MORPHOLOGICAL AND PHYSIOLOGICAL
STUDIES OF THE LIMULUS POLYPHEMUS
HEART: MICROSCOPIC ANATOMY OF
THE CARDIAC GANGLION AND ROLE OF
THE CARDIOREGULATORY NERVES

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
CHARLES ROBERT BURSEY
1969

 

341‘.

This is to certify that the

thesis entitled
MORPHOLOGICAL AND PHYSIOLOGICAL
STUDIES OF THE LIMULUS POLYPHEMUS
HEART: MICROSCOPIC ANATOMY OF
THE CARDIAC GANCLION AND ROLE OF
THE CARDIOREGULATORY NERVES

presented by

CHARLES ROBERT BURSEY

has been accepted towards fulfillment
of the requirements for

Ph. D. degree in ZOOlOgy

 

Date XL}; 3I,IQACI
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L I B H A R Y

Michign State
University

 

  
  

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ABSTRAC T

MORPHOLOGICAL AND PHYSIOLOGICAL STUDIES OF THE
LIMULUS POLYPHEMUS HEART: MICROSCOPIC ANATOMY
OF THE CARDIAC GANGLION AND ROLE OF THE
CARDIOREGULATORY NERVES

 

By

Charles Robert Bursey

The morphology of the cardiac ganglion of Limulus

polyphemus (L) was examined by reconstructions from stained

 

serial sections. This ganglion is composed of two distinct parts:

a fiber tract extending the entire length of the heart and a cellular
portion underlying the fiber tract. The cellular portion extends
continuously from ostia III to the posterior terminus of the heart.
The mean number of ganglion cell bodies is 231. Most of the
ganglion cells are located among the glial elements of the cellular
portion. The greatest density of cells is found in segments five and
six. Six nerve cell types are recognized: 1) large pigmented uni-
polar cells approximately 120 u in diameter with distinct connective
tissue capsules around them; 2) large pigmented bipolar cells

approximately 120 u in length which are also encapsulated;

Charles Robert Bursey

3) pigmented multipolar cells approximately 80 u in diameter which
are free of capsules; 4) small pigmented bipolar cells approximately
40 u in length which are encapsulated but which are found exclusively
within the fiber tract; 5) non-pigmented multipolar cells approxi-
mately 30 u in diameter which are scattered among the connective
tissue elements of the cellular portion; and 6) small non-pigmented
cells approximately 7-10 u in diameter.

The cardioregulatory nerves were studied by electrical
stimulation. Nerves 7 and 8 are predominately inhibitory and
nerve 8 contains a few acceleratory fibers. Nerves 9-11 are pre-
dominately acceleratory but they contain a few inhibitory fibers.
Nerve 9 probably has more of these inhibitory fibers since inhibi-
tion produced by stimulation of nerve 9 is greater than that produced
by stimulation of nerves 10 and 11. Stimulation of nerve 6 or the
pericardial nerve does not affect heart rate. The cardioregulatory

nerves enter the ganglion directly.

MORPHOLOGICAL AND PHYSIOLOGICAL STUDIES OF THE

LIMULUS POLYPHEMUS HEART: MICROSCOPIC ANATOMY

 

OF THE CARDIAC GANGLION AND ROLE OF THE

CARDIOREGULATORY NERVES

By

Charles Robert Bursey

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Zoology

1969

ACKNOWLEDGMENTS

I wish to express my appreciation to Dr. Ralph A. Fax
who directed this investigation and whose advice, criticism, and
assistance has been invaluable during this study. Grateful
acknowledgment is made to Drs. T. Wayne Porter, .R. N. Band,
and Ronald E. Monroe, who served as members of my guidance
committee, and also to Mrs. B. Henderson for assistance in

obtaining materials.

ii

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES
IN TRODUC TION

Anatomical Background .
Physiological Background .

MATERIALS AND METHODS

Histological Studies
Physiological Studies .
Extract Studies '

RESULTS

Gross Morphology of the Ganglion

Cells of the Cardiac Ganglion
Pigmented unipolar cells
Pigmented large bipolar cells
Pigmented multipolar cells
Pigmented small bipolar cells .
Non-pigmented multipolar cells
Small non-pigmented cells

Variation in Pigmented Cells

Role of the Cardioregulatory Nerves

Tissue Extracts . . . .

DISCUSSION .

Morphology of the Cardiac Ganglion
Regulation of the Neurogenic Heart

iii

Page

vi

15

15
17
22

23

23
27
28
33
41
42
45
46
46
51
70

72

72
75

Page

Anatomy of cardioregulatory nerves . . . . . . . . 76
Function of cardioregulatory nerves . . . . . . . . 77
Neurohormonal regulation . . . . . . . . 80
Function of the Cells of the Cardiac Ganglion . . . . . . 81
CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . 85
LIST OF REFERENCES . . . . . . . . . . . . . . . . . 86

iv

LIST OF TABLES

Table Page

1. Distribution of total cells by segment and cell
typesforsevenhearts .............. 48

Figure

10.

LIST OF FIGURES

A semi-diagrammatical section of L. poly-
phemus showing the general plagement of
organs and the system of nerves connecting
the cardiac ganglion with the ventral nervous
system (Modified from Patten and Reden-
baugh, 1900)

Representative sections of the cardiac ganglion
of L. polyphemus

 

A pigmented unipolar cell from the fifth heart
segment . . .

A pigmented unipolar cell bulging from the
ganglion

A pigmented unipolar cell lying within the
ganglion

Frontal section of the ganglion in heart seg-
ment 5

A section of the unfixed cardiac ganglion viewed
through a fluorescent microscope

The absorbancy spectrum of the pigment when
measured in vivo

The distribution of the various pigmented cell
types along the length of the ganglion

A pigmented large bipolar cell lying adjacent
to the fiber tract in the fifth heart segment .

vi

Page

10

25

30

30

32

32

35

35

37

40

Figure

11. A pigmented multipolar cell lying within the
cellular portion of the ganglion .

12. A pigmented small bipolar cell lying within the
fiber tract . . . . . . . . . .

13. Non-pigmented multipolar cells (arrows) are

found within the cellular portion of the ganglion
and adjacent to pigmented cells

14. The distribution of unipolar and bipolar cells
along the ganglion

15. The relationship between heart length and diameter
of the unipolar cells

16. Change in heart rate as a result of stimulation
of nerves 7-11 at their origins

17. Change in heart rate as a result of stimulation
of nerve 10 at its origin for one and two
minute stimulation periods

18. Change in heart rate as a result of stimulation
of nerve 9 at its origin .

19. Change in heart rate as a result of stimulation
of nerve 9 in three different locations

20. Change in heart rate as a result of stimulation

of fused nerve 7 and 8 and nerves 9-11 as
they enter the cardiac ganglion

vii

Page

40

43

43

50

53

57

62

64

67

69

INTRODUC TION

Many of the arthropod hearts that have been investigated
have nerve cells which initiate the rhythmic contractions of the
heart, while the cardiac muscle cells themselves possess no
inherent rhythmicity. The nerve cells are located in a cardiac
ganglion which lies on the heart and which receives nerves from the
ventral ganglion that are capable of modifying the activity of these
cells. This situation occurs in a number of Crustacea, in limulus
and in several arachnids.

The crustacean heart has received the most attention and
the morphology of many representative cardiac ganglia has been
well described (see Bullock and Horridge, 1965, for review). The
arachnids have not received as much attention and little morpho-
logical data are available (Zwicky and Hodgson, 1963; Sherman and
Fax, 1968; Sherman, 31:31. , 1969). The heart of limulus has
become the classic example of the neurogenic heart, but little has
been published about the anatomical organization of its cardiac
ganglion. Most of what is known about the morphology of this

ganglion is based upon highly selective methylene blue stain or has

been obtained as a secondary objective of some specific physiological
study.
Milne-Edwards (1873) noted nerve elements associated with

the Limulus polyphemus heart, but Patten and Redenbaugh (1900)

 

were the first to describe the gross anatomy of the heart and the
cardiac ganglion. Nukada (1925), using methylene blue stains,
described three nerve cell types within the ganglion. Nukada con-
sidered the largest of the cell types to be autorhythmic since their
distribution correlated with the distribution of pacemaker regions
reported earlier by Carlson (1904c). From a more physiological
standpoint, Heinbecker (1936) attempted to correlate cell types with
electrical activity in the ganglion. By progressively sectioning the
ganglion, he was able to show that spontaneously active sections
always contained at least one large cell. Fedele (1942) described
five nerve cell types in the ganglion and attempted to describe cellu-
lar interaction on a morphological basis.

The physiology of the cardioregulatory nerves has been
most studied in the Crustacea. The distribution, ramification, and
function of these nerves is known for a number of species (Carlson,
1905a; Smith, 1947; Maynard, 1953, 1961a; Florey, 1960). The
presence of pericardial organs and the influence of these organs on

heart rate has been discussed by several authors (Carlisle and

Knowles, 1959; Belamarich, 1963; Cooke, 1966). In the arachnids,
the cardioregulatory nerves of scorpions have been found to
originate from the cerebral ganglion (Police, 1902, 1903), and
Zwicky (1968) has been able to trace part of the pathway of these
nerves by transection. On the basis of electrical stimulation
experiments of the brain of spiders, Carlson (1905a, 1909) sug-
gested that cardioregulatory fibers are present. Wilson (1967) and
Legendre (1968) show that nerves enter the cardiac ganglion, but it
is yet to be determined whether these nerves are in fact cardio-
regulatory in nature. Patten and Redenbaugh (1900) were the first
to describe the presence of segmental cardiac nerves in limulus,
but Carlson (1905b) described the system in its entirety and studied
by electrophysiological methods the relationship of this system to
the heart. Heinbecker (1933) described the effects of electrical
stimulation of both cardioinhibitory and cardioacceleratory nerves
upon the activity of the cardiac ganglion. Pax and Sanborn (1964,
1967a, 1967b) and Fax (1969) have studied the various components
of the cardioregulatory system in limulus. No pericardial ramifica-
tions of the cardioregulatory nerves have been described in spiders,
scorpions or limulus.

Since the heart of the decapod crustaceans has been so well

studied and since limulus has been shown to have a number of

characteristics in common with the Crustacea, a comparison of these
two hearts seems in order. The specific number, type, and arrange-
ment of cells within the limulus ganglion has not yet been reported
nor has the structure of the ganglion itself been eXamined. These
features are well known in the Crustacea. The cardioacceleratory
nerves in limulus have not been traced farther than the pericardial
area. In order to analyze ganglionic aspects of cardioregulation, it
is necessary to know the relationship of the cardioacceleratory nerves
to the cardiac ganglion. Do these nerves enter the ganglion proper
as in the case of the Crustacea or do they form a neuro-hemal struc-
ture which is in turn responsible for cardioacceleration?

The question, stated simply, is whether or not the limulus
cardiac ganglion is similar in structure and function to the cardiac
ganglion of the Crustacea. This study was undertaken to provide

some of this information.

Anatomical Background

 

The anatomical studies of Patten and Redenbaugh (1900)

are the best account of the general anatomy of the L. polyphemus

 

heart and its innervation. The heart is located directly beneath the
carapace just dorsal to the intestine. It extends from a point mid-
way between the lateral eyes to the seventh pair of entapophyses in

the opisthosoma. It has the general appearance of a segmented tube

and attains a length of about 15 cm in an adult, fully one -half the
length of the body. There are eight pairs of ostia on the dorso-
lateral surfaces of the heart. These ostia divide the heart into nine
unequal segments.

Eleven arteries are given off from the heart, three from
the anterior end, and one pair from each of the four anterior seg-
ments. Blood is carried from each gill in a large branchio-cardiac
canal to a pericardial sinus. From this sinus blood enters the heart
through the ostia.

In the opisthosoma the large pericardial sinus around the
heart is enclosed by a membranous connective tissue pericardium.
Ventral to the heart, it is a well-defined layered membrane which
attaches to the bases of the branchial cartilages, but lateral to the
heart it becomes thinner and joins the connective tissue lining of the
branchio-cardiac canals. Dorsally the pericardium is attached to
the carapace at successive entapophyses. In the prosoma the ventral
pericardium is similar to that in the opisthosoma and is continuous
with it. Dorsal to the heart the opisthosomal extensor muscles lie
immediately above the heart and there is no pericardium interposed
between these muscles and the heart.

The heart is held in position in the pericardial sinus by

suspensory connective tissue fibers. Laterally, eight pairs of

connective tissue supports, the alary muscles of Van der Hoeven,
arise from the lateral edge of the heart at the level of each ostium.
Each of these supports fuses distally with the pericardium. The
ventral surface of the heart is attached to the pericardium throughout
its entire length by numerous connective tissue fibers, but dorsally
the heart is suspended only at certain points. In the opisthosoma
there is a band of connective tissue fibers opposite each of the ostia
while in the prosoma two masses of connective tissue provide sup-
port: one between the second and third pair of ostia, and the other
between the first and second pair of ostia.

The heart itself is composed of three layers: an inner
muscle layer, a median basement membrane, and an outer layer of
thick connective tissue fibers. This outer layer of fibers anastomoses
with the connective tissue fibers suspending the heart in the peri-
cardial sinus. These connective tissue fibers also surround the
cardiac ganglion.

The cardiac ganglion extends almost the entire length of the
heart and occupies the outer mid-dorsal aspect. The ganglion has
its greatest width in the fourth, fifth and sixth segments and it
becomes progressively narrower both anteriorly and posteriorly.
The ganglion forms the center of the intrinsic cardiac nervous com-

plex which is confined mainly to the dorsal and lateral sides of the

heart. Numerous anastomosing and branching nerve bundles are
given off by the ganglion to the myocardium. These branches come
to form more or less definite tracts at the lateral edges of the heart,
which are known as the lateral cardiac nerves. These lateral nerves
are much smaller than the ganglion and they contain no nerve cell
bodies.

The intrinsic nervous complex is connected with the central
nervous system by an extensive system of nerves. A pair of nerves
is given off from the dorsal surface of the brain just above each pair
of nerves to the walking appendages. These nerves go dorsally to
innervate the integument, the viscera, and the dorsal musculature
with the posterior three pairs of nerves sending fibers to the cardiac
ganglion. The anterior pair of nerves from each of the abdominal
ganglia takes a similar course and makes connections similar to
those made by nerves from the dorsal side of the brain. Since these
nerves innervate structures dorsal to the level of the brain and
ventral cord, Patten and Redenbaugh called them ”hemal" nerves
to distinguish them from the ventral nerves of the walking appendages
and gills, which they designate the "neural" nerves.

Of the dorsal nerves arising from the brain only the poste-
rior three pairs connect with the cardiac ganglion (hemal nerves 6-8

of Patten and Redenbaugh). The cardiac branches of the seventh and

eighth dorsal nerves fuse on each side to form a large nerve which
passes to the dorsal hypodermis. Branches of this nerve join the
cardiac ganglion between ostia III and IV. The main body of this
nerve goes to supply the large opisthosomal extensor muscles and to
make up the pericardial nerve which is a relatively large nerve trunk
that runs parallel to the heart on each side just outside the lateral
pericardium (see Figure 1). The sixth dorsal nerve sends branches
to the opisthosomal extensor muscles and the cardiac ganglion.
These branches join the cardiac ganglion near ostia II.

The dorsal segmental nerves from the abdominal ganglia
(hemal nerves 9—13 of Patten and Redenbaugh) send branches to the
digestive tract and integument. These nerves continue dorsally and
before penetrating the dorsal pericardium to connect with the cardiac
ganglion, each nerve sends a communicating branch to the pericardial
nerve.

There is considerable individual variation in the exact
position of entrance of the segmental cardiac nerves into the cardiac
ganglion. Generally the cardiac nerves from the five abdominal
ganglia enter the cardiac ganglion opposite the five pairs of posterior
ostia (Carlson, 1905b). The fused seventh and eighth nerve enters
the cardiac ganglion sometimes at the level of the second pair of

ostia, sometimes in the middle of the third segment and sometimes

Figure 1. -- A semi-diagrammatical section of L. polyphemus

 

showing the general placement of organs and the system of nerves con-
necting the cardiac ganglion with the ventral nervous system (Modified

from Patten and Redenbaugh, 1900).

 

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just posterior to the third pair of ostia (Carlson, 1905b). The
connections of the sixth nerve appear consistently at the level of

ostia II (Fax, 1964).

Physiological Background

 

Carlson, (1904a, 1904b) found that the limulus heart when

exposed from the dorsal side in situ or when removed from the

 

animal continues to beat for hours. The heart appears to contract
simultaneously throughout its entire length. Section of the cardiac
ganglion results in incoordination of the beating in the two ends of

the heart. The lateral nerves are not essential to coordination or
conduction (Carlson, 1904a). The whole heart and the lateral nerves
may be sectioned, leaving only the ganglion intact, and the coordina-
tion of both ends will be maintained. Removal of the cardiac ganglion
causes immediate and permanent cessation of beating. The heart
beat in limulus, therefore, has its genesis in the cardiac ganglion,
and is thus neurogenic.

Carlson' s indirect evidence of the neurogenic origin of the
heart beat was supported by Hoffman (1911), who was able to obtain
with the use of a string galvanometer a continuous series of rapidly
(tenths of a millisecond) oscillating waves separated by periods with

no wave activity. Garrey (1912) verified Hoffman' s findings of the

12

oscillatory character of the electrocardiogram of limulus and studied
the modification of normal activity under the influence of changes in
temperature and of drugs. However, Nukada (1918), Hoshino (1925),
Dubussion (1930) and Monnier and Dubussion (1931) could not find
oscillatory discharges from the cardiac ganglion and they described
a series of slow waves similar to that of the vertebrate heart. It
remained for Rijlant (1931) to resolve these disagreements. By
using an improved cathode ray oscilloscope, he was able to record
the oscillatory character of the electrical activity from the isolated
cardiac ganglion. This electrical activity consists of periodic
changes in potential lasting 1. 2 seconds. These bursts of activity
are separated by periods of quiescence of 2 seconds. Each of these
potential changes is composed of a series of waves. Three different
types of activity were obtained: 100 to 200 fast waves; 10 to 30 slow
waves; or simultaneously fast and slow waves. The maximum action
potential obtained was 150 to 200 uV while the normal value was

100 uV.

Under normal conditions the activity starts in the region
corresponding to the fifth segment and is conducted up and down the
ganglion at an average speed of 75 cm/sec (Edwards, 1920; Pond,
1921; Rijlant, 1931). The first sign of the beginning of a burst is a

sharp initial potential change which is immediately followed by a

13

series of slow waves each lasting about 100 msec and repeating about
ten times during each burst (Rijlant, 1932). The first of the slow
waves has the largest amplitude and succeeding waves are progres-
sively damped until undetectable. Superimposed on the slow waves
is a series of fast waves (spikes) appearing initially at a high fre-
quency but rapidly declining in parallel with the decline in amplitude
of the slow waves (Heinbecker, 1936). Armstrong, gall. (1939) and
Prosser (1943a) showed that the slow potentials are largest in seg-
ment five and spread more slowly than the spikes.

The cardiac ganglion is connected with the ventral ganglion
at the level of each of the five posterior pairs of ostia and to the
brain at the level of the second and third pairs of ostia. Cardio-
inhibitory nerves are carried in the last two pairs of dorsal nerves
arising from the brain; stimulation of either of these two nerves at
points where they emerge from the brain results in a decrease in
heart rate and strength of beating (Carlson, 1905b; Fax and Sanborn,
1964). Cardioacceleratory nerves are carried in the segmental
cardiac nerves that arise from each of the five abdominal ganglia.
Stimulation of the ventral cord or any of the abdominal ganglia
results in an increased rate of heart beat (Carlson, 1905b ; Hein-
becker, 1933). However, the abdominal nerves have been shown to

also carry inhibitory nerves (Fax, 1969). The characteristics of

14

inhibition found in the abdominal cardiac nerves are similar to those
for the cardioinhibitory nerves arising from the brain.

Heinbecker (1933) recorded the electrical activity in the
cardiac ganglion during stimulation of the inhibitory nerves. Such
stimulation lowered the amplitude of the slow potentials, decreased
the rate of the fast discharge and shortened the total duration of each
burst. Stimulation of the accelerators increased the amplitude of the
slow waves, increased the frequency of the fast discharge and length-

ened the burst duration (Heinbecker, 1933).

MATERIALS AND METHODS

Mature male and female Limulus polyphemus with body

 

lengths between 25 and 30 cm were used for these studies. They
were shipped by air express from Gulf Specimen Company, Panacea,
Florida, and maintained at first in a cold box in moist excelsior at
5° C. Pax and Sanborn (1967a) report that responses of animals so
maintained did not vary for at least six weeks. Later, the animals
were maintained in running artificial sea water at 15° C. These
animals remained active for long periods of time and on several

occasions trilobite larval stages were found.

Histological Studie s

 

Preparation of the cardiac ganglion for histological section
requires isolation of the heart. The method for heart isolation has
been previously described (Fax and Sanborn, 1967a). After the heart
was isolated, a glass tube (6 mm CD) was inserted into the lumen of
the heart to prevent distortion during fixation. At this time most of

the loose connective tissue around the ganglion was dissected away.

15

16

A heart supported by the glass tube was then placed in
Gilson' s fixative for 12 hours (Gray, 1954). Other fixatives, notably
formalin, Bouin' s and Zenker' s, were used; however, none were
satisfactory since most of the cytoplasmic detail was lost.

Each heart was washed in tap water for 30 minutes after
fixation. The glass tube was then removed and the heart was cut
into pieces approximately 2 cm in length. These were dehydrated
in graded alcohols, cleared in xylene and embedded in paraffin by
usual methods. Serial frontal sections and serial cross sections
were cut at 7 u and stained by Bretschneider' s method (Gray, 1954).
Bretschneider' 3 method was modified by the addition of 0.2%
Chromotrope 2R (Allied Chemical) to the counterstain in order to
enhance nucleolar staining. Ganglia wholemounts stained by the
reduced methylene blue method (Pantin, 1946) were also examined.
Fresh frozen 10 11 sections were used to monitor the amount of
shrinkage and distortion caused by fixation.

The serial frontal sections were used in a reconstruction of
the cardiac ganglion. A section was projected by a microslide pro-
jector utilizing a 10X objective and traced on paper. Each subsequent
section was then superimposed on the previous tracing until the entire
ganglion was reconstructed. By this method the types of cells present
and their distribution along the length of the ganglion was determined

for seven hearts.

17

Some of the cells in the cardiac ganglion contain a yellow
pigment which is strongly autofluorescent. The total number of
these cells can be easily counted under a fluorescent microscope.
For this procedure, ganglia were removed from isolated hearts,
placed on a glass slide, dried in a desiccator jar under vacuum for
24 hours and then examined under a Leitz fluorescent microscope
utilizing a mercury vapor lamp with 400 mu filters. The pigment
was also observed m with a Leitz microspectrometer and a
Photovolt microphotometer and the resultant absorbance spectrum

plotted.

PhjLSi olongcal Studies

 

Stimulation of segmental nerves 6-11 was performed at
three different levels: 1) ventrally as the nerves leave the central
nervous system; 2) pericardially, both above and below the peri-
cardial nerve; and 3) dorsally as the nerves enter the cardiac
ganglion. Two different animal preparations were used: 1) hearts
isolated from the animals and 2) intact hearts within partially dis-
sected animals.

The preparation used for ventral stimulation of cardio-
regulatory nerves has been previously described (Fax, 1969) and
was followed in part. The gills, genital opercula and legs were

removed. A longitudinal dorso-ventral cut through the entire

18

thickness of the animal was made about 4 cm on either side of the
midline for a length approximating the underlying heart. Transverse
cuts were made through the animal at the anterior and posterior
boundaries of the heart. This leaves a rectangular block of tissue
which includes most of the musculature, the digestive tract and the
ventral nervous system; and preserves the heart and pericardium

as well as the cardioregulatory nerves. This rectangular block was
pinned ventral side up in a shallow tray. The abdominal nerve cord
and the brain were exposed along their entire length and nerves 6-11
were cut free at their point of emergence and dissected free of under-
lying tissue laterally to the point where they turn dorsally. The
ventral cord and brain were then removed. A portion of the ventral
abdominal muscles and the intestine were removed to expose the
heart from ostia VIII to the posterior terminus. The mechanical
activity of the heart was monitored at the eighth segment. Stimula-
tion was accomplished by lifting the nerve onto a pair of platinum
hook electrodes.

Pericardial stimulation was accomplished by continued dis-
section of the above preparation. The ventral abdominal muscles
and the digestive tract were removed so that the ventral pericardium
was exposed in its entirety. The pericardium was slit unilaterally

at the level of the entrance of the branchio-cardiac canals into the

l9

pericardium and the pericardial nerve from the hinge of the carapace
to the pericardial branch of the eleventh segmental nerve was exposed.
By following the pericardial branch of the segmental nerve to the
main nerve tract, the segmental nerves were easily located. Stimu-
lation was accomplished by lifting the nerve onto the hook electrodes.
The mechanical activity was monitored at the eighth segment.

The preparation used for dorsal stimulation of the cardio—
regulatory nerves was prepared as follows: The dorsal exoskeleton
was sawed through just lateral to the underlying heart and these cuts
were joined by transverse anterior and posterior cuts. The rectan-
gular piece of isolated exoskeleton overlying the heart was then
removed by lifting and scraping it free of the underlying tissues.

The exoskeleton must be separated carefully from the underlying
tissue so that the segmental nerves to the heart are not destroyed.
The entire heart and the overlying tissue were removed and placed
in a tray dorsal side up. The heart was stretched to its original
length and pinned in place. Segmental nerves 9-11 were dissected
from the tissue overlying the heart. These nerves are most easily
found by first locating the pericardial nerve and following it to the
area in which the cardiac branch crosses the pericardial nerve.

The fused nerve 7 and 8 is found anterior to the overlying tissue and

does not require dissection. Nerve 6 lies in a mass of connective

20

tissue at ostia II and must be dissected free. These nerves were
lifted onto the hook electrodes for stimulation. Mechanical activity
was monitored at the second heart segment.

Carlson (1907a) reported that heart wall tension and intra-
lumenal pressure are factors that influence both the rate and the
strength of beating of the heart. For the isolated heart, longitudinal
tension approximating that of the intact heart was obtained by
stretching the heart to a length equal to that present before removal
from the animal. In those preparations used for ventral and peri-
cardial stimulations, the pericardium and the suspensory ligaments
remain intact and thus the longitudinal tension is not disturbed. In
order to approximate the normal intra-lumenal pressure, a gravity-
fed reservoir of Chao' s saline solution (Chao, 1933) was connected
by cannula to the posterior end of the heart. Later this system was
replaced by a peristaltic perfusion pump (and artificial sea water.
The hearts were perfused at a rate of 15 ml/min, the route of per-
fusion fluid being that of normally expelled blood. Perfusion was
begun as soon as the preparation was pinned to the tray. All experi-
ments were conducted at room temperature (20-260 C).

Records of the mechanical activity of the hearts were
obtained by the use of an E & M myograph "A" transducer. Since

the main component of tension development in the heart is radial due

21

to the circularly directed muscle fibers, the transducer was attached
laterally to the heart muscle. The transducer output was recorded
on an E & M Physiograph.

Stimulation was accomplished by a Grass S 4 stimulator and
isolation unit. Fax (1969) reported a mean rheobase of 2. 5 V with a
chronaxie of 0. 5 msec for the abdominal segmental nerves. Because
of the relatively high threshold and long chronaxie, he routinely used
a stimulus strength of 5 V with a duration of 3 msec and found that at
all stimulus frequencies maximum increase in rate occurred only
after 30—40 seconds of stimulation and the maximum rate change
during this time was produced by stimulus frequencies between 5
and 10/sec. Thus one and two minute stimulations of all nerves in
all areas studied were carried out at 5/sec at 5 V (duration 3 msec).
Fax and Sanborn (1964) note that maximum slowing of the heart due
to stimulation of nerves 7 and 8 occurs with stimulation frequencies
between 10 and 80/sec at 40 V and 1 msec duration. Thus nerves 7
and 8 were also stimulated in this study at stimulus strengths of 6 V
for 5 msec duration and 20/sec. Increase in either voltage or‘dura-
tion during a stimulation period did not change the on-going pattern,
showing that all stimulations were above threshold.

Since normal heart rates in limulus vary greatly from

animal to animal (Pax and Sanborn, 1964), each animal is used as

22

its own control. All rates in this study are expressed as change in
heart rate. The change in rate is determined by the difference
between the experimentally altered heart rate and the control rate,
which is that rate during the minute prior to stimulation. Thus
positive values are indicative of excitation and negative values are
indicative of inhibition. The averaged rate for each ten second

period is plotted.

Extract Studies

 

Since the effects of secretions from the pericardial organs
upon the heart beat of crustaceans is well known (Belamarich, 1963;
Belamarich and Terwilliger, 1966; Cooke, 1966), various tissues

of _L_. polyphemus (muscle, ventral cord, brain, heart, hypodermis,

 

pericardial tissue) were tested to determine what effect they might
have on heart rate. These tissues were macerated in Chao' s saline
by mortar and pestle (10 mg tissue per 1 ml saline). The filtrate
from the various tissues was tested on the isolated heart, which,

in this case, was dissected free of all extraneous tissues. Aliquots
of approximately 20 cc were used to bathe the heart or were perfused
through the heart. The mechanical activity of the heart was moni-

tored at segment two as previously described.

RESULTS

Gross MorphologLof the Ganglion

 

Under low magnification (30X), the unfixed cardiac ganglion
appears as a large bundle of intertwining nerve fibers with inter-
mingled masses of cells. A few individual large cell bodies are
discernible by the presence of pigment within them and at various
levels along the ganglion some of these cell bodies bulge from the
surface of the ganglion.

In cross sections from the middle segments of the heart,
it is apparent that the ganglion consists of two morphologically dis-
tinct areas: a ventral region containing most of the ganglionic cell
bodies, and a dorsal region composed primarily of nerve fibers
(Figures 2c, d, e). The ventral region I have designated the cellular
portion of the ganglion; the dorsal region the fiber tract.

The cellular portion makes up the bulk of the ganglion. It
is continuous from ostia III to the posterior terminus of the heart.
This cellular portion contains most of the cell bodies of the ganglion

as well as a number of connective tissue elements. Some nerve

23

24

Figure 2. -- Representative sections of the cardiac ganglion

of L. poLyphemus.

 

A.
B.
C

D.

E.

Sagittal section from the fifth heart segment.
Cross-section through second segment.
Cross-section through fourth segment.
Cross-section through seventh segment.

Cross—section through eighth segment.

In A dorsal is to the left. A large unipolar cell is visible. The

area to the left of this cell is the fiber tract; the area to the right

the cellular portion. In B the entire cross-section is fiber tract

but in C, D, and E the cellular portion can be seen underlying the

fiber tract.

Calibration is 200u.

25

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2

26

fibers of various diameters are also found throughout this region of
the ganglion.

The fiber tract extends the entire length of the heart and
has approximately the same cross sectional area throughout segments
three through eight. It consists mainly of nerve processes and con-
nective tissue elements. The number of fibers within a segment
varies greatly. This variation is due to fibers arriving from the
cellular portion and to branches leaving the fiber tract. Generally,
30-40 fibers are found in segment one and 80-120 fibers are found in
segments two and three. Posterior to ostia III, the number of fibers
increases to 200—300, with the largest number of fibers occurring
in segment five. The number of fibers decreases posterior to seg-
ment five and in the posterior half of segment eight and in segment
nine the number of fibers again approaches 30-40.

The oval nuclei of fibroblasts are easily recognized in the
fiber tract and hemocytes which tend to aggregate in intra-ganglionic
spaces are often seen. Anterior to ostia III isolated groups of small
cells are scattered among the fibers, while posterior to ostia III the
fiber tract is free of these cell groups. A few individual small
bipolar cells are also found in the fiber tract. A connective tissue
layer surrounds the cellular portion and in sagittal sections it can
be seen that processes from the cell bodies in the cellular portion

pass through this layer to enter the fiber tract (Figure 2a).

27

The nerve processes leave the fiber tract in bundles of
various sizes and spread over the myocardium. There is an almost
segmental arrangement of these bundles as they leave the ganglion.
The largest bundle is usually found in the fifth segment and contains
80 to 100 fibers. It may run anteriorly or posteriorly and may
occupy either the left or right side. The contralateral bundle is
smaller in size, but there are usually more bundles on that side.
These tracts may bifurcate immediately or they may travel several
millimeters before branching. Segments three through eight give
rise to at least one bundle of large size containing 40-80 fibers, one
or two bundles of medium size containing 20-40 fibers and several
smaller bundles of 5-20 fibers. All bundles branch randomly and
small bundles of one to five fibers are found throughout the cardiac
muscle mass. Individual fibers have not yet been traced to individ-
ual muscle cells (see Abbott, gt_a_l. , 1969). The bundles that
remain on the surface of the heart come to form more or less
definite tracts at the lateral edges of the heart. These lateral nerves
contain 80-100 fibers when counted at their greatest diameter,

which is in the fifth segment.

Cells of the Cardiac Ganglion

 

In addition to the fibroblasts of the connective tissue ele-

ments, six types of cells are clearly recognizable by morphological

28

characteristics. A description of each of these cell types follows.

Cell sizes are expressed for a heart of 15 cm length.

1) Pigmented unipolar cells. -- The pigmented unipolar

 

cells have cell bodies approximately 120 u in diameter which may be
either round or oval (Figures 3, 4, 5). They give rise to a single
large process approximately 40 u in diameter. This process often
bifurcates soon after leaving the cell (Figure 3). Processes from
different unipolar cells often cross one another with little if any
connective tissue between them (Figure 6). These fibers can be
traced for some distance and pass eventually into the fiber tract.

The unipolar cell is invested by a connective tissue capsule.
This capsule may be thick (up to 10 u) as when the cell body bulges
from the ganglion or it may be thin (2 u) as when the cell body lies
within the ganglion. In either case the capsule thins as it approaches
the process and does not appear to continue along the process. Con-
nective tissue fibers of the capsule anastomose with other connective
tissue fibers to form the fibrous network seen throughout the cellu-
lar portion of the ganglion.

Around the cell body and within the capsules of the unipolar
cells, there are numerous small cells, approximately 7-10 u in
diameter. These small cells bear a constant relationship to the

process of the unipolar cell. In cells that bulge from the ganglion,

29

Figure 3. --A pigmented unipolar cell from the fifth heart

segment. The large axon can be seen to bifurcate a short distance

from the cell body. Small non-pigmented cells (arrows) are always

present within the capsule of the unipolar cell. Calibration is 30 u.

Figure 4. --A pigmented unipolar cell bulging from the

ganglion.

Calibration is 40 11.

Here the small non-pigmented cells surround the process.

 

 

 

30

 

Figure 3

7'!

 

 

31

Figure 5. --A pigmented unipolar cell lying within the
ganglion. Here the small non-pigmented cells occur at the side
opposite the process. Pigment granules are visible (arrow).

Calibration is 50 11.

Figure 6. -- Frontal section of the ganglion in heart
segment 5. Three unipolar cells are visible. Note the manner in
which the axons of two of the unipolar cells come to lie adjacent to

each other. Calibration is 100 u.

32

 

33

they aggregate around the process (Figure 5); while in those cells
which lie within the cellular portion, the small cells lie at the end
opposite the process (Figure 4).

The nucleus of the unipolar cell is round or oval and
occupies the center of the cell; it contains scattered chromatin and
one nucleolus. The cytoplasm is finely granular becoming reticular
near the cell membrane. No cytoplasmic inclusions are visible.
However, there are pigment granules at the periphery just below the
cell membrane. Thesegranules are approximately 1 u in diameter
and in the unipolar cells they are usually concentrated in one large
group. These granules are responsible for the fluorescence seen in
all pigmented cells (Figure 7). At least four and perhaps five bands
of absorbance (Figure 8) are seen in in vivo determinations: 590,
570, 510, 490 and 470 mu.

The unipolar cells occur in all segments of the heart except
anterior to ostia III (Figure 9c). They are most numerous in seg-
ments five and six. The size of the cells varies with position,
becoming smaller in segments seven and eight. The mean number
of unipolar cells was 124 with a range of 93 to 163 in the seven

hearts studied .

2) Pigmented large bipolar cells. -- The pigmented large

 

bipolar cells are approximately 120 u in length and 90 u in width

34

Figure 7. -- A section of the unfixed cardiac ganglion viewed
through a fluorescent microscope (excitation wave length 400 mu).
The fluorescence of a large pigmented bipolar cell (arrow) and

several unipolar cells can be seen. Calibration is 100 u.

Figure 8. -- The absorbancy spectrum of the pigment when
measured in vivo. There are two major absorbancy bands at 510
and 490 mu. Three other bands possibly exist at 590, 570 and

470 mu. The arrows mark these bands.

 

Absorbent:

35

Figure 7

IIIIIIIIIIIIII

 

 

IIIII'I'II

III II

 

 

 

lllll|lllIIllll|llllllllljllllllll
a a
3

v

Wave Length my

Figure 8

36

Figure 9. -- The distribution of the various pigmented cell
types along the length of the ganglion. Each bar represents the mean

determined from seven different hearts. The vertical lines give the

range.
A. Total number of pigmented cells.
B. The multipolar cells.
C. The unipolar cells.
D. The large bipolar cells.
E. The small bipolar cells.

37

 

 

d I

«Jae-JfiIfiwailqu.‘

1m

 

 

 

 

 

 

 

 

 

 

 

 

 

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mjmu “.0 ammZDZ

II
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hI -
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F F [
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D b bIPIPIFHPLbbbihlp) FD? bILIDDDIPI PRbbP’PP-D b3?
0 O o 0 0 O 0 0 o 0 o 0 0 o o 0 0 0 O 0 O 0 0 0
l 0 9 8 7 6 5 4 3 2 l l 5 4 3 2 l 5 4 3 2 I 2 l

SEGMENT

Figure 9

38

(Figure 10). They have two large processes of equal size. These
processes are sometimes seen to bifurcate, although bifurcation has
not been found within 200 u of the cell body. The long axes of the
bipolar cells are parallel to the length of the ganglion. The bipolar
cells exhibit varying degrees of connective tissue investment. When
lying adjacent to the fiber tract or at the surface of the cellular
portion, they have a heavy capsule (10-15 u thick) and are almost
always fusiform. When lying within the cellular portion, they have
less connective tissue investment (up to 5 u) and may assume a more
contorted form. The capsule is formed from several layers of con-
nective tissue. It maintains the same thickness over the cell body
and continues at this thickness along the processes for some distance.

The round or oval nucleus contains scattered chromatin and
at least one nucleolus. The cytoplasm is finely granular becoming
reticulated toward the cell membrane. No cytopolasmic inclusions
were noted. Aggregations of pigment granules are found at both
poles of the cell.

While this cell and the previously described unipolar cell
are of the same size, the bipolar cell is easily distinguished from
the unipolar cell. Grossly the pyriform appearance of the unipolar
cell contrasts with the fusiform appearance of the bipolar cell. No

small cells of the type seen within the capsules of the unipolar cells

39

Figure 10. --A pigmented large bipolar cell lying adjacent

to the fiber tract in the fifth heart segment. Calibration is 40 11.

Figure 11. -- A pigmented multipolar cell lying within the
cellular portion of the ganglion. Two processes are visible in the
photograph: a large process at center right; a smaller process at

upper left. Non-pigmented cells and processes of other pigmented

cells appear in the photograph also. Calibration is 20 u.

40

 

 

 

41

are found within the capsules of the bipolar cells. And the capsule
of the unipolar cell does not continue along the process as it does
on the processes of the bipolar cell.

The pigmented large bipolar cells are present in all seg-
ments posterior to ostia III (Figure 9d). They are by far the most
variable in number among the various cell types. The mean total
number of these cells in the seven hearts studied was 55 with a range

from 18 to 132..

3) Pigmented mufipolar cells. -- The pigmented multipolar

 

cells are approximately 60-80 11 in diameter (Figure 11). These

cells occur in the cellular portion and are located well away from

the fiber tract. A few connective tissue fibers are seen surround-
ing these cells, but there is no distinct capsule. Small non-pigmented
cells often lie in close proximity and are occasionally seen to abut
this cell.

The nucleus of the multipolar cell is round and exhibits
scattered globular chromatin and at least one nucleolus. The cyto-
plasm is finely granular with no distinct inclusions. The pigment
granules are concentrated in several groups close to the cell mem-
brane and near the processes.

These cells are distributed evenly along the ganglion

posterior to ostia III and are the most constant in number from one

42

heart to the next (Figure 9b). The mean number of these cells was
21 in the seven hearts studied with a range of 13 to 31.

In my examination of methylene blue stained ganglia, I have
been unable to count more than 30 large stainable cells in any one
ganglion, although numerous small cells were visible. This num—
ber corresponds most closely with the number of pigmented multi-
polar cells counted by other means. Moreover the cells with
connective tissue capsules, namely, the pigmented unipolar and
bipolar cells, do not appear to be stained by methylene blue since
in several instances these cells, though unstained, were visible due
to their pigment inclusions. Thus it seems that only the unencapsu-

lated cells are stained by methylene blue.

4) Pigmented small bipolar cells. -- The pigmented small

 

bipolar cells are approximately 40 u in length and are always fusi-
form (Figure 12). They lie exclusively within the fiber tract and
like the large bipolar cells, they are invested by a heavy capsule.
Their long axes are parallel to the long axis of the ganglion.

The round nucleus usually has two large nucleoli and small
inclusions which have a staining affinity similar to that of the
nucleoli. Scattered chromatin is also present in the nucleus. The

cytoplasm is finely granular and more deeply staining than that of

43

Figure 12. -- A pigmented small bipolar cell lying within

the fiber tract. Calibration is 20 u.

Figure 13. -- Non-pigmented multipolar cells (arrows) are
found within the cellular portion of the ganglion and adjacent to
pigmented cells. Here two of these cells lie adjacent to a unipolar
cell. Small non-pigmented cells are also present. Calibration is

30 u.

 

44

 

 

45

the large bipolar cell. No cytoplasmic inclusions are found.
Pigment granules are limited to the polar regions.

These cells may be found in all segments of the heart
(Figure 9e). They averaged 31 in the seven hearts studied. The

range was 22 to 47.

5) Non-pignented multipolar cells. -- The non-pigmented

 

multipolar cells are approximately 20-30 u in diameter (Figure 13).
They have a rounded or oval appearance with their processes push-
ing out between the small non-pigmented cells which always surround
the multipolar cell body. These multipolar cells are found through-
out the cellular portion of the ganglion and are often found in close
association with the pigmented multipolar cells. They also often
occur in isolated groups anterior to ostia III in the fiber tract.

The nucleus is round with extensive chromatin and in most
cases there are two or more nucleoli. The cytoplasm stains more
darkly than that of the pigmented cells and it has a heterogeneous
granular appearance with distinct granules forming at the periphery.
Darkly staining cytoplasmic inclusions are often seen.

No attempt was made to count these cells, but the number
of cells of this type greatly exceeds that of any of the pigmented

cell types severalfold.

46

6) Small non-pigmented cells. -- The small non-pigmented

 

cells are approximately 7-10 u in diameter and are by far the most
numerous of cells within the ganglion (Figure 13).. They occur
within the capsules of the pigmented unipolar cells and are seen
filling the interstices around the non-pigmented multipolar cells in
the cellular portion of the ganglion. Anterior to ostia III, they can
be found in the fiber tract where they surround the non-pigmented
multipolar cells.

These cells have large nuclei which may assume various
shapes from round to kidney-shaped. No distinct nucleolus can be
seen and the sparse granular chromatin is scattered throughout the
nucleus. Occasionally enlarged nuclei with chromatin in strands are
seen. The cytoplasm of this cell is extremely pale staining in
comparison to that of the other cells and darkly staining inclusions
are often present in the cytoplasm. Frequently only the nucleus is
conspicuous. The cell form is highly irregular, but no processes
can be seen.

It was impossible to count these cells.

Variation in Pigmented Cells

 

The numbers and types of pigmented cells were determined
on a segment to segment basis by reconstructivedrawings. The

mean total number of pigmented cells for the seven hearts studied

47

was 231 with a range of 171 to 305. This mean total cell number
agrees well with that obtained from the five hearts studied by the
fluorescent microscope method: 229 cells with a range of 190 to
265.

The total number of pigmented cells within the ganglion is
not related to the length of the ganglion (Table 1). Of the seven
ganglia I studied, the shortest had the largest number of pigmented
cells, while the longest was third in number. There is also no
apparent relationship between the numbers of individual cell types
and the length of the heart. The small bipolar cells and the multi-
polar cells are most constant in numbers from one ganglion to the
next. The large bipolar cells are the most variable in number.

Table 1 also shows the segment by segment cell totals in
the seven hearts studied. There is no consistent pattern of cell
distribution within the ganglion. The greatest concentration of cells
occurs in the middle segments, but the segment containing the most
cells in any one heart may be either segment four, five or six.

Figure 14 presents cell placement for unipolar and bipolar
cells in the seven hearts studied. Each vertical bar represents the
number of cells within 5% of the total length of a segment. As can
be seen from this figure, there is no relationship between the cell

placement in the ganglion and external heart features such as ostia.

48

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49

Figure 14. -- The distribution of unipolar and bipolar cells
along the ganglion. Each square represents one cell.
A. Unipolar cells.

B. Bipolar cells .

NUMBER

HEART

50

1

-II-

 

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Figure 14

 

51

Nor does the cell pattern appear to be consistent from ganglion to
ganglion. Rather the cells appear to be random in placement but
most numerous in segments four, five and six (see Figure 9a also).

The cells of the cardiac ganglion also exhibit much indi—
vidual variation in size within a single cell type. In a single heart
of 18 cm length, the unipolar cells varied from an average diameter
of 115 u to 155 u within the middle segments (Figure 15). However,
cell size within a ganglion is influenced by position since those cells
in segments four, five and six are on the average larger than those
of seven, eight and nine. Cell size is also influenced by heart
length. Figure 15 presents data on the relationship between heart
length and the size of the unipolar cells in segment five for ten
hearts. As can be seen from this figure, the cell size increases as
the length of the heart‘increases.

It is also to be noted from Figure 15 that cell size is con-
sistent whether measured from fresh frozen sections or fixed stained
sections, showing that measurable shrinkage did not occur during

processing.

Role of the Cardiorej'ulatory Nerves

 

The segmental nerves have been shown to connect the brain
and ventral cord with the cardiac ganglion (Carlson, 1905b). Stimu-

lation of these nerves near their origins in the brain or ventral cord

52

Figure 15. -- The relationship between heart length and
diameter of the unipolar cells. Each vertical line represents the
range in cell diameters measured for ten different cells chosen at

random from segment five.

53

 

 

 

 

 

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1) o
g N
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J 1 J I I l l
O O O o o O O
.9 1 51‘ 2 °° 0 "
vaalw 'aaiawvm 113:)

12 13 14 15 lb I7 18 I9
HEART LENGTH, CENTIMETERS

II

10

Figure 15

54

causes a change in heart rate; nerves 7 and 8 are inhibitory (Carlson,
1905b; Fax and Sanborn, 1964) while nerves 9-13 are predominantly
acceleratory (Carlson, 1905b; Fax, 1969). Several questions arise
from these observations: Do the cardioregulatory fibers have direct
connections with the ganglion? Is a neurohormonal system involved?
Is there a combination of direct connections and neurohormonal
systems? Do the segmental nerves carry both cardioinhibitory and
cardioacceleratory fibers? What is the arrangement of the cardio-
regulatory fibers? Are the pericardial nerves involved in cardio-
regulation?

Partial solution of these questions can be carried out by an
examination of the segmental cardiac nerves and an isolated heart
preparation. Since stimulation of the segmental nerves at their
origins produces a change in heart rate, what change occurs when
these nerves are stimulated at various locations along their tracts
and when various branches are stimulated? If the response pattern
in the heart'is observed during these stimulations, the fibers which
are responsible for cardioregulation can be traced. If stimulation
of the segmental nerves to an isolated heart preparation in which
extraneous tissues have been removed produces the same response
pattern as before, then the segmental nerves carry fibers which

enter the cardiac ganglion and which are responsible for the

55

inhibition and acceleration seen when the ventral nervous system
is stimulated.

In this study nerves 7 and 8 were stimulated ventrally near
their origins in the brain and dorsally as they enter the cardiac
ganglion. Likewise, the segmental nerves 9-11 were stimulated
ventrally near their origins in the ventral cord, pericardially as
they cross the pericardial nerve and dorsally as they enter the
cardiac ganglion. The results of such stimulation were compared
with one another.

Stimulation of the segmental cardiac nerves 7 and 8 at
their origins (5 V, 3 msec, 5/sec) resulted in a slowing of the heart
rate (Figure 16). At least three phases of response can be seen:

1) time for maximum inhibition, 2) minimum rate and 3) recovery
time. The most rapid change in heart rate occurs before the first
30 seconds of stimulation.

The mean response to stimulation of nerve 7 is an inhibited
rate throughout the stimulation period. In one individual the
response did not occuruntil after 30 seconds of stimulation. In all
other animals, inhibition begins immediately and establishes the
new rate within the first 30 seconds of stimulation. The response
does not end with cessation of stimulation since the heart rate

requires several seconds to return to the original rate. In one

56

Figure 16. -- Change in heart rate as a result of stimulation
of nerves 7-11 at their origins (stimulus strength: 5 V, 3 msec,
5/sec). The arrows mark the duration of stimulation. The vertical
lines give the range.

A. Nerve 7 (N = 6)

Nerve 8 (N = 7)
Nerve 9 (N = 6)

Nerve 10 (N = 10)

P199!”

Nerve 11 (N = 7)

A [AYE (Boots/ Min)

II
.M.”

57

 

r

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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d
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. I
. II:

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t

 

l J l l 1
2 I 4 5 I 7
MINUTES

Figure 1 6

—

 

58

individual the heart established a new rate one beat per 'minute
faster than the original rate. In other animals the heart rate
returned to the original level and maintained that rate or after
several minutes began a slower rate. The mean deviation from the
original rate is less than one beat per minute for the entire five
minutes following stimulation.

The mean response to stimulation of nerve 8 differs
markedly from that of nerve 7. Inhibition begins immediately at the
onset of stimulation and reaches maximum inhibition at 30 seconds.
However, from this time on the mean heart rate returns toward the
original rate. At the end of the stimulation period inhibition is one-
half the value of maximum inhibition. The mean heart rate here
as in stimulation of nerve 7 requires several seconds to recover.
Not all individuals return to the original rate. The extremes in the
responses to stimulation of nerve 8 during the recovery phase (are
an increase of seven beats per minute before returning toward the
original rate and a maintained depression of three beats per minute.
This variation suggests that the response to stimulation of nerve 8
is more or less an individual pattern. However, there is a char-
acteristic feature in all response patterns except one. The rate
after cessation of stimulation increases and then decreases. Indeed,

themean response shows an increase of one beat per minute before

59

returning to the original rate. These differences in response
patterns to stimulation of nerves 7 and 8 suggest that there is a
basic difference in the fibers that make up these nerves.

Since Pax and Sanborn (1964) observed that stimulus
frequencies of 10-80/sec produced maximum response of the
inhibitory nerves, nerves 7 and 8 were also stimulated at 20/sec.
The response is similar to that seen when these nerves are stimu-
lated at 5/ sec except that there is greater inhibition of the heart
rate. In both cases the inhibition is closely coupled to stimulation
with maximum inhibition occurring within a few seconds after stimu-
lation begins and a return toward original rates immediately after
release from stimulation. The response to stimulation of nerve 8
again shows a post-stimulation increase but it is not detectably dif-
ferent from that obtained by stimulation at 5/ sec.

Stimulation of the segmental cardiac nerves 9-11 at their
origins increased the heart rate (Figure 16). The response patterns
to stimulation of nerves 9-11 resemble one another. Three phases
of response can be seen: 1) rise time, 2) maximum rate and
3) recovery time. The most rapid change in mean rate occurs
during the first 30-40 seconds of stimulation with a lessened increase
in rate during the remainder of stimulation. Maximum mean rate

is not reached until 10-20 seconds following cessation of stimulation.

60

Individually, however, the maximum rate may be reached during the
last few seconds of stimulation and this rate is then maintained for
several seconds after cessation of stimulation. It is also to be
noted that an initial inhibition is present in some individuals. In
these individuals the maximum rate always occurs after cessation
of stimulation. But these two response characteristics do not
always occur together since maximum rate may occur after cessa-
tion of stimulation in an individual in which initial inhibition does not
occur. Recovery requires several minutes, usually between four
and ten, and it occurs in a very nearly exponential pattern. Recovery
also shows much variation; some hearts return to the original level
quickly and establish a new level of beating at a slower rate; while
other hearts may establish a slightly faster rate and not return to
the original rate at all. The response pattern is the same whether
the stimulation lasts for one or two minutes (Figure 17).

Individual nerves cause relatively consistent responses
with similar stimulations over long periods of time. Figure 18
presents the results of repeated ventral stimulation of nerve 9 over
a two hour period. The initial rate and the maximum rate varied
over the range of four beats per minute during the various stimula-
tions; however, the recovery phase shows less variation.

Figure 19 displays the mean response to stimulation of

nerve 9 when stimulated in three different locations: 1) at its

61

Figure 17. -- Change in heart rate as a result of stimulation
of nerve 10 at its origin (stimulus strength: 5 V, 3 msec, 5/sec)
for one and two minute stimulation periods. The vertical lines give
the range.

A. Nerve 10 stimulated for 2 minutes (N = 10)

B. Nerve 10 stimulated for 1 minute (N = 5)

62

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 18. -- Change in heart rate as a result of stimulation
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origin, 2) at the level of the pericardial nerve and 3) at the level of
the cardiac ganglion. There are no detectable differences in the
responses. Nerves 10 and 11 give similar results when stimulated
in the corresponding locations.

Figure 20 displays the responses to stimulation of fused
nerve 7 and 8 and nerves 9-11 when stimulated as they enter the
cardiac ganglion. There is little difference in the mean responses
to stimulation of these nerves dorsally and the responses when they
are stimulated ventrally (see Figure 16). The ranges of responses
overlap to the extent that the responses in both instances are
essentially the same.

My observations essentially confirm Carlson! s (1905b)
observation that nerves 7 and 8 carry inhibitory fibers and Pax and
Sanborn' s (1964) observation that fused nerve 7 and 8 enters the
ganglion. Also Pax' s (1969) observation that nerves 9-13 carry
excitatory fibers is confirmed. These experiments show, however,
that the rise time, maximum rate and recovery phase of the response
to stimulation of nerves 9-11 are essentially the same no matter
where stimulation occurs; thus cardioacceleratory fibers like
cardioinhibitory fibers enter the ganglion and their effect appears
to be directly on the ganglion and not through some intermediary

neurohormonal system.

66

Figure 19. -- Change in heart rate as a result of stimulation
of nerve 9 in three different locations (stimulus strength: 5 V,
3 msec, 5/sec). Arrows mark the duration of stimulation. The

vertical lines give the range.

A. Ventral stimulation (N = 5)
B. Pericardial stimulation (N = 8)
C. Dorsal stimulation (N = 6)

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68

Figure 20. -- Change in heart rate as a result of stimulation
of fused nerve 7 and 8 and nerves 9-11 as they enter the cardiac
ganglion (stimulus strength: 5 V, 3 msec, 5/sec). The duration of
stimulation is marked by arrows. The vertical lines give the range.

A. Fused nerve 7 and 8 (N = 9)

B Nerve 9 (N = 6)

C. Nerve 10 (N = 8)

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70

Two other nerves were also stimulated: segmental nerve 6
and the pericardial nerve. Segmental nerve 6 was non-responsive
when stimulated both at its origin and where it joins the ganglion.
This confirms Carlson's (1905b) observation that nerve 6 is non-

responsive when stimulated at its origin; but not Pax' s (1966) obser-

“=2

vation that nerve 6 was inhibitory when stimulated near its point of
entry into the ganglion. This difference can probably be explained

since fused nerve 7 and 8 enters the ganglion at various locations.

I “-11:51

The number of branches of this nerve entering the ganglion also
varies; on occasion two and sometimes three branches instead of
the usual one are found. It is possible that one of these branches
entered the ganglion near nerve 6 and Fax stimulated this combina-
tion. While nerve 6 does not apparently function in cardioregulation,
its function is still unclear.

Stimulation of the pericardial nerve and the pericardial
branches of the segmental nerves does not affect heart rate. It does,

however, cause contractions of the opisthosomal extensor muscles.

Tissue Extracts

 

There have been no reports of pericardial organs in

E. polyphemus; and in my observations of the pericardial region,

 

I could find no nerve trunks or endings such as those described in

71

the Crustacea. The pericardial area is richly supplied with nerves
of one to five fibers, many of which branch from the segmental
nerves. Other small nerves branch from the pericardial nerve.
Since stimulation of nerves in this area was previously shown to be

ineffective in altering heart rate, tissue extracts were utilized to

tat-’abuw —M‘
. . .1 - . ‘-
’.

determine if some tissue might contain a substance capable of affect-
ing heart rate.
Of the experiments utilizing tissue extracts, only two
tissues show consistent effects upon the heart. Tissue extracts

(filtrate of 10 mg tissue per 1 ml saline) of ventral cord and brain
depress heart rate by four to ten beats per minute while extracts of
the pericardial tissues increase heart rate by eight to twelve beats
per minute. Boiling the extract and applying the recovered filtrate
produced the same results as fresh materials. These results sug-
gest that some substances are present which have effects upon the
heart, but further work is necessary to determine if they are part

of an organized system affecting heart rate.

DISCUSSION

Morpoholggy of the Cardiac Ganglion

 

The organization of the cardiac ganglion of L... polyphemus

 

differs markedly in many respects from that of other arthropods for
which comparative information is available. In the crustaceans,
arachnids, and limulus the heart is tubular and a cardiac ganglion lies
on the dorsal midline. In isopods and decapods this ganglion lies on the
inner surface of the heart; but in the amphipods, stomatopods, scorpi-
ons, spiders and limulus, it lies on the outer surface of the heart.

No further comparisons can be made with the arachnids,
since quantitative data have not been reported. In the malacostracans
which have been studied, the total number of cells is usually small.

In amphipods and isopods there are only six neurons in the ganglion
(Alexandrowicz, 1954);'in stomatopods the number of neurons is 14
(Irisawa and Irisawa, 1957); and six neurons are present in the

mysidicean Praunus flexuosus (Alexandrowicz, 1955). The decapod

 

cardiac ganglion also contains a limited number of cells; five large
and four small cells have been found in lobsters and crabs

(Alexandrowicz, 1932), but eight large and eight small cells have

72

 

73

been found in the crayfish Astacus potamobius (Alexandrowicz,

 

1929) and only five cells in the shrimp Alpheus spp (Suzuki, 1935).

 

The cardiac ganglion of _I___.. polyphemus differs markedly from that

 

of the malacostracans with respect to both the total number of cells

as well as the number of cell types. The total number of cells in

s

the ganglia I studied averaged 231 and there were six cell types.
Not only is the total cell number in malacostracans small,

it is also remarkably constant for any particular species. In the

 

lobster Homarus americanus, for example, in only three of about .. ~

 

150 cardiac ganglia the total number of cells was 10 rather than
nine (Maynard, 1961). The total number of cells in the cardiac
ganglion of limulus ranged from 171 to 305 in the twelve hearts
studied.

In the malacostracan cardiac ganglion the large cells are
usually anterior and small cells posterior. The large cells range
up to 200 u in large decapods and are generally multipolar; the small
cells are about 100 u and are always multipolar, although they often
have one large process (Bullock and Horridge, 1965). The arrange-

ment in g. polyphemus is different. Six morphological cell types

 

are distinguishable and, unlike the situation in the malacostracan
cardiac ganglion, unipolar and bipolar cells are the dominant cell
types. Moreover, the cell types are not segregated on any morpho-

logical basis, but lie randomly along the length of the ganglion.

74

In the crustaceans the cardiac ganglion consists mainly of
a limited number of cell bodies, their branched axons, and neuropile.
The neuropile of the ganglion is regionally clumped and surrounded
by glial cells (Alexandrowicz, 1932). In limulus there is a separa-
tion of the cardiac ganglion into only two recognizable regions; the
cellular portion and the fiber tract. Unlike the crustacean ganglion,
neuropile and cell body regions are not clearly segregated.

Thus in all aspects of the cardiac ganglion discussed above,

 

namely, the total cell number, variability in cell numbers, distribu- ....
tion of cell types along the ganglion, and arrangement of the com-
ponents of the ganglion, limulus differs markedly from that of the
malacostracans. In light of such differences, the question may be
raised as to whether there is a functional difference between these
cardiac ganglia. The answer to such a question must await a
physiological analysis of the various cell types.

Also from a broader viewpoint, the limulus cardiac
ganglion appears to be different from many other arthropod nervous
structures. In those systems thoroughly analyzed on a morpho-
logical basis, there appears to be a constancy in cell number as
well as cell arrangement, so that specific neurons can be identified
from animal to animal and the three dimensional distribution of cell

bodies and connectives can be mapped. This has been possible in

75

such structures as the lobster cardiac ganglion (Maynard, 1953;
Hagiwara and Bullock, 1957; Hartline, 1967), the crayfish ventral
nerve cord (Kendig, 1967), the locust thoracic ganglion (Wilson,
1961) and the cockroach metathoracic ganglion (Cohen, 1967). Such
a specific arrangement of elements apparently does not occur in the

limulus cardiac ganglion. There is a large variation in total cell

 

numbers as well as a large variation in the total number of each cell

type from one ganglion to the next.

Inu- _
‘3 .;-~ ‘1'. .4.»

Regulation of the Neurogenic Heart

 

Numerous factors are capable of altering the activity of
arthropod hearts (see Krijgsman, 1952, for review). The usual
endpoint used in evaluation of activity is either a change in rate or
in amplitude of contraction. Interpretation of the results of such
changes is complicated since there may be many sites of action:
pacemaker cells, motor cells, coordinating system or regulation
system of the ganglion or neuromuscular junctions or muscle itself.

The similarity between the activity of the crustacean heart
and that of limulus is amplified by the similarity of factors modulat-
ing the activity of the heart. There is positive correlation between
temperature and the rate,of beating (Carlson, 1906b; Garrey, 1920,
1932a; Seiwell, 1930). Distension of the heart increases the heart rate

(Carlson, 1904c, 1907a; Garrey, 1930; Dubisson, 1931; Isquierdo,

76

1932). The effects of electrical stimulation have been reported
(Samojloff, 1930; Garrey, 1932b; Garrey and Knowlton, 1934;
Bullock, 311—211. , 1943), a variety of ions have been tested for their
effects (Carlson, 1906c, 1906d, 1906e; Prosser, 1943b; Prosser
and Brown, 1961) and numerous pharmacological agents which
cause a change in both rate and strength of beating have been
reported (Carlson, 1904a, 1906f, 1907b; Welsh, 1939, 1942;
Prosser, 1942; Smith, 1947; Fax and Sanborn, 1967a, 1967b;
Abbott, 321. , 1969). While these studies have described many
factors modulating heart action, no study has yet been made con-

cerning the problems of intraganglionic relationships.

Anatomy of cardioregulatory nerves. -- The similarity of

 

the anatomy of the cardioregulatory nerves of the Crustacea and
limulus is also strikingly similar. Cardioregulatory nerves in
decapods were indicated early by several investigators (Dogiel,
1876; Plateau, 1880; Conant and Clark, 1896). Carlson (1905a,
1906a) studied the nerves of Panulirus s2. and came to the con-
clusion that two nerve pairs from the subesophageal ganglion inner-
vate the heart, the anterior pair inhibitory and the posterior pair
acceleratory. The entrance of the inhibitory nerves into the hearts
of various crabs was observed by Alexandrowicz (1932), Heath

(1941), Wiersma and Novitski (1942) and Smith (1947). Maynard

 

77

(1953) observed in Panulirus s2. that the extrinsic cardioregulatory
nerves consist of two pairs of cardioacceleratory nerves (posterior)
and one pair of cardioinhibitory nerves (anterior) which arise in the
anterior portion of the thoracic ganglion. These nerves run dorsally
and laterally to join the pericardial plexus where fibers from each
come together to form the dorsal nerve which runs to the cardiac
ganglion. This anterior-posterior arrangement of cardioregulatory
nerves is the same as found in limulus. However, the cardioregu-
latory nerves of limulus enter the ganglion individually rather than

forming a single nerve.

Function of cardioregulatory nerves. -- Stimulation of

 

inhibitory nerves in both decapod Crustacea and limulus produces
similar effects. Heart rates in both groups are decreased within a
few seconds of application of above threshold stimuli and return to
the original rate a few seconds after cessation of the stimulus. In
the case of the crustacean, Florey (1960) reported that frequencies
of stimulation less than 15/sec were ineffective and frequencies
above 35/sec result in heart standstill. In the range of frequencies
between 15-35/sec the decrease in heart rate varies almost expo-
nentially as the frequency of stimulation is increased. Pax and
Sanborn (1964) report that frequencies as low as 2. 5/sec cause

slowing of the heart of limulus and that as frequency is increased

 

78

the heart rate reaches a maximum depression at a stimulation
frequency of 15/sec and remains at that level up to a stimulation
frequency of 90/sec. Frequencies above 90/sec were found to be
less effective in rate depression and even greater stimulation
frequencies accelerate the heart. Both Smith (1947) and Florey
(1960) report heart stOppage in the Crustacea and Carlson (1905b)
reports similar occurrences in limulus, but Pax and Sanborn (1964)
were unable to produce heart standstill.

When stimulating nerves 7 and 8 with frequencies of 80-
100/sec, Pax and Sanborn (1964) report an acceleration of heart
rate and suggest that some excitatory fibers are contained in the
inhibitory nerves of limulus. Both Terzuolo and Bullock (1958) and
Florey (1960) describe post—inhibitory excitation; but they do not
discuss it. Later Florey (1968) showed that in the crayfish

Pacifiastacus leniusculus some individuals may have both inhibitory

 

and acceleratory fibers in a single nerve rather than the usual
separate nerves. Thus the presence of a post—stimulatory increase
may indicate that acceleratory fibers are present in the predomi-
nantly inhibitory nerves of both limulus and the Crustacea. The
range of responses in nerve 7 as reported here indicates that inclu-
sion of acceleratory fibers probably occurs only rarely. In nerve 8,

however, the slope of the mean response and the post-stimulatory

 

79

increase indicates that probably most animals have acceleratory
fibers in this nerve.

Excitation is similar in limulus and the Crustacea. It does
not start abruptly as does inhibition, but develops gradually and

reaches a maximum only after some seconds of stimulation. The

.— g
‘9
‘1

heart rate remains elevated after cessation of stimulation and
returns slowly to the original rate. Both the acceleration and the

after-effects are much more pronounced in limulus than in the

Q '3‘. ARM
l Q ~

Crustacea.

Limulus also shows two other differences in response to
stimulation of the acceleratory nerves; often there is an initial
inhibition and a post—stimulatory increase. The initial inhibition
seen in nerves 9-11 is similar to that seen in nerves 7 and 8. In
both cases the inhibition is closely coupled to stimulation with inhi-
bition occurring immediately. Pax (1969) examined the frequency—
response characteristics of both sets of nerves and found that the
acceleratory nerves like the inhibitory nerves give marked inhibitory
effects only with stimulus frequencies in excess of 10/sec. Thus
the inhibition reported here is below the maximum that might be
expected. The post-stimulatory increase occurs during the 10-20
seconds after stimulation which is the main period of recovery from

the influence of inhibition. Thus this increase suggests that the

80

ganglion has been released from inhibition and the full effect of
stimulation of the acceleratory fibers is then seen.

Initial inhibition and the post-stimulatory increase are
evidence that inhibitory fibers are present in the abdominal nerves.
Since the inhibitory effects are more marked in nerve 9, it appears
that more fibers are present in this nerve or lower thresholds are
present. The number of inhibitory fibers is not known but must
vary from individual to individual since the range of responses is

large.

Neurohormonal regulation. -- In recent years a great deal

 

of attention has been focused on the brachyuran pericardial organs
discovered and described by Alexandrowicz (1953). Because of the
location of the pericardial organs in relation to the heart, Alexandro-
wicz and Carlisle (1953) believed that secretions of the pericardial
organs could be involved with regulation of the heart. Maynard
(1961a, 1961b) demonstrated that cell groups from the ventral
ganglia send axons to the pericardial organs by way of the first
seven segmental nerves and suggested on morphological evidence
that at least three types of fibers are present. Maynard and
Maynard (1962) described a number of bipolar cells within these
organs. Various substances have been proposed as the active

principle (Belamarich, 1963; Belamarich and Terwilliger, 1966)

 

81

and Cooke (1966) has reported the sites of action of pericardial
organ extract on the lobster heart. All this work has not yet
demonstrated that the heart is the primary target of pericardial
organ substances. Indeed, Cottrell and Osborne (1969) suggest that
placement of neurosecretory systems near the heart is ideally
situated for the release of a substance causing the general stimula-
tion of an animal. Release of such a substance would first acceler—
ate the heart and thus promote its own distribution throughout the
rest of the animal; whereas, release of the substance elsewhere
would take longer for distribution and initiation of stimulation would
be delayed. Thus the primary target of the pericardial organs of
Crustacea may not be the heart and secretions of these organs may
not function in normal cardiac regulation.

No such organs have yet been described for limulus, but
extracts of tissues from the pericardial area consistently caused an
increase in heart rate. It cannot yet be stated whether this increase
is due to the activity of a neurohormonal system or due to a general

cytoplasmic substance of the pericardial tissues.

Function of the Cells of the Cardiac Ganglion

Morphology of a structure is at best only a small part of
an overall understanding of a structure. An equally important

aspect of the structure is function. The function of the cardiac

 

82

ganglion is initiation of the heart rhythm. However, since the
ganglion is composed of many smaller components, it is necessary
to examine each of these with regard to function. Function for the
cells of the cardiac ganglion can be suggested if the data from the
distribution of the pigmented cells (Figure 9) and some data from
previous physiological studies are used.

With the establishment of the neurogenicity of the limulus

heart (Carlson, 1905b), early workers began looking for the pace-

 

maker and its constituents. Garrey (1930) was the first to examine
the ganglion systematically for its pacemaker. He noticed that with
section of the heart, each piece maintains the same rate as the
original but the pieces no longer beat synchronously. Continued
cutting of the ganglion produces contractile segments except anterior
to ostia III and posterior to ostia VIII. Prosser (1941) was able to
record slow waves externally from the ganglion. These slow
potentials are largest in segments four, five and six, less in seg-
ments three, seven and eight and not present in segments one, two
or nine.

Since the limulus ganglion shows a morphological change
at ostia III, two aspects of cardiac ganglion organization are sug-
gested by the work of Carlson, Garrey and Prosser. First, pace-

maker cells probably lie within the cellular portion of the ganglion.

83

Secondly, since small bipolar cells and non-pigmented cells are found
anterior to ostia 111, they probably do not participate in ganglion
activity initiation.

Carlson (1905b) demonstrated that when the intra—cardiac
pressure is increased there is an increase in heart rate. Garrey
(1930) noted that when the anterior end of the animal was raised, the
increased intra-cardiac pressure altered the character of the con- E

traction. These observations suggest the probability of a stretch

 

if.

receptor-pacemaker interaction. Bullock and Horridge (1965)
indicate that most of the stretch receptor cells studied thus far are
bipolar cells. There are two types of bipolar cells in limulus
cardiac ganglia; whether one or both are stretch receptors is an
open question.

This leaves only two cell types, the pigmented unipolar
and the pigmented multipolar, which may be involved in the produc-
tion of the heartbeat. Heinbecker (1936) found that when he cut the
ganglion into 3-6 mm pieces, only some of the pieces were spon-
taneously active. By histological examination of these pieces he
found that pieces showing spontaneous activity always contained at
least one large unipolar cell as well as several multipolar cells.
Pieces of ganglion containing only multipolar cells were not spon-
taneously active. On this basis Heinbecker concluded that the large

unipolar cells are responsible for the initiation of the heartbeat.

84

Heinbecker, however, did not obtain any sections in which
only the unipolar cell was present; in all cases there were at least
a few multipolar cells. Since the sample size in his experiments
was quite small and since he did not distinguish between pigmented
and non-pigmented cells, it is possible that a pigmented multipolar
cell was always present when the piece showed spontaneity and
absent when it did not. For this reason the evidence presented by

Heinbecker does not appear to clearly demonstrate that the unipolar

 

cell is necessarily responsible for the initiation of the heartbeat.
Obviously further physiological experimentation is needed before
specific functions can be assigned to each of the cell types found

within the cardiac ganglion of E. polyphemus.

CONCLUSIONS

This study provides the basis for future physiological

examination of the _L_. polyphemus cardiac ganglion, especially

 

electrophysiological studies of individual ganglionic cells. Since
the cell types, cell numbers and distribution along the ganglion are
now known, it will be possible to make comparisons of physiological
data from one heart to the next. Likewise, since the cardioregula-
tory nerves enter the ganglion directly, ventral nervous system
influence on the cardiac ganglion can now be examined by electro-

physiological methods.

85

LIST OF REFERENCES

 

LIST OF REFERENCES

Abbott, B. C., F. Lang and I. Parnas. 1969. Physiological
properties of the heart and cardiac ganglion of Limulus
polyphemus. Comp. Biochem. Physiol. 28:149-158.

 

Alexandrowicz, J. S. 1929. Recherches sur l'innervation du coeur
de l' écevisse (Potamobius astacus). Folia morph. 1:37-
67.

 

Alexandrowicz, J. S. 1932. The innervation of the heart of the
Crustacea. I. Decapoda. Quart. J. Micr. Sci. 15:182-
249. -

Alexandrowicz, J. S. 1953. Nervous organs in the pericardial
cavity of the decapod Crustacea. J. Marine biol. Ass.
U. K. 31:563-580.

Alexandrowicz, J. S. 1954. Innervation of an amphipod heart.
J. Marine biol. Ass. U. K. flz709-719.

Alexandrowicz, J. S. 1955. Innervation of the heart of Praunus
flexuosus (Mysidacea). J. Marine biol. Ass. U. K.
_3_4_:47—54.

Alexandrowicz, J. S. , ‘ and D. B. Carlisle. 1953. Some experi—
ments on the function of the peri cardial organs in Crustacea.
J. Marine biol. Ass. U. K. 2:175-192.

Armstrong, F., M. Maxfield, C. L. Prosser and G. Schoepfte.
1939. Analysis of the electrical discharge from the cardiac
ganglion of Limulus. Biol. Bull. , Woods Hole. 21:327.

Belamarich, F. A. 1963. Biologically active peptides from the
pericardial organs of the crab Cancer borealis. Biol.
Bull. , Woods Hole. 124:9-16.

 

86

 

87

Belamarich, F. A., and R. Terwilliger. 1966. Isolation and
identification of cardio-excitor hormone from the peri-
cardial organs of Cancer borealis. Amer. Zool. _6_:101—
106.

 

Bullock. T. H., H. S. Burr and L. F. Nims. 1943. Electrical
polerization of pacemaker neurons. J. Neurophysiol.
6:85-98.

Bullock, T. H., and G. A. Horridge. 1965. "Structure and
Function in the Nervous Systems of Invertebrates. "
W. H. Freeman and C. , San Francisco. 2 vols. 1722 pp.

Carlisle, D. B., and F. G. W. Knowles. 1959. "Endocrine
Control in Crustaceans. " Cambridge University Press,
Cambridge. 120 pp.

Carlson, A. J. 1904a. The nervous origin of the heart beat in
Limulus and the nervous nature of co-ordination in the
heart. Amer. J. Physiol. 2:67-74.

Carlson, A. J. 1904b. The nature of the action of drugs on the
heart. Science. Qz684-689.

Carlson, A. J. 1904c. Further evidence of the nervous origin of
the heart beat in Limulus. Amer. J. Physiol. 13:471-498.

Carlson, A. J. 1905a. Comparative physiology of the invertebrate
heart. Biol. Bull., Woods Hole. 8:123-169.

Carlson, A. J. 1905b. The nature of cardiac inhibition with special
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