Findings on the Electrograms of the Cicada's Heart (Cryptotympan a Japonensis Kato)

FINDINGS ON THE ELECTROGRAMS OF THE CICADA'S HEART (CRYPTOTYMPAN A JAPONENSIS KATO)

HIROSHI IRISAWA, AYA F. IRISAWA AND TETSUJI KADOTANI Department of Physiology, School of Medicine, Hiroshima University

Several works on the neurogenic hearts of arthropoda by Carlson (1) and Crescitelli (2, 9) have already shown that their electrocardiograms are markedly different from these of vertebrates. One of the characteristics of the electrograms of the arthropoda heart is that they show oscillatory changes of potentials which have been attributed to rhythmical discharges of the pacemakers (9, 5, 7). Some of the previous workers (4, 13) have shown that under normal con- ditions the electrogram exhibits simpler patterns. However, it seems that their work did not show anything about the pacemaker activities of the heart beat. The present paper deals with the experimental results of the localization of pacemakers in the cicada's heart.

METHODS

A schematic sketch of the cicada's heart (Cryptotymana japonensis Kato) is shown in figure 1. It consists of seven segments, each of which is stretched by a pair of alary muscles. The microscopic sturucture is illustrated in figure 2. The surface of heart is covered with several layers of cubic cells. Beneath these layers there is a layer of striated muscle fibers about 20ƒÊ in diameter which are loosely connected with each other unlike those of the mammalian myocardium. For electrical observations, two types of electrode were used. 1) A low- resistance microelectrode. originally reported by Tomita et al. (17), which consists of about a 5ƒÊ silver wire, covered with a glass tubing, with an outer diameter of 10-15ƒÊ. By means of this electrode the potential can be led off from much smaller areas, than those of previous workers who used such electrodes with tip diameters exceeding 100ƒÊ for the insect heart. The heart action potential was recorded by a cathode ray oscilloscope through a 4 stage CR-amplifier. Cicada was placed in Ringer solution, to which the indifferent electrode was connected. 2) In order to record the membrane potential of heart muscle fiber intracellular electrodes of the type employed by Nastuk and Hodgkin were used (14). They were filled with 3 M KCl solution by Tasaki's method and those with electrical resistances of about 20 Megohms were selected. For the recording a cathode follower preamplifier (954) with very small grid current

Received for publication December 23, 1956. *入沢宏 ,入沢彩,角谷哲司

150 ELECTROGRAMS OF THE CICADA'S HEART 151 was connected with the main amplifier. Both low-and high-resistance micro- electrodes were moved by a micromanipulator under the microscope.

FIG.2.•@ Photomicrograph of cicada's heart.

M: Striated muscle. Parallel fibers about20ƒÊ in diameter. A: Alary muscle attached to heart

surface. V: Valves located at ostium. FIG.1.•@ Schematic sketch of

cicada's heart. Roman numerals label each segment.

EXPERIMENTAL RESULTS 1) Electrogram from the intact heart: In order to observe the heart tube, the abdomen was opened, and the muscle and digestive organs were removed. Figure3shows recordings of intricate surface action potentials led off with a low resistance electrode from various parts along the heart. At the anterior part of heart, the recording consists of several small and one large action potential. Usually this tendency was observed almost every- where on the heart tube. In this instance, however, diphasic action potentials were led off from the5th and 6th segment, while slow monophasic potential changes were observed at the7th segment. From these pictures, it can be seen that the electrograms of intact cicada's heart are so complex, that it seems to be very difficult to analyse them as a whole.

2) Electrogram from the isolated heart: As the intricacy of the electrogram described above, seemed to be mainly due to the alary muscles, the isolation of the heart tube per se from the alary muscles was tried. No remarkable changes of heart contraction were observed as a result of the removal of the alary muscles. Figure4illustrates the surface action potentials of the extirpated heart which are simpler than those in figure3. As would be expected almost all of the action potentials were triphasic, except two tracings from the anterior part of heart. At the neighbourhood of the7th segment, the initial deflection 152 H. IRISAWA ET AL.

FIG.3.•@ Electrograms

from the intact cicada's heart. Right: Schematic

sketch of the cicada's heart. Left: Action potentials recorded from

the place indicated. Up- ward deflection in elec-

trograms is positive. Time maker: 0.3sec.

FIG.4.•@ Surface action potentials from isolated cicada's

heart. Recorded from indicat-

ed locations. ELECTROGRAMS OF THE CICADA'S HEART 153 of tracing is negative. This negative sign localized in and around the 7th segment. If the 7th segment is cutted off, this negative deflection would be observed only at the 2nd segment. In the isolated heart, another very special finding was obtained from a particular part near the end of the 7th segment. As shown in the bottom tracing of figure 4, this was a very regular positive monophasic action potential with a different frequency from that of other parts of heart, and it seems that this potential localized only at the 7th segment and was not conducted.

3) The action potentials from the anterior and posterior parts of heart tube: As mentioned in the preceding section, a very regular monophasic potential change was recorded from a localized area in the 7th segment. The same tracing was also found at the anterior end of the 1st segment, where the artery is enlarged in the form of an ampula. The beat frequencies of these two places were three times greater than at other parts of heart. Under the microscope very minute contractions corresponding to the electric changes were found there. A typical electrogram of the 7th segment illustrated in figure 5A consists

FIG.5.•@ Action potentials from the 7th

segment: A: Repetitive action potential corresponding to the local miniature con-

traction. B: A large action potential following after local contraction of the 7th seg-

ment. C-D: Similar tracing led off from the anterior end of heart tube. Many small

action potentials coincide with the contraction of anterior end of heart tube, while a larger one coincides with that of the 1st

segment.

of slow rising phase followed by a rapid spike potential. B, C and D are examples which were all led off from the anterior and posterior ends of the heart tube. One illustrates the small action potential which gradually develops into a large one, while other examples show large number of rapid small action potentials of the 7th segment which are preceded by a regular contraction of the 6th segment.

4) Determination of pacemakers by the transection method: In seeking the localization of pacemakers the transection method (1, 8b) was used. The heart 154 H. IRISAWA ET AL.

tube was severed into two parts with a sharp razor-blade at the level of the 3rd segment to the4th segment. From table1it is clear that both divided parts beat with nearly equal rhythm. It is suggested that the heart of cicada seems to have two or more than two pacemakers. When the heart tube was severed into more than two parts, the following tendencies were noticed.

TABLE1

TABLE2

TABLE3

Each number shows heart beat (frequency) per1minute. E. H.: exposed heart S. S. A.: semisection of alary muscle B. S. A.: bisection of alary muscle the Roman numerals (1st line: segment number, 1st column: case number)

√:location sectioning.

a) One group of the results is illustrated in table2, where the frequency of the2nd segment is quite similar to that of the normal heart beat. This fact shows that the2nd segment plays a dominant role in pacemaker activity. Es- pecially in the cases II, III and IV, after the heart tube was severed at the level of the3rd segment, the separated posterior part completly ceased to beat, while the anterior part continued to contract with a frequency equal to that of the intact heart. b) Another group (table3) shows a quite different tendency from the former. The examples here show that the posterior end beats more fre- ELECTROGRAMS OF THE CICADA'S HEART 155 quently than the anterior end, after the heart tube was divided into several parts. Among these posterior segments, the7th segment seems to play a dominant role in the pacemaker activity. This table also shows that the frequency of the contraction of the posterior segment does not show any remarkable difference from that of the intact heart. From these three tables, it seems that there are two pacemakers in the heart of cicada, one at the2nd segment and another at the7th segment. As the heart was further severed into smaller pieces, they usually ceased beating. The smallest unit required to maintain its rhythmical contraction is illustrated schematically in figure6. When the heart tube was severed like in figure A and B, the beat stopped, while in the case of C, this section can con- tinue to beat, with a frequency of from21to30per minute. This fact indicates that at least one complete segment is needed to keep the rhythmical contraction.

FIG.6.•@ Schematic sketch of heart tube, showing the places of transverse section.

With cuts as in A and B the middle segment ceases to beat. With cuts as in C the middle segment continues beating (see Text).

5) Reversal of heart beat: In many instances the contraction waves usually progress forwards, that is, from the posterior to anterior end. However, after a while the contraction reverses its direction and begins to progress backwards, It has already been described by Wigglesworth (22) that when the insect is at rest, its blood pressure is nearly equall or even less than the atomospheric pressure. So in the exposed heart it would be resonable to assume that the intracardiac pressure would not exceed greatly from the atomospheric pressure. From this reason, when the cicada's heart was exposed the direction of intracardiac fluid flow changes according as the various changes of position of whole body. For instances when the cicada brought into a vertical position, the body fluid in the heart tube flows to posterior end. This phenomenon suggests that there may be no effective impediment such as valves inside of the cicada's heart tube. It was also observed that India ink or other dye solution injected into the heart tube, did not flow into tissue spaces through the ostia of the heart. This fact shows that these side ostia play a valve like role and prevent the leaking off of intracardiac fluid into the tissue space through them. Further studies on this heart beat reversal were made by recording the electrogram of heart. A series of surface action potentials were led off from5th segment (figure7). At first the rhythm followed the pace of the7th segment, as shown in a-b of the figure. However, after five seconds, the direction of conduction is suddenly reversed and it followed the pace of the2nd segment. As illustrated in c-d curve in the figure, diphasic action potentials were recorded in place of the former two positive waves, but after a short period, again the 156 H. IRISAWA ET AL. pace of the7th segment became predominant (e), and recorded potentials returned to the form of a-b. After three seconds, however, the2nd segment again began to play a role of the pacemaker (f). Thus the two pacemakers seem to contend for the possession of the leadership in pacemaker activity (f-j). It was also confirmed by the gross observation with the naked eye that the changes of the initial deflection of electrical tracings corresponds with the reversal of heart beat.

FIG.7.•@ The reversal of

action potentials led off from

the5th segment. See from the bottom to top, descriptions

are in text. Arrow indicates the place of reveral.

FIG.8.•@ Localized cocaini-

zation of the7th segment. A: Before the application of co-

caine solution (control). B: 1 minute after the application of

the drug, the1st positive com-

ponent decreases its magnitude. C: 2minutes after the application of cocaine, the 1st dis- FIG.8 appears except for small pre-

potential.

FIG.7

6) Action of cocaine: By means of a fine glass tubing (tip diameter of about200ƒÊ) filled with0.5per cent cocaine, local application of it was made on the various parts of the heart tube. This caused the stoppage of contraction at the applied segment, and produced a heart block. This event is very similar to that in the previous experiment, in which the transverse-section of heart tube divided the heart into two seperately beating sections. After washing with physiological saline solution, the heart recovered from the block. Figure8 shows an example of local cocainization at the7th segment, while active electrode placed on the4th segment. The figure, at the top is the control curve which has two positive waves. After cocainization, the first positive waves in the control picture gradually decreased ( middle) and finally it changed into an initial negative deflection in electrogram, doubtlessly indicating a reversed heart beat. After washing with physiological saline solution to remove the cocaine, the curve returned to the former pattern. When cocaine was applied on the ELECTROGRAMS OF THE CICADA'S HEART 157

4th segment, contraction of the heart stopped completely and action potentials could not be led off from the heart. When the heart was washed by applying drops of Ringer Solution which could be removed by a Khali pipette, a small action potential rose from posterior end and gradually propagated over all through the heart. 7) Localized cooling: A cooling thermode was placed on the7th segment, and observed surface action potentials led off from the4th segment. The direction of contraction which formerly came from the posterior end was reversed and the pace of the arterial part took the leadership. An instance of this case is shown in figure9A, where the initial deflection of action potentials was changed, after the localized application of cooling thermode. it was also recognized that after the localized cooling of the2nd segment initial deflection of the action potential was reversed (see fig.9B). In this case the reversal of contraction wave also coincided with this reversal of the initial deflection of action potential.

FIG.9.•@ Effect of localized cooling of the heart tube on the direction of

propagation. A) Active electrode was

placed on the4th segment. a: direction of propagation of contraction waves from posterior to anterior. b-c: locali-

zed cooling of the7th segment, the1st

positive component disappeared. B) Active electrode placed just on the3rd

segment. a: control. propagated direction of contraction is from posterior to anterior. b-d: localized cooling of the 2nd segment, initial positive wave disappeared gradually. Time maker: 0.3sec .

8) Intracellular recording from cicada's heart: There are great difficulties in the usage of the ultra-microelectrode for cicada's heart, because many hard bronchial branches run over the heart surface. Some results were obtained, however, as follows: Intracellular action potentials were led off from the 2nd segment. This tracing shows a slow depolari- 158 H. IRISAWA ET AL. zation phase followed by rapid action potentials of about50mV. Contrary to the surface action potentials, this intracellular recording shows a very simple pattern (figure10A and B).

FIG.10.•@ Action potentials which

were recorded with an intracellular electrode. A) a-d: several instances

from the segment2. e: calibration

50mV, 20mV, 10mV and5 mV, bot- tom0.3sec. B) Action potentials from

segment7. a: zero potential, b, c, d: action potential, e: is withdrawn, f: calibration, 20mV, 10mV, 5mV.

DISCUSSION 1) From observation of the heart under a microscope, India ink solution dropped on the heart tube was noted to flow into the heart tube, and the major part of it flows usually in the anterior direction, while a part of it moves to and fro in the heart tube by the heart contraction. If the anterior end is placed higher than the posterior end, the ink flows from the anterior end to the posterior end, while when the anterior end is placed lower it flows reversely. This event suggests that the cicada's heart has a very different structure from that described formerly for insects heart (for instance in the cockroach by Marchal). So, it may be suggested that the location of the heart valves in cicada's heart may be more suitable for the reversal contraction than the heart of other insects that have been observed. 2) It is a known fact that pattern of the electrograms of arthropoda heart varies greatly, according to the position of the electrode (15). This fact is further supported , by our studies. The intricate picture of the surface action potentials of the intact cicada's heart becomes simpler after the removal of the alary muscle. In the grasshopper heart Crescitelli et al. (9) found the so-called oscillatory potential changes disappeared when they used an isolated heart. Among the surface action po- tontials of the cicada's heart electronegative deflection appeared limitedly at both the2nd and7th segment. The fact that the2nd segment shows a marked elctronegativity in its sur- ELECTROGRAMS OF THE CICADA'S HEART 159 face action potentials and that the contraction of2nd segment is stronger than any other part of the heart tube, suggests that this part is a pacemaker of the heart. However, the7th segment which also shows an electronegative deflection, has a role in pacemaker activity. 3) As illustrated in experiment4, after the transverse section of this tubular heart at the level of middle part, both of the separated parts displayed contractions with similar frequencies. This is one evidence of the presence of two pacemakers in this heart. In order to conclude whether this pacemaker activity is neurogenic or not, further experiments will be necessary. But Mclndoo (12) found a train of nerve cells on the cockroach heart, and Krijgsman et al. (10) also asserted that there was a neurogenic pacemaker of the insect heart. Unfortunately, there are yet no detailed morphological studies of cicada's heart and the present authors failed to confirm nervous elements at the2nd and7th segment of the cicada's heart. The physiological findings, however, which were mentioned above, suggested that the pacemakers of cicada's heart seemed to be localized at the2nd segment and the7th segment. The transverse section of the heart tube at the level of the2nd segment, resulted in no change of frequency of contraction at the2nd segment, while the beat of remaining part was lengthened or stopped. The same results were obtained on sectioning at the7th segment. 4) Localization of two pacemakers are clearly demonstrated by observing the action potentials which were led off from the 4th and 5th segment, and the change of initial deflection of action potentials were recognized. The reversal of heart beat was formerly described by Gerould (6) who observed it in the adult beetle (Prines laticolis). There was no marked difference between the backward and forward beats, but in the adult crane fly the number of forward beats exceeds the backward beats. The latter case seems to agree with that of the cicada's heart. Many instances, however, in exposed heart showed that the heart beat seems to have its origin at the2nd segment. When the activity of one pacemaker was paralysed by action of cocaine or cooling, the heart beat ceased or gave place to another intact pacemaker. All these experiments suggested that there are two pacemakers in the heart of cicada, one in the anterior end of heart tube and another in the posterior end and usually one of them seems to play a dominant role in the heart beat. 5) As to the oscillation which was observed in the electrograms of the intact heart of cicada, very similar records were previously described by Jahn- Crescitelli-Taylor (9) and Crescitelli-Jahn (2) in the grasshopper heart. They suggested that these oscillations were apparently composed from a number of component potentials. The present experiment shows, however, that these oscillations are only led off from the intact heart. When the alary muscles were removed, usually triphasic waves were obtained. This result coincide with the findings of the grasshopper heart (9). The authors have also studied the electrogram of the grasshopper heart and obtained a similar oscillation as described by Jahn et al. As to the origin of the oscillation further experiments are needed. With regard to the miniature contractions which were observed at both ends 160 H. IRISAWA ET AL.

of the heart tube, Wakabayashi and Hagiwara (19), and Hagiwara (8) also have found a miniature response of the sound muscle of cicada which was always accompanied by the corresponding mechanical changes. Their electrical records of miniature contraction are quite similar to that of the7th segment of cicada's heart. Hagiwara (8) suggested that these miniature potentials are due to ex- citation of the individual muscle fibers or of the smaller specialized membrane areas related to the nerve terminals. In the present case, similar potentials were led off from the pacemaker areas. Furthermore it was confirmed that the large action potentials were preceded by three or four contractions of this small area. 6) Intracellular recording of heart muscle was initally described by Wood- bury et al. (20, 21), on the frog heart. In the present case as described above, the fiber diameter is much smaller than that of the vertebrate heart, so we should be very careful about the results obtained by this method. Obtained values of membrane resting and action potentials were very small compared with those values of vertebrates. The simple monophasic action potential of the insect heart was very similar in its character to that of vertebrate cardiac muscle fiber, though the presence of overshoot of action potentials, was not confirmed exactly. It was also observed that the intracellular action potential of a single fiber does not show any sign of oscillation. This probably affords direct proof of the authors assumption, that the oscillation may be due to the sum of many small other components. One of the interesting findings in the intracellular recording is that, a slow depolarization preceeds the rapid depolarization phase. This sign seems to be similar to that of the pacemaker potential described by Draper and Weidmann (3), and Trautwein et al. (18). The present material has a seasonal limitation, as adult cicadas are to be found only in summer. They will be studied further when they can again be obtained.

SUMMARY Cicada's heart action potentials were studied by both surface and intracellular electrodes. 1) Surface electrograms indicate that the inricate tracings of the action potentials are due to the presence of alary muscle. After the removal of alary muscle, the picture became simpler. It was found that the negative deflection became pronounced at the2nd and the 7th segment. 2) From the results of transverse section, localized cocainization and localized cooling, it seems reasonable to assume that the cicada's heart has two pacemakers. 3) Intracellular recording from single fibers of cicada's heart show muscle action potentials of a very simple monophasic form, from which the oscillation of surface potential is concluded to be due to the sum of many other components.

We wish to thank Prof. Y. Nisimaru for his encouragemement, and also it is our privilege to acknowledge our sincere gratitude to Prof. Y. Katsuki, who has given much ELECTROGRAMS OF THE CICADA'S HEART 161

good advice throughout this study and Dr. L. A. Woodbury, Biostatistics Department, A. B. C. C. for his kind help, valuable advice and reading this paper in manuscript.

REFERENCES

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