Autonomic innervation of the human cardiac conduction system: changes from infancy to senility--an immunohistochemical and histochemical analysis

In order to study the changes in the pattern of autonomic innervation of the human cardiac conduction system in relation to age, the innervation of the conduction system of 24 human hearts (the age of the individuals ranged from newborn to 80 years), freshly obtained at autopsy, was evaluated by a combination of immunofluorescence and histochemical techniques. The pattern of distribution and density of nerves exhibiting immunoreactivity against protein gene product 9.5 (PGP), a general neural marker, dopamine beta-hydroxylase (DBH) and tyrosine hydroxylase (TH), indicators for presumptive sympathetic neural tissue, and those demonstrating positive acetylcholinesterase (AChE) activity, were studied. All these nerves showed a similar pattern of distribution and developmental changes. The density of innervation, assessed semiquantitatively, was highest in the sinus node, and exhibited a decreasing gradient through the atrioventricular node, penetrating and branching bundle, to the bundle branches. Other than a paucity of those showing AChE activity, nerves were present in substantial quantities in infancy. They then increased in density to a maximum in childhood, at which time the adult pattern was achieved and then gradually decreased in density in the elders to a level similar to or slightly less than that in infancy. In contrast, only scattered AChE-positive nerves were found in the sinus and atrioventricular nodes, but were absent from the bundle branches of the infant heart, whereas these conduction tissues themselves possessing a substantial amount of pseudocholinesterase. During maturation into adulthood, however, the conduction tissues gradually lost their content of pseudocholinesterase but acquired a rich supply of AChE-positive nerves, comparable in density to those of DBH and TH nerves. The decline in density of AChE-positive nerves in the conduction tissues in the elders was also similar to those of DBH and TH nerves. Our findings of initial sympathetic dominance in the neural supply to the human cardiac conduction system in infancy, and its gradual transition into a sympathetic and parasympathetic codominance in adulthood, correlate well with the physiologic alterations known to occur in cardiac rate during postnatal development. The finding of reduction in density of innervation of the conduction tissue with ageing is also in agreement with clinical and electrophysiological findings such as age-associated reduction in cardiac response to parasympathetic stimulation. Finally, our findings also support the hypothesis that, in addition to the para-arterial route, the parafascicular route of extension along the conduction tissue constitutes another pathway for the innervation of the conduction system of the human heart during development.     

Localisation and quantitation of autonomic innervation in the porcine heart I: conduction system

This study was prompted by the prospect of transgenic pigs providing donor hearts for transplantation in human recipients. Autonomic innervation is important for the control of cardiac dynamics, especially in the conduction system. Our objective was to assess the relative distribution of autonomic nerves in the pig heart, focusing initially on the conduction system but addressing also the myocardium, endocardium and epicardium (see Crick et al. 1999). Quantitative immunohistochemical and histochemical techniques were adopted. All regions of the conduction system possessed a significantly higher relative density of the total neural population immunoreactive for the general neuronal marker protein gene product 9.5 (PGP 9.5) than did the adjacent myocardium. A similar density of PGP 9.5-immunoreactive innervation was observed between the sinus node, the transitional region of the atrioventricular node, and the penetrating atrioventricular bundle. A differential pattern of PGP 9.5-immunoreactive innervation was present within the atrioventricular node and between the components of the ventricular conduction tissues, the latter being formed by an intricate network of Purkinje fibres. Numerous ganglion cell bodies were present in the peripheral regions of the sinus node, in the tissues of the atrioventricular groove, and even in the interstices of the compact atrioventricular node. Acetylcholinesterase (AChE)-containing nerves were the dominant subpopulation observed, representing 60–70% of the total pattern of innervation in the nodal tissues and penetrating atrioventricular bundle. Tyrosine hydroxylase (TH)-immunoreactive nerves were the next most abundant neural subpopulation, representing 37% of the total pattern of innervation in the compact atrioventricular node compared with 25% in the transitional nodal region. A minor population of ganglion cell bodies within the atrioventricular nodal region displayed TH immunoreactivity. The dominant peptidergic nerve supply possessed immunoreactivity for neuropeptide Y (NPY), which displayed a similar pattern of distribution to that of TH-immunoreactive nerve fibres. Calcitonin gene-related peptide (CGRP)-immunoreactive nerves represented 8–9% of the total innervation of the nodal tissues and penetrating atrioventricular bundle, increasing to 14–19% in the bundle branches. Somatostatin-immunoreactive nerve fibres were relatively sparse (4–13% of total innervation) and were most abundant in the nodes, especially the compact atrioventricular node. The total pattern of innervation of the porcine conduction system was relatively homogeneous. A substantial proportion of nerve fibres innervating the nodal tissues could be traced to intracardiac ganglia indicative of an extensive intrinsic supply. The innervation of the atrioventricular node and ventricular conduction tissues was similar to that observed in the bovine heart, but markedly different to that of the human heart. It is important that we are aware of these findings in view of the future use of transgenic pig hearts in human xenotransplantation.     

The distribution of terminal sympathetic nerve fibers in bundle branches and false tendons of the bovine heart. An immunohistochemical and catecholamine histofluorescence study

Summary The sympathetic innervation in false tendons as a whole and the distribution of the terminal sympathetic nerve fibers in the conduction tissue in the bundle branches is unclear. Therefore, in the present study, false tendons and bundle branch regions of the bovine heart were examined using tyrosine hydroxylase (TH) immunohistochemistry and the glyoxylic acid induced catecholamine (CA) fluorescence method for demonstration of sympathetic nerve fibers. Acetylcholinesterase (AChE) histochemistry was also applied. Some of the nerve fascicles in the false tendons were found to contain large numers of sympathetic nerve fibers and such nerve fibers formed plexuses in the walls of arteries and arterioles in these structures. In both false tendons and bundle branches sympathetic nerve fibers 1) were non-homogeneously distributed in the conduction tissue, most regularly occurring in the channels of extracellular space that are present within the bundles of Purkinje fibres. and 2) showed the same pattern of distribution in relation to Purkinje fibre bundle surfaces as the AChE-positive nerve branches. The observations show that there is a substantial sympathetic innervation in false tendons. The final distribution of the nerve fibers in these structures and in the bundle branches are discussed in relation to what is known of tissue morphology and the occurrence of sympathetic nerve influences in these regions. In the present study, previous CA-fluorescence observations of a marked sympathetic innervation in bundle branch regions, in terms of the presence of sympathetic nerve fibers in nerve fascicles and vessel walls, were also corroborated by the application of TH-immunohistochemistry.     

Spatial distribution of nerve processes and β-adrenoreceptors in the rat atrioventricular node

Atrioventricular (AV) nodal conduction time is known to be modulated by the autonomic nervous system. The presence of numerous parasympathetic and sympathetic nerve fibres in association with conduction tissue in the heart is well authenticated. In this study, confocal microscopy was used to image the distribution of antibodies directed against the general neuronal marker PGP 9.5, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and β₁ and β₂-adrenoreceptors. Serial 12 μm sections of fresh frozen tissue taken from the frontal plane of the rat atrioventricular node, His bundle and bundle branches were processed for histology, acetylcholinesterase (AChE) activity and immunohistochemistry. 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 immunoreactivity. 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 a preponderance in the circumferential transitional cell region. TH-immunoreactivity was uniformly distributed throughout the AV and ventricular conduction systems including the central midnodal and circumferential transitional cell regions. β₁-adrenoreceptors were found throughout the AV and ventricular conduction systems with a preponderance in the circumferential transitional cell region. β₂-adrenoreceptors were localised 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 β₂-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.     

Localisation and quantitation of autonomic innervation in the porcine heart II: endocardium, myocardium and epicardium

The immunological problems of pig hearts supporting life in human recipients have potentially been solved by transgenic technology. Nevertheless, other problems still remain. Autonomic innervation is important for the control of cardiac dynamics and there is evidence suggesting that some neurons remain intact after transplantation. Previous studies in the human heart have established regional differences in both general autonomic innervation and in its component neural subpopulations. Such studies are lacking in the pig heart. Quantitative immunohistochemical and histochemical techniques were used to demonstrate the pattern of innervation in pig hearts (Sus scrofa). Gradients of immunoreactivity for the general neural marker protein gene product 9.5 were observed both within and between the endocardial, myocardial and epicardial plexuses throughout the 4 cardiac chambers. An extensive ganglionated plexus was observed in the epicardial tissues and, to a lesser extent, in the myocardial tissues. The predominant neural subpopulation displayed acetylcholinesterase activity, throughout the endocardium, myocardium and epicardium. These nerves showed a right to left gradient in density in the endocardial plexus, which was not observed in either the myocardial or epicardial plexuses. A large proportion of nerves in the ganglionated plexus of the atrial epicardial tissues displayed AChE activity, together with their cell bodies. Tyrosine hydroxylase (TH)-immunoreactive nerves were the next most prominent subpopulation throughout the heart. TH-immunoreactive cell bodies were observed in the atrial ganglionated plexuses. Endocardial TH- and NPY-immunoreactive nerves also displayed a right to left gradient in density, whereas in the epicardial tissues they showed a ventricular to atrial gradient. Calcitonin gene-related peptide (CGRP)-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. Several important differences were observed between the innervation of the pig heart compared with the human heart. These differences may have implications for the function of donor transgenic pig hearts within human recipients.     

Nervous regulation of the tone of the retractor penis muscle in the goat

The nervous control of the retractor penis muscle (rp) was investigated in the anaesthetized goat. Also, isolated field stimulated strips of the muscle were studied. The noradrenaline (NA) and acetylcholine (ACh) content of the rp was determined, and histochemistry for adrenergic and acetylcholinesterase (AChE) positive nerves was performed. The muscle exhibited spontaneous activity that persisted after section of all nerves. There was, however, also a tendency of the activity to follow the general vasomotor tone, which disappeared after section of the sympathetic chains. The excitatory adrenergic nerves which innervate the muscle come from the sympathetic chains and run along the pudendal, the hypogastric and the pelvic nerves. The rp has a dense network of adrenergic fibres and is very sensitive to excitatory adrenergic stimulation. It has a fairly large NA content, which is higher in old goats (5.95 ± 0.42 μg g^-1) than in young goats (2.87 ± 0.78 μg g-¹). Inhibitory non-adrenergic non-cholinergic (NANC) innervation reaches it via the pelvic and the hypogastric nerves. The maximum inhibitory response is reached at low frequencies (2–4 Hz). Cholinergic prejunctional inhibition of the excitatory response to sympathetic chain stimulation was effected by simultaneous stimulation of the hypogastric nerves. In vitro experiments confirmed the presence of endogenous cholinergic muscarinic suppression of the excitatory adrenergic neurotransmission. Significant amounts of ACh (0.81 7 plusmn; 0.18 μg g^-1) are present in the muscle, and it contains strongly AChE positive nerve fibres and nerve cell bodies. It is concluded that the goat rp is innervated by sympathetic adrenergic excitatory nerves and parasympathetic NANC inhibitory nerves. It further has a direct sympathetic inhibitory NANC innervation, and an indirect inhibitory cholinergic innervation which at least in part is sympathetic.     

Reconsideration of the Autonomic Cranial Ganglia: An Immunohistochemical Study of Mid‐Term Human Fetuses

The cranial parasympathetic ganglia have been reported to paradoxically contain the sympathetic nerve marker, tyrosine hydroxylase (TH), in addition to neurons expressing parasympathetic markers such as vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (nNOS). However, the distribution of these molecules in the cranial ganglia of human fetuses has not yet been examined. Using paraffin sections from 10 mid‐term human fetuses (12–15 weeks), we performed immunohistochemistry for TH, VIP, and nNOS in the parasympathetic ciliary, pterygopalatine, otic, and submandibular ganglia, and for comparison, the sensory inferior vagal ganglion. The ciliary and submandibular ganglia contained abundant TH‐positive neurons. In the former, TH‐positive neurons were much more numerous than nNOS‐positive neurons, whereas in the latter, nNOS immunoreactivity was extremely strong. No or a few cells in the pterygopalatine, otic, and inferior vagal ganglia expressed TH. Ciliary TH neurons appeared to compensate for classically described sympathetic fibers arising from the superior cervical ganglion, whereas in the submandibular ganglion, nNOS‐positive neurons as well as TH neurons might innervate the lingual artery in addition to the salivary glands. Significant individual variations in the density of all these markers suggested differences in sensitivity to medicine affecting autonomic nerve function. Consequently, in the human cranial autonomic ganglia, it appears that there is no simple dichotomy between sympathetic and parasympathetic function. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.     

Cells migrating from the neural crest contribute to the innervation of the venous pole of the heart

Cells migrating from the neural crest are known to septate the outflow tract of the developing heart, and to contribute to the formation of the arterial valves, their supporting sinuses, the coronary arteries and cardiac neural ganglia. Neural crest cells have also been suggested to contribute to development of the venous pole of the heart, but the extent and fate of such cells remains unclear. In this study, in the mouse, it is shown that cells from the neural crest contribute to the parasympathetic and, to a lesser extent, the sympathetic innervation of the venous pole of the heart. Nerves within the venous pole of the heart are shown to be of mixed origin, with some being derived from the neural crest, while others have an alternative origin, presumably placodal. The neurons innervating the nodal tissue, which can exert chronotropic effects on cardiac conduction, are shown not to be derived from the neural crest. In particular, no evidence was found to support previous suggestions that cells from the neural crest make a direct contribution to the myocardial atrioventricular conduction axis, although a small subset of these cells do co-localize with the developing left bundle branch. We have therefore confirmed that cells from the neural crest migrate to the venous pole of the heart, and that their major role is in the development of the parasympathetic innervation. In addition, in some embryos, a population of cells derived from the neural crest persist in the leaflets of the atrioventricular valves, but their role in subsequent development remains unknown.     

Nerves in the intersphincteric space of the human anal canal with special reference to their continuation to the enteric nerve plexus of the rectum

In the intersphincteric space of the anal canal, nerves are thought to “change” from autonomic to somatic at the level of the squamous‐columnar epithelial junction of the anal canal. To compare the nerve configuration in the intersphincteric space with the configuration in adjacent areas of the human rectum, we immunohistochemically assessed tissue samples from 12 donated cadavers, using antibodies to S100, neuronal nitric oxide synthase (nNOS), and tyrosine hydroxylase (TH). Antibody to S100 revealed a clear difference in intramuscular nerve distribution patterns between the circular and longitudinal muscle layers of the most inferior part of the rectum, with the former having a plexus‐like configuration, while the latter contained short, longitudinally running nerves. Most of the intramural ganglion cells in the anal canal were restricted to above the epithelial junction, but some were located just below that level. Near or at the level of the epithelial junction, the nerves along the rectal adventitia and Auerbach's nerve plexus joined to form intersphincteric nerves, with all these nerves containing both nNOS‐positive parasympathetic and TH‐positive sympathetic nerve fibers. Thus, it was histologically difficult to distinguish somatic intersphincteric nerves from the autonomic Auerbach's plexus. In the intersphincteric space, the autonomic nerve elements with intrapelvic courses seemed to “borrow” a nerve pathway in the peripheral branches of the pudendal nerve. Injury to the intersphincteric nerve during surgery may result in loss of innervation in the major part of the internal anal sphincter. Clin. Anat. 26:843–854, 2013. © 2013 Wiley Periodicals, Inc.     

Ingrowth of sympathetic innervation occurs concomitantly with a decrease of ANP in the growing rat cardiac ventricles

The relationship between the immunohistochemical expression of atrial natriuretic peptide and the occurrence of sympathetic nerve fibres, as visualized by staining for tyrosine hydroxylase, in the growing rat heart was evaluated. Rats were investigated at four different stages from birth to 21 days postnatally. The effects of chemical destruction of sympathetic nerve terminals in neonatal rats on the cardiac atrial natriuretic peptide content were furthermore examined by use of radioimmunoassay. There was in principle a reciprocal pattern of immunoreaction for atrial natriuretic peptide and tyrosine hydroxylase positive innervation in the ventricular myocardium, atrial natriuretic peptide reaction becoming less and less pronounced with the ingrowth of innervation positive for tyrosine hydroxylase. Furthermore, in the peripheral Purkinje fibre network, there was a marked atrial natriuretic peptide immunoexpression and scarce or no nerve fibres throughout the examination period. The radioimmunoassay measurements showed that chemical sympathectomy lead to elevated cardiac levels of atrial natriuretic peptide. The study shows that sympathetic innervation grows into the ventricular parts concomitantly with the occurrence of a decline in atrial natriuretic peptide expression during development of the heart. Furthermore, it is shown that a reversion of the in growth of sympathetic innervation by destruction of cardiac sympathetic nerves at an early stage leads to increased levels of atrial natriuretic peptide in the heart. The results give new evidence to the phenomenon that the atrial natriuretic peptide levels in the ventricular myocardium and the peripheral parts of the conduction system are under influence of the presence of sympathetic innervation. Accepted: 25 July 2000     

Sensory components in the terminal innervation of the ovine cardiac conduction system

Neuromuscular relationships in the cardiac conduction system of the ovine heart were studied by means of light and electron microscopy. Fluorescence microscopy was used to demonstrate the distribution of adrenergic nerves. Histochemical methods demonstrated sites of acetylcholinesterase and nonspecific cholinesterase activity at both light and electron microscopic levels. Surgical denervation was used in an attempt to support the identification of different types of nerve endings. A dense plexus of nerves strongly positive for acetylcholinesterase was present in all parts of the conduction system. Nonspecific cholinesterase was found to be limited to large nerve bundles which were presumed to be preganglionic. A dense plexus of adrenergic nerve fibers was identified in both the SA and AV nodes, but such fibers were very sparsely distributed along the AV bundle and its branches. On the basis of morphology as demonstrated by electron microscopy, three types of nerve endings or varicosities associated with the specialized cells of the conduction system were identified. Cholinergic endings, presumably post-ganglionic parasympathetic, contained many nongranulated synaptic vesicles. Adrenergic or post-ganglionic sympathetic endings or varicosities contained granulated synaptic vesicles. Axonal enlargements containing numerous mitochondria and varying numbers of pleomorphic dense bodies were identified as sensory endings on the basis of their morphologic similarity to such endings found elsewhere in the animal body. These were found in 22% of the blocks examined by electron microscopy, were present in all parts of the conduction tissue, and were acetylcholinesterase positive. They did not show obvious signs of degeneration following vagotomies or thoracic sympathectomy, suggesting the possibility that they were endings of neurons intrinsic to the heart. No signs of degeneration were noted in the endomysial plexus following vagotomy either proximal or distal to the distal (nodose) ganglion. Degeneration of small fibers of the plexus was noted following thoracic sympathectomy. This study provides evidence suggesting a significant sensory innervation to the cardiac conduction system, with the cell bodies of the sensory neurons located in or near the heart.     

Cardiac autonomic denervation in congestive heart failure: comparison of Chagas' heart disease with other dilated cardiomyopathy

Congestive heart failure (CHF) is associated with activation of the cardiac sympathetic nerves. However, impairment of the sympathetic nerve terminals in patients with CHF has been indicated by studies showing reduction of cardiac norepinephrine uptake and stores. This investigation studies the histochemical evaluation of the sympathetic nerve terminals in CHF. The cardiac parasympathetic innervation was also studied to address the question of specificity of the presumed sympathetic denervation. Nineteen patients with CHF underwent cardiac transplantation or partial ventriculectomy, which provided the heart tissue. In 11 of them, the dilated cardiomyopathy was associated with Chagas' disease. Inflammatory process and fibrosis were studied histologically. The sympathetic and parasympathetic nerve fibers were visualized through histochemical techniques for, respectively, catecholamines and acetylcholinesterase activity. By using a computer-assisted morphometric program, the inflammation, fibrosis, and parasympathetic innervation were quantified. Moderate to severe fibrosing myocarditis characterized the hearts of the chagasic patients. In cardiomyopathies not associated with Chagas' disease, the inflammation was discrete, if present, but the amount of fibrosis was similar to that found in Chagas' cardiomyopathy. Reduction of both kinds of nerve terminals occurred in the heart of all patients. The parasympathetic denervation was proven to be more severe in chagasic cardiomyopathy. Our data on the heart innervation indicate a progressive autonomic denervation in heart failure. In Chagas' heart disease, the denervation seems to be more severe or rapid, probably because of the sustained inflammatory process.     

An immunocytochemical study of cutaneous innervation and the distribution of neuropeptides and protein gene product 9.5 in man and commonly employed laboratory animals

The cutaneous nerves of rat, cat, guinea pig, pig, and man were studied by immunocytochemistry to compare the staining potency of general neural markers and to investigate the density of nerves containing peptides. Antiserum to protein gene product 9.5 (PGP 9.5) stained more nerves than antisera to neurofilaments, neuronspecific enolase (NSE), and synaptophysin or histochemistry for acetylcholinesterase (AChE). Peptidergic axons showed species variation in density of distribution and were most abundant in pig and fewest in man. However, the specific peptides in nerves innervating the various structures were consistent between species. Nerve fibers immunoreactive for calcitonin gene-related peptide (CGRP) and/or vasoactive intestinal polypeptide (VIP) predominated in all the species; those immunoreactive to tachykinins (substance P and neurokinin A [NKA]) and neuropeptide tyrosine (NPY) were less abundant. Neonatal capsaicin, at the doses employed in this study, destroyed approximately 70% of CGRP- and tachykinin-immunoreactive sensory axons; whereas 6-hydroxydopamine (6-OHDA) at the doses employed resulted in a complete loss of NPY and tyrosine hydroxylase (TH) immunoreactivity without affecting VIP, CGRP, and tachykinins. Thus, this study confirms that antiserum to PGP 9.5 is the most suitable and practical marker for the demonstration of cutaneous nerves. Species differences exist in the density of peptidergic innervation, but apparently not for specific peptides. Not all sensory axons immunoreactive for CGRP and substance P/NKA are capsaicin-sensitive. However, all sympathetic TH- and NPY- immunoreactive axons are totally responsive to 6-OHDA; but no change was seen in VIP-immunoreactive axons, suggesting some demarcation of cutaneous adrenergic and cholinergic sympathetic fibers.     

Autonomic innervation of the developing heart: origins and function

Maintenance of homeostatic circulation in mammals and birds is reliant upon autonomic innervation of the heart. Neural branches of mixed cellular origin and function innervate the heart at the arterial and venous poles as it matures, eventually coupling autonomic output to the cardiac components, including the conduction system. The development of neural identity is controlled by specific networks of genes and growth factors, whereas functional properties are governed by the use of different neurotransmitters. In this review, we summarize briefly the anatomic arrangement of the vertebrate autonomic nervous system and describe, in detail, the innervation of the heart. We discuss the timing of cardiac innervation in the chick and mouse, emphasizing the relationship of the cardiac neural networks to the anatomical structures within the heart. We also discuss the variable contribution of the neural crest to vagal cardiac nerves, and summarize the main neurotransmitters secreted by the developing sympathetic and parasympathetic autonomic divisions. We provide an overview of the main growth factor and gene families involved in neural development, discussing how these factors may impact upon the development of cardiac abnormalities in congenital syndromes associated with autonomic dysfunction.     

Comparative Anatomy and Evolution of the Cardiac Innervation in New World Monkeys (Platyrrhini,E. Geoffroy, 1812)

The morphology of the autonomic cardiac nervous system (ACNS) was examined in 24 sides of 12 New World monkeys (Platyrrhini) of all four families to document the morphology systematically and to study the evolutionary changes of the ACNS in this primate lineage. We report the following: (1) Although several trivial intra‐ and inter‐specific variations are present, a family‐dependent morphology of the ACNS does not exist in New World monkeys. (2) The sympathetic ganglia in New World monkeys consist of the superior cervical, the middle cervical, and the cervicothoracic which is composed of the inferior cervical and first and second thoracic, and the thoracic ganglia starting with the third thoracic. The general cardiac nervous system is the sympathetic middle and inferior cardiac nerves and all parasympathetic vagal cardiac branches. (3) The morphology of the ACNS in the New World monkeys is almost consistent regardless of the number of vertebrae, the cardiac position and deviation (axis), and the great arterial branching pattern of the aortic arch, and it is very similar to that in the Old World monkeys, with only one difference: the superior cervical ganglion in the New World monkeys tends to be relatively smaller, higher, and provides a narrower contribution to the spinal nerves than in the Old World monkeys. The ACNS morphology exhibits significant evolutionary changes within the primate lineage from New and Old World monkeys to humans. The comparative morphology within the lineage is concordant with the phylogeny, suggesting that the primate ACNS preserves its evolutionary history in close alignment with phylogeny. Anat Rec, 292:670–691, 2009. © 2009 Wiley‐Liss, Inc.     

Atrial sympathetic and parasympathetic nerve sprouting and hyperinnervation induced by subthreshold electrical stimulation of the left stellate ganglion in normal dogs

BACKGROUND: Subthreshold electrical stimulation of the left stellate ganglion (LSG) can induce nerve sprouting and sympathetic hyperinnervation in canine ventricles. It is unclear whether a similar neural plasticity involving both sympathetic and parasympathetic innervation also exists in the atria. METHODS AND RESULTS: We applied subthreshold electrical stimulation at 20 Hz (0.45 ms pulse width) or 5 Hz (1.9 ms pulse width) to the LSG in 6 normal mongrel dogs. After 41+/-9 days, the hearts were harvested and the right and left atrium stained for synaptophysin (SYN), growth-associated protein 43 (GAP43), sympathetic nerve markers tyrosine hydroxylase (TH), and parasympathetic marker choline acetyltransferase (ChAT). Tissues from 6 additional healthy dogs were used as controls. The hearts from dogs with LSG electrical stimulation had a higher density of nerve structures immunopositive to the SYN, GAP43, TH, and ChAT (P<.01) in both right and left atria. Nerve density was equal in right and left atria. There were more TH-positive nerve structures than ChAT-positive nerve structures (P<.01) for both right and left atria. No atrial arrhythmia was observed at the second surgery. CONCLUSIONS: Continuous subthreshold electrical stimulation to the LSG induces both sympathetic and parasympathetic hyperinnervation in both right and left atria in normal dogs.     

Developmental regulation of parasympathetic nerve density by sympathetic innervation in the tarsal smooth muscle of the rat.

The developmental influence of sympathetic innervation on parasympathetic nerve density was investigated in the tarsal smooth muscle of the rat. Specificity of acetylcholinesterase staining as a marker for parasympathetic innervation was first determined by acute selective denervations. Excision of the ipsilateral superior cervical ganglion caused a 39% reduction in the density of acetylcholinesterase-positive nerves seven days later, indicating that sympathetic nerves contribute to cholinesterase-positive tarsal muscle innervation. Excision of the pterygopalatine ganglion concurrent with superior cervical ganglionectomy caused a virtually complete disappearance of acetylcholinesterase-positive innervation within seven days, indicating that non-sympathetic cholinesterase-positive fibers derive from the pterygopalatine ganglion and are presumed to be parasympathetic. Analysis of the control population indicated that parasympathetic nerve density did not vary significantly between males and females, between the superior and inferior muscles, or in rats studied at four and 12 months of age. The influence of sympathetic innervation on parasympathetic nerve density during postnatal development was examined by conducting surgical sympathectomies on postnatal day 5 and quantifying acetylcholinesterase-positive nerve density at four months of age. Neonatal sympathectomy caused a 46% reduction in cholinesterase-positive nerve density beyond that which occurred in acutely sympathectomized adult controls. It is concluded that sympathetic innervation is required for developing parasympathetic nerves to attain their normal density within the rat tarsal muscle. This finding is consistent with the idea that sympathetic nerves can exert positive effects on parasympathetic nerve outgrowth during development.     

Synaptic Connections of the Paracervical (Frankenhauser) Ganglion of the Rat Uterus Examined with the Electron Microscope after Division of the Sympathetic and Sacral Parasympathetic Nerves

The fine structure of the synapses in the paracervical (Frankenhauser) ganglion of the rat uterus was studied after sympathectomy and sacral parasympathectomy. Degenerative nerve endings to the ganglion cells were observed after division of both the sympathetic and parasympathetic nerves. No degeneration of the synapses to the chromaffin cells were seen. The results are interpreted as evidence of both sympathetic and parasympathetic innervation of the ganglion cells, which send their axons to the uterus, whereas the “chromaffin cells” are innervated by intraganglionic nerves.     

Distribution of catecholaminergic afferent fibres in the rat globus pallidus and their relations with cholinergic neurons

The topographical distribution of catecholaminergic nerve fibres and their anatomical relationship to cholinergic elements in the rat globus pallidus were studied. Peroxidase–antiperoxidase and two-colour immunoperoxidase staining procedures were used to demonstrate tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT) and choline acetyltransferase (ChAT) immunoreactivities, combined with acetylcholinesterase (AChE) pharmacohistochemistry. TH immunoreactive nerve fibres were seen to enter the globus pallidus from the medial forebrain bundle. The greatest density of such fibres was found in the ventral region of the globus pallidus, which was also characterized by the greatest density of ChAT immunoreactive neurons. TH immunoreactive nerve fibres showed varicose arborizations and sparse boutons, which were occasionally seen in close opposition to cholinergic structures. In all regions of the globus pallidus, there were also larger, smooth TH immunoreactive nerve fibres of passage to the caudate putamen. A smaller number of DBH immunoreactive nerve fibres and terminal arborizations were found in the substantia innominata, internal capsule and in the globus pallidus bordering these structures. A few PNMT immunoreactive nerve fibres in the substantia innominata and internal capsule did not enter the globus pallidus. Electron microscopy revealed TH immunoreactive synaptic profiles in the ventromedial area of the globus pallidus corresponding to the nucleus basalis magnocellularis of Meynert (nBM). These made mainly symmetrical and only a few asymmetrical synaptic contacts with dendrites containing AChE reaction product. The results indicate that cholinergic structures in the nBM are innervated by dopaminergic fibres and terminals, with only a very small input from noradrenergic fibres.     

Contribution of the cervical sympathetic ganglia to the innervation of the pharyngeal arch arteries and the heart in the chick embryo

In the chick heart, sympathetic innervation is derived from the sympathetic neural crest (trunk neural crest arising from somite level 10–20). Since the trunk neural crest gives rise to sympathetic ganglia of their corresponding level, it suggests that the sympathetic neural crest develops into cervical ganglia 4–14. We therefore tested the hypothesis that, in addition to the first thoracic ganglia, the cervical ganglia might contribute to cardiac innervation as well. Putative sympathetic nerve connections between the cervical ganglia and the heart were demonstrated using the differentiation markers tyrosine hydroxylase and HNK‐1. In addition, heterospecific transplantation (quail to chick) of the cardiac and trunk neural crest was used to study the relation between the sympathetic neural crest and the cervical ganglia. Quail cells were visualized using the quail nuclear antibody QCPN. The results by immunohistochemical study show that the superior and the middle cervical ganglia and possibly the carotid paraganglia contribute to the carotid nerve. This nerve subsequently joins the nodose ganglion of the vagal nerve via which it contributes to nerve fibers in cardiac vagal branches entering the arterial and venous pole of the heart. In addition, the carotid nerve contributes to nerve fibers connected to putative baro‐ and chemoreceptors in and near the wall of pharyngeal arch arteries suggesting a role of the superior and middle cervical ganglia and the paraganglia of the carotid plexus in sensory afferent innervation. The lower cervical ganglia 13 and 14 contribute predominantly to nerve branches entering the venous pole via the anterior cardinal veins. We did not observe a thoracic contribution. Heterospecific transplantation shows that the cervical ganglia 4–14 as well as the carotid paraganglia are derived from the sympathetic neural crest. The cardiac neural crest does not contribute to the neurons of the cervical ganglia. We conclude that the cervical ganglia contribute to cardiac innervation which explains the contribution of the sympathetic neural crest to the innervation of the chick heart. Anat Rec 255:407–419, 1999. © 1999 Wiley‐Liss, Inc.