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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. 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