every 4 sec.
INTRODUCTION THE MAJORblood vessels of the earthworm,
Lumbricus tewestris, consist of longitudinal dorsal and ventral vessels which run the length of the animal. Five pairs of lateral vessels (“hearts”) in the seventh to eleventh segment connect the dorsal and ventral vessels at the anterior. Blood flows anteriorly in the dorsal vessel through the lateral vessels to the ventral vessel. The blood then flows posteriorly in the ventral vessel eventually being carried back to the dorsal vessel by a series of smaller vessels and capillaries. The dorsal vessel and the lateral vessels are contractile and appear to be the major propulsive organs for the blood (Johnston & Johnson, 1902; Johnston, 1903). There have been relatively few physiological studies of the contractile vessels in the earthworm. Stiibel(l909) by means of visual observations on intact animals at room temperature recorded ten to fifteen contractions of the dorsal vessel per minute, each wave of contraction usually beginning at the posterior and moving anteriorly. He also observed that the lateral vessels usually beat at the same rate and in synchrony with the dorsal vessel. On the basis of a variety of experiments, particularly by ones in which local pressure was applied to the dorsal 627
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CHARLESR. FOURTNJXRANDRALPH
A. PAX
vessel and by stimulation experiments, Stiibel concluded that the rhythmic contractions of the dorsal vessel originate in the dorsal vessel itself and that they are probably produced by nerve cells in the walls of the dorsal vessel. Prosser & Zimmerman (1943) a1so examined the circulatory system in Lumbricus terrestris. In contrast to the findings of Stiibel they saw no synchrony between dorsal vessel activity and contractions in the lateral vessels. They and also Kiefer (1959) examined the responses of the contractile vessels to various pharmacological agents. The most notable of the drugs they tested was acetylcholine which caused an increase in the rate of contraction of the dorsal vessel. On the basis of these observations Prosser & Zimmerman concluded along with Stiibel that the contractions of these vessels may be of neural origin. To the best of our knowledge the electrical activity associated with contractions of these vessels has not been recorded nor have the exact relationships between the dorsal and lateral vessels been determined. For these reasons we have undertaken this investigation of the contractile blood vessels of the earthworm, Lumbricus terrestris. MATERIALS
AND
METHODS
Adult Lumbricus terrestris either collected locally or obtained from a commercial supplier (Wholesale Bait Co., Hamilton, Ohio, U.S.A.) were used in all experiments. Before use the animals were stored in “Buss-Bedding” (Buss Manufacturing Co., Lanark, Illinois, U.S.A.) in a cold room maintained at 12-15°C. Before dissection the animals were anesthetized with cold by placing them in an icebath for several minutes. The worms were then pinned out in a paraffin tray and the dorsal half of the body wall from the peristomium as far back as the clitellum was carefully removed. This operation exposes the five pairs of lateral vessels at the anterior and the dorsal vessel from anterior to the first pair of lateral vessels back as far as the anterior portion of the intestine. During the dissection and at all times after this, the preparation was covered with cold (15-16°C) earthworm saline (Pantin, 1948). During the dissection described above one must be certain that no major blood vessels are severed. If such does occur the contractions of the dorsal and lateral vessels decrease in rate, soon becoming irregular and often completely ceasing within 30 min. If none of the vessels are severed, regular contractions will continue for as long as 6 hr or more. The activity of the dorsal vessel and the lateral vessels was recorded by means of glass suction electrodes (I.D. 100 CL)applied to the vessels with a minimal amount of suction. The activity thus recorded was amplified by means of a Grass P15 A.C. preamplifier and displayed on either a Tektronix 502A oscilloscope or on a “Physiograph” (Narco Biosystems, Inc., Houston, Texas, U.S.A.). RESULTS
A. Observations
on intact animals
The rate of beating of the dorsal vessel was examined in six intact animals. In the posterior third of the earthworm the dorsal vessel can be easily discerned as a dark tortuous line. Contractions of the dorsal vessel are easily discernible by the temporary disappearance of this line. Animals which had been disturbed as little as possible as well as animals which had been agitated were examined for
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the rate of beating of their dorsal vessels. During these experiments temperatures between 18 and 19°C were maintained at all times. In order to make observations on animals disturbed as little as possible, the container in which the worms were kept was carefully removed from the cold room. The “Buss-Bedding” was carefully pushed aside until a worm was exposed for a short distance along its posterior region. By carefully directing a dim light onto this area and observing the area under a low power microscope it was possible to count the number of pulsations in the dorsal vessel. The rate of pulsations seen in these experiments was 11.0 & 2.1 beats/min ( x + S.D.). This number corresponds quite closely to the values given by Stiibel (1909). Following determination of the resting rate of pulsation of the dorsal vessel, animals were then removed from the “Buss-Bedding” and forced to locomote for several minutes. The rate of pulsations was then taken again. After the forced exercise the mean rate of pulsations was increased to 17.2 f 2.1 beats/min ( x& S.D.). B. Dorsal and lateral vessel activity
in dissected worms
In the dissected worms the dorsal vessel activity can readily be seen to consist of a wave of contraction beginning at the posterior and moving anteriorly over the region where the lateral vessels join the dorsal vessel. Generally the rate of contractions was less than in intact animals, averaging about 8 beats/min in good preparations. As mentioned previously, in preparations where a major vessel had been severed, rates were generally much lower, often less than 1 beat/mm and activity soon ceased altogether. In a few rare instances waves of contraction moving posteriorly over the dorsal vessel were seen. Since these reverse waves appeared only in damaged preparations or in preparations which were obviously deteriorating it seems unlikely that such reverse waves occur in intact animals. Activity in each of the lateral vessels consists of a wave of contraction which begins dorsally and passes ventrally to the ventral vessel. The relationship between contractions in the lateral vessels and contractions of the dorsal vessel is variable. In some cases there is a clear correlation between the two, each of the lateral vessels contracting in turn as the wave of contraction in the dorsal vessel sweeps by them. In other instances the lateral vessels beat in an unto-ordinated manner and each at a rate independent of that in the dorsal vessel. Often one or more lateral vessels may be completely quiescent while others may be vigorously beating, but later in time, the formerly quiescent lateral vessels may be beating, while the lateral vessels which were formerly beating are quiescent. Figure 1, an example of the electrical activity recorded from lateral vessel IV, illustrates well how the activity in one of the lateral vessels varies over time. C. Electrical activity
in lateral and dorsal vessels
The patterns of electrical activity recorded from the dorsal vessel and the lateral vessels is given in Fig. 2. The pattern of activity recorded from the dorsal
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CHARLESR. FOURTNERAND RALPH
A. PAX
FIG. 1. A “Physiograph” recording of the electrical activity in lateral vessel IV showing the variability in the activity over time. Time mark, 1 min.
vessel varies depending on the site from which the recording is taken. Recordings from the dorsal vessel over the intestine show that each wave of contraction in this region is accompanied by a complex electrical waveform lasting 1.5-2.5 set (Fig. 2A and B). Generally there is an initial fairly large potential which is followed by a variable number of smaller slow potentials. The initial potential
I FIG. 2. Oscilloscope records of the electrical activity associated with dorsal and lateral vessel contractions. A and B, dorsal vessel just posterior to gizzard; C, dorsal vessel over crop ; D, lateral vessel V ; E, lateral vessel IV. Calibration : A and B, 8 mV, 1 set; C, 4 mV, 1 set; D, 4 mV, 10 set; E, 15 mV, 5 sec.
may be simple or consist of two or more apparently summating waves. The number and size of the later potentials is variable from contraction to contraction. At times they may be nearly as large as the first potential, at other times they may be recorded as only very small oscillations. Recordings from the dorsal vessel over the region anterior to the crop show a different wave form (Fig. 2C). Here the wave from is generally much simpler, consisting of only one or two waves with considerably faster rise times than the waves seen over the intestine.
CONTRACTILE BLOODVESSELSOF THE EARTHWORM LUMBBICLJS
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Recordings from the lateral vessels show still another pattern (Fig. 2D and E). Here the activity consists of a very slowly rising potential. In most cases the wave is a fairly simple smooth wave which, after reaching a peak, declines slowly to its original level (Fig. 2E). In a few instances more complex wave forms are seen. In these one sees on the slow rising phase of the potential a superimposed faster wave (Fig. 2D). D. Conduction velocity in the dorsal vessel In order to determine the velocity with which the wave of contraction in the dorsal vessel is propagated, pairs of suction electrodes were placed at measured distances at various points along the dorsal vessel. In a total of ten animals the mean conduction velocity measured at various points posterior to lateral vessel Posterior to lateral vessel V the conduction V was 2.4 & O-3 cm/set ( x it: S.D.). velocity is uniform, the conduction velocity not being significantly different whether one measures it over the anterior region of the intestine, over the region of the crop-gizzard or between the crop-gizzard and lateral vessel V. When one determines the conduction velocity anterior to the fifth pair of lateral vessels, however, one does see significant differences in the conduction velocity. Table 1 shows how the conduction velocity varies at the anterior end TABLE ~-CONDUCTION VELOCITIESAS MEASUREDOVER THE ANTERIORPORTIONSOF THE DORSALVESSEL Measurements made between dorsal vessel on anterior intestine and dorsal vessel On crop-gizzard Between lateral vessels Between lateral vessels Between lateral vessels Between lateral vessels
IV and V III and IV II and III I and II
Conduction velocity f S.D. (cm/set) 2.54 of:0.24 1.96 + 0.96 0.98 + 0.42 O-66 f 0.16 0.54 + 0.11
of the animal. In these experiments one suction electrode was placed on the dorsal vessel just anterior to the crop. The other electrode was placed successively on the dorsal vessel just posterior to lateral vessel V and between each of the lateral vessels. As can be seen from the table the conduction velocity markedly and progressively slows as the wave of contraction passes anteriorly over the region where the lateral vessels are located. The wave of contraction is slowed to such an extent that the total time required for the wave of contraction to travel from the dorsal vessel posterior to lateral vessel V to lateral vessel I is 3 set or more. This slowing in conduction velocity, because of the way in which it was recorded, might be due to a delay in propagation at each of the dorsal vessellateral vessel junctions or there might be a uniform decline in conduction velocity along the dorsal vessel. Because of the short distances between lateral vessels it
632
CHARLESR. FOURTNERANDRALPH A. PAX
was
possible in only a few instances to record the conduction velocity in the dorsal vessel between just two of the lateral vessels. Two measurements between lateral vessels II and III gave a conduction velocity of O-3 cm/set; two measurements between lateral vessels III and IV gave a conduction velocity of 0.4 cm/set; and two measurements between lateral vessels IV and V gave a conduction velocity of 1.2 cm/set. Thus it appears likely that there is a uniform decrease in conduction velocity along the anterior portions of the dorsal vessel, and not discrete delays at each of the lateral vessel-dorsal vessel junctions. E. Co-ordination between activity in the dorsal vessel and the lateral vessels As mentioned previously the co-ordination between activity in the dorsal vessel and the lateral vessels appears to be variable. In order to determine more precisely the relationship between the dorsal vessel and the lateral vessels, simultaneous electrical recordings were taken from the dorsal vessel and the lateral vessels. With such measurements one finds that there are several ways in which the activity in the dorsal vessel and that in the lateral vessels may be correlated.
FIG. 3. Oscilloscope records showing the relationship between the electrical activity associated with contractions of the dorsal vessel over the crop (lower traces) and lateral vessel V (upper traces). Calibration: 4 mV upper traces, 8 mV lower traces, 4 sec.
In the simplest case one sees a simple one-to-one correspondence between activity in the dorsal vessel and the lateral vessel. The first two contractions in Fig. 3A give one such example. For each wave of activity in the dorsal vessel there is a wave of activity in the lateral vessel. In other cases one finds that the lateral vessel possesses a rhythm of its own, contracting at a rate independent of the dorsal vessel activity. In such cases one often observes that even though the lateral vessel possesses its own rhythmicity,
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it can be reset by the activity in the dorsal vessel if the excitation from the dorsal vessel occurs sufficiently long after the endogenous contraction has occurred in the lateral vessel (Fig. 3C). If the excitation occurs at too short an interval after an endogenous contraction no visible contraction occurs in the lateral vessel, but one often records a small electrical potential which becomes smaller as it .comes closer and closer in time to an endogenous contraction. For example, in Fig. 3C three contractions of the dorsal vessel are shown. The first contraction occurs shortly after a spontaneous contraction in the lateral vessel and there occurs in the lateral vessel a small electrical potential. The second contraction of the dorsal vessel produces a full-sized contraction in the lateral vessel while the third contraction of the dorsal vessel occurs at such a short time after the lateral vessel contraction that no response of any kid is detectable in the lateral vessel. The particular type of relationship between contractions in the dorsal vessel and those seen in a particular lateral vessel does not remain constant over time. At one particular time a particular lateral vessel may be contracting at a rate nearly double that of the dorsal vessel. Later in time this same lateral vessel may contract completely in co-ordination with the dorsal vessel or it may cease contracting completely for a period of time. The particular kind of relationship between the dorsal vessel and a particular lateral vessel at a particular time is not seen for all the lateral vessels at the same time. Typically one finds that one or two of the lateral vessels may be contracting rather vigorously and rapidly at a particular time while the other lateral vessels may not be contracting at all. Later in time one will find that the opposite situation may hold. Also there is no evident co-ordination between the beating of the lateral vessels of a pair; at any particular time one vessel may be beating at a very regular rate whereas the contralateral vessel is completely quiescent.
F. Eflects of intralumenal pressure As mentioned in the Materials and Methods section, if during the initial dissection of these animals any of the larger vessels are severed, there follows rather quickly an irregular slow beating of the dorsal vessel and eventually complete cessation of activity. This observation leads one to believe that possibly the amount of intralumenal pressure in the dorsal and lateral vessels has an important influence on the rate of beating. In order to test this possibility experiments were performed in which the intralumenal pressure was either increased or decreased. In order to test whether decreased intralumenal pressure has an effect on the rate of the contraction, the dorsal vessel was occluded by sucking a short length of the dorsal vessel into a small capillary. This was sufficient to block blood flow in the dorsal vessel. In almost every case within 1 min after the occlusion was applied there occurred anterior to the occlusion a marked decrease in the rate of the contractions, generally to a rate less than one-half of the pre-occlusion rate. When the occlusion was removed the rate again rather quickly returned
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CHARLES R. FOURTNER AND RALPH A. PAX
to its former value. In a few instances at the time the occlusion was produced or removed a temporary excitation was seen but this excitation lasted for less than 30 set and the overall result of the occlusion was always a depression in activity. Figure 4 gives one example of such experiments.
A
‘I
FIG. 4. A tracing of a “Physiograph” record showing the effects of occlusion of the dorsal vessel between lateral vessels IV and V on lateral vessel IV activity. At the upward arrow the dorsal vessel was occluded, at the downward arrow the occlusion was removed. Calibration: 1 min.
The effects of increased intralumenal pressure were also tested in a few instances. In order to do this a fine glass capillary cannula was inserted into the dorsal vessel just anterior to the crop. The dorsal vessel is quite small and in only a small percentage of cases was there successful insertion of the cannula into the dorsal vessel. Insertion of the cannula as such had no observable effect on the rate of the contractions. When, however, the intralumenal pressure was increased by forcing saline under pressure into the vessel, a marked effect on the rate of contraction was seen. Figure 5 shows a typical example of the results
4
4
record showing the effects of increased intralumenal FIG. 5. “Physiograph” pressure on activity in lateral vessel V. At the upward arrow pressure was increased by injecting saline into the dorsal vessel approximately 1 cm posterior to lateral vessel V. At the downward arrow the injection was stopped. Calibration: 1 min.
In this example the rate of contraction of the lateral vessel of such experiments. from which the recordings were taken had become very low and irregular. When the intralumenal pressure was increased there occurred a marked increase in the rate. When the pressure was released the rate immediately reverted to its former value. Thus the change in rate appears to be a response to the increased pressure and not to the saline as such. G. Ejfects of electrical stimulation of the dorsal vessel In order to examine some of the electrical characteristics of the dorsal and lateral vessels, a series of experiments was performed in which the dorsal vessel was electrically stimulated while the activity in the dorsal vessel or lateral vessels
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was recorded. In these experiments a suction electrode placed on the dorsal vessel in the region of the crop-gizzard or the anterior portion of the intestine was used as a stimulating electrode. Stimuli consisted of square wave pulses (l-10 V, 3 msec). Stimulation of the dorsal vessel in this way elicits waves of contractions in the dorsal vessel. These waves are similar in conformation to those seen during spontaneous contractions and they are conducted both anteriorly and posteriorly For example in one preparation the elicited from the point of stimulation. waves had a velocity in the anterior direction of 2.6 cm/set posterior to lateral vessel V; 2.2 cm/set measured between lateral vessels IV and V; 1.2 cm/set measured between lateral vessels III and IV; 0.8 cm/set measured between lateral vessels II and III ; and O-6 cm/set measured between lateral vessels I and II. Elicited waves in the posterior direction over the dorsal vessel in the region of the crop-gizzard and intestine had a velocity of 24 cm/set. The rate at which contractions can be elicited by electrical stimulation is quite low. The maximum rate of stimulation in any case which gave a one-to-one correspondence between stimulations and contractions was one stimulus every 4.2 sec. In most cases the maximum rate at which one obtained a one-to-one correspondence between stimulation and contractions was one stimulus every 5.0 to 5.5 sec. When stimuli above these rates were given one obtained contraction waves in response to every other stimulus.
50
H
40
M
4.5
6. A tracing of a “Physiograph” record showing the effects of electrical stimulation of the dorsal vessel posterior to the gizzard on lateral vessel V activity. Stimulus artifacts are marked by dots; the numbers below the record indicrlte the intervals between successive shocks, Stimulus: 8 V, 3 msec. Calibration: 10 sec. FIG.
The ability of stimulation of the dorsal vessel to elicit contractions in the lateral vessels was also examined. Here results essentially the same as those obtained when recording from the dorsal vessel were obtained. Figure 6 gives one example of such stimulation. In this particular case spontaneous contractions of the dorsal vessel had dropped to less than one per minute. When stimuli at intervals of 5.0 set were applied there resulted from each stimulus, a contraction of the lateral vessel. When the interval was dropped to 4-O set only every second stimulus was effective. When the interval was raised to 4.5 set, a one-to-one relationship was again established after several stimuli. DISCUSSION
The results we have reported here confirm for the most part the observations of Stiibel(l909). All parts of the dorsal vessel appear to have the ability to initiate
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CHARLES R. FOURTNER AND RALPH A. PAX
contractions but under normal circumstances there is a wave of contraction which originates at the posterior of the animal and moves forward, The degree of stretch on the walls of the dorsal vessel appears to be important in determining whether a particular section of the vessel may initiate a contraction. Thus if there is excessive loss of blood from the vessels the rate of pulsations in the dorsal vessels becomes quite low or ceases altogether. Also when intralumenal pressure in the dorsal vessel is reduced by means of an occlusion of the dorsal vessel, one observes a decrease in the rate. In like manner if the amount of intralumenal pressure is increased by means of saline injection one observes an increase in the rate of the pulsations. From these observations it appears likely that the amount of intralumenal pressure is an important variable determining the rate of pulsation of the dorsal vessel. Although stretch of the vessel walls appears to be important in determining the rate at which the pulsations occur, stretch of itself does not appear necessary for conduction of a contraction wave once initiated. Stiibel (1909) observed that if by pressure he prevented contraction of the dorsal vessel over several segments, waves of contractions could still be propagated through the quiescent region. Previous investigators who have observed the pulsations of the blood vessels in the earthworm, L. tewestris, and other earthworm species have given conflicting reports on the degree of co-ordination between dorsal vessel and lateral vessel activity. Thus Stiibel (1909) reported that lateral vessel activity was closely co-ordinated with dorsal vessel activity, but Prosser & Zimmerman (1943), working with dissected worms, reported that they could not observe any co-ordination between beating in the dorsal vessel and the lateral vessels. Johansen & Martin (1965), working with the giant earthworm, Glossoscolex giganteus, also reported a lack of synchrony between dorsal vessel activity and lateral vessel activity. They did indicate, however, that the lateral vessels all beat in synchrony with one another. In our experiments we observed that at times there is a co-ordination between lateral vessel and dorsal vessel activity, while at other times there is no obvious co-ordination visible. From our electrical recordings of the activity in the lateral vessels it appears likely that each of the lateral vessels possesses an inherent rhythmicity but that each of the lateral vessels can be driven by excitation arriving via the dorsal vessel. A major difference between the experiments done by Stiibel and the other investigators mentioned above as well as our own, is that Stiibel made his observations on intact animals. It is quite possible that in the intact animal there is a one-toone correspondence between activity in the dorsal vessels and the lateral vessels, but in the process of dissection of the worms preparatory to recording, there is sufficient disruption of the circulatory system so that lateral vessel activity may become unto-ordinated with that in the dorsal vessel. Stiibel (1909) as well as Prosser & Zimmerman (1943) concluded that it is likely that the waves of contraction in the dorsal vessel in the earthworm are neurogenic and not myogenic, that is, that they are conducted by way of nerve
CONTRACTILE BLOODVESSELSOF THE EARTHWORM LVMBRICVS
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cells rather than muscle cells. Several lines of evidence-none of them unequivocal-however, lead us to conclude that conduction in the dorsal vessel is probably myogenic. The extremely slow conduction velocity of the contraction wave, the conformation of the electrical activity associated with the contraction wave, the exceedingly long refractory period following a contraction wave and the ability of the dorsal vessel to conduct waves both anteriorly and posteriorly, are all characteristics of a myogenically conducted wave and not a neurogenically conducted wave. SUMMARY
1. The contractile dorsal and lateral blood vessels of the earthworm, Lumbricus ten-e&s, have been examined. 2. In intact resting animals the rate of contraction of the dorsal vessel is 11 beats/min; in “exercised” animals, 17 beats/min. 3. In dissected animals the contractions of the dorsal vessel average 8 beats/ min, each contraction consisting of a peristaltic wave passing anteriorly. 4. The velocity of the propagated wave is a uniform 2.4 cm/set posterior to the lateral vessels but becomes progressively less in the region of the lateral vessels. 5. Lateral vessel contractions consist of peristaltic waves passing from the dorsal to the ventral vessel. 6. Each lateral vessel possesses an endogenous rhythmicity but can be driven by activity in the dorsal vessel. 7. Intralumenal pressure influences the rate of beating of the dorsal vessel, decreased pressures decreasing the rate; increased pressures increasing the rate. 8. Electrical stimulation of the dorsal vessel elicits propagated contraction waves but there is a long refractory period following each contraction so that the dorsal vessel cannot be driven in a one-to-one manner at a rate greater than once every 4-5 sec. Acknowledgement-This Research Grant HE-09718
investigation was supported in part by Public Health Service from the National Heart and Lung Institute. REFERENCES
JOHANSENK. & MARTIN A. W. (1965) Circulation in a giant earthworm, Glossoscolex gigunteus-I. Contractile processes and pressure gradients in the large blood vessels. J. exp. Biol. 43, 333-347. JOHNSTONJ. B. (1903) On the blood-vessels, their valves, and the course of blood in Lumbricus. Biol. Bull. mar. biol. Lab., Woods Hole 5, 74-84. JOHNSTONJ. B. & JOHNSONS. W. (1902) The course of blood flow in Lumbricus. Am. Nat. 36, 317-328. KIEFER G. (1959) Pharmakologische Untersuchungen fiber den Automatismus der Lateralherzen des Regenwurmes Lumbricus terrestris Linne. 2. wiss. Zool. 162, 356-367. PANTINC. F. A. (1948) Notes on Microscopical Technique for Zoologists. Cambridge University Press.
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PROSSERC. L. & ZIMMERMANG. L. (1943) Effects of drugs on the hearts of Arenicolu and Lumbricus. Physiol. Zoiil. 16, 77-83. STUBELH. (1909) Studien zur vergleichenden Physiologie der peristaltischen BewegungenIV. Die Peristaltik der Blutgefasse des Regenwurmes. Pfltigers Arch. ges. Physiol. 139, l-34. Key Word Index-Earthworm; activity; circulatory system.
Lumbricus terrestris; contractile blood vessels; rhythmic
Physiol., 1972, Vol. 42A, pp. 627 to 638. Pergamon Press. Printed in Great Britain
THE CONTRACTILE BLOOD VESSELS OF THE EARTHWORM, LUMBRICUS TERRESTRIS CHARLES
R. FOURTNER
and RALPH
A. PAX
Department of Zoology, Michigan State University, East Lansing, Michigan 48823 (Received 11 October 1971)
Abstract-l.
In intact resting earthworms the rate of contraction of the dorsal vessel is 11 beats/mm; in “exercised” animals, 17 beats/min. 2. In dissected animals the rate averages 8 beats/min, each contraction consisting of a peristaltic wave passing anteriorly. 3. The contraction wave has a uniform velocity of 2.4 cm/set posterior to the lateral vessels but the velocity becomes progressively less in the region of the lateral vessels. 4. Each lateral vessel possesses an endogenous rhythmicity but can be driven by activity in the dorsal vessel. 5. Increased intralumenal pressure increases the rate of beating of the dorsal vessel; decreased intralumenal pressure decreases the rate. 6. Electrical stimulation of the dorsal vessel elicits propagated contraction waves but there is a long refractory period following each contraction so that the dorsal vessel cannot be driven in a one-to-one manner at a rate greater than once every 4 sec.
INTRODUCTION THE MAJORblood vessels of the earthworm,
Lumbricus tewestris, consist of longitudinal dorsal and ventral vessels which run the length of the animal. Five pairs of lateral vessels (“hearts”) in the seventh to eleventh segment connect the dorsal and ventral vessels at the anterior. Blood flows anteriorly in the dorsal vessel through the lateral vessels to the ventral vessel. The blood then flows posteriorly in the ventral vessel eventually being carried back to the dorsal vessel by a series of smaller vessels and capillaries. The dorsal vessel and the lateral vessels are contractile and appear to be the major propulsive organs for the blood (Johnston & Johnson, 1902; Johnston, 1903). There have been relatively few physiological studies of the contractile vessels in the earthworm. Stiibel(l909) by means of visual observations on intact animals at room temperature recorded ten to fifteen contractions of the dorsal vessel per minute, each wave of contraction usually beginning at the posterior and moving anteriorly. He also observed that the lateral vessels usually beat at the same rate and in synchrony with the dorsal vessel. On the basis of a variety of experiments, particularly by ones in which local pressure was applied to the dorsal 627
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CHARLESR. FOURTNJXRANDRALPH
A. PAX
vessel and by stimulation experiments, Stiibel concluded that the rhythmic contractions of the dorsal vessel originate in the dorsal vessel itself and that they are probably produced by nerve cells in the walls of the dorsal vessel. Prosser & Zimmerman (1943) a1so examined the circulatory system in Lumbricus terrestris. In contrast to the findings of Stiibel they saw no synchrony between dorsal vessel activity and contractions in the lateral vessels. They and also Kiefer (1959) examined the responses of the contractile vessels to various pharmacological agents. The most notable of the drugs they tested was acetylcholine which caused an increase in the rate of contraction of the dorsal vessel. On the basis of these observations Prosser & Zimmerman concluded along with Stiibel that the contractions of these vessels may be of neural origin. To the best of our knowledge the electrical activity associated with contractions of these vessels has not been recorded nor have the exact relationships between the dorsal and lateral vessels been determined. For these reasons we have undertaken this investigation of the contractile blood vessels of the earthworm, Lumbricus terrestris. MATERIALS
AND
METHODS
Adult Lumbricus terrestris either collected locally or obtained from a commercial supplier (Wholesale Bait Co., Hamilton, Ohio, U.S.A.) were used in all experiments. Before use the animals were stored in “Buss-Bedding” (Buss Manufacturing Co., Lanark, Illinois, U.S.A.) in a cold room maintained at 12-15°C. Before dissection the animals were anesthetized with cold by placing them in an icebath for several minutes. The worms were then pinned out in a paraffin tray and the dorsal half of the body wall from the peristomium as far back as the clitellum was carefully removed. This operation exposes the five pairs of lateral vessels at the anterior and the dorsal vessel from anterior to the first pair of lateral vessels back as far as the anterior portion of the intestine. During the dissection and at all times after this, the preparation was covered with cold (15-16°C) earthworm saline (Pantin, 1948). During the dissection described above one must be certain that no major blood vessels are severed. If such does occur the contractions of the dorsal and lateral vessels decrease in rate, soon becoming irregular and often completely ceasing within 30 min. If none of the vessels are severed, regular contractions will continue for as long as 6 hr or more. The activity of the dorsal vessel and the lateral vessels was recorded by means of glass suction electrodes (I.D. 100 CL)applied to the vessels with a minimal amount of suction. The activity thus recorded was amplified by means of a Grass P15 A.C. preamplifier and displayed on either a Tektronix 502A oscilloscope or on a “Physiograph” (Narco Biosystems, Inc., Houston, Texas, U.S.A.). RESULTS
A. Observations
on intact animals
The rate of beating of the dorsal vessel was examined in six intact animals. In the posterior third of the earthworm the dorsal vessel can be easily discerned as a dark tortuous line. Contractions of the dorsal vessel are easily discernible by the temporary disappearance of this line. Animals which had been disturbed as little as possible as well as animals which had been agitated were examined for
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the rate of beating of their dorsal vessels. During these experiments temperatures between 18 and 19°C were maintained at all times. In order to make observations on animals disturbed as little as possible, the container in which the worms were kept was carefully removed from the cold room. The “Buss-Bedding” was carefully pushed aside until a worm was exposed for a short distance along its posterior region. By carefully directing a dim light onto this area and observing the area under a low power microscope it was possible to count the number of pulsations in the dorsal vessel. The rate of pulsations seen in these experiments was 11.0 & 2.1 beats/min ( x + S.D.). This number corresponds quite closely to the values given by Stiibel (1909). Following determination of the resting rate of pulsation of the dorsal vessel, animals were then removed from the “Buss-Bedding” and forced to locomote for several minutes. The rate of pulsations was then taken again. After the forced exercise the mean rate of pulsations was increased to 17.2 f 2.1 beats/min ( x& S.D.). B. Dorsal and lateral vessel activity
in dissected worms
In the dissected worms the dorsal vessel activity can readily be seen to consist of a wave of contraction beginning at the posterior and moving anteriorly over the region where the lateral vessels join the dorsal vessel. Generally the rate of contractions was less than in intact animals, averaging about 8 beats/min in good preparations. As mentioned previously, in preparations where a major vessel had been severed, rates were generally much lower, often less than 1 beat/mm and activity soon ceased altogether. In a few rare instances waves of contraction moving posteriorly over the dorsal vessel were seen. Since these reverse waves appeared only in damaged preparations or in preparations which were obviously deteriorating it seems unlikely that such reverse waves occur in intact animals. Activity in each of the lateral vessels consists of a wave of contraction which begins dorsally and passes ventrally to the ventral vessel. The relationship between contractions in the lateral vessels and contractions of the dorsal vessel is variable. In some cases there is a clear correlation between the two, each of the lateral vessels contracting in turn as the wave of contraction in the dorsal vessel sweeps by them. In other instances the lateral vessels beat in an unto-ordinated manner and each at a rate independent of that in the dorsal vessel. Often one or more lateral vessels may be completely quiescent while others may be vigorously beating, but later in time, the formerly quiescent lateral vessels may be beating, while the lateral vessels which were formerly beating are quiescent. Figure 1, an example of the electrical activity recorded from lateral vessel IV, illustrates well how the activity in one of the lateral vessels varies over time. C. Electrical activity
in lateral and dorsal vessels
The patterns of electrical activity recorded from the dorsal vessel and the lateral vessels is given in Fig. 2. The pattern of activity recorded from the dorsal
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CHARLESR. FOURTNERAND RALPH
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FIG. 1. A “Physiograph” recording of the electrical activity in lateral vessel IV showing the variability in the activity over time. Time mark, 1 min.
vessel varies depending on the site from which the recording is taken. Recordings from the dorsal vessel over the intestine show that each wave of contraction in this region is accompanied by a complex electrical waveform lasting 1.5-2.5 set (Fig. 2A and B). Generally there is an initial fairly large potential which is followed by a variable number of smaller slow potentials. The initial potential
I FIG. 2. Oscilloscope records of the electrical activity associated with dorsal and lateral vessel contractions. A and B, dorsal vessel just posterior to gizzard; C, dorsal vessel over crop ; D, lateral vessel V ; E, lateral vessel IV. Calibration : A and B, 8 mV, 1 set; C, 4 mV, 1 set; D, 4 mV, 10 set; E, 15 mV, 5 sec.
may be simple or consist of two or more apparently summating waves. The number and size of the later potentials is variable from contraction to contraction. At times they may be nearly as large as the first potential, at other times they may be recorded as only very small oscillations. Recordings from the dorsal vessel over the region anterior to the crop show a different wave form (Fig. 2C). Here the wave from is generally much simpler, consisting of only one or two waves with considerably faster rise times than the waves seen over the intestine.
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Recordings from the lateral vessels show still another pattern (Fig. 2D and E). Here the activity consists of a very slowly rising potential. In most cases the wave is a fairly simple smooth wave which, after reaching a peak, declines slowly to its original level (Fig. 2E). In a few instances more complex wave forms are seen. In these one sees on the slow rising phase of the potential a superimposed faster wave (Fig. 2D). D. Conduction velocity in the dorsal vessel In order to determine the velocity with which the wave of contraction in the dorsal vessel is propagated, pairs of suction electrodes were placed at measured distances at various points along the dorsal vessel. In a total of ten animals the mean conduction velocity measured at various points posterior to lateral vessel Posterior to lateral vessel V the conduction V was 2.4 & O-3 cm/set ( x it: S.D.). velocity is uniform, the conduction velocity not being significantly different whether one measures it over the anterior region of the intestine, over the region of the crop-gizzard or between the crop-gizzard and lateral vessel V. When one determines the conduction velocity anterior to the fifth pair of lateral vessels, however, one does see significant differences in the conduction velocity. Table 1 shows how the conduction velocity varies at the anterior end TABLE ~-CONDUCTION VELOCITIESAS MEASUREDOVER THE ANTERIORPORTIONSOF THE DORSALVESSEL Measurements made between dorsal vessel on anterior intestine and dorsal vessel On crop-gizzard Between lateral vessels Between lateral vessels Between lateral vessels Between lateral vessels
IV and V III and IV II and III I and II
Conduction velocity f S.D. (cm/set) 2.54 of:0.24 1.96 + 0.96 0.98 + 0.42 O-66 f 0.16 0.54 + 0.11
of the animal. In these experiments one suction electrode was placed on the dorsal vessel just anterior to the crop. The other electrode was placed successively on the dorsal vessel just posterior to lateral vessel V and between each of the lateral vessels. As can be seen from the table the conduction velocity markedly and progressively slows as the wave of contraction passes anteriorly over the region where the lateral vessels are located. The wave of contraction is slowed to such an extent that the total time required for the wave of contraction to travel from the dorsal vessel posterior to lateral vessel V to lateral vessel I is 3 set or more. This slowing in conduction velocity, because of the way in which it was recorded, might be due to a delay in propagation at each of the dorsal vessellateral vessel junctions or there might be a uniform decline in conduction velocity along the dorsal vessel. Because of the short distances between lateral vessels it
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CHARLESR. FOURTNERANDRALPH A. PAX
was
possible in only a few instances to record the conduction velocity in the dorsal vessel between just two of the lateral vessels. Two measurements between lateral vessels II and III gave a conduction velocity of O-3 cm/set; two measurements between lateral vessels III and IV gave a conduction velocity of 0.4 cm/set; and two measurements between lateral vessels IV and V gave a conduction velocity of 1.2 cm/set. Thus it appears likely that there is a uniform decrease in conduction velocity along the anterior portions of the dorsal vessel, and not discrete delays at each of the lateral vessel-dorsal vessel junctions. E. Co-ordination between activity in the dorsal vessel and the lateral vessels As mentioned previously the co-ordination between activity in the dorsal vessel and the lateral vessels appears to be variable. In order to determine more precisely the relationship between the dorsal vessel and the lateral vessels, simultaneous electrical recordings were taken from the dorsal vessel and the lateral vessels. With such measurements one finds that there are several ways in which the activity in the dorsal vessel and that in the lateral vessels may be correlated.
FIG. 3. Oscilloscope records showing the relationship between the electrical activity associated with contractions of the dorsal vessel over the crop (lower traces) and lateral vessel V (upper traces). Calibration: 4 mV upper traces, 8 mV lower traces, 4 sec.
In the simplest case one sees a simple one-to-one correspondence between activity in the dorsal vessel and the lateral vessel. The first two contractions in Fig. 3A give one such example. For each wave of activity in the dorsal vessel there is a wave of activity in the lateral vessel. In other cases one finds that the lateral vessel possesses a rhythm of its own, contracting at a rate independent of the dorsal vessel activity. In such cases one often observes that even though the lateral vessel possesses its own rhythmicity,
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it can be reset by the activity in the dorsal vessel if the excitation from the dorsal vessel occurs sufficiently long after the endogenous contraction has occurred in the lateral vessel (Fig. 3C). If the excitation occurs at too short an interval after an endogenous contraction no visible contraction occurs in the lateral vessel, but one often records a small electrical potential which becomes smaller as it .comes closer and closer in time to an endogenous contraction. For example, in Fig. 3C three contractions of the dorsal vessel are shown. The first contraction occurs shortly after a spontaneous contraction in the lateral vessel and there occurs in the lateral vessel a small electrical potential. The second contraction of the dorsal vessel produces a full-sized contraction in the lateral vessel while the third contraction of the dorsal vessel occurs at such a short time after the lateral vessel contraction that no response of any kid is detectable in the lateral vessel. The particular type of relationship between contractions in the dorsal vessel and those seen in a particular lateral vessel does not remain constant over time. At one particular time a particular lateral vessel may be contracting at a rate nearly double that of the dorsal vessel. Later in time this same lateral vessel may contract completely in co-ordination with the dorsal vessel or it may cease contracting completely for a period of time. The particular kind of relationship between the dorsal vessel and a particular lateral vessel at a particular time is not seen for all the lateral vessels at the same time. Typically one finds that one or two of the lateral vessels may be contracting rather vigorously and rapidly at a particular time while the other lateral vessels may not be contracting at all. Later in time one will find that the opposite situation may hold. Also there is no evident co-ordination between the beating of the lateral vessels of a pair; at any particular time one vessel may be beating at a very regular rate whereas the contralateral vessel is completely quiescent.
F. Eflects of intralumenal pressure As mentioned in the Materials and Methods section, if during the initial dissection of these animals any of the larger vessels are severed, there follows rather quickly an irregular slow beating of the dorsal vessel and eventually complete cessation of activity. This observation leads one to believe that possibly the amount of intralumenal pressure in the dorsal and lateral vessels has an important influence on the rate of beating. In order to test this possibility experiments were performed in which the intralumenal pressure was either increased or decreased. In order to test whether decreased intralumenal pressure has an effect on the rate of the contraction, the dorsal vessel was occluded by sucking a short length of the dorsal vessel into a small capillary. This was sufficient to block blood flow in the dorsal vessel. In almost every case within 1 min after the occlusion was applied there occurred anterior to the occlusion a marked decrease in the rate of the contractions, generally to a rate less than one-half of the pre-occlusion rate. When the occlusion was removed the rate again rather quickly returned
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CHARLES R. FOURTNER AND RALPH A. PAX
to its former value. In a few instances at the time the occlusion was produced or removed a temporary excitation was seen but this excitation lasted for less than 30 set and the overall result of the occlusion was always a depression in activity. Figure 4 gives one example of such experiments.
A
‘I
FIG. 4. A tracing of a “Physiograph” record showing the effects of occlusion of the dorsal vessel between lateral vessels IV and V on lateral vessel IV activity. At the upward arrow the dorsal vessel was occluded, at the downward arrow the occlusion was removed. Calibration: 1 min.
The effects of increased intralumenal pressure were also tested in a few instances. In order to do this a fine glass capillary cannula was inserted into the dorsal vessel just anterior to the crop. The dorsal vessel is quite small and in only a small percentage of cases was there successful insertion of the cannula into the dorsal vessel. Insertion of the cannula as such had no observable effect on the rate of the contractions. When, however, the intralumenal pressure was increased by forcing saline under pressure into the vessel, a marked effect on the rate of contraction was seen. Figure 5 shows a typical example of the results
4
4
record showing the effects of increased intralumenal FIG. 5. “Physiograph” pressure on activity in lateral vessel V. At the upward arrow pressure was increased by injecting saline into the dorsal vessel approximately 1 cm posterior to lateral vessel V. At the downward arrow the injection was stopped. Calibration: 1 min.
In this example the rate of contraction of the lateral vessel of such experiments. from which the recordings were taken had become very low and irregular. When the intralumenal pressure was increased there occurred a marked increase in the rate. When the pressure was released the rate immediately reverted to its former value. Thus the change in rate appears to be a response to the increased pressure and not to the saline as such. G. Ejfects of electrical stimulation of the dorsal vessel In order to examine some of the electrical characteristics of the dorsal and lateral vessels, a series of experiments was performed in which the dorsal vessel was electrically stimulated while the activity in the dorsal vessel or lateral vessels
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was recorded. In these experiments a suction electrode placed on the dorsal vessel in the region of the crop-gizzard or the anterior portion of the intestine was used as a stimulating electrode. Stimuli consisted of square wave pulses (l-10 V, 3 msec). Stimulation of the dorsal vessel in this way elicits waves of contractions in the dorsal vessel. These waves are similar in conformation to those seen during spontaneous contractions and they are conducted both anteriorly and posteriorly For example in one preparation the elicited from the point of stimulation. waves had a velocity in the anterior direction of 2.6 cm/set posterior to lateral vessel V; 2.2 cm/set measured between lateral vessels IV and V; 1.2 cm/set measured between lateral vessels III and IV; 0.8 cm/set measured between lateral vessels II and III ; and O-6 cm/set measured between lateral vessels I and II. Elicited waves in the posterior direction over the dorsal vessel in the region of the crop-gizzard and intestine had a velocity of 24 cm/set. The rate at which contractions can be elicited by electrical stimulation is quite low. The maximum rate of stimulation in any case which gave a one-to-one correspondence between stimulations and contractions was one stimulus every 4.2 sec. In most cases the maximum rate at which one obtained a one-to-one correspondence between stimulation and contractions was one stimulus every 5.0 to 5.5 sec. When stimuli above these rates were given one obtained contraction waves in response to every other stimulus.
50
H
40
M
4.5
6. A tracing of a “Physiograph” record showing the effects of electrical stimulation of the dorsal vessel posterior to the gizzard on lateral vessel V activity. Stimulus artifacts are marked by dots; the numbers below the record indicrlte the intervals between successive shocks, Stimulus: 8 V, 3 msec. Calibration: 10 sec. FIG.
The ability of stimulation of the dorsal vessel to elicit contractions in the lateral vessels was also examined. Here results essentially the same as those obtained when recording from the dorsal vessel were obtained. Figure 6 gives one example of such stimulation. In this particular case spontaneous contractions of the dorsal vessel had dropped to less than one per minute. When stimuli at intervals of 5.0 set were applied there resulted from each stimulus, a contraction of the lateral vessel. When the interval was dropped to 4-O set only every second stimulus was effective. When the interval was raised to 4.5 set, a one-to-one relationship was again established after several stimuli. DISCUSSION
The results we have reported here confirm for the most part the observations of Stiibel(l909). All parts of the dorsal vessel appear to have the ability to initiate
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CHARLES R. FOURTNER AND RALPH A. PAX
contractions but under normal circumstances there is a wave of contraction which originates at the posterior of the animal and moves forward, The degree of stretch on the walls of the dorsal vessel appears to be important in determining whether a particular section of the vessel may initiate a contraction. Thus if there is excessive loss of blood from the vessels the rate of pulsations in the dorsal vessels becomes quite low or ceases altogether. Also when intralumenal pressure in the dorsal vessel is reduced by means of an occlusion of the dorsal vessel, one observes a decrease in the rate. In like manner if the amount of intralumenal pressure is increased by means of saline injection one observes an increase in the rate of the pulsations. From these observations it appears likely that the amount of intralumenal pressure is an important variable determining the rate of pulsation of the dorsal vessel. Although stretch of the vessel walls appears to be important in determining the rate at which the pulsations occur, stretch of itself does not appear necessary for conduction of a contraction wave once initiated. Stiibel (1909) observed that if by pressure he prevented contraction of the dorsal vessel over several segments, waves of contractions could still be propagated through the quiescent region. Previous investigators who have observed the pulsations of the blood vessels in the earthworm, L. tewestris, and other earthworm species have given conflicting reports on the degree of co-ordination between dorsal vessel and lateral vessel activity. Thus Stiibel (1909) reported that lateral vessel activity was closely co-ordinated with dorsal vessel activity, but Prosser & Zimmerman (1943), working with dissected worms, reported that they could not observe any co-ordination between beating in the dorsal vessel and the lateral vessels. Johansen & Martin (1965), working with the giant earthworm, Glossoscolex giganteus, also reported a lack of synchrony between dorsal vessel activity and lateral vessel activity. They did indicate, however, that the lateral vessels all beat in synchrony with one another. In our experiments we observed that at times there is a co-ordination between lateral vessel and dorsal vessel activity, while at other times there is no obvious co-ordination visible. From our electrical recordings of the activity in the lateral vessels it appears likely that each of the lateral vessels possesses an inherent rhythmicity but that each of the lateral vessels can be driven by excitation arriving via the dorsal vessel. A major difference between the experiments done by Stiibel and the other investigators mentioned above as well as our own, is that Stiibel made his observations on intact animals. It is quite possible that in the intact animal there is a one-toone correspondence between activity in the dorsal vessels and the lateral vessels, but in the process of dissection of the worms preparatory to recording, there is sufficient disruption of the circulatory system so that lateral vessel activity may become unto-ordinated with that in the dorsal vessel. Stiibel (1909) as well as Prosser & Zimmerman (1943) concluded that it is likely that the waves of contraction in the dorsal vessel in the earthworm are neurogenic and not myogenic, that is, that they are conducted by way of nerve
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cells rather than muscle cells. Several lines of evidence-none of them unequivocal-however, lead us to conclude that conduction in the dorsal vessel is probably myogenic. The extremely slow conduction velocity of the contraction wave, the conformation of the electrical activity associated with the contraction wave, the exceedingly long refractory period following a contraction wave and the ability of the dorsal vessel to conduct waves both anteriorly and posteriorly, are all characteristics of a myogenically conducted wave and not a neurogenically conducted wave. SUMMARY
1. The contractile dorsal and lateral blood vessels of the earthworm, Lumbricus ten-e&s, have been examined. 2. In intact resting animals the rate of contraction of the dorsal vessel is 11 beats/min; in “exercised” animals, 17 beats/min. 3. In dissected animals the contractions of the dorsal vessel average 8 beats/ min, each contraction consisting of a peristaltic wave passing anteriorly. 4. The velocity of the propagated wave is a uniform 2.4 cm/set posterior to the lateral vessels but becomes progressively less in the region of the lateral vessels. 5. Lateral vessel contractions consist of peristaltic waves passing from the dorsal to the ventral vessel. 6. Each lateral vessel possesses an endogenous rhythmicity but can be driven by activity in the dorsal vessel. 7. Intralumenal pressure influences the rate of beating of the dorsal vessel, decreased pressures decreasing the rate; increased pressures increasing the rate. 8. Electrical stimulation of the dorsal vessel elicits propagated contraction waves but there is a long refractory period following each contraction so that the dorsal vessel cannot be driven in a one-to-one manner at a rate greater than once every 4-5 sec. Acknowledgement-This Research Grant HE-09718
investigation was supported in part by Public Health Service from the National Heart and Lung Institute. REFERENCES
JOHANSENK. & MARTIN A. W. (1965) Circulation in a giant earthworm, Glossoscolex gigunteus-I. Contractile processes and pressure gradients in the large blood vessels. J. exp. Biol. 43, 333-347. JOHNSTONJ. B. (1903) On the blood-vessels, their valves, and the course of blood in Lumbricus. Biol. Bull. mar. biol. Lab., Woods Hole 5, 74-84. JOHNSTONJ. B. & JOHNSONS. W. (1902) The course of blood flow in Lumbricus. Am. Nat. 36, 317-328. KIEFER G. (1959) Pharmakologische Untersuchungen fiber den Automatismus der Lateralherzen des Regenwurmes Lumbricus terrestris Linne. 2. wiss. Zool. 162, 356-367. PANTINC. F. A. (1948) Notes on Microscopical Technique for Zoologists. Cambridge University Press.
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PROSSERC. L. & ZIMMERMANG. L. (1943) Effects of drugs on the hearts of Arenicolu and Lumbricus. Physiol. Zoiil. 16, 77-83. STUBELH. (1909) Studien zur vergleichenden Physiologie der peristaltischen BewegungenIV. Die Peristaltik der Blutgefasse des Regenwurmes. Pfltigers Arch. ges. Physiol. 139, l-34. Key Word Index-Earthworm; activity; circulatory system.
Lumbricus terrestris; contractile blood vessels; rhythmic