Download morphological and immunohistochemical patterns of the intrinsic

LITHUANIAN UNIVERSITY OF HEALTH SCIENCES
MEDICAL ACADEMY
Kristina Rysevaitė
MORPHOLOGICAL AND
IMMUNOHISTOCHEMICAL PATTERNS
OF THE INTRINSIC GANGLIONATED
NERVE PLEXUS IN THE MOUSE HEART
Doctoral Dissertation
Biomedical Sciences,
Biology (01 B)
Kaunas, 2011
The dissertation has been prepared during the period of 2007–
2011 at the Institute of Anatomy, Medical Academy, Lithuanian
University of Health Sciences
Scientific Supervisor:
Prof. Dr. Neringa Paužienė (Medical Academy, Lithuanian
University of Health Sciences, Biomedical Sciences, Biology –
01 B)
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CONTENTS
LIST OF FREQUENTLY USED ABBREVIATIONS ............................. 5
INTRODUCTION........................................................................................ 7
Actuality of the Study .............................................................................. 7
Aim and Objectives.................................................................................. 8
Originality and Implications .................................................................... 8
1. REVIEW OF LITERATURE ................................................................. 9
1.1. Innervation of the Heart .................................................................... 9
1.2. Neurotransmiters in Cardiac Innervation ........................................ 11
1.3. Physiology of Intrinsic Cardiac Neurons ........................................ 14
1.4. Intrinsic Cardiac Nerve Plexus ....................................................... 16
1.5. Immunohistochemical Characterization of Intrinsic Cardiac
Neurons .................................................................................................. 23
1.6. Immunohistochemistry of Nerves and Nerve Fibers in Intrinsic
Cardiac Nervous System........................................................................ 25
1.7. Autonomic Control and Innervation of Mammalian Cardiac
Conduction System ................................................................................ 27
1.8. Studies on Intrinsic Cardiac Nervous System by Kaunas
Anatomists ............................................................................................. 33
2. MATERIALS AND METHODS .......................................................... 34
2.1. Material ........................................................................................... 34
2.2. Methods........................................................................................... 34
2.2.1. Total heart preparations .................................................... 34
2.2.2. Thorax-dissected preparations .......................................... 34
2.2.3. Whole-mount preparations................................................ 35
2.2.4. Immunohistochemistry...................................................... 35
2.3. Microscopic Examinations and Measurements............................... 37
2.4. Statistical Analysis.......................................................................... 38
3. RESULTS ............................................................................................... 39
3.1. Distribution of TH-IR and ChAT-IR Nerve Fibers in the
Intrinsic Cardiac Nerves......................................................................... 39
3.2. Access of Mediastinal Nerves into the Mouse Heart ...................... 39
3.3. Architecture and Topography of the Itrinsic Cardiac Nerve
Plexus .................................................................................................. 39
3.4. Morphology of Mouse Cardiac Ganglia ......................................... 40
3.5. Distribution of Immunochemically Distinct Intrinsic Cardiac
Ganglia and Neurons.............................................................................. 41
3.6. ChAT-IR Neurons........................................................................... 41
3.7. TH-IR Neurons and Small Intensively Fluorescent (SIF) Cells ..... 42
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3.8. Distribution of ChAT-IR and TH-IR Nerve Fibers......................... 43
3.9. Distribution of ChAT-IR and TH-IR Nerve Fibers in the
Sinuatrial and Atrioventricular regions .................................................. 44
3.10. Distribution of SP-IR and CGRP-IR Nerve Fibers ....................... 52
4. DISCUSSION ......................................................................................... 65
CONCLUSIONS ........................................................................................ 71
PUBLICATIONS ....................................................................................... 72
RERERENCES .......................................................................................... 74
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LIST OF FREQUENTLY USED ABBREVIATIONS
ACh – acetylcholine
AChE – acetylcholinesterase
AV – atrioventricular
AVN – atrioventricular node
cChAT – conventional form of ChAT
CGRP – calcitonin gene related peptide
ChAT – choline acetyltransferase
CHT – high-affinity choline transporter
DBH – dopamine beta hydrohylase
DRA – dorsal right arial subplexus
DVM – dorsal motor nucleus of the vagus
ENP – epicardiac neural plexus
GNPHH – ganglionated nerve plexus of the heart hilum
HCN4 – hyperpolarization activated cyclic nucleotide-gated potassium
channel 4
HH – hilum of the heart
ICG – intrinsic cardiac ganglia
ICNS – intrinsic cardiac nervous system
ICNs – intrinsic cardiac neurons
INP – intrinsic neural plexus
IR – immunoreactive
IVC – inferior vena cava
LA – left atria
LAu – left auricle
LC – left coronary subplexus
LD – left dorsal subplexus
LOM – ligament of Marshall or neural fold of the left atrium
LPV – left pulmonary vein
MD – middle dorsal subplexus
MPV – middle pulmonary vein
NA – nucleus ambiguus
NOS – nitric oxide synthase
NE – norepinephrine
NPY – neuropeptide Y
pChAT – choline acetyltransferase of a peripheral type
PGP 9.5 – protein gene product 9.5
PVs – pulmonary veins
RA – right atria
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RAu – right auricle
RPV – right pulmonary vein
RV – right ventral subplexus
SA – sinoatrial
SAN – sinoatrial node
SIF – small intensively fluorescent cell
SP – substance P
TH – tyrosine hydroxylase
VAChT – vesicular acetylcholine transporter
VC – vena cava
VLA – ventral left atrial subplexus
VRA – ventral right arial subplexus
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INTRODUCTION
Actuality of the Study
The intrinsic cardiac nervous system plays a crucial role in the regulation of heart rate, atrioventricular nodal conduction, and inotropism of
atria and ventricles (Baumgart and Heusch, 1995; Cifelli et al., 2008; Feigl,
1998; Gorman et al., 2000; Randall et al., 2003; Tsuboi et al., 2000).
Intrinsic ganglionated cardiac plexus integrates input from multiple sources
including vagal efferent and afferent neurons, extrinsic sympathetic and
spinal sensory neurons. The balance between the stimulatory sympathetic
and inhibitory parasympathetic inputs are important for control of cardiac
function (Ardell et al., 1991; Pauza et al., 1997b; Smith, 1999). It is widely
recognized that autonomic nervous system modulates the cardiac electrophysiology of the heart and influences the genesis of cardiac arrhythmias or
sudden cardiac death (Racker and Kadish, 2000).
The recent elucidation of the complete mouse genome in combination
with transgenesis and gene targeting in embryonic stem cells have opened
excellent opportunities to breed numerous genetically modified mouse lines
for experimental modeling and investigation of molecular mechanisms of
the role of sympathetic-parasympathetic imbalance in cardiac arrhythmia
predisposition (Kanazawa et al., 2010; Koentgen et al., 2010). The mouse,
as an animal model, is widely used in cardiovascular researches. It have
been developed transgenic mouse models, in which specific genes involved
in cardiac development, are modified (Feintuch et al., 2007). It is reported
that neuronal populations within the intrinsic cardiac neurons of adult mice
and human are competently comparable as they exhibit a similar neurochemical phenotype manifested predominantly by choline acetyl-transferase
and tyrosine hydroxylase (Mabe et al., 2006). Morphologically, the intrinsic
cardiac nervous system corresponds to the neural ganglionated plexus,
which is frequently subdivided according to layers of heart wall into epicardial, myocardial and endocardial (Marron et al., 1995). Cardiac neuroanatomical investigations have demonstrated that intrinsic cardiac neural
plexus may be considered as a complex of distinct ganglionated subplexuses
(Pauza et al., 2000). Intrinsic ganglia related to particular subplexuses are
distributed at specific atrial or ventricular regions around the sinuatrial node,
the roots of caval and pulmonary veins, and near the atrioventricular node
(Arora et al., 2003; Batulevicius et al., 2008; Pauza et al., 2002b; Pauza et
al., 2000). In contrast to human and very few species of laboratory animal
hearts, neuroanatomy of the mouse heart was rather poorly examined for a
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long while. In spite of very few investigations to this date (Ai et al., 2007;
Hoard et al., 2008; Hoard et al., 2007; Mabe et al., 2006; Maifrino et al.,
2006), both the distribution of sympathetic, vagal and sensory nerve fibers
within the mouse intrinsic cardiac nerve plexus was almost unknown.
Aim and Objectives
The aim of the present study was to determine the structural organization of the intrinsic neural plexus in the total, non–sectioned mouse heart,
identifying the immunohistochemistry of nerve fibers and neurons located
within this intrinsic neural plexus.
The objectives of the study:
1. To seek out the neural sources and neural pathways, by which
mediastinal nerves supply the mouse heart.
2. To ascertain the structural organization of the mouse cardiac neural
plexus.
3. To assess the ganglion size and the number of ganglionic cells in the
mouse hearts in order to compare this animal model with others.
4. To identify the distribution of cholinergic, adrenergic and peptidergic
neural structures in the whole-mount mouse heart preparations using
double immunohistochemical labeling.
Originality and Implications
This is the first detailed anatomical investigation of intrinsic ganglionated nerve plexus in the total (i.e., non-parceled and non-sectioned) mouse
heart. This study demonstrates for the first the distribution of cholinergic,
adrenergic and peptidergic nerve fibers and neurons in the whole-mount
preparation of the mouse heart. The technique of the whole-mount preparation allows precise identifying and mapping of the all intrinsic cardiac
ganglia. We also identified their immunohistochemical properties and interconnections of intrinsic cardiac neurons within the atria, interatrial and
interventricular septa. This study mapped in detail the distribution of the
mouse intrinsic cardiac nerves and ganglia and this may help in attempts to
stimulate and/or to ablate selectively the functionally distinct intrinsic neural
pathways for investigations of arrhythmic heart. The neuroanatomy of the
mouse heart demonstrated in this study facilitates further investigations with
this animal model and, thereby, it should increase our knowledge of
physiologic roles of distinct intrinsic nerves and individual intrinsic ganglia.
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1. REVIEW OF LITERATURE
1.1. Innervation of the Heart
Parasympathetic innervation of the heart is carried by the Xth cranial
(vagus) nerve, which originates in the medulla oblongata. In the human, it is
the superior, inferior, and thoracic branches of the vagus nerve that
innervate the heart (Kawashima, 2005). The nerves supplying the heart join
in the cardiac plexus, an accumulation of mixed neurons located cranial and
dorsal to the heart. In the human heart, left and right-sided cardiac plexuses
surround the brachiocephalic trunk and the aortic arch, respectively, and
form part of a larger cardiac plexus that lies between the aorta and pulmonary trunk (Kawashima, 2005; Pauza et al., 1997a; Pauza et al., 2000).
Nuclei in the ventral lateral medulla project via the vagus nerve to
postganglionic neurons in the cardiac nerve plexus. Physiological, viraltracing, and degeneration studies showed that neurons of the dorsal motor
nucleus of the vagus (DVM) and of the nucleus ambiguus (NA) innervate
the heart (Standish et al., 1994). Furthermore, it has been shown that the
ganglion cluster located at the left atrium adjacent to the inferior vena cava
receives input from both the DVM and NA, (Massari et al., 1995; Standish
et al., 1995) whereas, the ganglion cluster located at the right pulmonary
vein-left atrial junction receives fibers from the NA only (Massari et al.,
1994).
A submacroscopic anatomical investigations of the human extrinsic
cardiac nervous system showed that (1) the superior, and middle cervical,
and the cervicothoracic (stellate) ganglia, composed of the inferior cervical
and 1st thoracic ganglia, were mostly consistent sources of symphatetic
imputs to the heart; (2) the superior, middle, and inferior cardiac nerves
innervated the heart by simple following the descent great arteries; (3) the
thoracic cardiac nerve in the posterior mediastinum followed a complex
course because of the long distance to the middle mediastinum; (4) the
cranial cardiac nerve and branch tended to distribute into the heart medially,
and the caudal cardiac nerve and branch tended to distribute into the heart
laterally; (5) the mixing positions (cardiac plexus) of the sympathetic
cardiac nerve and the vagal cardiac branch, as well as the definitive
morphology of brachial arteries with the recurrent laryngeal nerves, tended
to differ on both sides (Kawashima, 2005). The dorsal right atrial, right
ventral and middle dorsal subplexuses of the sheep intrinsic cardiac nervous
system (ICNS) receive the main extrinsic neural input from the right cervicothoracic and right thoracic sympathetic T2 and T3 ganglia as well as from
the right vagal nerve. The left dorsal subplexus is supplied by sizeable
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extrinsic nerves from the left thoracic T4–T6 sympathetic ganglia and the left
vagal nerve (Saburkina et al., 2010). Convergence of inputs from extrinsic
cardiac (vagal and cardiopulmonary (CPN)) nerves on intrinsic cardiac
neurons was investigated in the pig (Smith, 1999).
There is a degree of asymmetry in the distribution of preganglionic
symphatetic neurons in the cat medulla (Massari et al., 1995; Massari et al.,
1994). Injection of a retrograde tracer into the atrioventricular (AV)
ganglion of the cat results in the labeling of twice as many cells on the left
side of the medulla as on the right side (Massari et al., 1995), whereas
injection of a retrograde tracer into the sinoatrial (SA) ganglion showed
asymmetrical distribution of labeled preganglionic neurons (Massari et al.,
1994).
Preganglionic neurons form synapses on postganglionic neurons in
autonomic ganglia, and there is a substantial degree of "convergence" in the
parasympathetic nervous system. However, there is considerable diversity in
the degree of convergence and divergence among species, also among
different autonomic ganglia and individuals of a single species (Wang et al.,
1995). The cardiac ganglion of the cat appears to be a degree of divergence
rather than convergence: one preganglionic neuron projects on average to 13
or 32 postganglionic neurons (Wang et al., 1995). Similarly, studies by
Massan and colleagues (Massari et al., 1995) indicate that a small number of
neurons in the medulla are capable of controlling significant numbers of
postganglionic cardiac neurons.
The ultrastructural development of the cardiac ganglia in the chicken
can be divided into three phases: (1) migration and aggregation of
neuroblasts on days 3.5-5; (2) differentiating ganglia, days 5-10; (3)
maturing ganglia, days 11 to hatching. The development of cholinergic mechanisms precedes that of adrenergic mechanisms. As a consequence the
parasympathetic-cholinergic control becomes functional and plays a role in
cardiac function earlier than the sympathetic-adrenal neural control
(Baptista and Kirby, 1997).
In the mouse, nerve cells are first seen in the dorsal mesocardium at 10.5
days after fertilization. Well developed nerve tracts that can be identified
using the neurofilament marker NF160D, extend through this region and
reach the heart by 12.5 days after fertilization (Hildreth et al., 2008).
Innervation of the outflow tract also occurs later in the mouse, with the first
neural elements first seen between the separating aorta and pulmonary
trunk. Staining against tyrosine hydroxylase (TH) and the parasympathetic
neuron marker vasoactive cholinesterase transporter protein (VAChT)
reveals the presence of sympathetic and parasympathetic neurons within the
main nerves innervating the arterial and venous poles of the heart at 11.5
10
days after fertilization and 12.5 days after fertilization (Hildreth et al.,
2008). Sympathetic innervation has been shown to occur at later stages
compared with parasympathetic innervation in both avian and mammalian
species (Kirby et al., 1980; Shoba and Tay, 2000). In the mouse, autonomic
efferent innervation precedes sensory innervation, as shown by the later
appearance of calcitonin gene related peptide (CGRP) positive nerve fibers
in comparison to neuropeptide Y (NPY) immunoactivity in the rat (Shoba
and Tay, 2000).
It has been proposed that cardiac parasympathetic neurons from the
DVM and the NA project their axons to the intrinsic cardiac neurons and
that the neurons from the DVM regulate cardiac inotropism, while those in
the NA are related to heart rate control (Armour, 2008; Gatti et al., 1995).
Some studies have shown the importance of the intrinsic cardiac ganglia in
modulating relay between extrinsic autonomic nerves and the nodal tissues
of the cardiac conduction system. Specifically, autonomic inputs from the
left and right vagal branches are regulated to varying degrees by different
cardiac ganglia, revealing complex interlinking pathways in the control of
heart rate. These pathways have been revealed in studies of atrial
fibrillation, which can be induced through electrical stimulation of autonomic nerves or cardiac ganglia (Patterson et al., 2005; Scherlag et al.,
2005a; Scherlag et al., 2005b). A study in the dog (Hou et al., 2007)
revealed that the right and left vagal trunks exert sympathetic control over
heart rate, but that both inputs are modulated through specific, interlinking
pathways, with these pathways differing between each node. There is,
however, a degree of variation in these pathways between different
individuals. Failure completely to attenuate the autonomic responses
following ablation of these ganglia suggested that the modulation involves
other ganglia within the cardiac network (Hou et al., 2007).
1.2. Neurotransmiters in Cardiac Innervation
Regulation of cardiac function by the autonomic nervous system plays
crucial roles in the response of the organism to external stimulation. The
sympathetic nervous system increases heart rate and the force of contraction
via effects on the function of the sarcoplasmic reticulum within the cardiomyocytes and on ion channel activity. Preganglionic sympathetic axons
from neurons in the T1-T5 spinal segments project to secondary sympathetic neurons that are located in the sympathetic chain, as well as the mediastinal and intrinsic cardiac ganglia (Armour, 2008; Horackova et al.,
1999; Kawashima, 2005; Richardson et al., 2006). Most of these sympathetic neurons contain TH, which is required for the synthesis of norepi11
nephrine (Maslyukov et al., 2006). The parasympathetic system, in contrast,
counteracts the action of the sympathetic nerves, slowing the heart rate.
Intrinsic parasympathetic neurons are primarily located within the atrial
epicardial ganglia and these neurons contain choline acetyltransferase
(Mabe et al., 2006), which is obligatory for the synthesis of acetylcholine
(ACh). Postganglionic cholinergic axons control cardiac function by
releasing ACh, which has a direct inhibitory influence on cardiomyocytes
and a prejunctional inhibitory effect on sympathetic axons (Weihe et al.,
2005). Under normal conditions, the balance between these two opposing
systems is well maintained. The local environment plays an important role
in determining the specific neurotransmitter used by presumptive autonomic
postganglionic neurons.
Most sympathetic neurons express norepinephrine (NE) as a primary
neurotransmitter, along with a range of peptides that modulate signaling.
Norepinephrine binds to a range of adrenergic receptors, broadly classified
as a and b. Under normal circumstances, a high affinity b-adrenoceptor
binds to mul-tiple G proteins, activating adenylyl cyclase to produce cAMP.
This then improves excitation-contraction coupling within the cardiac
muscle, increasing heart rate and force. Norepinephrine is produced from its
precursor, hydroxylase, dopamine b-hydroxylase, and phenylethanolamine
N-methyl transferase, all found within the cell body of sympathetic neurons.
Once produced, the NE is transported along the axon to nerve terminals
where it is stored. In all species studied to date, maturation of the
sympathetic innervation and onset of function occurs late in the fetal, or
early in the neonatal, heart. Despite this, response to catecholamines, in the
form of tachycardia, occurs well before birth, suggesting that b-adrenergic
recaptors must be present during the fetal period, perhaps as early as 9.5
days after fertilization in the mouse (Liu et al., 1999). Indeed, although few
TH positive neurons are found in the heart until late in gestation, myocardial
cells expressing TH, dopamine beta-hydroxylase, and phenylethanolamine
N-methyl transferase are found interspersed throughout the myocardium in
the mouse early in its gestation. By mid-gestation, they have localized to the
regions of formation of the sinus and atrioventricular nodes (Ebert and
Thompson, 2001). These data are supported by the finding that, following
sympathectomy, TH activity can still be detected in the chick heart (Stewart
and Kirby, 1985). Although the roles of these myocardially produced catecholamines remain unclear, the fact that the heart can respond to these factors from early in gestation suggests that they may be playing an important
role. A wide range of cotransmitters are found within the sympathetic nervous system, functioning alongside norepinephrine. Although the mechanisms underlying the specification of these cotransmitters remain unclear,
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cell lineage plays an important role. Of these cotransmitters, the adenosinergic system, using adenosine, is the earliest functionally responsive system in the rodent heart, with activation of the adenosine receptor slowing
the heart rate from 8 days after fertilization in the rat embryo (Porter and
Rivkees, 2001). Adenosine inhibits NE release from sympathetic nerve
endings and is important both as a vasodilator and for its anti-arrhythmic
properties. Similar to catecholamines, cardiac adenosine receptors are
functional well before sympathetic innervation occurs. It has been suggested
that fetal plasma may be the source early in gestation (Sawa et al., 1991).
Neuropeptide Y (NPY) is the most abundant peptide in the heart, being
found in postganglionic sympathetic neurons synapsing on postganglionic
parasympathetic neurons in the cardiac ganglia, endocardium, and
myocardium (Palmiter et al., 1998). It has been shown to inhibit release of
catecholamines and regulate the vasoconstrictor action of norepinephrine.
Neuropeptide Y has also been shown to produce vasoconstriction of
coronary vessels in many species, including human (Michel et al., 1989).
Neuropeptide Y is first seen late in development in the region of the sinus
and atrioventricular nodes and in the intrinsic cardiac ganglia. Levels
increase in the immediate postnatal period, and then remain steady
thereafter. Some studies have suggested that the peptide can produce longterm inhibition of the responses to vagal stimulation in the sinus and
atrioventricular nodes (Potter, 1987). Neuropeptide Y has been suggested to
play a crucial role as a neuromodulator in the complex interactions that take
place between different branches of the sensory and autonomic innervation
of the heart (Horst, 2000).
Acetylcholine (ACh) is the main neurotransmitter released by
preganglionic and postgangionic vagal nerve terminals. Two types of cholinergic receptor have been described, namely muscarinic and nicotinic. Muscarinic receptors are the main type found on cardiac effector cells, whereas
nicotinic receptors are found on intrinsic parasympathetic neurons. Acetylcholine is mainly synthesized at the nerve endings where it is stored until
release (Loffelholz and Pappano, 1985). Acetycholine can generally be
detected in the heart at an earlier stage than can norepinephrine, reflecting
the earlier onset of parasympathetic innervation than sympathetic ingrowth
(Pappano, 1977).
Vasoactive intestinal peptide (VIP) is a peptidergic cotransmitter in
cholinergic parasympathetic neurons. Neurons expressing this peptide are
localized primarily in relation to blood vessels in microvascular beds (Della
et al., 1983), and to the sinus and atrioventricular nodes and coronary
vessels (Weihe et al., 1984). The peptide has direct effects on heart rate. In
the dog, it has been reported to be twice as potent as norepinephrine in
13
increasing heart rate (Rigel, 1988). It has also been suggested to modulate
the activity of acetylcholine and norepinephrine (Ferron et al., 1985).
Somatostatin immunoreactivity is associated with intramural parasympathetic neurons, localizing to nerve fibers in the myocardium, endocardium, and the conduction system. The actions of somatostatin, slowing
the heart rate and decreasing cardiac output, closely resemble those caused
by vagal stimulation. It exerts its suppressing cardiac effects by acting on
calcium channels excited by b-adrenoceptor activity (Diez et al., 1985)
and/or by inducing the release of acetylcholine from intracardiac parasympathetic neurons (Wiley et al., 1989), but may also modulate sympathetic neurotransmission by acting at a postjunctional site.
The neurotransmitters substance P (SP) and calcitonin gene-related
peptide (CGRP) act on the heart via the sensory-motor pathway. Both
transmitters are stored in granules within the nerve terminal, are coreleased
on stimulation, and interact with parasympathetic nerves (Armour et al.,
1993). SP-containing nerve fibers are found surrounding blood vessels
within the myocardium, and within the sinus and atrioventricular nodes
(Crick et al., 1994). Substance P, a potent vasodilator, although it has no
effect on heart rate, may control parasympathetic activity (Corr, 1992).
Calcitonin gene-related peptide is scarce in the human heart (Crick et al.,
1994). It is a potent vasodilator of peripheral blood vessels, including the
coronary arteries. In the mouse, it is expressed in sensory nerves within the
heart, appearing around the time of birth. These, with other data, suggest
that the sensory innervation of the heart occurs later than autonomic
innervation (Gordon et al., 1993).
The myocardium also secretes endocrine factors that regulate the cardiac
nervous system. The two most abundantly expressed proteins are the atrial
and brain natriuretic peptides, which exert an inhibitory effect on sympathetic input to the heart. These factors are mainly expressed in the atria,
although they are also expressed in other regions of the heart. They have
additional roles, such as diuretic and natriuretic homeostatic function in the
kidney, and inhibitory effects on the renin-angiotensin-aldosterone system
(McGrath and de Bold, 2009; McGrath et al., 2005). A number of other
nonadrenergic noncholinergic transmitters have been localized within the
heart of various species, including opioids, neurotensin, and peptide
histidine isoleucine, albeit that their actions remain poorly defined.
1.3. Physiology of Intrinsic Cardiac Neurons
It has been shown that neurons with distinct electrophysiological
behavior differ in their number of dendritic processes and patterns of
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synaptic inputs (Edwards et al., 1995; Klemm et al., 1997). Many cardiac
neurons are synaptically coupled to each other and/or excited by stimulation
of extrinsic nerves. However, approximately 10% of intrinsic cardiac
neurons appear to lack a synaptic input as determined by staining wholemount preparations of guinea pig cardiac plexus with antibodies to
synaptophysin (Klemm et al., 1997).
Histological studies have shown that a complex network of neurons
exists within the mammalian cardiac ganglion. Intrinsic parasympathetic
neurons are not the only neurons present and there is evidence suggesting
that sensory neurons, interneurons, and efferent neurons are found in
intracardiac ganglia. Furthermore, the presence of sympathetic fibers
(Smith, 1999) and afferent nerve fibers (Hardwick et al., 1995) suggests that
there is potential for interaction between these elements. Sympathetic nerve
terminals are often found in close proximity to parasympathetic nerve
terminals (Randall et al., 1965). This anatomical arrangement of sympathetic and parasympathetic nerve terminals makes it possible for
transmitters released from the nerve terminals of one division to diffuse
readily to terminals of the other division, as well as to cardiac muscle cells.
Small intensely fluorescent (SIF) cells, which contain catecholamines, have
also been shown to be present within mammalian intracardiac ganglia
(Hassall and Burnstock, 1986; Jacobowitz et al., 1967; Seabrook et al.,
1990). Therefore, both the soma and axon terminal of parasympathetic
neurons may be under the physiological influence of catecholamines.
Sympathetic and parasympathetic (vagal) nervous systems exert
antagonistic effects on the heart and interaction between the two systems is
well established (Levy, 1971). In addition to parasympathetic efferent and
sensory afferent neurons, there also exists a population of interneurons
within the cardiac ganglia. The axons of these cells may reside within its
particular ganglion or travel out into another ganglion cluster. This
arrangement has been found in most species, including the dog (Xi et al.,
1991), guinea pig (Steele et al., 1996), and rabbit (Papka, 1976). Interneurons within cardiac ganglia mediate lateral interactions between various
ganglion neurons and allow a convergence of different inputs. Electrical
stimulation of the stellate ganglion or the vagosympathetic trunk produces
responses in ganglion cells, with variable latencies indicating polysynaptic
connections (Gagliardi et al., 1988).
Visceral cardiac afferent neurons also exist in cardiac ganglia, which are
identified by the presence of the neuropeptides SP and CGRP (FrancoCereceda, 1988). The presence of these afferents in close association with
parasympathetic neurons has led to postulation of the existence of a local
reflex circuit within the heart. Spontaneous activity has been demonstrated
15
in canine cardiac neurons (Gagliardi et al., 1988). The spontaneous firing is
entrained to events in the cardiac and respiratory cycles even with
disconnection from the central nervous system, although the amount of
activity is substantially less. These observations strongly suggest that
cardiac afferent fibers transmit cardiopulmonary information to ganglion
cells in a local reflex arc as well as through higher centers.
The sensory neurons associated with cardiac function have been
identified inside the nodose, C1-T4 dorsal root, mediastinal and intrinsic
cardiac ganglia (Armour, 2008; Foreman, 2007; Hopkins and Armour,
1989; Horackova et al., 1999). Recent findings suggest that intrinsic sensory
cardiac neurons are also involved in local neural circuits via their axonal
projections to efferent neurons distributed within the same or neighboring
intrinsic ganglia (Armour, 2008). Possibly, this diversity of neurons
composes an integrative neuronal network, which modulates extrinsic
autonomic projections to the heart and mediates local cardiac reflexes
(Armour, 2008; Armour and Ardell, 1994). In addition, the integrative
function of the intrinsic cardiac neurons is under the tonic influence of
neurons from the insular cortex, brainstem and spinal cord (Armour, 2008;
Armour and Ardell, 1994). To define sensory neuronal subpopulations,
immunohistochemistry for CGRP and SP are commonly used in recent
anatomical investigations. These both neuropeptides have been employed to
identify peptidergic class of nociceptors, although CGRP is clearly
expressed in some non-nociceptive neurons as well (Lawson et al., 2002;
Lawson et al., 1996; Lawson et al., 1993). Although SP is considered as a
pain transmitter, receptors for this neuropeptide are expressed not only on
neurons, but also on the surface of cardiomyocytes, endothelial cells and
immunocytes, such as lymphocytes and macrophages (Church et al., 1996;
Cook et al., 1994; Goode et al., 1998; Ho et al., 1997). Furthermore, it was
demonstrated that SP contributes to a dilated cardiomyopathy and is
essential for the pathogenesis of encephalomyocarditis viral myocarditis
(D'Souza et al., 2007; Robinson et al., 2009). Both the CGRP and SP play a
role of counter regulator in hypertension and coronary flow (Robinson et al.,
2009).
1.4. Intrinsic Cardiac Nerve Plexus
The extensive network of neuronal cell bodies receiving parasympathetic vagal input and comprising the intrinsic cardiac ganglia of the
mammalian heart has long been known (Meiklejohn, 1914; Woollard,
1926). These ganglia could well represent the final common pathway
through which the diverse, extrinsic neural signals to the heart are modified
16
before being transmitted to the effector tissues, yet the precise function of
the network and the way in which it mediates vagal input are largely
unknown. A detailed description of the location, distribution, and projections of the intracardiac ganglia has been provided for the heart in
numerous ma-mmalian species (Armour et al., 1997; Arora et al., 2003;
Baptista and Kirby, 1997; Batulevicius et al., 2003; Batulevicius et al.,
2004; Batulevicius et al., 2005; Batulevicius et al., 2008; Ellison and Hibbs,
1976; Hoover et al., 2004; Hoover et al., 2009; Horackova et al., 1999;
Horackova et al., 2000; Yuan et al., 1994; King and Coakley, 1958; Leger et
al., 1999; Moravec and Moravec, 1984; Pardini et al., 1987; Parsons et al.,
1987; Pauza et al., 1997a; Pauza et al., 2002b; Pauza et al., 1999; Pauza et
al., 2000; Pauza et al., 1997b; Pauziene et al., 2000a; Pauziene et al., 2000b;
Saburkina and Pauza, 2006; Saburkina et al., 2009; Saburkina et al., 2010).
Cardiac ganglia are located in epicardium and within the myocardium or endocardium (Batulevicius et al., 2003; Batulevicius et al., 2005; Pauza et al.,
2002a; Pauza et al., 2000; Saburkina et al., 2010). Whereas most of the ganglia are epicardial, the septal ganglia are located on the inner surface of the
atria. Cardiac ganglionic cells located in the right atrium are associated with
control of the sinoatrial node and neurons in the region of the inferior vena
cava modulate AV conduction (Armour and Ardell, 1994). The exact anatomical distribution of the intracardiac ganglia varies among species (Pauza et
al., 2002a).
Cardiac ganglia are distributed in different regions of the atria in a
number of mammalian species, surrounding the SA node, around the roots
of the vena cava and pulmonary veins, interatrial septum, and in the
proximity of the AV node. A typical cardiac ganglion consists of neurons,
satellite cells and SIF cells. The mammalian cardiac ganglia contain
unipolar, bipolar and multipolar neurons with differing dimensions and
shapes. The neurons and satellite cells of the cardiac ganglia originate from
neural crest cells that migrate to the heart. Upon arriving in the outflow tract
the cells segregate into parasympathetic neurons and supporting cells to
form the cardiac ganglia (Baptista and Kirby, 1997).
According to Armour et al. (1997) the human intrinsic cardiac nerve
plexus is distributed extensively, most of its ganglia being located on the
posterior surfaces of the atria and superior aspect of the ventricles. Atrial
ganglia in the human heart were identified on 1) the superior surface of the
right atrium, 2) the superior surface of the left atrium, 3) the posterior
surface of the right atrium, 4) the posterior medial surface of the left atrium
(the latter two fuse medially where they extend anteriorly into the interatrial
septum), and 5) the inferior and lateral aspect of the posterior left atrium.
Ventricular ganglionated plexuses were located in fat 1) surrounding the
17
aortic root, 2) at the origins of the right and left coronary arteries (the latter
extending to the origins of the left anterior descending and circumflex
coronary arteries), 3) at the origin of the posterior descending coronary
artery, 4) adjacent to the origin of the right acute marginal coronary artery,
and 5) at the origin of the left obtuse marginal coronary artery (Armour et
al., 1997). Pauza et al. (2000) concluded that the human heart is innervated
by seven subplexuses: the right atrium was innervated by two subplexuses,
the left atrium by three, the right ventricle by one, and the left ventricle by
three subplexuses. The highest density of epicardiac ganglia was identified
near the heart hilum, especially on the dorsal and dorsolateral surfaces of the
left atrium, where up to 50% of all cardiac ganglia were located (Pauza et
al., 2000). The study of epicardiac ganglia in the human fetuses showed that
the topography and structural organization of epicardiac neural plexus were
typical for hearts of adult human. The largest ganglion number comprising
77% of all counted ganglia was identified on the dorsal atrial surface. Fetal
epicardiac plexus in gestation period of 15-40 weeks contained 929±62
ganglia (Saburkina and Pauza, 2006).
A three-dimensional description of the distribution and organization of
the canine intrinsic cardiac nervous system was developed in order to
characterize its full extent physiologically. Collections of ganglia associated
with nerves, i.e., ganglionated plexuses, were identified in specific locations
in epicardial fat and cardiac tissue. Distinct epicardial ganglionated plexuses
were consistently observed in four atrial and three ventricular regions, with
occasional neurons being located throughout atrial and ventricular tissues.
One ganglionated plexus extended from the ventral to dorsal surfaces of the
right atrium. Another ganglionated plexus, with three components, was
identified in a fat on the left atrial ventral surface. A ganglionated plexus
was located on the mid-dorsal surface of the two atria, extending ventrally
in the interatrial septum. A fourth atrial ganglionated plexus was located at
the origin of the inferior vena cava extending to the dorsal caudal surface of
the two atria. On the cranial surface of the ventricles a ganglionated plexus
that surrounded the aortic root was identified. This plexus extended to the
right and left main coronary arteries and origins of the ventral descending
and circumflex coronary arteries. Two other ventricular ganglionated
plexuses were identified adjacent to the origins of the right and left marginal
coronary arteries (Yuan et al., 1994). Later on, it was performed study on
whole canine hearts to highlight the differences of intrinsic neural plexus
(INP) in dog and human. Pauza et al. (2002b) identified 13 locations
between the canine ascending aorta and pulmonary trunk, around the pulmonary veins, and on every side of the superior vena cava, through which
mediastinal cardiac nerves accessed the canine heart. Intrinsic nerves from
18
these locations extended within the canine epicardium by seven neuronal
subplexuses. Intrinsic nerves and ganglia were found to be widely
distributed in topographically consistent atrial and ventricular regions. The
canine right atrium, including the sinoatrial node, was innervated by two
subplexuses, the wall of the left atrium by three, and the right and left
ventricles by two subplexuses. Depending on the age of the animal, the
number of intrinsic ganglia per one canine heart might range from 400 up to
1,500. By taking into account the ganglion size and potential approximate
number of neurons residing inside a ganglion of a certain size, it was
estimated that on average about 80,000 intrinsic neurons are associated with
the canine heart. A comparative analysis of the morphological patterns of
the canine and human intrinsic cardiac neural plexuses showed that the
topography of these plexuses may be considered as quite similar, but the
structural and quantitative differences of the intrinsic cardiac neural
subplexuses between dogs and humans are significant (Pauza et al., 2002a;
Pauza et al., 2002b; Pauza et al., 1999; Pauza et al., 2000).
Sheep is routinely used in experimental cardiac electrophysiology and
surgery, however it is noted a possible distinct neural control of the
ventricles in the human and sheep hearts (Saburkina et al., 2010). Intrinsic
cardiac nerves extend from the venous part of the ovine heart hilum along
the roots of the cranial (superior) caval and the left azygos veins to both
atria and ventricles via five epicardial routes: the dorsal right atrial, middle
dorsal, left dorsal, right ventral, and ventral left atrial nerve subplexuses.
Intrinsic nerves proceeding from the arterial part of the heart hilum along
the roots of the aorta and pulmonary trunk extend exclusively into the
ventricles as the right and left coronary subplexuses. Sheep hearts contained
an average of 769±52 epicardial ganglia. Cumulative areas of epicardial
ganglia on the root of the cranial vena cava and on the wall of the coronary
sinus were the largest of all regions (Saburkina et al., 2010). Despite
substantial interindividual variability in the morphology of ovine epicardiac
neural plexus (ENP) right-sided epicardial neural subplexuses supplying the
sinuatrial and atrioventricular nodes are mostly concentrated at a fat pad
between the right pulmonary veins and the cranial vena cava. This finding is
in sharp contrast with a solely left lateral neural input to the human
atrioventricular node, which extends mainly from the left dorsal and middle
dorsal subplexuses (Pauza et al., 2000; Saburkina et al., 2010).
The nerves entered the porcine epicardium at five sites: (1) ventromedially to the origin of the superior vena cava, (2) dorsally to the origin of
the superior vena cava, (3) among the pulmonary veins, (4) dorso-medially
to the origin of the left azygos vein, and (5) ventrally to the left pulmonary
vein (Batulevicius et al., 2008). Numerous ganglia and interconnecting
19
nerves were found to be concentrated in an epicardial fat in five atrial and
six ventricular regions of the porcine heart (Arora et al., 2003). The five
atrial ganglionic fields were identified (1) the ventral right atrial, (2) the
right vena cava-right atrial, (3) the dorsal atrial, (4) the interatrial septal, and
(5) the left superior vena cava-left atrial ones. Six ventricular ganglionic
fields were identified in close proximity to the (1) roots of the aorta and
pulmonary artery (craniomedial), extending along the left main coronary
artery to the (2) ventral interventricular and (3) circumflex coronary arteries.
(4) ganglionic fields were also identified around the origin of the dorsal
interventricular coronary artery as well as the (5) right main and (6) right
marginal coronary arteries. Isolated neurons were identified scattered
throughout the cranial interventricular septum (Arora et al., 2003). Other
investigators divided the porcine ENP according to the neural pathways
(subplexuses): the left atrium received nerves by four subplexuses, left
ventricle by three subplexuses, right atrium and right ventricle each by two
subplexuses (Batulevicius et al., 2008). The estimated total number of the
intrinsic ganglia per porcine heart was 362±52. About 55% of ganglia per
porcine heart were accumulated on the left atrium while 36% on the right
atrium. The percentage of ganglia within the porcine ventricular and paraaortic regions was 7.6% and 1.6%, respectively (Batulevicius et al., 2008).
The nerves entering the guinea pig heart were found both in the arterial
and venous part of the heart hilum (Batulevicius et al., 2005). The nerves
from the arterial part of the heart hilum proceeded into the ventricles, but
the nerves from the venous part of the hilum formed a nerve plexus of the
cardiac hilum located on the heart base. Within the guinea pig epicardium,
intrinsic nerves divided into six routes and proceeded to separate atrial,
ventricular and septal regions (Batulevicius et al., 2005). The intracardiac
neurons from adult guinea pigs were amassed within 329±15 ganglia. The
hearts of young guinea pig contained significantly fewer ganglia, only
211±27 (Batulevicius et al., 2005). Of all identified neurons in the guinea
pig heart, 85-90% were located in ganglia (ganglionic neurons), the rest
being isolated (individual neurons) (Horackova et al., 1999). The
remarkable similarity was found in the architecture of the intracardiac nerve
plexuses between guinea pig and rat (Batulevicius et al., 2003; Batulevicius
et al., 2004; Batulevicius et al., 2005). Extrinsic nerves entered the rat heart
in both the arterial and venous parts of the cardiac hilum. Extracardiac
nerves entering the rat heart were found amid aorta and pulmonary trunk as
well as along both right and left cranial veins. The nerves from the arterial
part of the heart hilum extended directly to the ventricles but the nerves
from the venous part of the hilum interconnected among themselves
forming a nerve plexus of the cardiac hilum on the heart base. Within the rat
20
epicardium, intrinsic nerves clustered into six routes by which they
selectively projected to different rat heart regions. Ventral wall of the
ventricles was supplied by three neural subplexuses, dorsal ventricular wall
by one subplexus; each atrium received nerves from two distinct
subplexuses (Batulevicius et al., 2004). Also, the distributions of cardiac
ganglia and vagal efferent projections to cardiac ganglia in mice and rats
were quite similar both qualitatively and quantitatively (Ai et al., 2007).
Using the tracer into the left NA, cardiac ganglia of different shapes and
sizes were marked in the sinoatrial (SA) node, atrioventricular (AV) node,
and lower pulmonary vein (LPV) regions on the dorsal surface of the atria.
In each region, several ganglia formed a ganglionated plexus. The plexuses
at different locations were interconnected by nerves. Vagal efferent fibers
ramified within cardiac ganglia, formed a complex network of axons, and
innervated cardiac ganglia with very dense basket endings around individual
cardiac principal neurons (Ai et al., 2007).
The human intrinsic cardiac ganglia (ICG) range in size from those
containing a few neurons to large ganglia measuring up to 0.5 x 1 mm
(Armour et al., 1997). The human heart was estimated to contain more than
14,000 neurons. Neuronal somata varied in size and shape. Many axon
terminals in intrinsic cardiac ganglia contained large numbers of small,
clear, round vesicles that formed asymmetrical axodendritic synapses,
whereas a few axons contained large, dense-cored vesicles (Armour et al.,
1997). The canine intrinsic cardiac nervous system contains a variety of
neurons interconnected via plexuses of nerves. Intrinsic cardiac ganglia
range in size from ones comprising one or a few neurons along the course of
a nerve to ones as large as 1 x 3 mm estimated to contain a few hundred
neurons. Intrinsic cardiac neuronal somata vary in size and shape, up to 36%
containing multiple nucleoli. Electron microscopic examination
demonstrated typical autonomic neurons and satellite cells in intrinsic
cardiac ganglia (Yuan et al., 1994). Many of their axon profiles contained
large numbers of clear, round, and dense-core vesicles. Asymmetrical
axodendritic synapses were common (Yuan et al., 1994).
Approximately 3,000 neuronal somata were estimated to compose
intrinsic cardiac nervous system (ICNS) of the pig (Arora et al., 2003).
Some ganglia contained more than 100 neurons. Neuronal somata had
dimensions of roughly 33.1 (short axis) by 46.3 (long axis) microms (Arora
et al., 2003). Other authors calculated, that on average, the porcine heart
contained about 12,000 intrinsic neurons (Batulevicius et al., 2008).
An estimate number of neuronal somata in guinea pig individual ganglia were 100-300 (Horackova et al., 1999). The total number of the intracardiac neurons estimated per atria was 1,510±251 (Leger et al., 1999), in
21
the whole heart were counted 2,321±215 neurons, and this number did not
differ significantly between young and adult animals. In adult guinea pigs
approximately 60% of the intracardiac neurons were distributed within ganglia of not more than 20 neurons, but the ganglia of such size accumulated
only 45% of the neurons in young animals (Batulevicius et al., 2005).
Ganglia in the guinea pig heart contained three sub-populations of neurons:
approximately 80% of ganglionic neurons were large – 15-40 microm diameters, approximately 20% had smaller diameters - less than 15 microm and
5% of ganglionic neurons were small – less than 20 microms (Horackova et
al., 1999).
The total number of intrinsic cardiac neurons in old rats was 6,576±317
(Batulevicius et al., 2003). The juvenile animals contained significantly
fewer such neurons, only 5,009±332. Approximately 70% of all intracardiac
neurons were amassed within the heart hilum, while 30% of the neurons
were distributed epicardially. Within the interatrial septum, only 11±11
neurons were identified in the juvenile and 6±4 neurons in old rats
(Batulevicius et al., 2003).
The morphological organization and structure-function correlation of
mammalian intracardiac ganglion cells using conventional intracellular
microelectrode techniques were applied to the tissue whole mount preparation of the canine intracardiac ganglia (Xi et al., 1991). Somata were elongated (mean 62 x 40 microns) and had 2-12 primary dendrites restricted
within the ganglion. Almost half of the neurons had either a short axon that
was traced only within the ganglion or no axon distinguishable. Authors
suggest that these neurons may have been intraganglionically active
neurons. The other cells had a long axon that either coursed out of the
ganglion to peripheral cardiac tissue or exited the ganglion via interganglionic nerve to innervate more remote cardiac tissue or cells in other intracardiac ganglia. Interaction between neurons was suggested by the close
proximity of processes from different neurons. Previously defined electrophysiological cell types (R-, S-, and N-cells), which were significantly
different in their passive and active membrane properties, had different morphological features of the somata but not the axonal or dendritic processes.
Intraganglionic or long axon neurons were not associated with a particular
electrophysiological cell type (Xi et al., 1991). Most neuronal somata of
porcine intrinsic cardiac nervous system were multipolar, a small population
of unipolar neurons being identified in atrial and ventricular tissues (Arora
et al., 2003). A morphological study of neurons in the nerve plexus that lies
beneath the pulmonary arteries on the myocardium of the left atrium of rats
and guinea pigs revealed at least two major types of neuron: unipolar
(61.2%) and multipolar (38.8%). The neuron somata exhibit no significant
22
difference in their length or width (Pauza et al., 1997b). Classification of
nerve cells in the terminal plexus of the rat AV junction according to their
three-dimensional (3-D) morphology confirmed that they could be divided
into three categories: (1) large unipolar neurons with axonal projections
directed toward the interventricular junction, (2) large unipolar or bipolar
neurons, and (3) small multipolar interneurons (Moravec and Moravec,
1998). It is demonstrated that there are diverse populations of cardiac
ganglion cells in the guinea pig and that some of these neurons may act as
interneurons within the intrinsic cardiac plexuses (Steele et al., 1994).
Neurons from adult mouse heart, maintained in culture, were primarily
unipolar, and 89% had prominent neurite outgrowth after 3 days. Many
neurites formed close appositions with other neurons and nonneuronal cells.
Neurite outgrowth was drastically reduced when neurons were kept in
culture with a majority of nonneural cells eliminated (Hoard et al., 2007).
1.5. Immunohistochemical Characterization of Intrinsic Cardiac
Neurons
The studies of autonomic ganglia have shown that specific combinations
of neuropeptides and other potential neurotransmitters distinguish different
functional types of neurons (Hoover et al., 2009). Therefore it is highly
likely that vagal transmission in the heart is modified by sympathetic,
sensory and intrinsic neurons and those cardiac ganglia are complex
integrators of convergent neuronal activity rather than simple relays. The
findings demonstrate that the human intrinsic cardiac neuronal somata has a
complex neurochemical anatomy, which includes the presence of a dual
cholinergic/nitrergic phenotype for most of its neurons, the presence of
noradrenergic markers in a subpopulation of neurons, and innervation by a
host of neurochemically distinct nerves (Hoover et al., 2009). Most human
intrinsic cardiac neuronal somata displayed immunoreactivity for the
cholinergic marker ChAT and the nitrergic marker neuronal nitric oxide
synthase (NOS). A subpopulation of intrinsic cardiac neurons also stained
for noradrenergic markers. While the most intrinsic cardiac neurons
received cholinergic innervation evident as punctate immunostaining for the
high affinity choline transporter (CHT), some lacked cholinergic inputs
(Hoover et al., 2009). AChE and TH-immunoreactive (IR) cell bodies were
observed in the atrial ganglionated plexuses in the pig heart (Crick et al.,
1999a). Experiments demonstrated that guinea pig cardiac ganglia contain
prominent pericellular baskets of varicose nerve terminals of sympathetic
and sensory origin (Steele et al., 1994). The results indicate that all
postganglionic neurons in guinea pig cardiac ganglia are likely to utilize
23
acetylcholine as a neurotransmitter, regardless of their functional role in
circuitry of cardiac innervation, and each of these neurons is likely to
receive cholinergic input (Mawe et al., 1996).
The patterns of coexistence of neurochemicals in guinea pig cardiac
ganglion cells were examined using a multiple-labelling immunohistochemistry. Many neurons were found to contain the somatostatin immunoreactivity with various combinations of immunoreactivity for SP, and NOS
(Mawe et al., 1996; Steele et al., 1994), also neurons were protein gene
product 9.5 (PGP 9.5) immunoreactive, exhibiting ChAT, TH, NPY, VIP
immunoreactivity (Horackova et al., 1999; Parsons et al., 2006). The study
of distribution of cholinergic and adrenergic neurons in whole-mount preparations of the guinea pig atria showed that all neuronal somata expressing
PGP 9.5-IR also expressed ChAT-IR, suggesting that these neurons were
cholinergic. No neuronal somata expressed TH-IR or contained detectable
amines but these elements were expressed by somata of small cells
throughout the atria, primarily associated with ganglia (Leger et al., 1999).
Whereas, previously it was reported that immunoreactivity for ChAT was
also observed in a large proportion of the small TH-IR neurons that exist in
guinea pig cardiac ganglia (Mawe et al., 1996). The cell bodies of the rat
intramural ganglion cells localized between the right and left branches of the
bundle of His were TH-IR and dopamine beta hydrohylase immunoreactive
(DBH-IR) (Moravec et al., 1990).
The neurochemistry of intracardiac neurons of the rat intrinsic ganglia
was investigated in whole-mount preparations. This technique allowes to
study the morphology of ganglionated nerve plexus found within the atria as
well as of individual neurons. The results of this study indicated a moderate
level of chemical diversity within the intracardiac neurons of the rat. Such
chemical diversity may reflect functional specialisation of neurons in the
intracardiac ganglia (Richardson et al., 2003). The VIP, CGRP, pituitary
adenylate cyclase-activated peptide (PACAP), SP and TH immunoreactivity
was observed in nerve fibres within the ganglia, but never in neuronal
somata of the rat cardiac ganglia (Richardson et al., 2003). Catecholaminehandling intrinsic ganglion neurons were observed as SIF cells and largediameter neurons (Slavikova et al., 2003). The SIF cells are most probably
dopaminergic and serotonergic neurons, whereas large-diameter intrinsic
cells seem to represent a subpopulation of norepinephrine – and/or epinephrine-secreting neurons (Slavikova et al., 2003). Moravec and colleagues
investigations show that ganglion cells of sulcus terminalis as well as the
epicardial ganglia enclosed between the superior vena cava and ascending
aorta are VIP-IR and TH-negative, but NPY-IR and DBH-IR (Moravec et
al., 1990).
24
To defined the basic phenotypic properties of the dissociated neurons,
cells from adult mouse heart were maintained in culture (Hoard et al., 2007).
All neurons in coculture showed immunoreactivity for a full complement of
cholinergic markers, but about 21% also stained for tyrosine hydroxylase
(Hoard et al., 2007). Disruption of cholinergic function in diabetic mice
cannot be attributed to a loss of cardiac cholinergic neurons and nerve fibers
or altered cholinergic sensitivity of the atria. Instead, the decreased responses to vagal stimulation might be caused by a defect of preganglionic
cholinergic neurons and/or ganglionic neurotransmission. The increased
density of cholinergic nerves observed at the sinoatrial node of diabetic
mice might be a compensatory response (Mabe and Hoover, 2011).
1.6. Immunohistochemistry of Nerves and Nerve Fibers in Intrinsic
Cardiac Nervous System
It is reported that the neonatal human heart possesses a rich supply of
autonomic nerves (Chow et al., 1995). The atria possess at least two populations of nerves, presumably sympathetic and vagal, whereas the walls of
the ventricles are innervated principally by presumptive sympathetic nerves
(Chow et al., 1995). Numerous PGP-IR nerves were found in the atrial
myocardium, forming perivascular plexuses and lying in close apposition to
myocardial cells. Fewer PGP-IR nerves were found amongst the myocardium of the ventricles. Both DBH-IR and TH-IR nerves demonstrated a
similar pattern of distribution as that of PGP-IR nerves; in the atria,
however, they were less numerous, while in the ventricles, their density
approximated to that of PGP-IR nerves. The density of AChE positive
nerves in the walls of the atria was less than that of PGP-IR nerves although
their distribution patterns were similar. In the ventricles, AChE positive
nerves were rarely observed (Chow et al., 1995). Later, it was reported that
there were more AChE-IR nerves in the subendocardial area than in the
subepicardial area of the myocardium. In the atrium, AChE-positive nerves
were more numerous than TH-IR nerves. By contrast, there were more THpositive nerves than AChE-positive nerves in the ventricle. Predominancy
of the distribution density at the anterior to the posterior wall of the ventricle
was observed for TH-positive nerves (Kawano et al., 2003). Moreover,
peptidergic, nitrergic, and noradrenergic nerves provided substantial
innervation of intrinsic cardiac ganglia (Hoover et al., 2009).
The predominant neural subpopulation displayed AChE activity,
throughout the endocardium, myocardium and epicardium in the pig heart.
A large proportion of nerves in the ganglionated plexus of the atrial
epicardial tissues displayed AChE activity, together with their cell bodies.
25
Tyrosine hydroxylase immunoreactive nerves were the next most prominent
subpopulation throughout the heart. Endocardial TH- and NPYimmunoreactive nerves also displayed a right to left gradient in density,
whereas in the epicardial tissues they showed a ventricular to atrial gradient
(Crick et al., 1999a).
Varicose nerve fibers that were immunoreactive for ChAT were
abundant in guinea pig cardiac ganglia, with every cardiac neuron lying in
close apposition to one or more labelled varicosities. ChAT-IR nerve fibers
were also observed in large vagosympathetic fiber bundles, in interganglionic fiber bundles, and passing individually within the myocardium
(Mawe et al., 1996). Amine- and TH- containing varicosities were also
present in guinea pig ganglia, representing potential sites for adrenergic
modulation of ganglionic neurotransmission (Leger et al., 1999). Highaffinity choline transporter (CHT) and AChE immunoreactive nerve fibers
and nerves were very abundant in the sinus and AV nodes, bundle of His,
and bundle branches. Both markers also delineated a distinct nerve tract in
the posterior wall of the right atrium. AChE-positive nerve fibers were more
abundant than CHT-IR nerves in working atrial and ventricular myocardium. CHT-IR nerves were rarely observed in left ventricular free wall.
Both markers were associated with numerous parasympathetic ganglia that
were distributed along the posterior atrial walls and within the interatrial
septum, including the region of the AV node (Hoover et al., 2004). The
distribution of CHT-IR nerve fibers and parasympathetic ganglia in the
guinea pig heart suggests that heart rate, conduction velocity, and automaticity are precisely regulated by cholinergic innervation. In contrast, the
paucity of CHT-IR nerve fibers in the left ventricular myocardium implies
that vagal efferent input has little or no direct influence on ventricular
contractile function in the guinea pig (Hoover et al., 2004).
Cholinergic innervation of the rat heart was studied using various
cholinergic markers including AChE, VAChT, choline acetyltransferase of a
peripheral type (pChAT) and the conventional form of ChAT (cChAT). The
density of pChAT-positive fibers was very high in the conducting system,
high in both atria, the right atrium in particular, and low in the ventricular
walls. pChAT and VAChT immunoreactivities were closely associated in
some fibers and fiber bundles in the ventricular walls (Yasuhara et al.,
2007). Yasuhara and colleagues indicate that intrinsic cardiac neurons
homogeneously express both pChAT and cChAT and innervation of the
ventricular walls by pChAT- and VAChT-positive fibers provides morphological evidence for a significant role of cholinergic mechanisms in ventricular functions (Yasuhara et al., 2007). Nerve fibres showing DBH-IR and
TH-IR were present in the ganglia; some of these fibres being closely
26
associated with the ganglion cells or with the cells showing TH-IR
(Forsgren et al., 1990).Cholinergic nerve fibers in the mouse heart were
abundant in the sinus and atrioventricular nodes, ventricular conducting
system, interatrial septum, and much of the right atrium, but less abundant
in the left atrium. The right ventricular myocardium contained a low density
of cholinergic nerves, which were sparse in other regions of the working
ventricular myocardium. Some cholinergic nerves were also associated with
coronary vessels (Mabe et al., 2006).
Calcitonin gene-related peptide immunoreactive nerves were the most
abundant peptide-containing subpopulation after those possessing NPY
immunoreactivity. They were most abundant in the epicardial tissues of the
ventricles of the pig heart (Crick et al., 1999a). Populations of axons containing SP were observed in guinea pig cardiac ganglia. Intrinsic axons
containing SP-IR were very rare. The regions of the heart with the densest
innervation by axons of intrinsic neurons were the cardiac valves, the atrioventricular node and the sino-atrial node (Steele et al., 1996). Nerve fibres
showing SP-IR, CGRP-IR were present in the rat ganglia; some of these
fibres being closely associated with the ganglion cells or with the cells
showing TH-IR (Forsgren et al., 1990).
1.7. Autonomic Control and Innervation of Mammalian Cardiac
Conduction System
The so-called specialized tissues within the heart are the sinus node, the
atrioventricular conduction system, and the Purkinje network (Anderson et
al., 2009). Atrioventricular (AV) nodal conduction time is known to be
modulated by the autonomic nervous system. Parasympathetic stimulation
slows conduction through the AVN via the hyperpolarizing effects of the
neurotransmitter acetylcholine (Mazgalev et al., 1986), and sympathetic
stimulation accelerates AV nodal conduction via the effects of norepinephrine. Many studies have shown that autonomic modulation of the
sinoatrial node (SAN) causes the pacemaker site to shift in many species.
Sympathetic stimulation typically induces the SAN pacemaker to shift
toward the superior vena cava, whereas parasympathetic stimulation shifts
the leading pacemaker toward the inferior vena cava (Boineau et al., 1989;
Fedorov et al., 2006; Shibata et al., 2001). Clinically, the AV junctional
rhythm is evident in patients with sick sinus syndrome, in those receiving
isoproterenol infusion, and in patients subsequent to slow pathway ablation
(Matsushita et al., 2001). In normal heart, the AVN is located near the apex
of the triangle of Koch, and has at least two inputs, each with unique
electrophysiologic properties. The atrioventricular node has electrical
27
connection between the atria and ventricles, serving as the gateway to the
His-Purkinje system (Moe et al., 1956). The specialized tissues of the AVN,
its transitional cells, and inputs are collectively termed the atrioventricular
(AV) junction (Billette, 2002). Proximal atrioventricular bundle, atrioventricular node, and distal atrioventricular bundle are distinct anatomic
structures with unique histological characteristics and innervation (Racker
and Kadish, 2000). It has long been known that the AV junction can act as a
pacemaker (Watanabe and Dreifus, 1968). The pacemaking activity of the
AV junction now has been demonstrated to reside predominantly in the slow
pathway (Dobrzynski et al., 2003), which is an AVN input located within
the isthmus between the tricuspid annulus and the coronary sinus ostium in
both rabbits and humans (Inoue and Becker, 1998; Medkour et al., 1998).
Autonomic control of the AV junction has been the subject of numerous
studies. Structurally, the distribution of sympathetic and parasympathetic
neurons, as well as the distribution of β-receptors, has been documented in
rat AV junction (Petrecca and Shrier, 1998). Functionally, autonomic modulation of AV junctional conduction properties is well documented. Rate
control during atrial fibrillation has been attempted with vagal stimulation
of the AV junction using subthreshold stimulation, which excites intracardiac neurons without causing myocyte contraction (Mazgalev et al.,
1999). The feasibility of the subthreshold stimulation has been tested with
regard to energy requirements for chronic neurostimulation in canines (Soos
et al., 2005) and catheter placement to achieve parasympathetic stimulation
in humans (Schauerte et al., 2001). Despite many structural and functional
studies, few have examined the subthreshold stimulation technique and
quantitative immunohistochemistry to investigate autonomic control and
innervation of the AV junctional pacemaker in the same preparation
(Dobrzynski et al., 2003; Hucker et al., 2007).
The morphology of the human atrioventricular node, atrioventricular
bundle and bundle branches is described by serial sections of tissue bounded
by the ostium of the coronary sinus, the pars membranacea, the septal leaflet
of the tricuspid valve and the atrial and ventricular septa (Roberts and
Castleman, 1979). Later on serial semithin and thin sections of the interatrial
septum and atrioventricular junction of adult rat were examined in light and
electron microscopes (Moravec and Moravec, 1984). It was observed rings
of specialized tissue mainly in hearts from rats, mice, and guinea pigs,
negative for connexin 43 (Cx43) but positive for hyperpolarization activated
cyclic nucleotide-gated potassium channel 4 (HCN4). Each ring takes its
origin from an inferior extension of the atrioventricular node. The rightward
ring runs around the vestibule of the tricuspid valve, whereas the leftward
ring encircles the mitral valve. On returning toward the atrial septum, the
28
tricuspid ring crosses over the penetrating part of the atrioventricular conduction system, reuniting with the mitral ring to form a superiorly located
retroaortic node. The atrioventricular conduction system itself continues
beyond the origin of the right and left bundle branches, forming an aortic
ring that ascends toward the retroaortic node but fails to make contact
because of the intervening area of aortic-to-mitral valvar fibrous continuity.
Rings of conduction tissue take their origin from inferior extensions of the
atrioventricular node, passing rightward and leftward to encircle the orifices
of the tricuspid and mitral valves and reuniting to form an extensive
retroaortic node. Thus, a ring with morphologic features justifying a definition of specialized conduction tissue surrounds the atrioventricular
junctions, although its function has yet to be established (Yanni et al.,
2009). It is generated an interactive 3D model of the atrioventricular
junctions in the mouse heart using the patterns of expression of T-box
transcription factor (Tbx3), HCN4, connexin 40 (Cx40), Cx43, connexin 45
(Cx45), and sodium channel Nav1.5, which are important for conduction
system function. Authors found extensive figure-of-eight rings of nodal and
transitional cells around the mitral and tricuspid junctions and in the base of
the atrial septum. The rings included the compact node and nodal extensions. The majority of the atrial components, including the atrioventricular
rings and compact node, are derived from the embryonic atrioventricular
canal. The atrioventricular bundle, including the lower cells of the atrioventricular node, in contrast, is derived from the ventricular myocardium.
No contributions to the conduction system myocardium were identified
from the sinus venosus, the epicardium, or the dorsal mesenchymal protrusion (Aanhaanen et al., 2010).
According to the light microscope data, two kinds of ganglia can be distinguished at the level of the interatrial septum (Moravec and Moravec,
1984). Those of the first kind are composed of large pale cells with voluminous nuclei. Those of the other kind resemble acinuslike clusters of small
osmiophilic cells. Another small ganglion is invariably associated with the
distal edge of the bundle of His. At the electron-microscope level, two types
of ganglionic cells are found in the meshes of the peri- and intranodal
plexus: 1) small neurons (10 microns) with richly developed neuropiles, and
2) large 5-OH-dopamine contrasted neurosecretory cells (up to 25 microns)
containing electron-dense vesicles typical of sympathetic neurons. Numerous glomeruli with dendrodendritic and axodendritic connections are also
found in the vicinity of the specialized tissue; and, in the nodal interstitium,
several clusters of small chromaffin cells (5 microns) and a network of
multipolar satellite cells similar to the interstitial cells of Cajal can be
distinguished (Moravec and Moravec, 1984). These data suggest that the
29
microanatomical and cytological organization of the terminal innervation of
the node of Aschoff-Tawara and of the bundle to His resembles that of the
myenteric plexus (Moravec and Moravec, 1984).
Left vagal projections to the SA and AV nodal regions of the dog course
primarily along and between the right pulmonary artery and left superior
pulmonary vein (Ardell and Randall, 1986). Right vagal projections to the
SA and AV nodal regions are somewhat more diffuse but concentrate
around the right pulmonary vein complex and adjacent segments of the right
pulmonary artery (Ardell and Randall, 1986). PGP 9.5-IR nerves were present in large numbers in the sinus node, AV node, and penetrating atrioventricular bundle, in moderate numbers in the branching bundle, and
occasionally in the bundle branches. Small numbers of DBH-IR and TH-IR
nerves were seen in the sinus and AV nodes, mainly perivascularly; there
were few in the penetrating and branching bundles and none in the bundle
branches. A few perivascular NPY-IR nerves were seen only in the sinus
node. VIP-IR, CGRP-IR, and SP-IR nerves were not seen. Pseudocholinesterase activity was found in the conduction tissue, whereas occasional
acetylcholinesterase positive nerves were found only in the sinus and AV
nodes (Chow et al., 1993). A considerable innervation of the human cardiac
conduction system is present at birth, although, by comparison with the
results of other studies on adult tissue, the mature pattern has not yet been
established. Thus, it is still in the process of maturation, especially with
regard to the acquisition of various neurotransmitters (Chow et al., 1993).
Crick and co-workers (Crick et al., 1994) analyzed the innervation of the
human conduction system and distinguished the nerve subpopulations
according to their peptide and enzyme content. Nerve fibers and fascicles
displaying immunoreactivity for PGP 9.5 were more numerous in the sinus
and atrioventricular nodes than in the penetrating bundle, bundle branches,
and adjacent myocardium. The relative density of innervation was greater in
the central region of the sinus node than in the peripheral regions. Nerve
densities were also higher in the transitional region of the atrioventricular
node compared with its compact region (Crick et al., 1994). AChE positive
nerves were the main subtype identified in the sinus and atrioventricular
nodes, representing half to two thirds of the stained area occupied by PGP
9.5 immunoreactive nerves. NPY immunoreactive nerves represented the
main peptide-containing subpopulation and occurred throughout the conduction system, displaying a similar pattern of distribution and relative density
to those demonstrating TH immunoreactivity. Nerve fibers showing
immunoreactivity for VIP, somatostatin, SP, or CGRP exhibited distinct patterns of distribution and comprised a relatively minor component of the
innervation, the percentage of stained area being 10- to 40-fold lower than
30
that occupied by NPY- and PGP 9.5-IR nerves, respectively (Crick et al.,
1994). The AV junction of the rabbit is very densely innervated with both
cholinergic and adrenergic neurons. The posterior AV nodal extension was
similar to the compact AVN as determined by morphologic and molecular
investigations. In particular, both the posterior extension and the compact
node express the pacemaking channel HCN4 and neurofilament 160
(Hucker et al., 2005). In the rabbit heart, AV junction conduction, reentrant
arrhythmia, and spontaneous rhythm are governed by heterogeneity of expression of several isoforms of gap junctions and ion channels, and these properties are regulated by the autonomic nervous system. Uniform neurofilament expression suggests that AV nodal posterior extensions are an integral
part of the cardiac pacemaking and conduction system (Hucker et al., 2005).
AChE positive and TH-IR nerves were the main subtypes identified in
guinea pig sinuatrial and atrioventricular nodes, representing 40-45% of the
stained area occupied by PGP 9.5-IR nerves. AChE-positive nerves were the
dominant subtype identified in the left and right bundle branches, but were
equal in proportion to TH-immunoreactive nerves in the penetrating bundle.
NPY-IR nerves represented the main peptide-containing subpopulation in
the nodal tissues, displaying a similar pattern of distribution and relative
density to those nerves demonstrating TH immunoreactivity (Crick et al.,
1996). Substance P and CGRP immunoreactive nerves were present
throughout the conduction system and represented the main peptidecontaining subpopulation in the ventricular conduction tissues (Crick et al.,
1996). The distribution and density of nerve subpopulations in the guinea
pig conduction system differ from those observed in the human conduction
system, which suggests that the guinea pig may be an inappropriate model
for comparative functional studies (Crick et al., 1996).
Three-dimensional (3-D) morphology of neurons of the terminal nerve
plexus of the atrioventricular junction was examined in a scanning electron
microscope. Distributions of different cell types encountered as well as their
relations to different structures of the atrioventricular specialized tissue were
also studied (Moravec and Moravec, 1998). Most neurons were found
disseminated in a thin connective tissue layer separating different segments
of the atrioventricular conductive tissue from the interventricular septum.
Sometimes, they formed small pluricellular ganglia (up to 5 neurons) but,
frequently, they occurred isolated in the terminal ramifications of the
intramural nerve plexus of specialized tissue. Some intranodal neurons
could also be identified. According to their 3-D morphology, nerve cells of
the perinodal ganglionated plexus were divided into three categories (1)
large unipolar neurons were scattered throughout the atrioventricular
junction. Their long and thin axonal projections were often directed towards
31
the interventricular septum. (2) Large pseudounipolar or bipolar neurons
were located at a few specific loci, namely all along the bundle of His and
its bifurcation into the right and left bundle branches. Frequently, they
occurred solitary and immersed amongst strands of surrounding muscle
cells. Only occasional synaptic impacts could be identified on the surface of
neuronal bodies of these bipolar neurons. On the other hand, their dendritic
varicosities were richly innervated. Due to their irregular shape, intimate
association with muscular elements and their topographical superposition
with occasional spindle-like structures, these nerve cells recall prospective
sensory neurons involved in integration of mechanical and neural stimuli to
the heart. (3) Small multipolar interneurons could be identified in the
retronodal ganglion and within right and left bundle branches (Moravec and
Moravec, 1998).
The presence of numerous parasympathetic and sympathetic nerve fibres
in association with conduction tissue in the rat heart is well authenticated
(Petrecca and Shrier, 1998). It was found that the AV and ventricular conduction systems were more densely innervated than the atrial and ventricular myocardium as revealed by PGP 9.5-IR. Furthermore, the transitional
cell region was more densely innervated than the midnodal cell region,
while spatial distribution of total innervation was uniform throughout all
AV nodal regions. AChE-reactive nerve processes were found throughout
the AV and ventricular conduction systems, the spatial distribution of which
was nonuniform exhibiting a paucity of AChE-reactive nerve processes in
the central midnodal cell region and preponderance in the circumferential
transitional cell region. The TH-IR was uniformly distri-buted throughout
the AV and ventricular conduction systems including the central midnodal
and circumferential transitional cell regions. Beta1-adrenoreceptors were
found throughout the AV and ventricular conduction systems with preponderance in the circumferential transitional cell region. Beta2-adrenoreceptors were localized predominantly in AV and ventricular conduction systems with a paucity of expression in the circumferential transitional cell region. These results demonstrate that the overall uniform distribution of total
nerve processes is comprised of nonuniformly distributed subpopulations of
parasympathetic and sympathetic nerve processes. The observation that the
midnodal cell region exhibits a differential spatial pattern of parasympathetic and sympathetic innervation suggests multiple sites for modulation of
impulse conduction within this region. Moreover, the localisation of cardiomyocyte beta2-adrenergic receptors (beta2-ARs) in the AV conduction system, with an absence of expression in the circumferential transitional cell
layer, suggests that subtype-specific pharmacological agents may have
distinct effects upon AV nodal conduction (Petrecca and Shrier, 1998).
32
1.8. Studies on Intrinsic Cardiac Nervous System by Kaunas
Anatomists
The advanced neuromorphological studies of intrinsic cardiac nervous
system in Kaunas were initiated by R. Stropus and his colleagues (Stropus
and Vaicekauskas, 1978; Stropus, 1979; Stropus et al., 1982). They studied
the age-related changes in cholinergic and adrenergic nerve elements of the
human heart and their status in cardiovascular pathology (Abraitis et al.,
1981; Stropus, 1979), also the characteristics of myocardial cholinergic and
adrenergic innervation and its differences between species (Stropus and
Vaicekauskas, 1978; Stropus, 1979; Stropus et al., 1982). Together with the
development of surgical treatment of cardiac arrythmias and management of
myocardial ischemia, there was renewed the interest in morphology of the
intrinsic cardiac nervous system. Consequently, Pauza with his co–workers
defined the hilum of the heart and suggested to recognize a novel anatomical term – hilum cordis (Pauza et al., 1997a). He improved histochemical acetilcholinesterase technique and have analyzed the topography, morphology, distribution, and variability of epicardiac neural plexus (ENP) in
the human and experimental animals (Batulevicius et al., 2003; Batulevicius
et al., 2005; Batulevicius et al., 2008; Pauza et al., 2002a; Pauza et al.,
2002b; Pauza et al., 2000; Pauza et al., 1997b; Saburkina et al., 2010). In
addition, prenatal development of the human epicardiac ganglia was investtigated (Saburkina and Pauza, 2006; Saburkina et al., 2009; Saburkina et al.,
2005). The cited studies demonstrated the total ENP, and provided a morphological basis for an understanding how intrinsic ganglia and nerves are
structurally organized within the heart. Some works provided evident implications for selective denervation and electrophysiology of the sinoatrial
node in dog (Pauza et al., 1999), and for the use of rat heart in electrophysiological (Batulevicius et al., 2004).
In addition to macroscopic investtigations, the ultrastructure of intrinsic
cardiac ganglionic cells (Pauziene and Pauza, 2003) and intracardiac nerves
(Pauziene et al., 2000b) were described in detail on the healthy human. The
later studies provided baseline information on the normal ultrastructure of
intracardiac ganglia and nerves in healthy humans which might be useful for
assessing and interpreting the degree of damage of ganglionic cells in
autonomic neuropathy of the human heart.
33
2. MATERIALS AND METHODS
2.1. Material
Twenty adult C57BL/6J-linear mice of both sexes were used to examine
the immunohistochemistry of intrinsic cardiac neural plexus and twenty five
mice were used for histochemical acetylcholinesterase (AChE) staining.
Mice were obtained from the vivarium of the Lithuanian university of the
health sciences, Veterinarian academy. Animals were deeply anesthetized
with ether and euthanized by cervical dislocation in accordance with local
and state guidelines for the care and use of laboratory animals (Permission
No. 0133).
2.2. Methods
2.2.1. Total heart preparations
After thoracotomy, the mouse body was perfused with 0.01 M
phosphate buffered saline (PBS), consisting of 8.06 mM Na2HPO4, 1.94
mM NaH2PO4, and 137 mM NaCl in high purity distilled H2O (pH 7.4), at
room temperature. In order to stain the cardiac nerve plexus, fifteen mouse
hearts were prepared for histochemical acetylcholinesterase (AChE)
staining, as described previously (Pauza et al., 2000). The flabby atrial walls
were distended in situ via transmyocardial injection of a 20% warm water
solution of gelatin into the atria and ventricles. Once the injected gelatin
jelled, the heart was removed from the chest and immersed in a chamber
filled with room temperature PBS. Subsequently, the remains of the
pericardium, pulmonary arteries and mediastinal fat were gently separated
from the heart base. The prepared hearts were prefixed for 30 min at 4oC in
4% paraformaldehyde solution in 0.01 M phosphate buffer (pH 7.4). After
prefixation, the heart preparations were washed for 12 hours at 4oC in PBS
containing hyaluronidase (0.5 mg/100 ml, Sigma–Aldrich, USA) and placed
for 3 hours at 4oC in Karnovsky-Roots medium as described previously
(Pauza et al., 2000). Finally, heart reparations stained for AChE were fixed
and stored in 4% paraformaldehyde solution in 0.01 M phosphate buffer
(pH 7.4).
2.2.2. Thorax-dissected preparations
We microdissected ten mice chests to stain the vagal nerves, the
sympathetic chains, their ganglia and the branches linked
34
macroanatomically to the intrinsic cardiac nerve ganglionated plexus. After
perfusion with PBS at room temperature and injecting 20% warm water
solution of gelatin into the heart and great vessels, the anterior and lateral
walls of the chest cavity were carefully dissected out at the rib necks and the
vertebral column was cut out at the twelfth thoracic vertebra. The thoraxdissected preparations were placed into a chamber filled with room
temperature PBS, where lungs, pericardium and pulmonary arteries were
carefully separated from the heart base. Vagal and sympathetic chain nerves
of both sides and their branches extending toward the heart were
microdissected using a Stemi 200 CS stereoscopic microscope (ZEISS,
Gottingen, Germany) at 12.5x magnification. The prepared thorax-dissected
preparations were fixed, washed, stained histochemically for AChE and
stored according to the protocol described above.
2.2.3. Whole-mount preparations
After euthanasia, the mouse chest was opened and the heart was
perfused with 0.01 M phosphate-buffered saline (PBS; 0.9% NaCl, pH 7.4),
via a cannula inserted into the left ventricular cavity. The heart was then
removed and placed into a chamber containing cold 0.01M PBS. The
ventricles were separated from the atria along the atrioventricular groove,
preserving the roots of the aorta and pulmonary trunk. Thereafter, the
interatrial septum and both auricles were gently separated from atrial walls.
The root of the left cranial vein and the lateral atrial walls were cut
longitudinally, and the atria with the roots of the aorta and pulmonary trunk
were then stretched flat and pinned on a silicone pad, inside a shallow dish
with 1 ml of 0.01M PBS. The separated interatrial and intervetricular septa
and auricles were flattened and pinned on the same silicone pad as the
whole-mount preparation. Subsequently, the preparation was fixed for 30
min in 4% paraformaldehyde solution in 0.01M PBS at room temperature
and then washed 3 times for 10 minutes in 0.01 M PBS to remove the
paraformaldehyde fixative.
2.2.4. Immunohistochemistry
The cleansed specimens were subsequently dehydrated through a 30o,
50 , 70o, 90o, 96o and 3 times 100o ethanol series and placed for 2 h in
Dent's solution containing dimethylsulfoxide and 100o ethanol in a 1:4 (v/v)
ratio, which optimizes the penetration of antibodies into the whole-mount
heart walls. To enhance the fluorescence contrast, the specimens were then
bleached overnight in a solution of 30% hydrogen peroxide and Dent's
o
35
solution in 1:4 (v/v) ratio. Afterwards, preparations were rehydrated through
a graded ethanol series, washed 2 times for 10 min in 0.01 M PBS and
permeabilized for 30 min with 0.5% Triton-X (Carl Roth, Karlsruhe,
Germany) in 0.01 M PBS. Preparations were then incubated for 2 h in 5%
normal donkey serum (NDS) in 0.01 M PBS at room temperature in a
humid chamber to block non-specific antibody binding. Next, preparations
were washed three times for 10 min in 0.01M PBS and incubated in a
mixture of two primary antibodies for 48 h in a dark humid chamber at +4oC
(Table 2.2.4.1). After three 10 min-washes in 0.01M PBS, the whole-mount
heart preparations were incubated in an appropriate combination of
secondary antibodies for 4 h in a dark humid chamber on a shaker stage at
room temperature (Table 2.2.4.1). All antibodies were diluted in 0.01M
PBS. Thereafter the specimens were again washed in 0.01M PBS, incubated
for 2 h in a dimethylsulfoxide and PBS mixture, 1:4 (v:v) ratio, then
stretched on microscopic slide and mounted in Vectashield Mounting
Medium with DAPI (Vector Laboratories, California, U.S.A.). A cover slip
was placed on the tissue and then sealed with clear nail polish. Both positive
and negative controls were used.
Table 2.2.4.1. Primary and secondary antisera used in the study.
Antigen
Host
Dilution
Catalog number
Supplier
Primary
ChAT
TH
TH
CGRP
SP
PGP9.5
HCN4
Goat
Rabbit
Sheep
Rabbit
Rabbit
Rabbit
Rabbit
1:100
1:500
1:800
1:4000
1:800
1:2000
1:100
AB144P
AB152
AB1542
AB5920
20064
AB1761
AB5808-200UL
Chemicon*
Chemicon*
Chemicon*
Chemicon*
ImmunoStar**
Chemicon*
Chemicon*
Secondary
Cy3 anti Goat
Cy3 anti Rabbit
FITC anti Rabbit
FITC anti Sheep
Donkey
Donkey
Donkey
Donkey
1:300
1:300
1:100
1:100
AP180C
AP182C
AP182F
AP184F
Chemicon*
Chemicon*
Chemicon*
Chemicon*
*Chemicon International, Temecula, California, USA;
**ImmunoStar Incorporation, Wisconsin.
36
2.3. Microscopic Examinations and Measurements
The neural structures visualized histochemically for AChE were
examined stereoscopically at 12.5× magnification using a stereomicroscope
Stemi 200 CS (Zeiss, Gottingen, Germany) applying a transient light from
optic fiber illuminators (Zeiss, Gottingen, Germany). To identify the
external morphology of certain ganglia, preparations were additionally
analyzed using a contact microscope LUMAM K-1 (Lomo, St Petersburg,
Russia) at 10x magnification. The stereoscopically observed ganglia and
nerves were photographed at 4x using a digital camera Axiocam HRc
(Zeiss, Gottingen, Germany). The adjustments of final images and
measurements of cardiac ganglia were performed using AxioVision 4.7.1
software (Zeiss, Jena, Germany).
The whole-mount preparations were examined by a fluorescent
microscope AxioImage Z1 fitted with an Apotome and fluorescence filter
sets No 38HE, 43HE, and 49 (Karl Zeiss, Gottingen, Germany).
Fluorescent images were taken using a monochrome camera AxioCam
MRm (Zeiss, Gottingen, Germany) at 50x, 100x, 200x, 400x
magnifications. Digital enhancement of the taken images was kept to a
minimum, but images, if it was necessary, could be sharpened, lightened or
adjusted in another way using AxioVision software (v. 4.7.1, Karl Zeiss,
Gottingen, Germany). The sectioned optically stacks of images were
projected into a final image using a special extended focus module available
in the above mentioned AxioVision software.
In order to assess the relationship between the ganglion areas and the
number of neurons residing within them and, thereby, to approximate the
total number of intrinsic neurons per heart, the ganglion area was measured
on images taken at 50x or 100x magnifications using a special module
installed with AxioVision software (v. 4.7.1, Karl Zeiss, Gottingen,
Germany).
The number of neurons inside of intrinsic cardiac ganglia on the wholemount preparations was estimated in the 0.3-0.5 micron-thick optical
sections by counting exclusively those nerve cells that contained wellvisible nuclei. The latter rule was followed in order to escape an
overestimation of the number of neurons resided inside of intrinsic cardiac
ganglia as it was evident that some neuron nuclei appear in more than one
optical section. The measurements of long and short axis of neurons, was
also measured using special measuring module of AxioVision software.
37
2.4. Statistical Analysis
All quantitative results were expressed as mean ± standard error of the
mean (SEM). The “n” represents the number of samples from which
quantitative data were obtained. Linear regression was used to quantitatively
determine the relationship between the number of neurons residing within a
ganglion and its area. Statistical analysis of neuron soma size was
performed with both the paired and independent Student's tests using Origin
software (v.6.1, OriginLab Corporation, Northampton, MA, USA).
Significance was accepted at p<0.05.
38
3. RESULTS
3.1. Distribution of TH-IR and ChAT-IR Nerve Fibers in the Intrinsic
Cardiac Nerves
Immunohistochemistry for TH and ChAT performed after histochemical
staining of intrinsic cardiac nerves and ganglia for AChE (see Materials and
Methods) clearly demonstrated the coincident AChE distribution with both
the TH and ChAT-positive nerve fibers (Figure 3.1.1). Although AChE positivity or intensity of staining was not precisely measured in this study, it
was obvious that intraneural staining for AChE occurs even in nerves that
involve principally the TH immunoreactive nerve fibers (Figure 3.1.1). In
general, the ChAT immunoreactive nerve fibers did prevail in comparatively thinner epicardial nerves that were the most intensely stained by
brown precipitates of histochemical reaction for AChE. All intrinsic cardiac
ganglia examined in the present study contained the ChAT immunoreactive
neurons and were well stained for AChE.
3.2. Access of Mediastinal Nerves into the Mouse Heart
Extrinsic nerves entered the rat heart both in arterial and venous part of
the heart hilum (Figures 3.2.1 to 3.3.2). In the arterial part of the heart
hilum, the nerves entered heart in the fat on the left side between aorta and
pulmonary trunk. In the venous part of the heart hilum, the left and right
cervicothoracic and second thoracic sympathetic ganglia as well as both
vagal nerves were obvious neural sources from which extrinsic nerves extended toward and reached the mouse heart hilum. The right sympathetic
and vagal nerves entered the heart hilum at the right cranial vein, whereas
the left nerves entered at the left cranial vein (Figures 3.2.1 to 3.3.3).
3.3. Architecture and Topography of the Itrinsic Cardiac Nerve
Plexus
At the limit of the venous portion of the heart hilum, beneath the bifurcation of the pulmonary trunk and within fatty connective tissue, the extrinsic cardiac nerves form a ganglionated nervous plexus that consistently
contains right and left atrial clusters of ganglia (Figures 3.2.1 to 3.3.3). The
right-sided nerves enter the ganglionic cluster located on the right atrium at
the right cranial vein on the ventral and cranial aspects of the interatrial
groove (Figures 3.3.1 and 3.3.2d). From these ganglia, epicardial nerves
39
spread mainly onto the ventral, dorsal, and lateral surfaces of the right
atrium, which we have termed the right ventral and dorsal right atrial neural
pathways or nerve subplexuses, respectively (Figures 3.3.1; 3.3.2d, and
3.3.3). The right ganglionic cluster presumably supplies the interatrial
septum, as some fine nerves derived from these ganglia penetrated into
dorsal aspect of interatrial septum (Figure 3.3.2a). The left-sided nerves
access the left atrial ganglionic cluster at the left cranial vein, and from them
epicardial nerves proceed onto (1) the ventral surface of the left atrium as
the ventral left atrial neural pathway and (2) the dorsal surfaces of the left
atrium and both ventricles as the left dorsal neural pathway or subplexus
(Figures 3.2.1; 3.3.1a and 3.3.3). In all preparations examined, the vast majority of ventral left atrial subplexal nerves made consistent anastomoses
with right ventral subplexal nerves on the ventral aspect of the interatrial
groove (Figure 3.3.1a). In the heart shown in Figure 3.3.1a, both right and
left atrial ganglionic clusters intercommunicated by thin commissural
nerves. Similarly to some fine nerves derived from the right ganglionic
cluster, a few nerves derived from the left dorsal neural subplexus extended
toward and penetrated into the dorsal aspect of interatrial septum as well
(Figure 3.3.2a). Moreover, sparse extrinsic cardiac nerves attaining the
mouse heart through the arterial part of the heart hilum extended directly
into the epicardium of the ventricles. In all examined hearts, these nerves
passed between the roots of the aorta and the pulmonary trunk, were not
ganglionated, and passed along the anterior interventricular groove as the
left coronary neural pathway (Figures 3.2.1 and 3.3.1d). The distribution of
cardiac nerves and the number of ganglia in whole heart as well as in
separate subplexuses varied substantially from heart to heart (Figure 3.3.3).
3.4. Morphology of Mouse Cardiac Ganglia
Intrinsic ganglia in the mouse heart varied significantly in appearance,
but the majority of them were oval in shape (Figures 3.3.1 and 3.3.2). The
size of these ganglia was extremely variable and ranged from those that
were poorly observable with a dissecting microscope to ganglia that were
discernible with the naked eye (Table 3.4.1). Multifold variability was characteristic for both the cumulative ganglion area and the number of cardiac
ganglia (Table 3.4.1). Usually, nerve cells inside a ganglion were densely
packed in two- to three-cell layers, but certain ganglia contained neurons
bedded in four- to five-cell layers (Figures 3.3.1b, c; 3.32b, c, e). Numerous
interganglionic nerves interlinked the mouse cardiac ganglia (Table 3.4.1
and Figure 3.3.1a). Evidently, the large ganglia possessed more interganglionic nerves than the small ganglia (Figures 3.3.1b, c; 3.3.2b, c). In fact,
40
there was a strong correlation between the number of interganglionic nerves
and the cardiac ganglion area (Figure 3.4.1).
3.5. Distribution of Immunochemically Distinct Intrinsic Cardiac
Ganglia and Neurons
Immunohistochemical findings confirmet the prevoisly data on
distribution of intrinsic cardiac gandlia (ICG) obtained by a histochemistry
for AchE. The distribution of ganglionic cells positive for ChAT and/or TH
was reproducible from animal to animal. Intrinsic cardiac ganglia (ICG)
varied in shape and size and were mostly and consistently located at the
limits of the venous part of the heart hilum, near the vicinity of pulmonary
vein roots (Figures 3.5.1; 3.9.1).
Very few solitary ganglia, consisting from 3 to 10 neurons were distributed epicardially on the dorsal side of the left azygos vein root and close
to the roots of the aorta and pulmonary trunk (Table 3.5.1; Figures 3.6.1;
3.6.2d-f; 3.6.3a-c). Some of such small ganglia were inside of the nerves.
We observed individual intrinsic neurons as well (Figure 3.6.3d-f). They
were usually attached to epicardial nerves extending along the root of the
left cranial vein and/or on the ventral side of the right cranial (superior
caval) vein. The ICG situated around the roots of three pulmonary veins
were regularly interconnected by thin nerves that formed a ring-like
ganglionated neural chain thread (Figure 3.5.1). Some ganglia in this chain
were connected with commissural nerves that extended across the chain
(Figures 3.5.1; 3.91).
Since most of the ICG had clear borders, an assessment of the ganglion
number was simple and reliable. The whole-mount mouse heart preparations
had 11 to 30 ICG that were very variable in size. Cardiac ganglia could be
approximately classified as large (0.50–1.50 mm2), medium (0.01–0.50
mm2), and small (0.001–0.05 mm2). The smallest ICG could involve only 3
neuronal somata, whereas the largest ICG enclosed up to 700 neurons. On
average, a mouse cardiac ganglion contained 87±9 neurons (Table 3.5.1).
Based on the direct interdependence of the ganglion area and the number of
neurons (Figure 3.5.3), the total number of intrinsic cardiac neurons in the
mouse was estimated to be 1082±160 (Table 3.5.1).
3.6. ChAT-IR Neurons
ChAT immunoreactive neurons were observed in the all examined
ganglia (Figures 3.5.1a-c; 3.6.1; 3.6.2; 3.6.3). The cholinergic neurons
showed ChAT immunoreactivity throughout their somata but not in nuclei.
41
The immunohistoreactivity to ChAT was characteristic of 83% of the
neurons identified within the intrinsic cardiac ganglia (Table 3.6.1). These
cholinergic neurons were surrounded by baskets of varicose neural terminals
which expressed ChAT more intensely than the perinuclear regions of
cholinergic ganglionic cells (Figures 3.6.2a-f; 3.6.3d-f; 3.10.1e-f). The
ChAT immunoreactive nerve fibers were evident in the nerves which
accessed the heart and those which interconnected the large ganglia located
at the root of the pulmonary veins (Figure 3.5.1).
The ChAT positive neuronal somata varied in size and shape (Figures
3.6.2a-f; 3.6.3d-f; 3.10.1e-f). Their long axis was 20±0.3 µm, but
occasionally they could be remarkably larger (31.9 µm) or smaller (12.9
µm) in the length of their long axis (Table 3.6.1).
3.7. TH-IR Neurons and Small Intensively Fluorescent (SIF) Cells
The TH-IR was evenly distributed throughout the neuronal body, but
some neurons were more intensive in than others (Figure 3.6.3). Howerer
the TH-IR was independent from somata size (Figures 3.6.2a-f; 3.6.3). The
presence or absence of TH-IR does not affect the staining intensity and
staining pattern for ChAT-IR (Figure 3.6.2).
We detected neurons that were immunoreactive for TH only (Figure
3.6.2d-f), but they were rare and amounted only 3% of all neurons counted
in the heart. We also found ChAT positive neurons that were immunoreactive for TH (Figures 3.6.2a-f; 3.6.3). The biphenotypic neurons were
ubiquitously present and amounted to about 14% of all ganglionic cells
(Table 3.6.1). Bifenotypic neurons were found both in the large (Figure
3.62a-c) and in the small ICG (Figures 3.6.2d-f; 3.6.3). All the TH-IR
neurons usually had neuronal processes and were positive for PGP 9.5,
regardless of their size (Figure 3.6.2g-h). Compared with the purely ChATIR neurons, the TH immunoreactivity was evenly distributed throughout the
neuronal body. However, TH reactivity varied among individual neurons
and was not dependent on the neuron location inside the ganglion.
Evidently, the weakly positive TH neurons were significantly larger than
cholinergic neurons (Table 3.6.1).
The purely TH positive neurons were relatively rare compared to other
intrinsic cardiac neuron types (Table 3.6.1). However, the purely TH
positive neurons were considerably larger, than the biphenotypic neurons.
The mean length of the long and short axis of bifenotypic neurons were
21±0.4 µm and 14±0.4 µm, respectively. The mean length of the long and
short axis of TH-IR neurons were 22±0.7 µm and 15±0.3 µm res-pectively.
42
Thus, the TH-IR neurons were found to be the largest enes in the mouse
heart (Table 3.6.1).
We also observed small intensely fluorescent cells (Figure 3.6.2a-c).
These cells displayed very strong TH-IR were smaller than other TH-IR
nerve cells were grouped into small clusters of 3-8 cells and were dispersed
within large ganglia or separately on the atrial and ventricular walls.
Commonly, SIF cells were more frequent on the left atrium close to the
roots of the pulmonary veins and at the trunk of the left coronary artery.
Table 3.5.1. The mean number and range of neurons located inside an
intrinsic ganglion, solitary intrinsic neurons and the total number of
intrinsic cardiac neurons identified in 13 whole-mount mouse heart
preparations.
Parameter
On average
Range
Neuronal number per ganglion
87 ± 9
3 – 678
Number of solitary neurons per heart
19 ± 3
8 – 30
The approximate neuronal number per
heart
1082 ± 160*
600 – 1938
*Approximations were based on a strong correlation between ganglion area and neuronal
number shown on Fig. 3.5.3.
3.8. Distribution of ChAT-IR and TH-IR Nerve Fibers
Extrinsic nerves access the mouse heart on the arterial and venous
sections of the heart hilum (HH) (Figure 3.5.1). In the venous part, the
accessing nerves were mainly concentrated in two locations, i.e., at the
medial side of the right cranial vein root and on the anterior side of the left
cranial vein root (Figure 3.5.1). At the boundary of the HH, the accessing
nerves formed a chain-like ganglionated neural plexus with nerve extensions
that passed epicardially to the atria and ventricles (Figure 3.5.1). The heart
accessing nerves were predominantly TH-IR (Figure 3.8.1a-c), but some
ChAT-IR fibers were present as well (Figure 3.8.1a-c). In the arterial part of
the HH, the TH-IR and ChAT-IR axons inside the accessing nerves entered
the heart on the left side between the aorta and the pulmonary trunk (Figure
3.5.1) and extended epicardially to the left ventricular wall. The meshwork
of TH-IR and ChAT-IR fibers in the mouse heart could be observed in the
myocardium (Figure 3.8.2). In the epicardial neural plexus, the nerves were
mixtures of ChAT-IR and TH-IR axons (Figures 3.5.1; 3.8.1d-e; 3.8.4).
43
However, the TH-IR axons were more abundant than ChAT-IR axons in the
epicardial nerves traveling along the left cranial vein (Figures 3.6.3d-f;
3.8.1), coronary sinus and on the anterior sides of the atria and ventricles.
Despite the fact, that also, there were nerves, exceptionally positive to
ChAT (Figure 3.6.3a-c). The whole-mount heart preparations showed
substantial regional differences in the density of ChAT-IR nerve fibers. It
was evident, that ChAT-IR nerve fibers dominated in the nerves interconnecting the chain of intrinsic cardiac ganglia (ICG) on the heart base
(Figure 3.5.1). Moreower, the right atrial nerves contained the predominant
ChAT-IR nerve fibers, especially in the sinuatrial region (Figure 3.5.1f).
The wall of the left atrium was distinguished by a lower density of ChAT-IR
nerves compared to the right atrial wall, on which the thick nerves with
predominance TH-IR of fibers was observed (Figure 3.5.1d, g). In the myocardial layer, there were numerous varicosities and spine-like processes in
the preterminal and terminal parts of both the ChAT-IR and TH-IR axons
(Figure 3.8.3). Nevertheless, it was obvious that some axonal terminals
contained a mixture of neurotransmitters, i.e. they were simultaneously
immunoreactive to TH and ChAT (Figure 3.8.3). Interestingly, such biphenotypic axons were not identified within the epicardial or myocardial nerves
and neural bundles, in which TH-IR and ChAT-IR axons proceeded
separately and parallely.
3.9. Distribution of ChAT-IR and TH-IR Nerve Fibers in the Sinuatrial
and Atrioventricular regions
Immunohistochemistry demonstrates that the SA region extended to the
anterior, posterior and even medial side of the right cranial (superior caval)
vein as well as extended down and reached the caudal vein. Along these
sites, the density of ChAT-IR nerve fibers gradually decreased, but
remained greater than in the left atrial walls. Both the sinuatrial and
atrioventricular node regions contained meshworks of ChAT-IR nerve fibers
that were particularly dense and extended beyond the typical sites of these
nodes (Figures 3.9.1-3.9.3). The ChAT-IR nerve fibers in the
atrioventricular nodal meshwork occupied the entire lower part of the
interatrial septum and proceeded along the possible location of the His
bundle and its right and left branches within the interventricular septum
(Figure 3.9.3d-f). The density of the ChAT-IR neural meshwork close to the
typical sinuatrial node region was not equal everywhere on the right cranial
vein root walls (Figure 3.5.1).
44
Fig. 3.1.1. Microphotographs showing the presence of acetylcholinesterase
(AChE) within intrinsic cardiac nerves composed predominantly of tyrosine
hydroxylase (TH)-positive (adrenergic) and a few choline acetyltransferase
(ChAT)-positive (cholinergic) nerve fibers.
The image was taken from a whole-mount heart preparation that was immunohistochemically double labeled for ChAT (a) and TH (b) and subsequently stained histochemically for AChE (c).Note the sharp contrast of AChE staining compared with ChAT
labeling. Boxed areas within each image are enlarged in the upper right insets showing the
appearance of cholinergic and adrenergic nerve fibers within AChE-positive nerves of
mouse heart.
45
Fig. 3.2.1. Diagrams summarizing the distribution and morphology of
distinct intrinsic ganglionated nerve subplexuses in 15 mouse hearts as seen
from the ventral (left) and dorsal (right) sides on a pressure-inflated heart
stained histochemically for acetylcholinesterase. Dotted lines demarcate
limits of the heart hilum. Thick arched arrows indicate the course of neural
subplexuses. Polygonal areas indicate the main locations of intrinsic
ganglia in mouse heart. Note the left ganglia at the root of the left pulmonary vein and the right ganglia distributed above the interatrial septum at
the roots of right cranial (RCV) and middle pulmonary (MPV) veins.
Abbreviations: Ao – aorta; CS – coronary sinus; LAu – left auricle; LV – left ventricle; PT
– pulmonary trunk; RA – right atrium; RAu – right auricle; RV – right ventricle. Veins:
CV – caudal (inferior caval); LCV – left cranial (left azygos); LPV – left pulmonary; MPV
– middle pulmonary; RCV – right cranial (superior caval); RPV – right pulmonary. Neural
subplexuses: DRA – dorsal right atrial; LC – left coronary; LD – leftdorsal, RV – right
ventral; VLA – ventral left atrial.
46
Fig. 3.3.1. Macrophotographs showing the location and morphologic
pattern of right ventral (a, d), ventral left atrial (a), and left coronary (d)
nerve subplexuses in the mouse heart stained histochemically for
acetylcholinesterase. Boxed areas in a and d are enlarged using a contact
microscope and shown as insets in b and c. Note the right ganglia situated
at the root of the right cranial vein and the left ganglia on the cranialdorsal aspect of the left atrium. Black arrowheads indicate some cardiac
nerves. White arrowheads indicate ganglia. White solid arrows indicate
nerves accessing the heart hilum and originating from the right ventral (RV)
and left coronary (LC) neural subplexuses. Black thin arrows indicate
interganglionic nerves. White thin arrows indicate some neurons located
inside ganglia. Dashed lines demarcate the limits of the heart hilum.
Abbreviations: Ao – aorta; CA – conus arteriosus; IS – ventral interatrial groove; LAu –
left auricle; LCV – left cranial vein; MPV – middle pulmonary vein; PT – pulmonary
trunk; RAu – right auricle; RCV – right cranial vein; SAN – sinuatrial nodal zone. Neural
subplexuses: LC – left coronary; RV – right ventral; VLA – ventral left atrial.
47
Fig. 3.3.2. Location, course, and structure of left dorsal (LD) and dorsal
right atrial (DRA) neural subplexuses in a mouse heart stained histochemically for acetylcholinesterase. Boxed areas b and c in a, e in d, and f in e
are enlarged using a contact microscope and shown as insets b, c, e, and f,
respectively. Black arrowheads in a point to nerves that penetrate into interatrial septum and in d one that proceeds toward the region of the sinuatrial
node (SAN). White arrowheads indicate some ganglia. White solid arrows
indicate nerves entering the ganglionated nerve plexus of the heart hilum.
Black thin arrows indicate interganglionic nerves. White thin arrows indicate some neurons. Dashed line demarcates the heart hilum. In d, note right
ganglia situated at the root of the right cranial vein on the interatrial
groove.
Abbreviations: CS – coronary sinus; CV – orifice of caudal (inferior caval) vein; IS – interatrial groove; LAu – inferior surface of left auricle; LCV – left cranial vein; LPV – orifice
of left pulmonary vein; LV – left ventricle; MPV – orifice of middle pulmonary vein; RA –
right atrial wall; RCV – root of right cranial (superior caval) vein; RPV – orifice of right
pulmonary vein.
48
Fig. 3.3.3. Macrophotographs illustrating the structural variability of the
left dorsal neural subplexus in two mouse hearts stained histochemically for
acetylcholinesterase. White arrowheads indicate some ganglia. Black
arrowheads indicate topographically comparable nerves at the coronary
sinus. Dashedlines demarcate limits of the heart hilum.
Abbreviations: CS – coronary sinus; LCV – left cranial (left azygos) vein; LPV – left
pulmonary vein; LV – left ventricle; MPV – middle pulmonary vein. Note persistent
location of ganglia indicated by white arrowheads.
Table 3.4.1. Number of intrinsic ganglia, their sizes, cumulative areas, and
number of interganglionic nerves in 10 mouse hearts stained
histochemically for acetylcholinesterase.
Parameter
Mean
Range
No. of ganglia per heart
19 ± 3
11–30
Ganglion size (in mm2)
0.026 ± 0.003
0.001–0.167
0.35 ± 0.05
0.19–0.63
9±1
3–24
Cumulative ganglion area (in mm2)
No. of interganglionic nerves per
ganglion
49
Fig. 3.5.1. Whole-mount preparation. Both atrial appendages, pulmonary
vein roots, and the major part of the ventricles were dissected in order to
flatten the atria. ChAT-IR or cholinergic neural structures are labeled in
red, TH-IR or adrenergic structures in green. Arrows indicate the extrinsic
nerves accessing the mouse heart, whereas arrowheads point to intrinsic
ganglia. The boxed areas in panel a are enlarged in the insets to illustrate:
(b) - the predominance of ChAT-IR ganglionic cells; (c) - the small ganglia,
and single neurons; (d) - the nerves accessing the heart on the anterior side
of the left cranial vein root in which the TH-IR nerve fibers predominate
compared to neighboring solitary ChAT-IR nerve fibers; (e) - the nerves
accessing the heart on the medial side of the right cranial vein root with
absolute predominance of TH-IR nerve fibers; (f) - the considerable differrence in density of the cholinergic neural meshwork on the right cranial
(superior caval) vein root (RCV) compared with the rest of right atrial wall;
(g) - the occurrence of ChAT-IR fibers together with TH-IR fibers in
epicardial nerves.
Abbreviations: PT – orifice of pulmonary trunk, AO – orifice of aorta, RCV – orifice of
right cranial vein, CV – orifice of caudal vein, LCV – orifice of left cranial vein, PVs –
orifice of left ant middle pulmonary veins, RPV – orifice of right pulmonary vein.
50
Fig. 3.5.2. Number of interganglionic nerves plotted against areas of 31
ganglia derived from 10 mouse hearts.
The straight line indicates linear regression of plotted data. Each point corresponds to one
of the analyzed ganglia. R – correlation coefficient at P<0.5.
Fig. 3.5.3. The number of neurons plotted against the area of 132 ganglia
from 15 mouse hearts.
The straight line indicates the linear regression of the plotted data. Each point corresponds
to one of the analyzed ganglia (some points overlap in the graph). R – correlation coefficient at P<0.0001.
51
Fig. 3.6.1. Microphotographs of the small intracardiac ganglia, found in the
myocardium that illustrate the predominance of the ChAT-IR (asterisks)
neurons and the singular biphenotypic neuron (arrowhead).
The presence or absence of TH-IR does not affect both the staining intensity and the
staining pattern for ChAT-IR neurons. Note the process of the bifenotypic axon (arrows) is
not immunoreactive to TH.
3.10. Distribution of SP-IR and CGRP-IR Nerve Fibers
SP-IR and CGRP-IR nerve fibers were the most abundant in the
mouse epicardium and in ganglia adjacent to the HH. The SP-IR and CGRPIR nerve fibers were thin and generally identified within the mixed nerves
and nerve bundles together with ChAT- and TH-IR nerve fibers (Figures
3.10.1d-f and 3.10.2). Frequently, both the SP-and CGRP-IR nerve fibers
were situated near blood vessels of varying sizes. We failed to observe
neuronal somata that were even dimly immunoreactive to SP and/or CGRP.
Inside the ganglia, SP- and CGRP-IR nerve fibers contained numerous
varicosities and appeared as if in contact with ganglionic cells (Figure
3.10.1d-f). On the other hand, many SP- and CGRP-IR nerve fibers simply
passed through the ganglia without having any specialized ending. Some
cardiac ganglia contained no SP-IR fibers. SP- and CGRP-IR nerve fibers
were also identified within the heart accessing nerves, intrinsic nerve
bundles and interganglionic nerves that passed between intrinsic cardiac
ganglia adjacent to the pulmonary vein roots. Within the myocardial neural
network, the SP- and CGRP-IR nerve fibers were considerably rarer than
cholinergic and adrenergic fibers, but they appeared to proceed jointly
within the common myocardial nerve bundles (Figure 3.10.2). Single SP-IR
fibers were found in the mycardium endocardial layer as well.
52
Fig. 3.6.2. a-c: Microphotographs illustrating the predominance of ChATIR ganglionic cells inside a ganglion that also contains the biphenotypic
(arrowheads) and small intensively fluorescent (SIF) cells (arrows) immunoreactive to TH. Note that immunostaining for ChAT (b) does not exhibit
the SIF cells (arrows), while only the double-channel fluores-cence of
simultaneous immunostaining for TH and ChAT (c) reveals reliably the
biphenotypic neurons (arrowheads) that were dimly seen in monochannel
fluorescence applied to display immunostaining for TH (a). d–f: Microphotographs showing three types of neurons in small ganglion of the mouse
heart identified applying monochannel (d, f) and double-channel (f)
fluorescence. Note the baskets of ChAT-IR neural terminals that surround
the neurons and contain numerous varicosities. White crosses indicate the
neuron that is exclusively positive to TH. Asterisks indicate neurons positive
only to ChAT. Diamonds indicate two biphenotypic nerve cells. g–i: Microphotographs confirming the neuronal origin of all ganglionic cells and
showing the diversity of sizes and staining intensities of TH-IR neurons
located within one ganglion. Note the intensely (diamonds) and weakly
(crosses) stained TH-IR neurons, which are positive for the general
neuronal marker PGP 9.5.
53
Fig. 3.6.3. Microphotographs from whole-mount preparation. Figures a-c
illustrate ChAT-IR (asterisks) neuron, and biphenotypic (arrowheads)
neurons in the exceptionally parasymphatetic nerve. Panels d-f illustrate the
single biphenotypic (arrowheads) neuron inside the mixed nerve. Note the
neuronal TH-IR process (white arrows) derived fom bifenotypic neuron. The
TH immunoreactivity differs from very intensive (a-c) to dim (d-f).
In addition, note the intensive ChAT immunoreactivity in the single axon and its
varicosities (black arrows).
54
Fig. 3.8.1. Microphotographs from the whole-mount mouse heart
preparation. a-c: The nerves accessing the heart on the anterior side of the
left cranial vein root, in which the TH-IR nerve fibers predominate (b) compared to neighboring solitary ChAT-IR (a) nerve fibers. d-f: The epicardial
nerves on the left cranial vein both the ChAT-IR and TH-IR nerve fibers.
Note the exceptionally TH-IR nerve (white arrows) in e and f panels.
55
Fig. 3.8.2. The meshwork of TH-IR (symphatetic) and ChAT-IR
(parasymphatetic) imunoreactive fibers in the mouse heart.
56
Fig. 3.8.3. Micrographs demonstrate the myocardial layer of the mouse
heart where numerous axonal varicosities and spine-like processes in
preterminal and terminal parts contain both the ChAT and TH-IR.
Arrowheads indicate preterminal and terminal parts of the ChAT-IR axons. Arrows –
axonal terminals containing a mixture of neurotransmitters, i.e. they are immunoreactive for
both the TH and ChAT.
57
Fig. 3.8.4. Micrographs demonstrate nerves consisting of a mixture of
ChAT-IR and TH-IR axons.The arrow indicates varicosities on TH-IR
axons.
58
Fig. 3.9.1. Whole-mount preparation of the mouse heart to demonstrate the
distribution of HCN4-IR conductive myocytes and ChAT-IR neural
structures. ChAT-IR neural structures are labeled in red. HCN4-IR cells are
labeled in green. Arrows indicate the epicardial nerves, arrowheads point
to intrinsic ganglia.
Abrevations: Ao - orifice of aorta; CV - orifice of caudal vein; LCV _ orifice of left cranial
vein, PT _ orifice of pulmonary trunk; LPV - orifice of left pulmonary vein; MPV - orifice
of middle pulmonary vein; RPV - orifice of right pulmonary vein; RCV - orifice of right
cranial vein., SAN – sinoatrial region; RAu – right auricle.
59
Fig. 3.9.2. Whole-mount preparation of the mouse heart to demonstrate the
distribution of HCN4-IR conductive myocytes and TH-IR neural structures.
HCN4-IR or conducting myocytes are labeled in red, TH-IR or sympathetic
structures in green. Arrows indicate the nerves in the mouse heart.
Abbreviations: RCV – orifice of right cranial vein, CV – orifice of caudal vein, SAN –
sinoatrial region; RAu – right auricle.
60
Fig. 3.9.3. Whole-mount preparation of the mouse interatrial (a-c) and
interventricular (d-f) septa illustrating the pathways of intrinsic nerves
proceeding toward the atrioventricular node region (a) as well as along the
His bundle and its right branch (d).
Note the predominance of ChAT-IR fibers in the region of the atrioventricular node (c), at
His bundle (e) and it right branch (f). The nerves (arrowheads) from epicardial ganglia
(solid white arrow) extend toward the atrioventricular node and form the dense neural
meshwork therein. The boxed areas in panel a are enlarged as the insets in b and c, while
ones in panel d – as the insets in e and f. Double arrowheads in a and b point out the
solitary ChAT-IR neuron located beneath the endocardium of the interatrial septum.
Asterisks in d track the blood vessels in the interventricular septum. Abbreviations: FO –
fossa ovalis, AVN - atrioventricular node region, CS – orifice of coronary sinus.
61
Fig. 3.10.1. Microphotographs illustrating the SP-IR nerve fibers in close
vicinity to atrial myocytes (a-c) and CGRP-IR nerve fibers between the
ChAT positive ganglionic cells (d-f).
Note the numerous varicosities and button-like terminal endings of the CGRP-IR nerve
fibers at ChAT-IR nerve cells. In a-c panels, asterisks point to two atrial myocytes, in
which myofibril striations are seen, while in d-f panels, arrowheads – the varicosities of
CGRP-IR nerve fiber.
62
Fig. 3.10.2. Microphotographs of the mouse intrinsic cardiac nerve
illustrating CGRP-IR nerve fibers (in red) between the TH positive nerve
fibers (in green)
63
Table 3.6.1. The number, percentage and size of the immunohistochemically distinct intrinsic cardiac neurons
identified in whole-mount preparations.
ChAT – IR
TH – IR
Biphenotypic (ChAT +TH -IR)
n
Mean ± SD
Range
n
Mean ± SD
Range
n
Mean ± SD
Range
PNumber of neurons
per one heart
13
583 ± 99
292 – 969
13
25 ± 5
10 – 45
13
97 ± 36
47 – 279
Percentage of neurons
per one heart
13
83 ± 2
76 – 88
13
3±1
2–6
13
14 ± 2
7 – 22
Long axis of neurons
(µm)
140
20 ± 0.3**
13 – 32
110
22 ± 0.7*
13 - 30
90
21 ± 0.4***
13 – 30
Short axis of neurons
(µm)
140
14 ± 0.2**
7 – 23
110
15 ± 0.3*
9 – 23
90
14 ± 0.4***
6 – 24
Area of neurons (µm2)
140
223 ± 6
94 – 560
110
260 ± 9*
99 – 647
90
240 ± 9
75 – 566
* - Differences between the axes and area of ChAT-IR and TH-IR neuronal somata were statistically significant at P < 0.05.
** - Differences between the axes of ChAT IR and biphenotypic neuronal somata were statistically significant at P < 0.05.
*** - Differences between the axes of TH IR and biphenotypic neuronal somata were statistically significant at P < 0.05.
4. DISCUSSION
This is the first detailed investigation of the anatomy of the intrinsic
ganglionated nerve plexus of the whole (i.e., nonparceled and nonsectioned)
mouse heart also this study for the first time demonstrates the distribution of
cholinergic, adrenergic and peptidergic (putative sensory) nerve fibers and
neurons in whole-mount preparation of the mouse heart. The technique of
whole-mount preparation allowed us to precisely identify and map all the
intrinsic cardiac ganglia in this species. This study also classified their
immunohistochemical properties and the interconnections of their neurons
in the atria and the interventricular septum.
This study revealed the intrinsic ganglionated nerve plexus using
histochemical staining for AChE. Some investigators consider this that
histochemistry for AChE is a specific method for visualization only of the
cholinergic (parasymphatetic) neural structures (Crick et al., 1996; Crick et
al., 1999b; Hoover et al., 2004; Kawano et al., 2003; Otake et al., 2009).
Simultaneous histochemical staining for AChE and immunohistochemical
staining for TH and ChAT performed in the present study confirmed earlier
findings by Koelle and colleagues (Koelle et al., 1987) who demonstrated
that AChE histochemistry according to Karnovsky and Roots (Karnovsky
and Roots, 1964) stains both the adrenergic (sympathetic) and cholinergic
(parasympathetic) neurons and nerves. Moreover, comparison of labeling
patterns of cholinergic marker – high affinity choline transporter (CHT), and
AChE suggests that AChE histochemistry overestimates the density of
cholinergic innervation in the heart (Hoover et al., 2004). Therefore, there is
no doubt that both parasympathetic and sympathetic neural structures were
clearly visualized in this investigation using AChE histochemistry according
to Karnovsky and Roots (Karnovsky and Roots, 1964). Nonetheless,
sympathetic nerve fibers have less AChE activity than cholinergic fibers
(Koelle et al., 1987; Saburkina et al., 2010). Moreover, recent findings
indicate the presence of multiple neurochemical phenotypes of mammalian
cardiac neurons. For example, a dual cholinergic/nitrergic phenotype for
intrinsic cardiac neurons (Hoover et al., 2009), adrenergic/cholinergic
phenotype for cardiac sympathetic neurons (Kanazawa et al., 2010; Vega et
al., 2010), and cotransmission of acetylcholine, norepinephrine, and
neuropeptide Y in sympathetic neurons co-cultured with target cardiac
myocytes (Vega et al., 2010) have been demonstrated. Consequently, lighter
or weaker staining of some intrinsic cardiac nerves by AChE histochemical
reaction products presumably indicates the predominance of noncholinergic
nerve fibers within those intrinsic cardiac nerves.
In previous reports using hearts from humans, dogs, pigs, guinea pigs,
rats, and sheep, the intrinsic nerve plexus was analyzed as a complex of
seven ganglionated nerve subplexuses, each of which threaded into the heart
at a particular site of the heart hilum, occupied a specific location on the
epicardium, extended to a restricted cardiac region, and included its own
ganglionated field, preganglionated and postganglionated nerves
(Batulevicius et al., 2003; Batulevicius et al., 2005; Pauza et al., 2000;
Pauza, 2008; Pauza et al., 2002c; Saburkina et al., 2010).
This study demonstrates that the structural organization of the mouse
intrinsic nerve plexus is similar to that of other mammals. However, the
particular intrinsic ganglionated nerve plexus of the heart hilum (GNPHH)
is distributed on the base of the mouse heart and its postganglionated nerves
extend toward both atria and ventricles. The preganglionated nerves access
the GNPHH at both the right and left cranial veins and join two ganglionic
clusters interconnected by interganglionic nerves into a ring or a chain of
ganglia that also are interlinked by commissural nerves on the base of the
left atrium. In general, the morphologic pattern of the mouse epicardial
ganglionated nerve plexus is similar to, but somewhat less complex and
dense than, those of larger mammals, which accords to the small size of the
mouse heart. Sources of extrinsic cardiac nerves in the mouse heart are
rather distinct from those that were identified in humans, apes and sheep
(Kawashima, 2005; Kawashima et al., 2008; Saburkina et al., 2010).
Preparations in this study revealed complete symmetry of the
sympathetic and vagal sources, whereas in humans and sheep the left and
right sympathetic and vagal cardiac nerves overlap in front of the heart and
the left and right nerves access the heart in the same locations (Kawashima,
2005; Saburkina et al., 2010). Using a whole-mount preparations, injected
with
tracer
1,1'-dioctadecyl-3,3,3',3'
tetramethylindocarbocyanine
methanesulfonate (DiI) and Fluoro-Gold (FG), separately for each atrium of
the mouse heart, Ai et al. (Ai et al., 2007) identified three discrete
ganglionated plexuses that were distributed, respectively, at (1) the entrance
of the right pulmonary vein to the left atrium and the junction of the right
cranial vein and right atrium, (2) the entrance of the left cranial and caudal
veins to the right atrium, and (3) the junction of the left pulmonary vein and
the left atrium. Contrary to that report, our observations with
immunohistochemical technique indicate that nerves extend to both atria
and ventricles from the entire GNPHH by distinct nerve pathways of
subplexuses. Moreover, some cardiac nerves reach the epicardium of the left
ventricle directly through the arterial part of the heart hilum. With respect to
the distribution of mouse cardiac ganglia, our findings also contradict the
report of Ai et al., (Ai et al., 2007) in which intrinsic ganglia distributed in
66
the region of sinuatrial node were mentioned. The present study does not
confirm the location of any ganglia above the terminal groove in the typical
region of the sinuatrial node. As evident in Figure 19, the most ganglia
revealed in this study were distributed at a distance from both the typical SA
and atrioventricular nodes on the heart base.
Comparing the present observations with our earlier results in rat hearts
(Batulevicius et al., 2003), it is evident that the architecture and topography
of the GNPHH in mice and rats are similar. In both species, the GNPHH
ganglia are concentrated at almost the same locations. Also, in both species
the epicardial nerves derived from the GNPHH extend topographically and
morphologically following analogous routes and supply nearly identical
atrial and ventricular regions. The most remarkable difference between mice
and rats is the nerve supply of the ventral surface of both ventricles. In rats,
abundant nerves extend to the ventral surface of both ventricles from the
arterial part of the heart hilum (i.e., they access the heart at the roots of the
ascending aorta and pulmonary trunk) (Batulevicius et al., 2003). In mice,
the main nerve supply of the ventral surface of both ventricles is the right
ventral pathway that gets underway from the right atrial cluster of ganglia in
the GNPHH.
The present findings suggest that, in general, the topography and
morphologic pattern of neural extensions from the mouse ganglionated
nerve plexus of the heart hilum correspond to ganglionated nerve
subplexuses in the human heart, and this correspondence substantiates use
of the mouse as a simple and reliable model in cardiac arrhythmia research.
Hopefully, the neuroanatomy of the mouse heart demonstrated in this pilot
study will facilitate more advanced investigations with this animal model
that will increase our knowledge of the physiologic roles of distinct nerve
pathways and individual intrinsic ganglia.
The technique of whole-mount preparation applied in the present
examination allowed to identify definitely all the intrinsic ganglia
disseminated in the mouse heart as well as to show immunohistochemical
properties and interconnections of their neurons. Earlier reports on the
mouse intrinsic cardiac plexus have described exceptionally the intracardiac
ganglionic cells in culture (Hoard et al., 2007), on atrial pieces (Ai et al.,
2007) and traditional histological sections (Hoard et al., 2008).
As expected, in the mouse heart most of the intrinsic neurons are
cholinergic. This confirms the classical point of view that the intrinsic
cardiac neurons are mainly cholinergic and, therefore, they play an
inhibitory role in cardiac regulation. Prior immunohistochemical studies
have indicated that all intrinsic cardiac neurons are exclusively
immunoreactive to ChAT in the guinea pig (Horackova et al., 2000; Leger
67
et al., 1999), rat (Richardson et al., 2003) and mouse (Hoard et al., 2007).
Despite the fact that most neurons observed in our study showed
immunoreactivity for a cholinergic marker, 13% of neurons were
nevertheless positive for both TH and ChAT; i.e. they were biphenotypic,
whereas quantitative evaluation of ganglia present in sections showed that
30% of mouse intrinsic cardiac neurons (ICNs) were TH positive (Hoard et
al., 2008), and neurons, maintained in culture included 21% TH-IR neurons
(Hoard et al., 2007). The TH patterns of fluorescence in the somata of these
neurons did not overlap with those of ChAT. Therefore, the distribution of
these particular neurons was reliably assessed within the intrinsic ganglia.
Our finding of biphenotypic neurons in the mouse heart is consistent with
studies on tissue sections and cultured intrinsic neurons derived from mouse
and human hearts (Hoard et al., 2008; Hoard et al., 2007; Weihe et al.,
2005). In addition, we report that in the mouse heart the presence of a
population of intrinsic neurons that are exclusively positive for TH. It
should be noted that isolated rat intracardiac neurons were found to be
responsive to noradrenaline application in vitro (Ishibashi et al., 2003; Xu
and Adams, 1993). Therefore, the adrenergic input on some cholinergic
neurons in the heart needs to be addressed in the future.
We have identified biphenotypic TH- and ChAT-IR axons in the mouse
atrial myocardium, contrary to the Hoard et al. report (Hoard et al., 2008).
Interestingly, immunochemically distinct axons contained numerous
varicosities and were closely juxtaposed with each other up to their
terminals. At the terminals, TH- and ChAT-IR axons usually split and
petered out on the target cells with button and spine like endings. Based on
the present observations, it is evident that TH is colocalized with ChAT in
neuronal somata, as well as in most terminal parts of the axons. This is in
sharp contradiction with the conclusion reached by (Hoard et al., 2007;
Hoard et al., 2008), who proposed that TH is synthesized by a certain
population of intrinsic cardiac neurons but is not transported to their
processes. We found intrinsic cardiac neurons with TH-IR proceses (Figure
11) and this suggest (Figure 11a-c), that some TH positive transport tyrosine
hydroxylase. TH-IR terminals did not appear to form pericellular baskets
around principal neurons, these observations are constant with previous
studies of TH-IR terminals in rat intrinsic cardiac ganglia (Forsgren et al.,
1990; Moravec et al., 1990). Intraganglionic sympathetic terminals provide
inputs to intrinsic cardiac neurons in the pig (Smith, 1999) and dog
(Gagliardi et al., 1988), while in the guinea pig, distinct pericellular baskets
of TH-IR terminals probably of sympathetic origin, surround some
intracardiac neurons (Steele et al., 1994).
68
In contrast to histological sections, the whole-mount heart preparation
enables transmural examination of the entire intrinsic nerve plexus. Using
this technique, we show that the density of the intrinsic nerves and fibers is
different in distinct heart regions. ChAT-IR fibers are especially densely
distributed in the sinuatrial node region, the right cranial (superior caval)
vein root, and the interatrial septum, at the site of the atrioventricular node.
The dense innervation of the sinuatrial node was demonstrated earlier by
histochemical staining of acetylcholinesterase in mouse, rat, rabbit, dog, pig,
human (Crick et al., 1996; Crick et al., 1994; Hoover et al., 2004; Kent et
al., 1974; Petrecca and Shrier, 1998) as well as by immunohistochemical
staining of ChAT (Hoover et al., 2004; Yasuhara et al., 2007) and choline
transporters in guinea pig (Hoover et al., 2004; Steele et al., 1994) mouse
(Mabe et al., 2006), and human (Chow et al., 1993; Crick et al., 1994).
These morphological findings correlate well with physiological studies
showing that vagal nerve stimulation activates the cholinergic input to the
sinuatrial node, causing a decrease in the heart rate (Randall et al., 1985).
TH and ChAT staining in rabbit atrioventricular node sections revealed
a heterogeneous distribution (Hucker et al., 2007). However, no
morphological data have been published to date about the innervation of the
intact mouse atrioventricular node. Here we found an extremely dense and
wide neural meshwork in the vicinity of the atrioventricular node, which
extended beyond its typical anatomical location. This is in line with recent
reports in laboratory animals, including mouse (Anderson et al., 2009;
Efimov et al., 2004; Hou et al., 2007; Yanni et al., 2009). Presumably, all
conductive cardiomyocytes distributed widely on the lower interatrial
septum are under extensive control of efferent nerve fibers that make up the
meshwork we have described here. The high density of innervation
demonstrates that AV conduction properties are very tightly regulated by
the autonomic nervous system and any study of AVN function should take
into consideration the fact of this control.
Yasuhara and colleagues (Yasuhara et al., 2007) demonstrated that the
rat ventricular myocardium contains a small number of ChAT-IR fibers.
Coronary blood vessels in the ventricular myocardium are also supplied by
ChAT-IR fibers. Our observations confirm that the innervation of the mouse
ventricles is carried by ChAT-IR nerve fibers that are always accompanied
by TH-IR fibers. Interestingly, vagal stimulation decreases ventricular
contractility in dogs (Armour and Ardell, 1994), pigs and humans (Lewis et
al., 2001), but does not affect ventricular contractility in rats (Takahashi et
al., 2003) or guinea pigs (Hoover et al., 2004). It is possible that the
cholinergic innervation in ventricular myocardium varies significantly
between species.
69
The SP- and CGRP-IR nerve fibers observed in the mouse cardiac
ganglia exhibited varicosities and free endings. We were unable to perform
double labeling of these antigens. However, we noticed the predominance of
CGRP-IR compared to SP nerve fibers. In the rat heart, SP and CGRP
colocalized in nerve fibers distributed inside intrinsic ganglia (Forsgren et
al., 1990; Moravec et al., 1990; Onuoha et al., 1999a; Onuoha et al., 1999b;
Richardson et al., 2003). There were no neuronal somata immunoreactive to
SP or CGRP in the mouse heart. This suggests that mouse intrinsic cardiac
neurons lack of neuropeptide diversity when compared to the guinea pig
(Horackova et al., 1999; Steele et al., 1994).
We have described and characterized the cholinergic, adrenergic and
biphenotypic intrinsic neurons of the mouse heart. Adrenergic, cholinergic
and peptidergic (SP- and CGRP-IR) nerve fibers extend to their targets in
mixed nerves and nerve bundles, but the mouse intrinsic cardiac neural
plexus contains nerves that contain either adrenergic or cholinergic axons.
Studies such as ours, which map in detail the distribution of the intrinsic
cardiac nerve fibers, may help in guiding attempts to selectively stimulate
and/or ablate functionally distinct intrinsic neural pathways in the
arrhythmic heart.
70
CONCLUSIONS
1. The left and right cervicothoracic and second thoracic sympathetic
ganglia as well as both vagal nerves are the neural sources from which
extrinsic nerves extended toward and reach the mouse heart hilum. The
extrinsic cardiac nerves access the mouse heart at (1) left coronary site,
(2) the right and (3) left cranial veins and interblend within the
ganglionated nerve plexus of the heart hilum.
2. Nerves and bundles of nerve fibers extend epicardially from the heart
hilum plexus to atria and ventricles by the left dorsal, dorsal right atrial,
right ventral, left ventral atrial and left coronary routes or subplexuses.
3. The majority of intrinsic cardiac ganglia are localized on the heart base
at the roots of the pulmonary veins. These ganglia are interlinked by
interganglionic nerves into the above mentioned nerve plexus of the
heart hilum.
4. Mouse hearts contains 19 ± 3 ganglia, giving a cumulative number of
neurons 1082 ± 160 per one heart. There is a strong correlation between
the number of interganglionic nerves and the cardiac ganglion area.
5. There are cholinergic, adrenergic and peptidergic nerve fibers in the
mouse heart. Most nerves and neuronal bundles of the mouse epicardial
plexus are mixed, some express either adrenergic or cholinergic
markers.
6. There are cholinergic, adrenergic and bifenotypic intrinsic neurons
within the mouse heart which express simultaneously both the
cholinergic and adrenergic neuronal markers. The small intensively
fluorescent cells are also present in the mouse heart, and these cells are
positive to tyrosine hydroxylase.
7. There are axonal terminals, containing a mixture of cholinergic and
adrenergic neurotransmitters, in the myocardial layer.
8. There are nerve fibers immunoreactive to substance P and calcitonine
gene related peptide, in the epicardium and within ganglia adjacent to
the heart hilum. Intrinsic cardiac neurons of the mouse are not
imunoreactive neither to substance P neither to calcitonine gene related
peptide.
71
PUBLICATIONS
Articles
1. Rysevaite K, Saburkina I, Pauziene N, Vaitkevicius R, Noujaim SF,
Jalife J, Pauza DH. Immunohistochemical characterization of the
intrinsic cardiac neural plexus in whole-mount mouse heart preparations.
Heart Rhythm. Heart Rhythm. 2011; 8(5):731-738
2. Rysevaite K, Saburkina I, Pauziene N, Noujaim SF, Jalife J, Pauza DH.
Morphologic pattern of the intrinsic ganglionated nerve plexus in mouse
heart. Heart Rhythm. 2011; 8(3):448-454.
Abstracts
1. Rysevaitė K, Saburkina I, Paužienė N, Pauža DH. Morphologic and
immunohistochemical analysis of the mouse intrinsic cardiac nervous
system. Anatomische Gesellschaft - 105th Annual Meeting: Hamburg,
March 26-29, 2010: Abstracts of Lectures and Posters. Hamburg,
2010.p. 22, no. 13.
2. Rysevaitė K, Pauža DH, Gegužis V, Paužienė N. Distribution of
Tyrosine Hydroxylase and Choline Acetyltransferase Positive Nerve
Fibers within Epicardial Ganglionated Nerve Plexus in Dog and Mouse.
Baltic Morphology 2009 - the 5th Biannual Scientific Conference:
Kaunas, Lithuania, August 27-28 2009, p. 28.
72
Acknowledgments
The work was supported by Lithuanian University of Health Sciences
Foundation.
I am sincerely grateful to Prof. Dainius.H. Pauža and Prof. Neringa
Paužienė for help, ideas, and advices. I am thankful to Dr. Inga Saburkina,
Dr. Sami F. Noujaim, Prof. Jose Jalife for help and advices writing articles.
Great thanks to Rima Masiene and Nida Rutkauskiene for assistance.
I am thankful to everybody who worked together, helped and advised.
Thanks to Arvydas Dapkunas and Pardeep Kumar for articles.Great thanks
to my family and relatives for support. Special thanks to my sisters, also
Edita and Vaclovas for help, care, encouragements and being together.
73
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