Download Is Embryonic Limulus Heart Really Myogenic? Experimental

AMER. Zoou 19:39-51 (1979).
Is Embryonic Limulus Heart Really Myogenic?
DANIEL GIBSON
AND
FRED LANG
lioston University Marine Program, Marine Biological Laboratory, Woods Hole,
Massachusetts 02543
SYNOPSIS. Although the neurogenic nature of the heartbeat in adult Limulus has been well
studied and is undisputed, we contest the reports that the embryonic heartbeat is
myogenic. This notion, based on histological, calorimetric, and drug studies, is challenged
by evidence from transmission electron microscopy and intracellular recording. The first,
infrequent heartbeats occur at the time of the third embryonic molt when only the anterior
portion of the heart tube is formed and functional. Contractions extend further caudad
concomitant with lumen formation in the rear heart segments. All lumen-containing heart
sections that we have examined, from the earliest on, have revealed neural elements in a
bundle at the dorsal midline of the heart. Axons 1/i.m or less in diameter are prevalent:
vesicle-filled terminal-like areas adjacent to muscle cells are often present as well, even in
the youngest beating hearts. Myocardial cells show excitatory postsynaptic potentials as
soon as heartbeat has begun, but they often fail to summate in the earlier stages so that
contractions are few. Resting potentials remain at —65 to —70 mV from the onset of
heartbeat until well after the larva has hatched, but heartbeat frequency, regularity,
depolarization height (never overshooting) and duration all increase as embryos get older,
probably as innervation of muscle fibers increases and coordination between pacemaker
and follower neurons improves. We have found no evidence that embryonic Limulus heart
passes through a myogenic phase and believe that it is neurally driven from the beginning.
INTRODUCTION
Experimental investigation of the heart
of the horseshoe crab Limulus polyphemus
was initiated over seventy years ago by A. J.
Carlson (1904) for the primary purpose of
elucidating the general principles of cardiac physiology. It was Carlson's contention that the vertebrate heart was
neurogenic, but since the nerve plexus was
difficult to study, or even find, general
principles were best studied using the
heart of another animal, such as Limulus
where the ganglion and myocardium were
easily separable. In this light, Carlson's
studies continued for a number of years
with his comparisons always sustaining his
belief in the basic similarity between the
hearts of vertebrates and Limulus.
Later work on vertebrate hearts proved,
This study was supported by N1H Grant RO 1
HL18267.
of course, that their myogenic nature was
very different from neurogenic invertebrate hearts. Indeed, this dichotomy had
become well entrenched in the literature.
More recently, however, it has been demonstrated that some arthropod hearts are
myogenic, much like vertebrate hearts. For
instance, the moth heart lacks a ganglion
and is myogenic (McCann, 1963). Similarly, while cockroach heart has an attached ganglion, heartbeat is apparently
unaffected by removal of this structure
(Miller, 1968; this volume). Still other invertebrate hearts, on present knowledge,
seem to defy classification into the simple
dichotomy. For example, the heart of the
leech is apparently driven by neurons
originating in the central nervous system
(CNS), but may be capable of myogenic
contractions in the absence of this extrinsic
pacemaker activity (Thompson and Stent,
1976). Likewise, the heart of Aplysia normally has a myogenic beat, but it can be
driven by regulatory neurons from the
40
DANIEL GIBSON AND FRED LANG
CNS (Koester and Dieringer, this volume).
Thus while some hearts seem to be
clearly neurogenic or myogenic, others
seem to fall into some intermediate category. In this regard, previous work
suggested that Limulus heart might also fall
into some intermediate category. Carlson
and Meek (1908), on the basis of histological evidence, reported that embryonic
Limulus heart lacked innervation at the
time of the first heartbeat and for approximately 10 days thereafter. They concluded from this that the initial heartbeats
were myogenic. This was supported in two
later studies: Crozier and Stier (1927)
found differing activation energies (/x) for
embryonic and adult Limulus hearts, and
Prosser (1942) demonstrated that cardioacceleration by acetylcholine develops
secondarily a week or more after the embryonic heartbeat begins. In both of these
studies, the authors concluded that the different behavior of older hearts was a
consequence of the changeover to neurogenic control.
Added credence for this hypothesis was
lent by experiments which demonstrated
that it is possible to induce myogenic activity in a deganglionated, adult Limulus
heart. For instance, Carlson (1907, 1908)
demonstrated that a quiescent deganglionated heart would again begin to beat if
placed in isotonic NaCI solution. This was
confirmed by later workers who demonstrated that the contractions were due to
overshooting, sodium dependent, spikes
(Lang, 1971«; Rulon et ai, 1971). Another
example of myogenic activity was reported
by Heinbecker (1933) who demonstrated
that a deganglionated adult Limulw, heart
would beat if it had been inflated. This was
confirmed by later studies which demonstrated that nonovershooting spikes were
the apparent cause of the activity (Lang,
1971a)In light of the above work we decided to
reinvestigate the problem of the nature of
the heartbeat in the early Limulus embryo.
We will first review the evidence of recent
investigations on the adult heart. Thus far,
we have found no basis for the hypothesis
that Limulus heart is ever myogenic under
physiological conditions.
THE NEUROGENIC ADULT HEART
Perhaps the earliest physiological experiments on Limulus were those of
Carlson (1904, 1905) which established the
neurogenic nature of the heartbeat. When
he cut the ganglionic cord and left the
muscle intact, he showed that the heart
established different rates of beat on either
side of the cut. If he cut the muscle completely, in several spots, leaving the ganglion intact, he showed that all sections of
the heart would beat synchronously. Upon
removal of the ganglionic chain the heart
beat ceases completely (Carlson, 1904).
More recent work using electrophysiological techniques has supported the
notion that the adult heart is neurogenic.
The intracellular electrical activity recorded from Lumulus heart does bear some
superficial resemblance to a vertebrate
myocardial action potential, but the underlying mechanisms are completely different. The latter is a myogenic action
potential produced by regenerative membrane conductance changes to several cations. In the case of Limulus, the myocardial
membrane is depolarized by excitatory
postsynaptic potentials (EPSPs) generated
by activity in ganglionic follower neurons
which innervate the myocardium. These
EPSPs summate to provide a rapid depolarization and a sustained plateau (Fig.
M;Lang^«/., 1967; Abbott Hal., \969a,b;
Parnas et ai, 1969). The neuromuscular
junctions are similar to those found in
some arthropod skeletal muscles (Fig. 2;
Lang, 1972).
Early work on the cardiac ganglion lead
to the suggestion that there were three cell
types, the largest of which are the
pacemaker cells. Later studies described
five cell types, two small and three large
(Bursey and Pax, 1970a). Intracellular recording from the large cells demonstrated
that they were follower cells; they fired repetitively during each heartbeat (Fig. \li;
Palese^w/., 1970; Lang, 1971/;). Small cells
were shown to be pacemaker cells; each
fired an overshooting action potential at
the beginning of every heartbeat and each
had a slowly depolarizing pacemaker potential between spikes (Fig. 1(7; Lang,
1971/;).
CARDIAC CONTROL IN EMBRYONIC LIMULUS
FIG. 1. Intracellular records of electrical activity in
adult Limulus heart. A, cardiac muscle fiber; B, follower neuron; C, pacemaker neuron. Chemical
synapses link these elements such that an overshooting spike in a pacemaker (C) initiates a burst of attenuated spikes in a follower cell (as in B), which
sends processes to the myocardium and elicits excitatory postsynaptic potentials in the muscle (A). The
EPSPs summate to provide a sustained depolarization
ONSET OF HEARTBEAT IN THE EMBRYO
Clutches of fertilized eggs were dug
from shoreline nests near Mashnee, Cape
Cod, and reared in standing seawater in
the laboratory. Although breeding in this
locality is restricted to late spring and early
summer (Cavanaugh, 1975), embryos kept
below 4.5°C will not develop further until
warmed (Crozier and Stier, 1927); we
maintained embryos for later use by this
method. Vital staining of eggs with neutral
red (Sekiguchi, 1960) made the heart
easier to discern as it developed. The dye
accumulated both in cells flanking the
heart and in particles in the pericardial
space; the latter moved and thus became
visible once beating began.
The temporal staging described by
Carl.son and Meek (1908) and Prosser
(1942) stated that heartbeat began after
41
which causes a contraction. Muscle fibers receive
input from many follower cells, which may in turn
have multiple pacemaker innervation. Note that extracellularly recorded ganglionic bursts (lower traces
A, B, C) coincide closely with follower cell discharge
(B) but precede muscle response (A) and lag behind
pacemaker spikes (C).A from Lang, 1971a; B,C from
Lang, 1971*.
three weeks (no rearing temperatures
given). However, we found great variability of developmental rate even between
eggs in the same clutch, and it was therefore necessary to seek developmental
markers that would foretell incipient cardiac activity. Horseshoe crabs molt four
times within the egg (Sekiguchi, 1970,
1973). We found that sporadic heartbeats
began just before the third embryonic
molt, which often occurs soon after the
tough outer chorion of the egg splits and
the inner egg membrane swells. Our
findings corroborated those of Crozier and
Stier (1927).
Whatever the initial pacemaker mechanism, the early beats of the heart are extremely sporadic in terms of the interval
between beats. In order to determine the
earliest time when hearts were capable of
contracting, we used a device to stimulate
42
DANIEL GIBSON AND FRED LANC
stimulus make or break; as expected, when
the cathode is closer to the heart, contraction is on make, and is maintained until the
stimulus is released. Several contractions
sometimes followed a single stimulus of
200 msec (TTX absent).
Only the anterior portion of the dorsal
heart tube contracted in early embryos;
visible movement extended caudad only as
far as the area which will later form the
hinge between the prosoma and opisthosoma (Fig. 4). This supports the finding
of Scholl (1977) that the heart tube hollows
out first anteriorly. All parts of the functional portion contract and relax in unison,
rather than as a peristaltic wave. However,
three to five days later (at 20°C), when the
caudal portion begins to beat, it is often out
of phase with the rostral portion, sometimes appearing to fill with blood that is
being forced out of the contracting anterior part. Gill movements do not begin
until the fourth embryonic molt, so they
can have no effect on blood movements or
heart rhythm in these early stages.
FIG. 2. Neuromuscular junction in adult Limulus
heart. Nerve terminals may embed in sarcoplasm and
form synapses on sarcolemma, as this one does, or
they may embed on arms of granular sarcoplasm.
NT: nerve terminal, arrowhead pointing to presynaptic membrane where vesicles are aligned; G:
glial cell; MF: muscle fiber; Ex: extracellular space.
From Lang, 1972.
the heart muscle directly. The intact embryo was placed dorsal side up over a
hole connecting two vertically stacked
seawater-filled polystyrene chambers (Fig3). An effective seal between hole and embryo was made with petroleum jelly, so that
any current passed between chambers
would have to pass through the embryo. A
15-volt, 1.5 mA current elicited contractions in those hearts old enough to beat
even in the presence of tetrodotoxin(TTX,
10"" g/l). Tests with older embryos and
larvae injected with tetrodotoxin (TTX,
10 " g/l) indicated that contractions were
due to direct depolarization of the muscle,
as TTX blocks nerve activity on Limnlm
heart, but does not affect muscle sodium
channels (Lang, 1971c/). Polarity determines whether contraction occurs on
ELECTRON MICROSCOPY
Yolk proved to embed and section
poorly in epoxy resins; thus it was neces-
- polystyrene
FIG. 3. Chamber used for passing current through
intact embryos to elicit heart contractions. Hearts first
respond to DC current pulses S 200 msec at the time
of the third embryonic molt when only the expanded
anterior portion (ht) has formed (see Fig. 4). With
polarity as shown, contraction is on stimulus make;
with cathode in lower chamber, heart contracts on
stimulus break.
CARDIAC CONTROL IN EMBRYONIC LIMULUS
FIG. 4. Transverse 1 fim sections of the. heart of
Limulus at the third embryonic molt, taken from the
areas indicated in the lower drawing. Arrowheads on
the upper drawing delimit the functional portion of
the heart, which encompasses sections AA and 4B,
where lumen has formed. In 4C, only a thickened
ectodermal cord is present; the coelom and heart will
form below it. Figures 5 and 6 are electron micrographs of the 4J4 and 4B regions. Drawings
modified from Patten, 1912.
sary to remove it from the vicinity of the
heart before fixation. The following procedure was used: The dorsum was isolated
by cutting around the lateral margin of the
embryo. This 1.5 mm diameter piece of
skin was fastened to a coverslip with
cyanoacrylate glue, and immersed in seawater with the free, internal surface facing
upward. The yolk filling this concavity was
removed using forceps and a stream of
seawater from a hypodermic syringe, exposing the heart, which is attached to the
dorsum. Preparations were fixed for one
hour at room temperature in 29c glutaraldehyde buffered with 0.1 M cacodylate at
pH 7.5; fixative and buffer wash contained
10 mM CaCl2/l and were adjusted to 1000
mOs/kg with sucrose. Fixation was followed by overnight buffer wash, one hour
postfixation in \'/( OsO, in identical buffer,
and dehydration in ethanol. While in
ethanol, the glue-backed tissue was easily
pared from the cover slip using a razor
blade. Specimens were embedded in
Epon-Araldite and sectioned with glass
knives on a Sorvall MT-2B "Porter-Blum"
ultramicrotome. Thick sections of 1-2 /x.m
were taken and stained with toluidine blue
for corroborative light microscopy. Silver
thin sections were collected on uncoated
copper grids, stained with uranyl acetate
followed by lead citrate, and examined
with a JEOL 100-S electron microscope.
Thick, transverse sections were taken
through the dorsum of an embryo whose
heart had just begun to beat, progressing
from front to rear (Fig. 4). The most
rostral section (Fig. AA) has a smaller,
rounder lumen than the middle section
(Fig. AB). This is in agreement with drawings by Kingsley (1893) of hearts sectioned
at about this stage. The most caudal section
(Fig. AC) contains no heart tube as yet, only
a dorsal thickened tissue cord. This of
course explains the absence of heartbeat
from the caudal region of earlier embryos.
Figure bA is a low-power electron micrograph of an anterior heart section; Figure bB shows the mid-dorsal portion of
this section at greater magnification. A
bundle of small (l£im) axons lies in this
region, contacting the single layer of
myoblasts that form the heart. Synaptic
vesicles 500 to 650 A are visible in axons,
although distinct synaptic regions are not
apparent. Other axons are visible at some
distance from the heart muscle, above a
group of cells which have prominent nuclei. The electron micrograph in Figure 6
is taken from the middle region of the embryo shown in Figure AB. Axons are present in the mid-dorsal region next to the
muscle, and can be identified by the
numerous microtubules. Extracellular
filaments of unknown function are found
around some of the axons (Gibson and
Lang, 1977, and in preparation).
We have not encountered any hearts
which have a lumen but are devoid of
axons in the mid-dorsal region; axons appear consistently even in the youngest
beating hearts. We are as yet unable to
identify the cell bodies from which the
44
DANIEL GIBSON AND FRED LANG
FIG. 5. Electron micrographs of t,he region of early
Limulus heart shown in Figure 4A. Under low
magnification {A) the heart tube in transverse section
appears as a single layer of irregularly shaped cells.
Axons can be seen along the dorsal midline (circle
and box; see bli). It 1$ not possible to tell whether the
prominent nuclei (n) in the region belong to neuron
somata. li: Axonal region from box in A. The axons
contain numerous microtubules (t). One process
which forms a junction with the muscle (m) contains
numerous clear vesicles (v) 500 to 650 A in diameter.
Myofilaments (f) in the muscle are very sparse at this
stage.
axons project. There are cells interspersed
between some axons (Fig. 5A), but there is,
as yet, no evidence that these represent
neuron cell bodies. When beating begins,
the lumen-containing portion of the heart
extends from about coelomic segment 5 to
segment 8 (segments as numbered by
Scholl, 1977). This would correspond to
the first four heart segments in the adult;
the ganglion overlying the first three of
these consists of a fiber tract with a sparse
scattering of cell bodies (Hursey and Pax,
1970c/). By homology, we would not expect
to find cell bodies over most of the length
of the functional heart in the early stages,
and this might explain why Carlson and
Meek (1908) did not find them. Serial thin
sections may reveal the location, probably
posterior, of the early ganglion cell bodies.
Regardless of the validity of segmental
homologies between embryonic and adult
heart, the ganglionic structure we have
seen is quite different I mm that ol the
adult ganglion. F.leinents ol the embryonic
CARDIAC CONTROL IN EMBRYONIC LIMVLCS
nerve cord contact the sarcolemma of the
muscle layer (Fig. 5/i), while in the adult,
tissue sheaths and a large cleft of 30 jam or
more separate nerve cord from muscle
(Fig. 7). Also, in the posterior two-thirds of
the adult ganglion where most cell bodies
are found, they occupy the ventral region
of the cord, lying between the nerve fibers
and the muscle (Fig. 7; see also liursey and
Pax, 1970/). The dorsal midline of the
adult heart appears to be physically isolated from direct contact with nerve libers
and terminals, which can approach this region only laterally; conversely, in early
embryos, neuromuscular contact along the
dorsal midline seems to be the rule.
While we have lotind no gap junctions
between niyoblasts in the embryonic heart,
it appears unlikely that enough cells are
innervated directly to produce a coordinated contraction. While neurons are
never lacking along the dorsal midline.
45
that is the only place we have found them.
An ultrastructural feature not directly
involved in the myogenic-neurogenic
question but of developmental interest is
the paucity of myofilaments in the cells of
early hearts which can nevertheless contract. This characteristic is apparent in the
niyoblasts of Figures 5B and 6. Orientation
of these few myofilaments is variable, and
some even run longitudinally in the heart
(Fig. (i). although it contracts as circular
muscle. In later stages, filaments proliferate and become transversely aligned (Gibson and Lang, in preparation).
INTRACKl.l.t'I.AR KKCOKD1NC
Embryonic myocardial tissue is initially a
thin (I to '.\ ptm) single layer of niyoblasts
which may move a distance several times its
thickness during contraction. Prolonged
impalement with glass microelectrodes is
46
DANIEL GIBSON AND FRED LANG
obviously difficult. The best results in
young embryos were obtained with
tungsten wire-suspended glass electrode
tips, resistance 20 to 40 Mfl. These "floating" microelectrodes (Woodbury and
Brady, 1956) were impaled through the
dorsal skin of the intact embryo. We impaled near the midline of the heart to take
advantage of its attachment to the body
wall, which minimizes movement of that
region. In older embryos and trilobite larvae, the chitinized dorsum was clipped off
and inverted in filtered seawater, exposing
the ventral aspect of the heart for impalement.
Figure 8A compares heartbeat recordings from a newly hatched "trilobite" larva
and an embryo at the first day of heartbeat.
The initial volley of excitatory postsynaptic
potentials (EPSPs) is so well-coordinated in
the trilobite heart that the compound nature of the upsweep, actually a summation
of EPSPs, is not discernible. However, the
heart is almost certainly neurogenic at this
. /
FIG. 6. Electron micrograph of mid-dorsal region of
early heart, as in Figure 4B. Several axons (a) lie
above a myoblast (m) which contains sparse bundles
of myofilaments (I), most in cross-section. Exlracellular filamenLs (e) appear in cell interstices at this stage.
CARDIAC CONTROL IN EMBRYONIC LIMULUS
47
FIG. 7. Transverse section, light micrograph, of cardiac ganglion overlying heart in adult Limulus.
Somata (s) lie ventral to the axons, and a wide cleft
and connective tissue sheaths (ts) separate ganglion
from heart muscle; this is in contrast to the close con-
tact of axons and muscle along the dorsal midline
during early heart development (see Figs. 5, 6). The
nerves which leave the ganglion in each segment of
the adult heart apparently become the major avenues
of contact.
stage as hyperpolarization of the muscle
fiber during spontaneous activity suggests
that the depolarization is entirely synaptic
(unpublished observations). The younger
heart shows three distinct EPSPs on the
beat rise. The recordings are similar in
several respects: resting potentials are -65
mV for both, depolarizations are undershooting and of composite nature, and rise
times are similar. The smaller peak depolarization, disjointed rise, and shorter
tetanic phase of the lower trace are what
we would expect from a fledgling
neurogenic heart if the number of driving
units is initially small or if they start out
poorly coordinated.
While the heart rhythm of later embryos
is regular, it is quite sporadic in early embryos. In the latter, this may be due to poor
coordination of neural discharge and
paucity of stimulating units. Figure 8li
compares a train of regular beats from a
trilobite heart to a train of EPSPs from an
early heart, where summations are infrequent.
Impalement of the tail region prior to
formation of the lumen has not revealed
any neural activity. We have not yet successfully recorded the first beats in this
area, when it often appears out of phase
with the functional anterior region of the
heart.
Poking the embryonic heart with an
electrode often elicits a contraction. Less
forceful mechanical stimuli can evoke
electrical activity without causing contraction of the muscle. Figure 9 shows what
appear to be volleys of EPSPs in ah impaled muscle cell (3 days post-third molt)
elicited by lightly tapping the preparation
chamber. After several taps had elicited
one burst each, rhythmic bursting began
on its own (Fig. 9). No observable contractions resulted from the small depolariza-
48
DANIEL GIBSON AND FRED LANG
FIG. 8. Comparison of intracellular records from
heart of newly-hatched "trilobite" larva (upper traces,
A and B) with those from heart of embryo at third
embryonic molt when beating begins (lower traces).
A: Resting potentials and rise times are similar, but
the younger heart shows three distinct EPSPs during
initial depolarization, a smaller peak depolarization
and a shorter plateau. It appears that the number of
driving units is smaller or more poorly coordinated.
tions (maximum 15 mV), but the result
indicated neural responsiveness to mechanical stimulation, either directly, or as
feedback from the myocardium (c/., Lang,
1971ft). Tangled nerve endings, resembling sensory structures, have been described in adult Limutu.s myocardium
(Fedele, 1942). These might serve as feedback transducers, although we have no ultrastructural evidence for their existence
in embryonic heart.
Drawings from Patten, 1912. B: EPSP bursts are well
coordinated in "trilobite" heart, producing regular
beat rhythm with only occasional out-of-phase EPSPs
between beats (arrow, upper trace); in younger
hearts, the muscle may receive many EPSPs (lower
trace), but few summate to produce contractions (arrow). B, lower trace only, was recorded in AC mode;
baseline dip is movement artifact.
Carlson and Meek (1908) were aware that,
with the available techniques, fine nerve
fibers would escape their detection. The
neural elements observed in the present
study were minute indeed. Crozier and
Stier (1927) emphasized that there is no
intrinsic reason that a myogenic heart must
have a different activation energy than a
neurogenic one. They only suggested it
was true in lAmulus because their experimental results showed a difference between /it-values for embryonic and adult
heart. The difference is no less real, but
DISCUSSION
should be attributed to other factors, since
It seems likely from both ultrastructural the lack of innervation suggested by
and electrophysiological evidence that Carlson and Meek was actually a conseeven the earliest beating hearts of Limulus quence of the lack of adequate resolving
embryos contain active neural elements. power.
However, it is not yet possible to state
We have not repeated Prosser's experiwhether early coordination of contraction ments using acetylcholine on intact emis .solely neurogenic, especially since an bryos, but it appears that this drug affects
element of myogenicity has been found in heart rate only in heroic doses. Prosser
stretched adult hearts (Heinbecker, 1933; (1942) used a 10~4 wt/vol solution in seaLang, 1971«).
water (= 6 x 10~4 M), usually potentiated
4
4
Our findings conflict with those from with 10" ( = 3.6 x 10 M) eserine. Garrey
earlier studies on Limulus embryos, but it (1942) found that concentrations of ACh
may be possible to reconcile the two views. less than 1:16,000 (3.5 x 10 ' M) seldom
CARDIAC CONTROL IN EMBRYONIC LIMULUS
accelerated heart rate unless potentiated
first with 10"4 (3.6 x 10~* M) eserine.
Limulus heart muscle iself is insensitive to
ACh (Garrey, 1942) and the excitatory
neuromuscular transmitter might be
glutamate (Abbottsa/., \969a,b). The cardiac ganglion, while responsive to high
doses of ACh, is not the only site of its
action in cardioregulation. Von Burg and
Corning (1971) found that perfusion of
the abdominal ganglia with ACh caused
cardioacceleration, apparently by stimulating dorsal nerves 9 - 1 3 , which send
49
excitatory and inhibitory fibers from the
abdominal ganglia to the cardiac ganglion
(Carlson, 1905; Pax, 1969; Bursey and
Pax, 19706; Von Burg and Corning, 1970).
Von Burg and Corning (1971) also used
high doses (10~2 M) in their drug studies.
Acetylcholinesterase has been identified as
a normal constituent of the cardiac ganglion (Stephens and Greenberg, 1973).
Whether acetylcholine is normally present
there as well remains to be investigated.
On the other hand, Townsel et al. (1976)
demonstrated ACh synthesis from radio-
__|5mV
25 ms
JUUUUUUVAJUUUU.
_j5mV
250 ms
FIG. 9. Intracellular record from heart of embryonic
stage midway between third and fourth molts. Tapping the preparation chamber causes EPSP bursts in
the impaled myocardial cell. A: Tap, recorded as
audio signal (lower trace) elicits burst with summation
of EPSPs. B: Three successive taps (top trace) induce
one burst each, followed by a train of spontaneous
bursts (lower traces). The heart had stopped its
spontaneous beating prior to these recordings; no
visible contractions resulted from the tap-induced
bursts. Possible mechanisms of tap sensitivity are discussed in text. Drawing from Patten, 1912.
50
DANIEL GIBSON AND FRED LANG
active precursors in the abdominal ganglion. It seems likely to us that the cardiac
ganglion becomes functional before its
regulatory nerves; their input is never essential to heartbeat, as excision of the adult
heart demonstrates. The secondary development of sensitivity to ACh (Prosser,
1942) could have been due to the development of competence of cholinergic fibers from the abdominal ganglia. Thus,
the site of action of the applied ACh could
have been either the abdominal ganglia,
the cardiac ganglion, or both.
Dopamine excitation of early hearts
would indicate the presence of a functional
cardiac ganglion, as Fetterer and Augustine (1977) have found that dopamine excites the adult ganglion but does not affect
the muscle directly. Our preliminary trials
with this drug have been equivocal, as have
other drug tests, because of the difficulty
of maintaining impalements for intracellular recording. Other methods of recording activity from the early hearts have
not yet proved feasible: the difficulties of
extracellular electrical recording or tension recording from a heart less than 100
fjbm wide are obvious; the natural vagaries
of the incipient heart rhythm make visual
records inconclusive.
The developing picture is that of a heart
driven by neural elements from the beginning. In spite of inherent difficulties,
pharmacological study and additional ultrastructural work are needed to complement our initial findings.
REFERENCES
Abbott, B. C , F. Lang, and I. Parnas. 1969a. Physiological properties of the heart and cardiac ganglion
of Limulus potyphemus. Comp. Biochem. Physiol.
28:149-158.
Abbott, B. C , F. Lang, I. Parnas, W. Parmley, and E.
Sonnenblick. 19696. Physiological and pharmacological properties of Limulus heart. In F. V.
McCann (ed.), Comparative physiology of the heart,
current trends. Experientia, Suppl. 15, pp. 232-243.
Birkhaiiser Verlag, Basel.
Bursey, C. R. and R. A. Pax. 1970a. Microscopic
anatomy of the cardiac ganglion of Limulus
polyphemus. J. Morph. 130:385-396.
Bursey, C. R. and R. A. Pax. 19706. Cardioregulatory
nerves in Limulus polyphemus. Comp. Biochem.
Physiol. 35:41-48.
Carlson, A. J. 1904. The nervous origin of the
heartbeat in Limulus and the nervous nature of
coordination in the heart. Amer. J. Physiol.
12:67-74.
Carlson, A. J. 1905. The nature of cardiac inhibition
with special reference to the heart of Limulus.
Amer. J. Physiol. 13:217-240.
Carlson, A. J. 1907. The relation of the normal heart
rhythm to the artificial rhythm produced by
sodium chloride. Amer. J. Physiol. 17:478-486.
Carlson, A. J. 1908. The conductivity produced in the
nonconducting myocardium of Limulus by sodium
chloride in isotonic solution. Amer. J. Physiol.
21:11-18.
Carlson, A. J. and W. J. Meek. 1908. On the mechanism of the embryonic heart rhythm in Limulus.
Amer. J. Physiol. 21:1-10.
Cavanaugh, C. M. 1975. Observations on mating behavior in Limulus polyphemus. Biol. Bull. 149:422
(Abstr.)
Crozier, W. J. and T. J. B. Stier. 1927. Temperature
and frequency of cardiac contraction in embryos of
Limulus.]. Gen. Physiol. 10:501-518.
Fedele, M. 1942. Sulla innervazione intracardiaca del
Limulus polyphemus. Arch. Zool. Napoli 30:39-137.
Fetterer, R. H. and G. A. Augustine, Jr. 1977.
Dopamine cardioexcitation in Limulus. Amer. Zool.
17:959 (Abstr.)
Carrey, W. E. 1942. An analysis of the action of
acetylcholine on the cardiac ganglion of Limulus
polyphemus. Amer. J. Physiol. 136:182-193.
Gibson, D. and F. Lang. 1977. Extracellular filaments
in developing Limulus heart. J. Gen. Physiol. 70:8a
(Abstr.)
Heinbecker, P. 1933. The heart and median cardiac
nerve of Limulus polyphemus. Amer. J. Physiol.
103:104-120.
Kingsley, J. S. 1893. The embryology of Limulus. Part
II. J. Morph. 8:195-268.
Koester, J. J., N. Dieringer, and D. E. Mandelbaum.
1979. Control of circulation in Aplysia. Amer. Zool.
19:703-116.
Lang, F. 1971a. Induced myogenic activity in the
neurogenic heart of Limulus polyphemus. Biol. Bull.
141:269-277.
Lang, F. 19716. Intracellular studies on pacemaker
and follower neurones in the cardiac ganglion of
Limulus.]. Exp. Biol. 54:815-826.
Lang, F. 1972. Ultrastructure of neuromuscular
junctions in Limulus heart. Z. Zellforsch. 130:481488.
Lang, F., B. C. Abbott, and 1. Parnas. 1967. Electrical
activity in muscle cells of Limulus heart. J. Gen.
Physiol. 50:2500. (Abstr.)
McCann, F. V. 1963. Electrophysiology of an insect
heart. J. Gen. Physiol. 46:803-821.
Miller, T. A. 1968. Role of cardiac neurons in the
cockroach heartbeat. J. Insect Physiol. 14:12651275.
Miller, T. A. 1979. Nervous venus neurohumoral
control of insect heartbeat. Amer. Zool. 19:7786.
Palese, V. J., J. L. Becker, and R. A. Pax. 1970. Cardiac ganglion of Limulus: Intracellular activity in
the unipolar cells. |. F.xp. Biol. 53:41 1-423.
Parnas, I., B. C. Abbott, and F. King. 1969. Elec-
CARDIAC CONTROL IN EMBRYONIC L/MULUS
trophysiological properties of Limulus heart and effect of drugs. Amer. J. Physiol. 217:1814-1822.
51
B15:153-162.
Stephens, L. B. and M. J. Greenberg. 1973. The
localization of acetylcholinesterase and butyrylPatten, W. 1912. The evolution of the vertebrates and their
cholinesterase in the heart, cardiac ganglion and
kin. P. Blakiston's Son & Co., Philadelphia.
the lateral and dorsal nerves of Limulus polyphemus.
Pax, R. A. 1969. The abdominal cardiac nerves and
J. Histochem. Cytochem. 21:923-931.
cardioregulation in Limulus polyphemus. Comp.
Biochem. Physiol. 28:293-305.
Thompson, W. J. and G. S. Stent. 1976. Neuronal
Prosser, C. L. 1942. An anlysis of the action of acetylcontrol of heartbeat in the medicinal leech. I. Gencholine on hearts, particularly in arthropods. Biol.
eration of the vascular construction rhythm by
Bull. 83:145-164.
heart motor neurons. J. Comp. Physiol. 111:261279.
Rulon, R., K. Hermsmeyer, and N. Sperelakis. 1971.
Regenerative action potentials induced in the Townsel, J. G., H. E. Baker, and T. T. Gray. 1976.
neurogenic heart of Limulus polyphemus. Comp.
Radiochromatographic screening for neurotransmitters in Limulus. Neuroscience Abstracts,
Biochem. Physiol. 39:333-355.
Vol. 2, p. 618. Society for Neuroscience, Sixth AnScholl, G. 1977. Beitrage zur Embryonalentwicklung
von Limulus polyphemus L. (Chelicerata, Xiphosura). nual Meeting, Toronto.
Zoomorphologie 86:99-154.
Von Burg, R. and W. C. Corning. 1970. Cardioregulatory properties of the abdominal ganglia
Sekiguchi, K. 1960. Embryonic development of the
in Limulus. Can. J. Physiol. Pharmacol. 48:333-341.
horseshoe crab studied by vital staining. Bull. Mar.
Sta. Asamushi 10:161-164.
Von Burg, R. and W. C. Corning. 1971. The effects of
Sekiguchi, K. 1970. On the embryonic moultings of
drugs on Limulus cardioregulators. Can. J. Physiol.
the Japanese horseshoe crab, Tachypleus tridentatus. Pharmacol. 49:1044-1048.
Sci. Rep. Tokyo Kyoiku Daigaku B14:121-128.
Woodbury.J. W. and A. J. Brady. 1956. Intracellular
Sekiguchi, K. 1973. A normal plate of the developrecording from moving tissues with a flexibly
ment of the Japanese horseshoe crab, Tachypleus
mounted ultramicroelectrode. Science 123:100tridentatus. Sci. Rep. Tokyo Kyoiku Daigaku
101.