topographical and morphological studies of the cardiac

Showa Univ. J. Med. Sci. 1(1.2), 7-.22, December 1989

Original

Topographical and Morphological Studies of the Cardiac

Ganglia in the Prenatal Rat

Keh-Min Liu1) and Seiichiro INOKUCHI2)

Abstract: The loci and morphology of cardiac ganglia in the heart of prenatal rats were investigated at days 15 (D15), 18 (D18), and 21 (D21) of gestation. Ganglion cells were estimated in serial transverse sections of the hearts and adjacent organs.

All ganglion cells were distributed above the coronary sulcus. Six to seven

various sized groups of ganglion cells were located: (1) between the right

superior vena cava and ascending aorta, (2) between the right pulmonary artery

and trachea, (3) between the right pulmonary artery and right bronchus, (4)

dorsal to the right atrium, (5) dorsal to the left atrium, (6) in the region of the

dorsal interatrial groove and interatrial septum, (7) in the region including the

left bronchus, right inferior pulmonary vein, and left superior vena cava. The

ganglia contained small, densely stained ganglion cells, some nerve fibers, small

unidentified cells and capillaries. The irregular shaped ganglion cells were

characterized by large, round nuclei, thin layers of cytoplasm, and a few short

processes. Ganglia from D 18 were encapsulated by connective tissue, and some

ganglion cells were surrounded by satellite cells. The mean nuclear diameter

of cardiac ganglion cells was 4.28ƒÊm at D15, 4.76ƒÊm at D18, and 6.05ƒÊm

at D21, their mean somal diameters were 5.79ƒÊm, 6.23 ƒÊm, and 7.58ƒÊm, and

their mean numbers were 1994.33•}102.31, 5771.65•}233.72 and 6430.37•}

630.64, respectively.

It is concluded from these results that cardiac ganglion cells can be found in

6 to 7 atrial regions in the prenatal rat and their size, number , and maturity increase from D15 to D21, but they migrate with the growth of the fetus.

Key words: fetus, cardiac ganglia, topography, morphology

Introduction

Autonomic control of cardiac functions is necessary for the maintenance of circulatory

homeostasis in changing environmental and behavioral conditions.1 Although several ana-

tomical reports have described the distribution of cardiac ganglia in the rat,1-6) mouse ,6,7) dog,6,8) cat,6,9) guinea pig,6,7) frog,10 and other species,4,6,11) most of these observations

were made on macroscopic preparations or on serial histological sections from adults.

While much is now known about the location of cardiac ganglion cells in adult animals,

little information is available about the prenatal distribution of cardiac ganglion cells in the

embryonic animal heart. Kirby and Stewart12 reported that by stage 11 of chick incubation,

1) Department of Anatomy, Kaohsiung Medical College, Kaohsiung, Taiwan, Republic of China.2) Department of Anatomy

, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan.

8 Keh-Min Liu, et al.

cranial neural crest cells from somite 1-3 migrated ventrolaterally to the conotruncal region

of the heart, and aggregated into scattered ganglia. Hoar and Hall13) reported that by

day 25 of gestation, the primordia of the guinea pig cardiac ganglia reached the peripheral

walls of the atria, and at day 26 of gestation, clusters of large round cells could be seen in

several areas of the heart. Hall14) described small ganglia distinguished in the rat mesen-

chyma dorsal to the heart at 141 days of gestation. The considerable species differences

in patterns of development of cardiac innervation reflect variations in the degree of the

neuronal control of the heart. Halll4 reported that at D15 1/2 , cardiac ganglia in the heart

of the fetal rat, can be recognized in most specimens, and at DI 61 , the ganglia are large.

However, there are no quantitative data about the number and size of cardiac ganglion cells

in different fetal development stages of the rat.

Until now, changes in the size of cardiac ganglion cells and their nuclei, and the ratio

of nucleus size to cell body size in the embryonic rat heart seem never to have been studied

during the various developmental stages.

The present study, undertaken to investigate the topographical distribution of cardiac

ganglia, reveals the morphological structures of the ganglion cell, and quantifies and com-

pares the number and size of ganglion cells in different developmental stages of the fetal rat.

Materials and Methods

1. Perfusion and Histology

Female adult Sprague-Dawley rats were mated with males of the same strain and isolated on the morning of finding a vaginal plug and a vaginal smear with sperm, which was de-signated as day zero of gestation. On days 15, 18 and 21, the pregnant rat was anesthetized

with sodium pentobarbital (40 mg/kg, i.p.).The whole chest of each fetal rat was then removed and immersed in fresh 10% neutral

formalin for 7 to 10 days. The heart and attached large blood vessels were carefully dissected and dehydrated in an ascending sequence of ethanol, and embedded in paraffin. Serial 10

p,m thick sections were cut transversely through the whole heart and its large blood vessels. The sections were mounted serially and stained with hematoxylin and eosin, or cresyl violet,

and covered with a coverslip. Quantitative analysis was routinely performed on hematoxylin

and eosin stained sections.

2. Topography and Morphology of Cardiac Ganglia

Under a light microscope, the appearance of a section varies from one specimen to

another due to the variable geometry of the fixed hearts and slightly different angles of the

cutting plane. The distribution of cardiac ganglia seemed to be in slightly different positions,

however, when referred to certain well defined anatomical structures, it was thus not difficult

to correlate ganglion cell groups in different specimens.

Therefore, the descriptions and loci of cardiac ganglia are all presented relative to ana-tomical landmarks. Furthermore, in each developmental stage, 6 transverse sections at dif-ferent levels of the heart were selected and displayed by camera lucida. All structures

with special reference to ganglion cell groups were clearly projected on the tracing paper and marked through a drawing tube to show characteristic distribution of the cardiac gan-

glia. The morphological structures of the cardiac ganglia and ganglion cells were observed and photographed.3. Dimension and Number of Cardiac Ganglion Cells

Cardiac Ganglia in Prenatal Rat 9

For estimating the diameter of the nucleus and the cell body of cardiac ganglion cells in

the fetal rat, the large aggregates of ganglion cells from Dl 5, Dl 8, and D21 were selected

and microphotographed at a magnification of 400•~. From the enlarged photographs (•~3400)

of each developmental stage, the areas of the ganglion cell bodies and nuclei were traced

with a Houston Hipad digitizer with a resolution of 0.1 mm. The diameter of the nucleus

and soma were then calculated using the formula d=•¬ (d = diameter). For each

stage, the data from 1000 ganglion cells were quantitatively analyzed by the Bioquant sys-tem (R & M Biometrics). To investigate changes in the ratio of nuclei to cell bodies of

cardiac ganglionic neurons in various developmental stages, the areas of nuclei and cell

bodies of 1000 cardiac ganglion cells were measured separately and compared.

To estimate the total number of ganglion cells in the fetal heart in the different develop-

mental stages of rats, the nuclei of cardiac ganglion cells, each with the nucleolus, were

counted through the whole series of transverse sections at 600•~ magnification. The number

of ganglion cells was estimated after the method of Abercrombie.15)

The Student's unpaired t test was used to evaluate differences and confidence limits be-

tween pairs of age groups.

Results

1. Topographical Distribution of Cardiac Ganglia;

The cardiac ganglion cells of fetal rats were located primarily in connective and fatty

tissues, which surrounded the great vessels on the superior part of the heart, and were

present dorsal to the heart among the atria, the bronchi, and the esophagus. Neuronal somata were rarely seen within the subepicardium or within the myocardium, and were

hardly ever seen below the level of the atria and coronary sulcus.

In general, 6-7 major groups of cardiac ganglion cells can be identified in fetal rats.

The presence of these groups is summarized in Table 1, illustrated in Fig. 1, and depicted in the different levels of the heart in Figures 2, 3, and 4. The diagrams in Figures 2, 3 and 4 are composites of pictures obtained from camera lucida drawings of transverse sec-tions of fetal rat hearts at D15, Dl 8 and D21, to show the characteristic distribution of

ganglion cells.

Table 1. The distribution pattern of cardiac ganglia in fetal rats in

three different stages of development.

* Results from the observation of 5 fetal rats .


Fig. 1. Ventral and dorsal views (A and B) of the distribution of cardiac ganglion cells in the heart of prenatal rat. The numbers and arrows indicate the different groups and locations of these cardiac ganglion cells.

The first or most rostral group of cardiac ganglion cells was located medial to the light superior vena cava and the dorsal aspect of ascending aorta. They were present just above the superior part of the right atrium, and first appeared on the transverse section about the level of the aortic arch. At their most rostral end, they were often located dorsally to the right superior vena cava and more caudally migrated ventrally around the medial aspect of the right superior vena cava (Figs. 2A, 2B, 3A, 3B, 4A).

The second group of cardiac ganglia was small, was located in an area dorsal to the

aortic arch, and appeared around the bifurcation of the pulmonary trunk. The ganglion

cells of this group extended between the trachea and right pulmonary artery. This group was observed in fetal rats at D15 and Dl 8 only (Figs. 2B, 3B).

The third group of cardiac ganglion cells was small, and was situated mainly dorsal to the right pulmonary artery. It appeared in fetal rats at D21 (Fig. 4B).

The fourth group of cardiac ganglion cells, a large aggregate, was located throughout an

extensive area on the dorsal aspect of the right atrium (Figs. 2C, 3C, 3D, 3E, 4B, 4F).

The rostral pole of these ganglion cells began in the connective tissue between the right

pulmonary artery and right atrium, spread caudally and extensively in the space formed by the posterior wall of the right atrium, right bronchus, right superior pulmonary vein, and

right inferior pulmonary vein. The fourth group finally terminated at the level of the

termination of the superior vena cava.

The fifth group of cardiac ganglion cells was posterior to the left atrium. This was a

large group and included cells that were located behind the mid- and caudal portions of the left atrium (Figs. 2C, 2D, 2E, 3C, 3D, 3E, 4D, 4E). It appeared first within the pos-

terior wall of the left atrium, left pulmonary artery, and left bronchus; and then extended

caudally between the posterior wall of the left atrium and left superior vena cava. The cells

of the fifth cardiac ganglion group terminated at the level of the entrance of the left superior versaa cava into the coronary sinus.

The sixth group of cardiac ganglion cells was in the region of the interatrial groove.


Fig. 2. Diagrams at 6 transverse levels of the heart of a D15 rat show the major regions in which ganglion cells were located. In this figure and figures 3 and 4, numbers refer to the ganglion cell group described in the text. Scale bars = 0.5 mm.


Fig. 3. Diagrams at 6 transverse levels of a rat heart at D18 show the major locations of cardiac ganglion cells. Scale bars=0.5 mm.

The rostral pole of these cells began in the connective tissue above the intersection of the right and left atria, extended caudally to the interatrial groove, and then in a dorsal and caudal direction to spread behind the interatrial septum and the entrance of the coronary

sinus into the right atrium (Figs. 2D, 3E, 3F, 4F).The seventh group of cardiac ganglion cells was in the inferior portion of the interatrial


Fig. 4. Diagrams at 6 transverse levels of the heart of a D21 rat show the major sites in which ganglion cells were located. Scale bars=0.5 mm.

septum, rostral to the A-V node. This group was very small and was only observed in the fetal rats of E21. The ganglion cells were embedded in the wall of interatrial septum and

surrounded by cardiac muscle (Fig. 4E).The eighth group of cardiac ganglion cells was located on the dorsal aspect of the left

atrium, close to the left superior vena cava and the coronary sulcus. This group was usually situated among the right inferior pulmonary vein, left bronchus, and left superior vena cava

(Figs. 2F, 3F, 4F).Throughout the study of all transverse sections of the whole heart, no ganglion cells


Fig. 5. A. Light micrograph of transverse section of the heart of a D15 rat shows cardiac

ganglion cells (arrowheads) located dorsal to the atria. H.E. stain, •~190. B. Higher magnification of part of Fig. 5A. The ganglion cell (arrow) consists of a large, round nucleus

and cytoplasm. H.E. stain. C. Higher magnification of cresyl fast violet-stained ganglion

cells from the heart of a D15 rat. Arrows indicate some of the ganglion cells.

Fig. 6. A. Light micrograph of transverse section of the heart of a D18 rat shows the cardiac

ganglion cells (arrowheads) located between the right atrium (RA), the right bronchus (RB)

, and the left inferior pulmonary vein (LIPV). H.E. stain, •~190. B. A higher mag-

nification of part of Fig. 6A. The irregular shaped ganglion cell (arrow) is characterized by

a large nucleus with condensed chromatin, and a thin layer of cytoplasm. Occasional satellite

cells can be seen. H.E. stain. C. Higher magnification of cresyl fast violet-stained ganglion

cells (arrows) from the heart of D18 rat.

Fig. 7. A. Light micrograph of transverse section of the heart of a D21 rat shows groups 4

and 7 of cardiac ganglion cells (arrows). H.E. stain, •~190. B. Higher magnification of

part of Fig. 7A. The ganglion cell (arrow) consists of a large, light stained nucleus and a

thin layer of darkly stained cytoplasm. Arrowheads indicate satellite cells of the ganglion

cell. H.E. stain. C. Higher magnification of cresyl fast violet-stained ganglion cells (arrows).

Arrowheads show the darkly stained satellite cells of the ganglion cell.

Cardiac Ganglia in Prenatal Rat

15

were observed on the ventral surface of the two atria or on the ventral interatrial groove.

2. Morphological Structures of Cardiac Ganglia:

The morphology of the cardiac ganglia of fetal rats varied considerably, ranging from

loosely dispersed to highly compacted structures and from 400•~500 pm in size down to

just a few ganglion cells. The intermediate power photographs of Figs. 5A, 6A, and 7A

illustrate a large group of cardiac ganglia in the posterior wall of the atrium. In the heart

of 15D fetal rats, the cardiac ganglia consisted of small, densely stained ganglion cells, which

were loosely distributed and were not surrounded by capsules (Fig. 5A). Some nerve fibers,

small unidentified cells and capillaries were interspersed among the ganglion cells. The gan-

glion cells of the cardiac ganglia can be identified by their round or oval shaped, large nuclei

and deeply stained nuclear membranes. The round nucleoli were uniformly darker than the

nucleoplasm and occasionally two in number. The nucleus was surrounded by a thin layer

of cytoplasm (Fig. 5B), which could be seen clearly by using cresyl violet stain (Fig. 5C).

Occasionally, some short processes extending from the cell body could be seen, but both the

cell body and processes lacked satellite cell covering.

In the 18D rat heart, the cardiac ganglion was composed of loosely aggregated, darkly stained cells (Fig. 6A). Sometimes, numerous ganglion cells forming a large aggregation were

surrounded by a thin layer capsule. Usually, the ganglion cells were interspersed with fine blood vessels and nerve fibers. The ganglion cells were characterized by round to oval nuclei with a darkly stained, thin nuclear membrane. The nuclei were large in relation to the cyto-

plasmic mass and tended to be located in eccentric positions (Fig. 6B). The darkly stained nucleoli were slightly ovoid or round, and were sometimes two in number. In addition to the nucleoli, there were usually several darkly stained chromatin granules spread within the

nucleus (Fig. 6B). Occasionally, cytoplasmic processes projected from the cell body.In the cresyl violet stained sections, the ganglion cells consisted of large, pale stained

nuclei and darkly stained cytoplasm. Some ganglion cells were surrounded by satellite cells

(Fig. 6C). In the heart of a 21 D fetal rat, the cardiac ganglion consisted of large ganglion cells, small

supporting cells, unmyelinated nerve fibers and capillaries. Ganglion cells were usually

grouped into variable sized clusters (Fig. 7A) and were encapsulated by thin walls. The ganglion cells had single, large nuclei which appeared pale and round in profile. They were central or eccentric and possessed one or more densely stained nucleoli. The shapes of the

ganglion cells varied, from round to oval or polygonal (Fig. 7B). Some processes arose from the cell bodies. The cresyl violet stained ganglion cells showed numerous deeply stained

granules in their cytoplasm (Fig. 7C). Usually, a ganglion cell was intimately surrounded by darkly stained satellite cells.

3. Size Distribution of Cardiac Ganglion Cells:

Initially, cells obtained from different topographic groups of ganglion cells were compared

for size. Because no differences were found, the ganglion cells from different groups were

pooled and reported together.

The distribution frequency of the nuclear diameters of cardiac ganglion cells from fetal

rats in three different developmental stages is shown in Fig. 8. These distribution patterns

are unimodal. The nuclear diameters of ganglion cells in fetal rats ranged from 2.37 to

6.48 ƒÊm at D l5, 2.73 to 7.53 ƒÊm at D18, and 3.17 to 10.04 ƒÊm at D21. At D15, most

of the nuclear diameters were less than 5 ƒÊm. At D18, approximately 80% of the nuclear

16 Keh-Min Lw, et al.

Fig. 8. The distribution of nuclear diameters of cardiac ganglion cells in the rat heart in

three different prenatal stages.

Table 2. Diameter of cardiac ganglion neuronal nucleus and cell body, and ratio of nucleus/cell

body from fetal rats in different stages of development.

a N/C=area of nucleus (pmt)/area of cell body (tem2) , b D15 vs . D21, p<0.05. C D18 vs . D21, p<0.05.

diameters were between 4ƒÊm and 6ƒÊm. At D21, nuclear diameters larger than 5ƒÊm were

much more common.

As shown in the Table 2, the mean nuclear diameters of ganglion cells, were 4.28•}0.68ƒÊ

m at D15, 4.76•}0.66 ƒÊm at D18, and 6.05•}0.97 ƒÊm at D21. There was no significant

difference in the mean nuclear diameters of Dl 8 and D15. However, there was a 25-30%

increase in the mean nuclear diameter of ganglion cells at D21. This mean nuclear diameter

of D21 was significantly greater than that of D15 or D18 (P<0.05).

The frequency distributions of somal diameters of cardiac ganglion cells from fetal rats

at D15, Dl 8 and D21 are shown in the Fig. 9. These distribution patterns were unimodal.

The somal diameters of ganglion cells in fetal rats ranged from 3.17 to 8.17 ƒÊm at Dl 5,

3.38 to 9.86 ƒÊm at Dl 8, and 3.33 to 11.92 ƒÊm at D21. At D15 and D18, many neurons


Fig. 9. The frequency distribution of diameters of cardiac ganglion cells in the rat heart in

three different prenatal stages.

were less than 7 ƒÊm in diameter, but at D21, the distribution pattern of somal diameters

skewed toward the large size.

The mean somal diameters of ganglion cells were 5.79•}0.86 ƒÊm at D15, 6.23•}0.85 ƒÊm

at D18, and 7.58•}1.33 ƒÊm at D21 (Table 2). There were no significant differences in the

mean diameters of cell bodies between D 15 and D 18, or Dl 8 and D21. However , it can be

clearly seen that the mean somal diameter increased greatly (about 33% between D15 and

D21).

To understand the developmental changes of the ratio of nuclei to cell bodies in the

prenatal stage, comparison of the nuclear area to the cell area of the cardiac ganglion cell bodies at D15, Dl 8 and D21 is shown in Fig. 10. All frequency distribution patterns were

unimodal and ranged from 30% to 80%. However, the ratio distribution of D21 rats

shifted toward higher percentage, i.e. in about 70% of the ganglion cells, the nucleus oc-

cupied 60-80% of the size of the cell.

The mean ratio, nucleus/cell body, of cardiac ganglion cells in fetal rats was 55.96% at D 15, 58.05% at Dl 8, and 62.32% at D2 1, as shown in Table 2. There was no dif-ference in the mean ratio of nucleus/cell body in cardiac ganglia between D15 and D18. By D21, the mean ratio of nucleus/cell body increased significantly when compared with

that of D15 or D18 (p<0.05).4. Numerical Distribution of Cardiac Ganglion Cells:

The numbers of cardiac ganglion cells in the different groups at three different develop-

mental stages are shown in Table 3. The number of ganglion cells (mean•}S.D.) was counted

according to the various groups described previously from 5 fetal rats at each stage, and

was corrected. As shown in Table 3, groups 4 and 5 were large, and consisted of many


Fig. 10. The frequency distribution of the ratio of nucleus/cell body of cardiac ganglion cells in the heart of fetal rat in three different prenatal stages.

Table 3. Comparison of the number of cardiac ganglion neurons in fetal rats in

three different stages of development.

a Each value represents the mean•}S .D. for 5 fetal rats.

b. C. d The correction factors for the crude counts of cardiac ganglion neurons in rats

at D15, D18 and D21 are 4.29, 4.76 and 6.05 respectively.

e D15 v .s. D18, p<0.001

f D15 vs . D21, p<0.001

large aggregates of cells (as many as 200 nucleoli in a section). Groups 1, 6, and 8 were

small groups, and were composed of scattered small clumps of cells. Groups 2, 3 and 7 were

counted only in Dl 5, Dl 8 and D21 fetal rats, respectively. In general, the number of car-

diac ganglion cells in each group increased with the growth of the fetal rats.The means of the total numbers of cardiac ganglion cells in the fetal rats were 1994 .33


at D15, 5771.65 at Dl 8 and 6430.37 at D21. The total number of cardiac ganglion cells

at Dl 8 and D21 was significantly greater than that at D l 5, but there was no significant dif-

ference between Dl 8 and D21.

Discussion

The present investigation presents a picture of the distribution and morphological structure

of ganglion cells in the heart of the prenatal rat, and estimates their numbers in different areas. Our results, obtained from different projections of 15 separate specimens each in three developmental stages, yield a composite picture portraying the developing pattern of cardiac

ganglia in the rat.

In prenatal rats, 6-7 groups of various sizes of cardiac ganglia were observed around

the base and the posterior atrial wall of the heart, but were never seen below the coronary

sulcus or in the ventricle even at day 21 of gestation. There are reports of ganglion cells in

the ventricle of the chick embryo,16,1'' but the significance of this species difference is

obscure.

In fetal rats, the different aggregates of cardiac ganglion cells were usually distinctly

separated, but the groups of ganglion cells were occasionally clumped together. The topo-

graphy of the cardiac ganglion cells in prenatal rats is roughly similar to that in the kitten,10' but is somewhat different from that of the adult rat.1-3,6)

Group 1 cardiac ganglia, appearing on the 15th day of gestation, was close to the orifice

of the vena cava superior. This group may have functional connection with the sino-atrial

node.9)

Group 2 cardiac ganglia appeared on D15 and D18, and disappeared on D21. This

change in distribution may be due to the migration of ganglion cells, and elongation of the

ascending aorta and aortic arch. Group 3 cardiac ganglia were observed on D21, these

small cardiac ganglia may be formed from part of group 2. When compared with the

description by Pardini et al.,1) this group 3 cardiac ganglia in the prenatal rats may cor-

respond to the small rostral part of the group 2 cardiac ganglia of the adult rat. However,

according to a report by Tsubota, ' no cardiac ganglia were found around the aortic arch,

the peripheral region of aorta, or the bifurcated region of the trachea in adult rats. This

might be explained by the variation among animals of the same species, 7) or the continuous

growth and downward migration of cardiac ganglia in the postnatal stage.

Groups 4 and 5 were large cardiac ganglia. During the three developmental stages ex-

amined, the cardiac ganglion cells in the posterior wall of the right atrium (group 4) increased in number to a peak at D 18 and then declined, whereas those in the posterior wall of the left atrium (group 5) increased continuously. According to a report on adult rat cardiac

ganglia,l' the number of ganglion cells in the posterior wall of the left atrium is greater than that in the posterior wall of the right atrium. It appears that the numerical distribution

patterns of the cardiac ganglia are determined in the prenatal stage.Group 6 was a medium size cardiac ganglion located in the superior interatrial septum.

Pardini et dl.1) reported that the largest group of cardiac ganglia was observed in the superior

interatrial septum, but in their report, the boundary of the superior interatrial septum in-

cluded the posterior walls of both atria, and was not restricted in the narrow interatrial

septum only. In fact, the superior interatrial septum is a shallow furrow, sometimes called the

interatrial groove, and is formed by the enfolding of the interatrial wall during development.


At D21, some cardiac ganglion cells are found sandwiched between the myocardial tissue

of the two atria in the lower portion of the interatrial septum (group 7) . This group may have originated from the downward migration of the ganglion cells of group 6, and is very close to the atrioventricular node. 3,9) The group 7 cardiac ganglia is the only group that

was surrounded by cardiac muscle. A similar group of cardiac ganglia was also found in the

adult rat, but it was included with a group posterior to the right atrium .1)Group 8 cardiac ganglia consisted of several small ganglia , and was located close to the

lower margin of the posterior walls of both atria . However, there was an area surrounded by groups 4, 5, and 8 which was devoid of ganglion cells . During prenatal development, the number of cardiac ganglion cells in group 8 increased . After postnatal development, the

group 8 cardiac ganglia may become associated with group 4 or group 5, as described in the adult rat.1

The initial establishment of autonomic innervation of the embryonic heart was first re-

ported by Hoar and Hall.13~ In their report, the cardiac ganglion cell was described only

as a "large round cell". In the present study, the mean diameter of the cardiac ganglionic

cell body in D15 to D21 ranged from 5.79 to 7 .58ƒÊm, and had a unimodal pattern of

distribution. Previous studies showed that in the submandibular ganglia of mouse and rat,

there were two subpopulations of neurons that differed in size and target organs .18) Similarly,

in the ciliary ganglia of chicken, the large ciliary cells (30-50 ƒÊm in diameter) and small

choroid cells (15-20 ƒÊm in diameter) occurred in equal numbers .19 From the present study,

in the three different developmental stages, the unimodal distribution of the size of cell

bodies and nuclei shows that no subpopulation of neurons can be identified in the prenatal

stage by light microscope.

The mean diameter of cardiac ganglion cells of prenatal rats was 7 .58 ƒÊm at D21, but

those of one-day-old and adult rats were 12-15 ƒÊm20 and 26.9 ƒÊm,1 respectively . From

these data, it is clear that the cells of cardiac ganglia of the rat grow continuously after birth .

Neuronal death may be a regulatory mechanism in the control of cell numbers in the

nervous system. Many neurons in the peripheral ganglia die during normal ontogeny .21) It was reported that the parasympathetic ciliary ganglion of the chicken , which initially con-sisted of about 6,000 neurons, diminished to about half between the 8th and 14th day of

incubation.22) In the present study, cell numbers in the cardiac ganglia increased dramati-

cally from Dl 5 to D21. It must thus be considered that if natural cell death also occurred

in the cardiac ganglia, it must have happened before D15. Rickenbacher and Mi ller16 re-

ported that the cardiac ganglia of the chick embryo growth in the number and size of cells continued until D15. Whether or not natural death of neurons occurs in the cardiac ganglia

of the prenatal rat should be investigated further.

Relations between the preganglionic origin and the cell number of the cardiac ganglia in

the prenatal rat should be considered simultaneously. During the prenatal period of the rat , vagal innervation is very important in the functional development of the heart.23) It has been reported that cell death in the dorsal motor nucleus (DMN), the site of origin of cardiac

vagal preganglionic motoneurons (CVPM), occurred between incubation day 8 and hatching , and resulted normally in a 37% loss of neurons. The peak loss of neurons occurred between

incubation days 8-12.24) However, there is recent anatomical and electrophysiological evi-dence that the nucleus ambiguus and the ventrolateral nucleus ambiguus are also primary

sites of origin of CVPM.25~ The continuous increase in the number of cardiac ganglionic


cells indicates that there is no direct relation between preganglionic origin and the number

of postganglionic neurons of cardiac ganglia.

It was reported by Wright26) that neuronal death began after cessation of proliferation and migration, and coincided with the time of anatomical and functional innervation of the synaptic target. In the rat, the first appearance of ACNE in cardiac ganglia is at day 4

postnatal.25) In the present investigation, the number of cells in the cardiac ganglia of the

prenatal rat at D21 was 6430, and that in the adult rat was approximately 3980.1) The 38% decrease of cells in the cardiac ganglia occurred after birth and may have coincided with the appearance of AChE. This result is in accordance with the observation of Purves and Lichtman22) that a substantial portion (25-75%) of the original neuronal population

dies in neonatal life. For example, during early postnatal life, a 30% decrease in the number

of cells in the superior cervical uanulia occurred.

Kirby et al.2 7' described that neural crest cells contributing to the cardiac ganglia mi-

grated in association with the preganglionic nerve, i.e., the cardiac branches of the vagus. In the rat, the parasympathetic fibers began to penetrate the atria around D14-15,28) al-

though, the first appearance of ACNE in the cardiac ganglia was at day 4 postnatal, and

nerve fibers containing AChE could be identified in the sino-atrial node at day 15 postnatal.25)

The dramatic increase in the number and size of cardiac ganglion cells in prenatal rats, plus

the postnatal data previously described21) indicate that parasympathetic innervation in the

prenatal rat myocardium was very immature, but it develops and grows rapidly during early

postnatal life. This suggestion is supported by the report of Finlay and Anderson29) that histological maturation of the cardiac ganglia occurred during the 4 days subsequent to the

appearance of ACNE 1n the ganglionic cells, whereas, even at 31 days, ACNE-positive

innervation of the heart had not yet reached the adult level.

Acknowledgement

The authors express their grateful appreciation to Prof. E. A. Ling, and Prof. W. C. Wong, for en-

couragement; and Mr. W. J. Liang and Ms. S. C. Wu, for skillful technical assistance.

References

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Abbreviations

AA: ascending aorta, AAH: aortic arch, DA: descending aorta , DU: ductus arteriosus, E: esophagus, F: foramen ovale, JVC: inferior vena cava , LA: left atrium, LAU: left auricle, LB: left bronchus, LIPV: left inferior pulmonary vein , LPA: left pulmonary artery, LV: left ventricle, LVN: left vagus nerve, LSVC: left superior vena cava , PT: pulmonary trunk, RA: right atrium, RAU: right auricle, RB: right bronchus , RIPV: right inferior pulmonary vein, RPA: right pulmonary artery, RSPV: right superior pulmonary vein, RSVC: right superior vena cava, RV: right ventricle, RVN: right vagus nerve , T: trachea.

[Received July 12, 1989: Accepted October 20, 1989]

topographical and morphological studies of the cardiac

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