J. exp. Biol. (1981), 93, 149-165
Wiith 10 figures
printed in Great Britain
INTRACELLULAR RECORDING OF ELECTRICAL
ACTIVITY IN MUSCLE CELLS OF INTACT AND
ISOLATED DORSAL BLOOD VESSEL OF THE
EARTHWORM LUMBRICUS
TERRESTRIS
BY C. D. DREWES, M. R. DENNEY,*
B. O'GARA AND E. P. VINING
Zoology Department, Iowa State University, Ames, Iowa, U.S.A. 50011
(Received 8 October 1980)
SUMMARY
1. Intracellular electrical records from muscle cells in intact dorsal blood
vessels of the earthworm, Lumbricus terrestris, showed a polarization rhythm
which consisted of: (a) a prolonged ramp of depolarization, (b) one or a series
of several non-overshooting spike potentials, (c) a sustained, hump-like
depolarization, and (d) a complete repolarization. The polarization rhythm
of the intact vessel was reset by intracellular current injection.
2. Simultaneous recordings of intracellular electrical and mechanical
activities during rhythmic pulsation waves of the intact vessel indicated that
the ramp-like depolarization corresponded temporally to a gradual filling of
the vessel with blood. Spike potentials occurred during the later portion of
filling, and constriction began near the peak of the slow, sustained depolarization.
3. Simultaneous intracellular and extracellular electrical records during
rhythmic pulsations of the intact vessel indicated a close temporal correspondence between spike potentials in both electrical records. The repolarization phase in intracellular records corresponded to a small, slow wave in
extracellular records.
4. A polarization rhythm was usually absent in isolated preparations of the
dorsal blood vessel, but in a few preparations rhythmic activity persisted for
up to 1 h. In quiescent isolated preparations membrane potentials were stable
(mean resting potential = — 667 mV), but active membrane responses
(non-overshooting spikes and sustained depolarizations), were evoked by
intracellular current injection or by stretching of the vessel.
5. Evidence from dual intracellular recordings in isolated dorsal vessels
suggest that muscle cells are electrically coupled.
6. The intracellular activity patterns in intact and isolated vessels, and the
responsiveness of cells to current injection and stretching, suggest that both
initiation and conduction of pulsation waves in the dorsal blood vessel are
myogenic.
INTRODUCTION
The propulsion of blood through the closed circulatory system of earthworms is
•diieved by rhythmic waves of constriction in the dorsal blood vessel and five pairs
^ * Present address: Space Engineering and Aero-research Laboratory, University of Washington,
Seattle, Washington 98195.
150
C. D. DREWES AND OTHERS
of lateral vessels ('hearts'). These waves normally originate in the most posterior
segments of the animal and propagate anteriorly along the dorsal vessel and into the
lateral vessels of segments 7-11. After leaving the lateral vessels, the blood enters the
non-pulsating ventral vessel and is distributed to anterior and posterior segments. In
segments posterior to the lateral vessels, blood is returned to the dorsal vessel via
segmentally arranged parietal and dorso-intestinal vessels (Johnston & Johnson, 1902;
Bahl, 1921). Valves in the dorsal vessel, as well as in the parietal and dorso-intestinal
vessels, increase the efficiency with which the blood is moved anteriorly (Johnston,
The histology of the dorsal vessel in earthworms has been described by a number of
early investigators (for review, see Hanson, 1949). These investigators all found that
the main muscle elements in the wall of the dorsal vessel consisted of a well-developed
layer of circular muscle fibres, although some also described a few longitudinally
oriented fibres. No evidence for the presence of nervous tissue in the wall of the dorsal
vessel was given. More recently, Hama (1960) studied the ultrastructure of the dorsal
blood vessel of Eisenia foetida and helped to clarify its cellular organization. Hama
found that the dorsal vessel consisted of: (1) an inner articular layer, (2) a lining of
endothelial cells (some endothelial cells contained longitudinally arranged myofilaments and were referred to as ' myoendothelial' cells), and (3) a well-developed
layer of circular muscle fibres containing numerous mitochondria. These histological
and ultrastructural studies suggest that the circular muscle cells are the main elements
involved in producing the rhythmic waves of constriction that propel blood forward
in the dorsal vessel.
The myogenic or neurogenic origin of rhythmic pulsations of earthworm blood
vessels has not been clearly established. Based on an analysis of extracellular electrical
activity (i.e. suction electrode recordings), Fourtner & Pax (1972) have argued that
the pulsation waves along the dorsal vessel are conducted by some type of myogenic
mechanism.
Our objective in the present study has been to examine cellular mechanisms underlying the rhythmic pulsations of the dorsal blood vessel in the earthworm, Lumbricus
terrestris. This has been done by: (1) analysis of intracellular recordings of electrical
activity of muscle cells in intact and isolated dorsal blood vessels, and (2) correlation
of this activity with extracellular electrical recordings and the mechanical activity of
the vessel.
MATERIAL AND METHODS
Earthworms, Lumbricus terrestris, were obtained locally and maintained in Magic
Worm Bedding (Amherst Junction, Wisconsin) at 15 °C. The animals were pinned to
a paraffin dish and an incision was made along the dorsal midline from just anterior
to the crop to just anterior to the clitellum. Two lateral cuts were made at each end
of the longitudinal incision, to permit the cut edges of the body wall to be spread and
pinned down. Care was taken to avoid cutting any major blood vessels because the
resulting blood loss usually caused irregular beating or eventual cessation of beating,
approximately segment 10 to segment 20) to expose the dorsal vessel. Two fine thready
Earthworm dorsal blood vessel
fter dissection the specimens were rinsed and bathed in a saline solution (Drewes &
Pax, 1974) at 13-17 °C.
All in vivo electrical and mechanical recordings were obtained from the portion of
the dorsal blood vessel directly over the gizzard. The special advantage of recording
from this region is that the vessel is almost completely free from connexions to underlying tissue and can therefore be elevated away from other tissue without disturbance
in function.
All intracellular recordings were obtained with 3 M-KCl-filled glass microelectrodes
(resistances 10-60 MD). Electrodes with resistances of 10-20 MQ were most effective
for experiments involving intracellular current injection. For in vivo recordings the
electrodes were mounted on a coiled Ag-AgCl wire, which permitted the electrodes to
'float' with the movements of the blood vessel or animal. To help stabilize the dorsal
vessel a 1 -o mm wide platform (a flattened and painted silver wire) was attached to a
micro-manipulator and inserted under the vessel. Then, by slightly raising the platform,
the vessel was lifted just away from the underlying tissue without disrupting blood flow
through the vessel. If the vessel was elevated too much, the mechanical disturbance
caused weakening or cessation of the pulsations in that region. The reference electrode
was an Ag-AgCl wire or, in some cses, an Ag-AgCl wire inserted into a brokentipped glass microelectrode filled with 3 M-KC1 in agar. The electrical signals were
led into a WP Instruments Model M4A or M701 microprobe system and then displayed on a storage oscilloscope. In a few instances (e.g. Fig. 3), the signals were
displayed on a chart recorder (Physiograph Four, Narco Biosystems).
Extracellular recordings from the blood vessel were obtained with glass suction
electrodes (outside diameter of fire-polished tips = 75-100 /tm), as described by
Fourtner & Pax (1972). The electrodes were carefully applied to the vessel because
excessive suction resulted in weakening of rhythmic pulsations and extracellular
electrical signals. The electrodes were connected to the differential inputs of a
Tektronix 5A22N amplifier (high-frequency filter setting = 100 Hz; low-frequency
filter setting = 01 Hz).
Mechanical movements of the vessel were monitored by attaching the supporting
platform for the vessel to a Narco Biosystems F50 microdisplacement transducer.
The connexions between the platform, transducer, and oscilloscope were arranged in
such a way that any ventrally directed (downward) force by the vessel upon the platform caused positive, or upward, deflexions on the oscilloscope screen. Because the
vessel was loosely connected to underlying tissue (by means of laterally arranged
parietal and dorso-intestinal vessels), and rilling of the vessel with blood, as well as
the constriction of the vessel, produced ventrally-directed forces on the platform.
Thus both filling and constriction phases of rhythmic pulsations were indicated as
upward deflexions on the oscilloscope screen (e.g. upper traces of Figs. 1, 4). Mechanical movements of the vessel were also monitored under a dissecting microscope to
note the durations of the constriction and filling phases of pulsations. Onset and
termination of these phases were displayed on one channel of the oscilloscope by
means of a hand-operated event marker (lower traces of Fig. 1 A, B).
Isolated preparations of a portion of the dorsal vessel were obtained by pinning the
imal down and making a dorsal midline incision just through the body wall (from
r
152
C. D. DREWES AND OTHERS
Fig. i. Mechanical events associated with rhythmic pulsations of the dorsal blood vessel over
the gizzard. In A, the duration of each constriction phase, as determined by direct observation
of the vessel, was indicated in the lower trace by the duration of each broad band (event
marker). Corresponding to each constriction phase was a large upward deflexion in the
mechanotransducer record (upper trace) obtained from the same location along the vessel.
In B the duration of the filling phase, which was several times longer than the constriction phase,
was indicated by each broad band in the lower trace. The gradual filling of the blood vessel,
which occurred throughout most of the filling phase, coincided with a ramp-like increase in
the baseline of the mechanical record. Note that at the end of some filling phases there was a
small, shoulder-like deflexion (arrow in lower mechanotransducer recording) which just
preceded the next constriction. The deflexion represents a brief period of rapid filling.
Time scale: 5 s.
were used to ligate the vessel at the anterior limit of the crop and the posterior limit
of the gizzard. The portion of the vessel between the ligations was then excised by
severing the vessel just anterior and posterior to the ligations and by severing any
attachments of the vessel to underlying tissue. The excised portion of the vessel was
then transferred to a small saline-filled dish. For examination of the electrical coupling
between cells in isolated preparations an additional recording microelectrode was
connected to a WP Instruments Model VFI voltage follower.
In some isolated preparations the effects of intraluminal volume changes (i.e.
stretch) on intracellular electrical activity were studied. This was done by making a
small incision in the wall of the vessel, inserting a glass cannula (tip diameter =
50-100 fim) into the lumen of the vessel, injecting a very small amount of saline, and
noting membrane potential changes.
RESULTS
Mechanical events during dorsal blood vessel pulsations
Transducer recordings of pulsations of the dorsal blood vessel were compared
visual observations of the timing of vessel movements (Fig. 1 A, B). The constrict!
Earthworm dorsal blood vessel
153
e (time during which the vessel changed from maximal to minimal diameter) of
each pulsation was relatively constant in duration, usually lasting 1-5-2-0 s. Each
constriction phase always corresponded temporally to a large upward deflexion of
similar duration in the transducer recording. Immediately after constriction ended
and during the entire time between successive constriction phases the vessel dilated
and became filled with blood (filling phase). The rate of filling and the total duration
of the filling phase were variable. The duration of the filling phase ranged from 5 to
15 s and was inversely related to the rate of rhythmic pulsations. Usually there was an
initial, rapid filling of the vessel with blood, but throughout most of the remainder of
the filling phase there was also a much slower and more gradual filling of the vessel.
This gradual filling corresponded temporally to a ramp-like increase in the baseline
of the mechanical record (Fig. 1 A, B). Occasionally, during some cycles of pulsations,
there was a brief period of rapid filling at the end of the filling phase. This later rapid
filling appeared as a small upward deflexion, or shoulder, which preceded the next
constriction phase in the mechanical record (Fig. 1 B).
Intracellular activity of the dorsal blood vessel in vivo
Considerable difficulty was encountered in maintaining penetration of muscle cells
in the dorsal vessel due to the small size of cells and the pulsatile movements of the
vessel. Also, due to the extreme sensitivity of the vessel to mechanical disruption, the
combined effects of the supporting platform and microelectrode in contact with the
vessel at any location often caused marked weakening or cessation of pulsations in
that region. Animals were discarded if pulsations failed to persist because of these
problems. Despite these difficulties a total of 32 measurements of the muscle cell
activity were obtained in seven animals. The mean 'resting' potential (i.e. maximum
potential during filling) was —56-9 mV+ 1-4 S.E.M.
The intracellular records from the dorsal vessel indicated that the membrane
potential of each muscle cell fluctuated in a complex but rhythmic manner (Fig. 2).
Although there were variations in wave form, the activity in all cells included the
following components: (a) a prolonged ramp of depolarization, (b) an initial spike or
series of several spike-like potentials, (c) a sustained hump-like depolarization, and
(d) a complete repolarization.
The initial ramp of depolarization ranged from 1 to 9 mV in amplitude (29 measurements in 7 animals). In all cases termination of the ramp was marked by a distinct
spike-like depolarization which ranged in amplitude from 5 to 38 mV (22 measurements, 6 animals). The repolarization which followed this spike was always incomplete; the amount of repolarization ranged from 10 to 60% of the initial spike
amplitude. In approximately two-thirds of the cells the initial spike was followed by
one or more, usually smaller, spike-like potentials. In the remaining one-third of the
cells only the initial spike was seen. In a few cells successive cycles of activity showed
different numbers of spike potentials (Fig. 6). In all cells the initial spiking activity
was followed by, or superimposed upon, a prolonged hump-like depolarization. The
total amplitude of this depolarization (difference between the membrane potential
at the end of the ramp and at the peak of depolarization) ranged from 22 to 46 mV
K measurements, 6 animals). In all cells the peak depolarization was never over-
154
C. D. DREWES AND OTHERS
A
J
Fig. 2. Rhythmic intracellular activity from three different muscle cells of the dorsal blood
vessel over the gizzard. These records, from three different animals, are representative of the
range and variation in the waveform of intracellular records obtained from intact vessels. In A,
a single large spike potential was followed by a slower hump-like depolarization. In B,
two small spike potentials preceded a large, slow hump-like depolarization. In C, a small
initial spike was followed after approximately 2 8 by a series of small spike potentials which
were superimposed on a hump of depolarization. Note in all of the records that a ramp of
depolarization preceded, and smoothly graded into, each initial spike potential. Time scale: 2 8.
Voltage scale: io mV.
shooting with respect to zero potential. Following the peak depolarization a complete
and relatively smooth repolarization occurred. Together the spike potentials and
sustained hump-like depolarization produced a net depolarization which lasted 2*o4-5 s (17 measurements, 4 animals).
The general appearance of the intracellular activity patterns, especially the ramp
of depolarization and smooth transition of the ramp into a spike potential, suggested
the possibility that the polarization rhythm in the muscle cells may be myogenic. If
the rhythm is myogenic, then its periodicity should be modifiable, or reset, by displacement of the membrane potential (i.e. current injection). In most animals it was
not possible to maintain penetration of cells for a sufficiently long time to examine the
effects of current injection. However, on three different occasions (three animals)
cell penetrations were maintained long enough to examine the intracellular rhythm
before, during and after current injection. In each case the results indicated that
polarization rhythm was reset by current injection, thus supporting the idea
the rhythm is myogenic (Fig. 3).
Earthworm dorsal blood vessel
155
Fig. 3. Chart recording of the change in rhythmic intracellular activity of the intact dorsal
vessel in response to intracellular current injection. A prolonged hyperpolarizing current
pulse (15 nA) was delivered through the recording electrode. The result was a slowing of the
rhythmic activity. Note that when the hyperpolarizing pulse was terminated, an anode-break
excitation response was apparently evoked. During stimulation the bridge circuit was slightly
unbalanced, thus accounting for the elevation in baseline. The spike potential at the beginning
of each depolarization cycle was indistinct because of the relatively slow writing speed of the
pen. Also, the waveforms of the potentials were slightly distorted because the recording was
made with a curvilinear writing system. Time scale: 2 s. Voltage scale: 10 mV.
Relationship of intracellular activity to mechanical events in vivo
The timing relationships between intracellular electrical events in vivo and movements of the dorsal vessel were determined by simultaneously recording mechanical
and intracellular electrical activity. Two examples of such recordings are shown in
Fig. 4. These records indicate that the gradual filling of the vessel corresponded
closely in time with the ramp-like depolarization of individual muscle cells.
In intact blood vessels the ramp of depolarization was always terminated by a spike
potential. This spike always occurred during the later stages of the gradual filling
(Fig. 4B), or during the brief, rapid filling which sometimes just preceded the constriction phase (Fig. 4 A). The onset of constriction occurred simultaneously with,
or just before, the peak of the sustained and slow depolarization. The complete
repolarization of the membrane potential occurred throughout the later portion of
the constriction phase.
Relationship between intracellular and extracellular electrical activity in vivo
Fig. 5 A is an example of the rhythmic extracellular activity recorded from the
dorsal vessel. Each cycle of activity consisted of one or more spike-like potentials
which ranged in amplitude from 20 /iV to a few mV. The exact waveform of the spike
potentials was variable; sometimes the potentials were biphasic (Figs. 5B, 6), but
more complex, multiphasic waveforms were also seen (Fig. 5 C). These spike potentials
appear to be the same as those obtained from the dorsal vessel by Fourtner & Pax
(1972). However, extracellular records indicated that during each pulsation cycle
there was, in addition to the spike potentials, a slower potential not described by
Fourtner & Pax. This slower potential occurred approximately 2-3 s after the large
extracellular spike potential (Figs. 5 A, 6).
Simultaneous records of extracellular and intracellular activity from the dorsal
vessel are shown in Fig. 6. These records indicate a close temporal correlation between
fid changes in transmembrane potential and extracellular electrical events. Specifily, the intracellularly recorded spike potentials corresponded to the large, extra-
§
i56
CD.
DREWES AND OTHERS
f\
J
Fig. 4. Correlation of mechanical activity (upper traces in A and B) with intracellular activity
(lower traces) of the dorsal blood vessel. In A, the first rapid upward deflexion in the mechanical
record (arrow) represents the brief, rapid filling of the vessel which sometimes preceded constriction of the vessel. The onset of this rapid filling coincided with a spike in the intracellular
record. The second and largest upward deflexion in the mechanical record represents the
constriction phase. The onset of constriction occurred just before the peak of slow depolarization. Throughout the later portion of the constriction phase the cell was repolarizing. In B, the
initial spike occurred during the later stages" of gradual filling. Note in A and B that the
gradual filling (indicated in the mechanical records as a gradual increase in the baseline)
corresponded to the ramp-like depolarization in intracellular records. Time scale: 2 s. Voltage
scale: 10 mV.
cellular spike potentials, while each complete repolarization corresponded to the small,
slow wave in extracellular records.
Responses to current injection and stretching in isolated preparations
To obtain more information regarding electrical properties of individual muscle
cells, intracellular recordings were obtained from isolated dorsal blood vessels.
Ligation and excision usually resulted in immediate cessation of rhythmic pulsation
waves in the vessels. However, rhythmic pulsations of the vessels could sometinM
Earthworm dorsal blood vessel
1
INU-
J
Fig. 5. Extracellular electrical activity from the dorsal blood vessel over the gizzard. In A, each
cycle of rhythmic activity consisted of a large spike potential followed after approximately
2 8 by a smaller and glower potential (arrow). In B and C, faster sweep speeds reveal some of
the variations in the waveform in the extracellular spike potential. Time scale: 5 s (A); <>•$ s
(B, C). Voltage scale: 20 /iV.
be re-initiated by either brief electrical stimulation (e.g. a depolarizing intracellular
current pulse) or mechanical stimulation (e.g. touching or stretching). Such rhythmic
pulsations often persisted for only a few cycles, but in a few vessels, especially those
within which a relatively large volume of blood was retained, rhythmic pulsations
persisted as long as one hour. An example of intracellular electrical activity from an
isolated preparation in which rhythmic pulsations persisted is shown in Fig. 7. With
the exception of the slower rate of rhythmic pulsations, the activity pattern in this
preparation was very similar to that seen in the intact vessel (e.g. Fig. 2 A). Also, as in
intact vessels (Fig. 3), the rate of rhythmic depolarizations in isolated preparations
was markedly altered and reset by depolarizing or hyperpolarizing current injection.
The eventual cessation of rhythmic pulsations in isolated preparations was accompanied by the absence of rhythmic changes in membrane potential. The mean resting
potential of muscle cells in these quiescent preparations was —667 mV± 2-4 S.E.M.
(n = 7 animals), or slightly greater than the value obtained in vivo. This difference
may be due to the fact that after penetration, resting membrane potentials in the
Biescent preparations gradually became several mV more negative before stabilizing
6
KXB93
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CD.
DREWES AND OTHERS
Fig. 6. Temporal correlation of extracellular (upper trace) and intracellular (lower trace) electrical events. Each spike potential in the extracellular record coincided with a spike potential in
the intracellular record. Also each small, slow potential (arrows) in the extracellular record
corresponded to the repolarization in intracellular records. Time scale: 2 8. Voltage scale:
20 fiV (upper trace); 10 mV (lower trace).
Fig. 7. Rhythmic intracellular activity in an isolated dorsal blood vessel. Each cycle of activity
consisted of a ramp of depolarization which graded smoothly into a non-overshooting spike
potential. The spike was followed by a slower and sustained hump of depolarization. Note the
similarity between this activity and that of some intact vessels (e.g. Fig. 2A). Time scale:
5 s. Voltage scale: 10 mV.
Earthworm dorsal blood vessel
Fig. 8. Responses of muscle cells in three different isolated preparations to intracellular
current injection. In A, the response to the larger of two prolonged hyperpolarizing pulses
was an anode-break excitation spike which occurred after the termination of the pulse. In B,
superimposed intracellular responses of a cell (lower trace) to two depolarizing and two
hyperpolarizing current pulses (upper trace) are shown. The higher level of depolarizing current
evoked a non-overshooting spike followed by a slow hump-like depolarization. This response
was accompanied by a weak wave of contraction originating at the point of stimulation. Note in
A and B the long time required to reach the final plateau of depolarization or hyperpolarization.
The time required to reach 63 % of the plateau of depolarization or hyperpolarization in these
two cells was approximately 07 s (A) and 00 s (B). The input resistances were approximately
7 Mft (A) and 4 MO (B). In C, spikes were evoked in response to two different levels of depolarizing current (bridge slightly unbalanced). The smaller current evoked a single non-overshooting
spike. The larger current evoked a similar spike followed by a sustained hump of depolarization, upon which a series of small spike potentials was superimposed. Approximately 4 s after
the onset of the stimulus, but well before the stimulus was terminated, a smooth and nearly
complete repolarization occurred. Time scale: 2 s. Voltage scale for intracellular records:
20 mV (A, C); 10 mV (B). Vertical scale for current monitor: 5 nA (A, B): ionA (C).
after approximately one minute. Presumably, the muscle cell membrane became sealed
around the electrode during this time. By comparison, penetrations in vivo rarely
lasted one minute and, because of movements, the membrane probably never sealed
around the electrode.
The occurrence of spontaneous spiking activity in isolated preparations suggested
P possibility that spike potentials originated intrinsically and that muscle cell
6-3
i6o
C. D. DREWES AND OTHERS
J
Fig. 9. Simultaneous intracellular recordings at two different locations along the isolated
dorsal vessel in response to intracellular current injection. In A, a hyperpolarizing current
pulse (upper trace) was delivered to one cell. The resulting change in membrane potential at
the stimulating electrode (lower trace) was approximately 8 mV, whereas the change measured
at 400 fun from the stimulating electrode (middle trace) was approximately 4 mV. In B, an
identical stimulus was delivered to the same cell, but the second recording electrode (middle
trace) was moved to a site 1500 /tm from the stimulating electrode. At this distance the change
in membrane potential was only about 1 mV. Based on the two measurements in A and B, as
well as another recording at 900 /tm, the space constant in this preparation was estimated to be
600 /an. Time scale: 2 s. Voltage scale for intracellular records: 10 mV. Scale for current
monitor: 10 nA.
membranes were electrically excitable. To test this possibility current injection experiments were done in 12 isolated preparations. Membrane potential changes were then
recorded in response to prolonged depolarizing or hyperpolarizing current pulses
delivered through the same microelectrode. The results from every preparation
indicated that the muscle cells in the dorsal vessel were electrically excitable. Electrically excited responses, in the form of anode-break excitation spikes, were consistently
evoked in every preparation after termination of large and prolonged hyperpolarizing
pulses (Fig. 8 A). In a few cases these spikes were accompanied by a sustained depolarization and a weak contraction of the vessel in the region of the microelectrode.
Spiking was observed in response to depolarizing pulses in six preparations. In
some cases these responses consisted of single non-overshooting spike potentials and
were accompanied by no visible contraction, but occasionally depolarizing pulses
evoked a spike which was followed by a sustained hump of depolarization (Fig.
Earthworm dorsal blood vessel
161
J
Fig. 10. Stretch-induced changes in muscle cell membrane potential in two different isolated
and cannulated preparations. In A, the three short horizontal bars below the intracellular
record indicate the times when slight stretching of the vessel occurred as a result of repeatedly injecting and then withdrawing a very small amount of saline through the cannula.
The longer bar indicates a prolonged stretching. In each case stretching caused a slight
depolarization whose time course closely paralleled the time course of stretching. In B, slight
increments of stretch, indicated by the first bar, resulted in a cumulative depolarization which
led to a suprathreshold response. The response consisted of a series of non-overshooting spike
potentials, a sustained depolarization (approximately 3 s duration), and a spontaneous repolarization. A vigorous contraction of the vessel accompanied this response and forced fluid out
of the vessel. A second injection of saline (second bar) produced a gradual depolarization and
a single spike potential. This spike, in turn, was followed by a series of spike potentials, a
sustained depolarization, and a repolarization similar to that evoked by the first stretch.
Note the similarity of these intracellular responses to some of the activity patterns seen
in vivo (e.g. Fig. 2A). Time scale: 5 8. Voltage scale: 10 mV.-
This sustained depolarization was accompanied by a visible wave of pulsation which
propagated away from the point of stimulation. In a few cases depolarizing pulses
evoked more complex responses, consisting of (a) an initial spike, (b) a sustained
depolarization upon which a series of small spike potentials was superimposed, and
(c) a spontaneous and nearly complete repolarization (Fig. 8C). Such responses
resembled the activity pattern seen in some intact vessels (Fig. 2C).
The fact that some preparations produced a propagated wave of pulsation in
jgsponse to intracellular current injection suggested the possibility of electrical
162
C. D. DREWES AND OTHERS
coupling between muscle cells. To test this possibility, intracellular recordings
obtained a short distance away from the site of current injection. In each test, in each
of 10 preparations, injection of depolarizing or hyperpolarizing pulses into one cell
resulted in attenuated, but measurable, changes in the membrane potential at distances
at least 15 mm from the site of stimulation. Fig. 9 shows records from one preparation
in which responses to hyperpolarizing current pulses were recorded at two different
distances from the site of intracellular stimulation. The conformation of these
responses indicated a passive electrotonic spread of the subthreshold membrane
potential changes. Given the small diameter and circular arrangement of most muscle
cells in the dorsal vessel (Johnston & Johnson, 1902; Hama, i960), it seems highly
unlikely that each set of simultaneous recordings was always obtained from a single
cell. The most likely explanation is that the electrodes were in different cells and
that muscle cells in the dorsal vessel were electrically coupled.
Intracellular responses to stretching (i.e. intraluminal volume increases) were
examined in six isolated and cannulated preparations. The responses to very slight
stretching of the vessel, brought about by injection of small amounts of saline through
the cannula and into the vessel, are shown in Fig. 10A. The responses consisted of
graded and subthreshold depolarizations, with time courses corresponding closely
to the time course of the volume changes.
In two preparations larger amounts of stretching evoked graded depolarizations
and suprathreshold responses (Fig. 10B). The waveform of these responses resembled
the waveforms of activity in intact preparations. That is, the response to stretch
consisted of (a) a large spike potential(s), (b) a sustained hump of depolarization, and
(c) a complete repolarization.
In conclusion, the intracellular records from isolated dorsal blood vessels indicated
that spontaneous rhythmic activity, resembling activity patterns in vivo, may occasionally persist for periods up to one hour. In quiescent and isolated preparations,
membrane responses resembling the activity patterns in vivo were evoked by intracellular current injection or by increases in intraluminal volume.
DISCUSSION
On the basis of their extracellular recordings Fourtner & Pax (1972) suggested that
the conduction along the dorsal blood vessel of the earthworm was myogenic. They
cited several lines of evidence which support this idea. These include: (1) slow conduction velocity of the contraction wave along the vessel (2-54 cm/s over the crop
and gizzard); (2) general conformation of the extracellular electrical activity associated
with each contraction wave; (3) long refractory period after a contraction wave;
and (4) ability of the vessel to conduct waves in both anterior and posterior directions.
However, none of these lines of evidence is unequivocal, since the strongest evidence
for neurogenicity or myogenicity is obtained from intracellular recordings of electrical events in the muscle cells. In particular the occurrence of slow, ramp-like
depolarizations during diastole, the occurrence of smooth sustained plateaux of depolarization, and the resetting of the polarization rhythm by intracellular current
injection are characteristics of intracellular activity in myogenic hearts (McCann
Earthworm dorsal blood vessel
163
0 6 6 , 1967). Our records of intracellular activity in the intact dorsal vessel are consistent with these features of myogenicity. Our records indicated that the polarization
rhythm of the vessel consisted of the following sequences of events: a slow ramp of
depolarization, a spike (or series of a few spike-like potentials), a sustained hump of
depolarization, and a final repolarization. In addition, the frequency of the polarization rhythm in intact or isolated vessels was markedly altered and reset by intracellular current injection into a muscle cell.
The specific mechanism by which the ramp of depolarization is produced in intact
vessels is not clear. The close correlation between the ramp and the gradual filling
of the vessel suggests that stretching may be an important factor in somehow inducing
the ramp. This idea is consistent with the observation that the polarization rhythm
(including the ramp) tended to persist in isolated vessels in which blood was retained
(Fig. 7), but did not persist in vessels without blood. Also, when slight stretching of
a quiescent vessel was produced by saline injection, graded depolarizations, and in
some cases spike potentials, were evoked (Fig. 10).
The demonstration that stretch can induce depolarization and spiking responses in
isolated preparations (Fig. 10) is consistent with the observations of Fourtner & Pax
(1972). They reported that occlusion of the earthworm dorsal vessel (by applying
external suction) decreased the rate of beating anterior to the occlusion. Conversely,
they found that an increase in intraluminal pressure (by cannulation) caused an
increase in the rate of beating. These excitatory effects of stretch, or pressure, on
electrical and mechanical activity of the earthworm dorsal vessel are similar to the
effects of stretch on the myogenic hearts of other invertebrates (for review, see
Irisawa, 1978).
The slow ramp of depolarization in the earthworm dorsal vessel was always terminated by the initiation of a non-overshooting spike. The smooth transition of the ramp
into the spike potential, as well as the resetting of spike rhythmicity by current
injection, provided evidence that the spikes originated intrinsically and that muscle
membranes were electrically excitable. This idea was further supported by our
observations that current injection in isolated blood vessels evoked spike potentials
(Fig. 8). However, it appeared that the effectiveness with which depolarizing pulses
evoked spikes varied from one cell to another. In many cells spikes were evoked by
hyperpolarizing pulses (anode-break excitation, Fig. 8 A), but not by depolarizing
pulses. These results suggest the possibility that differences exist in the functional
properties of the muscle cell membranes. Differences in membrane sensitivity to
depolarizing versus hyperpolarizing pulses have been noted in pacemaker and nonpacemaker cells of the cultured chick ventricle (Lehmkuhl & Sperelakis, 1967). The
consistency with which spikes could be evoked by current injection in isolated preparations of the earthworm dorsal vessel, and the relative ease of obtaining prolonged
intracellular records in these preparations, indicate that they would be favourable for
studies of the ionic basis of the non-overshooting spike potentials and the sustained
hump of depolarization.
In intact blood vessels the initial spike (or series of spikes) was always accompanied
by a sustained hump of depolarization, with the peak amplitude of the hump often
164
C. D . DREWES AND OTHERS
exceeding the peak during the spike(s). Several lines of evidence suggest that
sustained depolarization is not an artifact of microelectrode movement during constriction, but is probably a necessary factor for initiating a sustained and vigorous
contraction of the vessel. First, constriction of the vessel did not begin until well
after the slow hump of depolarization had begun; the onset of constriction corresponded to a time just before or at the peak of depolarization (Fig. 4). Secondly, a
slow potential (which occurred during the final repolarization that followed each
sustained depolarization) was detected in extracellular records even when no microelectrode was present (Fig. 5). Third, in isolated blood vessels marked contractions
of the vessel were not observed in response to intracellular current injection, unless
preceded by a sustained depolarization. The observations that small pulses of depolarizing current sometimes evoked only single spike potentials, but larger pulses
evoked spike potentials and sustained humps of depolarization, suggest that the
mechanisms underlying both the spike and the hump are separable and voltagedependent. Assuming this were the case in vivo, the initial spike potential may function in triggering the sustained hump of depolarization required for constriction.
The more frequent observation of sustained depolarization phases and vigorous
pulsations in intact, as compared to isolated vessels, may be the result of deterioration
of isolated preparations. However, another factor which may contribute to these
differences is the marked amount of stretching which exists in the intact vessel just
prior to sustained depolarization and constriction. Johanson & Martin (1965) concluded that filling of the hearts of the giant earthworm, Glossoscolex, is important in
determining the force of contraction and general performance of the hearts. Also in
some molluscan hearts a decrease in tension or pressure leads to loss or delay of the
sustained depolarization which normally follows each spike (Irisawa, Kobayashi &
Matsubayashi, 1961; Hill & Irisawa, 1967).
The combination of various electrophysiological properties of muscle cells in the
dorsal vessel is well suited for myogenic initiation and conduction of pulsation waves
along the vessel. The stretch sensitivity and electrical excitability of the vessel would
ensure that active membrane responses and associated depolarization-contraction
coupling would begin only after sufficient filling of the vessel had occurred. The
electrical coupling between cells would ensure that the membrane potential changes
in the cells of any particular region along the vessel would be coupled, thus producing
a smooth and synchronous contraction of the vessel in that region. Similar properties
of mechanical sensitivity, electrical excitability, and cell-to-cell electrical coupling
are seen in many vertebrate smooth-muscle cells, including the smooth-muscle cells
of some blood vessels (Creed, 1979; Holman & Nield, 1979; Keatinge, 1979).
In summary, the results of the present study support the conclusion that the
polarization rhythm of muscle cells in the earthworm dorsal blood vessel is myogenic.
This conclusion does not preclude the possibility of neural or humoral modulation
of the myogenic rhythm. For example, neural elements could modulate, or at times
override, a myogenic rhythm as demonstrated in the circular muscle cells of the leech
heart (Thompson & Stent, 1976). Some type of chemical modulation of contractility
in the earthworm dorsal vessel appears likely, in view of the pharmacological studies
of Prosser & Zimmerman (1943) and Kiefer (1959). Of particular importance to A
Earthworm dorsal blood vessel
165
Understanding of the circulatory physiology of earthworms will be future studies of
rhythmicity patterns in the most posterior portion of the earthworm dorsal vessel
(i.e. normal initiation site for pulsatile waves) as well as in the lateral vessels ('hearts').
We thank Drs R. A. Pax and S. Shen for critical reading of the manuscript.
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