Intrinsic Cardiac Ganglia Research Articles

Characterization of glutamatergic neurons in the rat atrial intrinsic cardiac ganglia that project to the cardiac ventricular wall

The intrinsic cardiac nervous system modulates cardiac function by acting as an integration site for regulating autonomic efferent cardiac output. This intrinsic system is proposed to be composed of a short cardio-cardiac feedback control loop within the cardiac innervation hierarchy. For example, electrophysiological studies have postulated the presence of sensory neurons in intrinsic cardiac ganglia (ICG) for regional cardiac control. There is still a knowledge gap, however, about the anatomical location and neurochemical phenotype of sensory neurons inside ICG. In the present study, rat ICG neurons were characterized neurochemically with immunohistochemistry using glutamatergic markers: vesicular glutamate transporters 1 and 2 (VGLUT1; VGLUT2), and glutaminase (GLS), the enzyme essential for glutamate production. Glutamatergic neurons (VGLUT1/VGLUT2/GLS) in the ICG that have axons to the ventricles were identified by retrograde tracing of wheat germ agglutinin–horseradish peroxidase (WGA–HRP) injected in the ventricular wall. Co-labeling of VGLUT1, VGLUT2, and GLS with the vesicular acetylcholine transporter (VAChT) was used to evaluate the relationship between post-ganglionic autonomic neurons and glutamatergic neurons. Sequential labeling of VGLUT1 and VGLUT2 in adjacent tissue sections was used to evaluate the co-localization of VGLUT1 and VGLUT2 in ICG neurons. Our studies yielded the following results: (1) ICG contain glutamatergic neurons with GLS for glutamate production and VGLUT1 and 2 for transport of glutamate into synaptic vesicles; (2) atrial ICG contain neurons that project to ventricle walls and these neurons are glutamatergic; (3) many glutamatergic ICG neurons also were cholinergic, expressing VAChT; (4) VGLUT1 and VGLUT2 co-localization occurred in ICG neurons with variation of their protein expression level. Investigation of both glutamatergic and cholinergic ICG neurons could help in better understanding the function of the intrinsic cardiac nervous system.

Differential Effects from Pharmacological and Electrical Stimulation of Rabbit Cardiac Ganglia

Cardiac autonomic innervation is complex due to the presence of an independent network of intrinsic cardiac ganglia, which are capable of differentially modulating cardiac function. Cardiac effects from these ganglia are studied using pharmacological or electrical stimulation and this was investigated in the current study. Langendorff perfused NZW rabbit hearts (2.5‐3kg) were used and ganglionic effects from left and right neuronal complexes (RNC) studied. Nicotine was used for pharmacological activation (0.1mg/10ul) and compared to electrical stimulation (20‐50Hz, 500‐700uA). Heart rate (HR) and atrioventricular conduction (AVC) was measured during control, muscarinic (atropine, 0.1uM), beta‐receptor (metoprolol, 1.8uM) and nicotinic (hexamthonium, 0.5uM) receptor blockade. Nicotine elicited mixed HR responses i.e. bradycardia alone, tachycardia alone and/or a mixed brady/tachycardia at each site. At the LNC, there was a smaller bradycardia (147.9±5.0 ‐ 142.2±5.6 bpm) but larger tachycardia (208.1±6.8 bpm) vs. the RNC (146.1±4.5, 131.5±5.4 [brady], 191.3±8.0 [tachy] bpm). Nicotine elicited AVC prolongation and/or shortening at each site. AVC prolongation was largest at the RNC (122.4±3.2 ‐ 129.5±3.6 ms) vs. the LNC (122.9±4.1 ‐ 126.2±4.3 ms). AVC shortening was indifferent. Only bradycardia and AVC prolongation was elicited by electrical stimulation. Responses were largest at the RNC. Bradycardia/AVC prolongation was blocked by atropine. Tachycardia/AVC shortening was blocked by metoprolol. Hexamthonium blocked all responses. These are the first data to show functional effects from ganglionic plexus stimulation in the rabbit with different responses from pharmacological vs. electrical stimulation.

Low level tragus nerve stimulation is a non-invasive approach for anti-atrial fibrillation via preventing the loss of connexins

Atrial fibrillation (AF) is the most common cardiac arrhythmia, but the mechanism of AF maintenance remains unclear. It has been found that gap junctional remodeling (connexin expression is downregulated and distributed) is closely associated with AF maintenance [ [1] Van der Velden H.M. Ausma J. Rook M.B. Hellemons A.J. van Veen T.A. Allessie M.A. Jongsma H.J. Gap junctional remodeling in relation to stabilization of atrial fibrillation in the goat. Cardiovasc. Res. 2000; 46: 476-486 Crossref PubMed Scopus (214) Google Scholar ]. Autonomic nervous system stimulation can exert anti-arrhythmia effects [ 2 Thunsiri K. Shinlapawittayatorn K. Chinda K. Palee S. Surinkaew S. Chattipakorn S.C. KenKnight B.H. Chattipakorn N. Application of vagus nerve stimulation from the onset of ventricular fibrillation to post-shock period improves defibrillation efficacy. Int. J. Cardiol. 2014; 176: 1030-1032 Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar , 3 He B. Lu Z. He W. Huang B. Jiang H. Similar effects of vagus nerve stimulation and atrial ganglionated plexi stimulation on ventricular effective refractory period and action potential duration in canine. Int. J. Cardiol. 2013; 168: 5116-5118 Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar ]. Recently, we reported that cervical low level vagal nerve stimulation (LL-VNS) could suppress AF induced high-frequency stimulation [ [4] Yu L. Scherlag B.J. Li S. Sheng X. Lu Z. Nakagawa H. Zhang Y. Jackman W.M. Lazzara R. Jiang H. Po S.S. Low-level vagosympathetic nerve stimulation inhibits atrial fibrillation inducibility: direct evidence by neural recordings from intrinsic cardiac ganglia. J. Cardiovasc. Electrophysiol. 2011; 22: 455-463 Crossref PubMed Scopus (104) Google Scholar ] and then we developed a non-invasive approach, low level tragus nerve stimulation (LL-TS), and found that it had the same effect on suppressing AF [ [5] Yu L. Scherlag B.J. Li S. Fan Y. Dyer J. Male S. Varma V. Sha Y. Stavrakis S. Po S.S. Low-level transcutaneous electrical stimulation of the auricular branch of the vagus nerve: a noninvasive approach to treat the initial phase of atrial fibrillation. Heart Rhythm. 2013; 10: 428-435 Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar ]. However, whether LL-TS could regulate Cx40 and Cx43 to suppress AF remains unknown. We hypothesized that LL-TS exerted anti-atrial fibrillatory effect via the downregulation of atrial Cx40 and Cx43.

A combined acetylcholinesterase and immunohistochemical method for precise anatomical analysis of intrinsic cardiac neural structures

A significant challenge when investigating autonomic neuroanatomy is being able to reliably obtain tissue that contains neuronal structures of interest. Currently, histochemical staining for acetylcholinesterase (AChE) remains the most feasible and reliable method to visualize intrinsic nerves and ganglia in whole organs. In order to precisely visualize and sample intrinsic cardiac nerves and ganglia for subsequent immunofluorescent labeling, we developed a modified histochemical AChE method using material from pig and sheep hearts. The method involves: (1) chemical prefixation of the whole heart, (2) short-term and weak histochemical staining for AChE in situ, (3) visual examination and extirpation of the stained neural structures from the whole heart, (4) freezing, embedding and cryostat sectioning of the tissue of interest, and (5) immunofluorescent labeling and microscopic analysis of neural structures. Firstly, our data demonstrate that this modified AChE protocol labeled intrinsic cardiac nerves as convincingly as our previously published data. Secondly, there was the added advantage that adrenergic, cholinergic and peptidergic neuropeptides, namely protein gene product 9.5 (PGP 9.5), neurofilament (NF), tyrosine hydroxylase (TH), vesicular monoamine transporter (VMAT2), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene related peptide (CGRP), and substance P may be identified. Our method allows the precise sampling of neural structures including autonomic ganglia, intrinsic nerves and bundles of nerve fibers and even single neurons from the whole heart. This method saves time, effort and a substantial amount of antisera. Nonetheless, the proof of specific staining for many other autonomic neuronal markers has to be provided in subsequent studies.

Atrial fibrillation increases sympathetic and parasympathetic neurons in the intrinsic cardiac nervous system.

Chronic atrial fibrillation (AF) leads to heterogeneous autonomic nerve innervation termed neural remodeling. The quantitative changes in neural density as a function of autonomic remodeling and its association with sustained AF has not been previously determined. Seven dogs (paced group) were chronically paced with electrodes sutured to the epicardium of left atrial appendages. Seven dogs (control animals) were not paced. All paced dogs developed sustained AF by 5 weeks of pacing. The fat pads on the atrial epicardium containing ganglionated plexuses (GP) were separated along with underlying myocardial tissue. Immunocytochemical techniques were used to identify the neurons immunoreactive to anti-tyrosine hydroxylase (TH) and anti-acetylcholine antibodies. After chronic AF, sympathetic and parasympathetic neurons in the atrial intrinsic cardiac ganglia increased significantly. In paced dogs, the density of sympathetic neurons was 3,022 ± 507 μm(2) /mm(2) in the right atrial GP (vs control P < 0.01), 8,571 ± 476 μm(2) /mm(2) in the ventral left atrial GP (vs control P < 0.0001)， 6,422 ± 464 μm(2) /mm(2) in the dorsal atrial GP (vs control P < 0.0001) and 5,392 ± 595 μm(2) /mm(2) in the inferior vena cava-inferior atrial GP (vs control P <0.0001), respectively. The density of parasympathetic neurons was 4,396 ± 877 μm(2) /mm(2) in the right atrial GP, 7,769 ± 465 μm(2) /mm(2) in the ventral left atrial GP， 7,016.47 ± 687 μm(2) /mm(2) in the dorsal atrial GP and 5,485 ± 554 μm(2) /mm(2) in the inferior vena cava-inferior atrial GP, respectively, which was higher than control cohorts in corresponding GP (P < 0.05). This study provides evidence for the remodeling in atrial intrinsic cardiac ganglia in the dogs with pacing induced AF. A significant increase of sympathetic and parasympathetic neurons was present in atrial intrinsic cardiac ganglia.

Immunohistochemical detection and gene expression of tyrosine hydroxylase and vesicular monoamine transporter type 2 in intrinsic cardiac ganglia of socially isolated rats

Social isolation induced a significant increase in resting heart rate and reduction in heart rate variability. Dysfunction of the intrinsic cardiac nervous system is implicated in the genesis of cardiovascular diseases. Previous evidence suggests that cardiac ganglia contain noradrenergic neurons. Thus, immunohistochemical expression of catecholaminesynthesizing enzyme tyrosine hydroxylase (TH) and vesicular monoamine transporter 2 (VMAT2) were analyzed, as well as the effects of social isolation stress on mRNA and protein levels of this enzyme and transporter in the intrinsic cardiac nervous system of adult rats. Our results indicate that cardiac ganglion neurons express TH and VMAT2 immunoreactivity. Chronic isolated stress of rats caused a decrease in TH mRNA and VMAT2 mRNA in the neurons of intrinsic cardiac ganglia. No significant alterations in the protein levels of TH and VMAT2 were observed in these neurons. These data indicate that the neurons of intrinsic cardiac ganglia express TH as well as VMAT2 but that social isolation stress does not change their protein levels.

Pheochromocytoma and paraganglioma: Diagnosis, genetics, management, and treatment

Paragangliomas are neuroendocrine tumours of the sympathetic and parasympathetic nervous systems originating from neural crest cells. Less than 1% of paragangliomas in the heart originate from intrinsic cardiac ganglia cells in the posterior wall of the atria, atrioventricular groove, and along the root of the great vessels. We describe the tumour characteristics, patient demographics, presentation, means of diagnosis, pathology correlation, management, and outcome in 11 patients with intrapericardial paragangliomas. To the authors' knowledge, this is the largest case series reported, with emphasis on multimodality imaging findings.

Distribution and morphology of calcitonin gene-related peptide and substance P immunoreactive axons in the whole-mount atria of mice.

The murine model has been used to investigate the role of cardiac sensory axons in various disease states. However, the distribution and morphological structures of cardiac nociceptive axons in normal murine tissues have not yet been well characterized. In this study, whole-mount atria from FVB mice were processed with calcitonin gene-related peptide (CGRP) and substance P (SP) primary antibodies followed by secondary antibodies, and then examined using confocal microscopy. We found: 1) Large CGRP-IR axon bundles entered the atria with the major veins, and these large bundles bifurcated into small bundles and single axons that formed terminal end-nets and free endings in the epicardium. Varicose CGRP-IR axons had close contacts with muscle fibers, and some CGRP-IR axons formed varicosities around principle neurons (PNs) within intrinsic cardiac ganglia (ICGs). 2) SP-IR axons also were found in the same regions of the atria, attached to veins, and within cardiac ganglia. Similar to CGRP-IR axons, these SP-IR axons formed terminal end-nets and free endings in the atrial epicardium and myocardium. Within ICGs, SP-IR axons formed varicose endings around PNs. However, SP-IR nerve fibers were less abundant than CGRP-IR fibers in the atria. 3) None of the PNs were CGRP-IR or SP-IR. 4) CGRP-IR and SP-IR often colocalized in terminal varicosities around PNs. Collectively, our data document the distribution pattern and morphology of CGRP-IR and SP-IR axons and terminals in different regions of the atria. This knowledge provides useful information for CGRP-IR and SP-IR axons that can be referred to in future studies of pathological remodeling.

How the pulmonary veins 'talk' to the sinoatrial node: new insights into an old mystery

This editorial refers to ‘Nerves projecting from the intrinsic cardiac ganglia of the pulmonary veins modulate sinoatrial node pacemaker function’ by M. Zarzoso et al ., pp. 566–575, this issue. The role of the autonomic nervous system (ANS) in the genesis of atrial arrhythmias has been extensively studied in the past,1,2 with both the parasympathetic and the sympathetic nervous system having been implicated in the genesis of atrial fibrillation (AF).3,4 The discovery over a decade ago of the role of the pulmonary veins (PVs) in the genesis of AF has generated renewed interest in understanding the autonomic profile of the atria, in order to understand the role of the ANS in the genesis of focal triggers in the PVs. Clinical and animal data suggest that these PV trigger and drivers appear to be at least partially modulated by the ANS,4–6 with different studies implicating either the sympathetic or the parasympathetic nervous system in the genesis and/or maintenance of focal AF. More recent studies postulate a dominant role for the parasympathetic nervous system (in focal AF), in part on account of the vagal or Bezold–Jarisch-like reflexes (e.g. sinus bradycardia, asystole, AV block, or hypotension) noted during radiofrequency ablation of the PV tissues in patients with paroxysmal focal AF.7 Indeed, some clinical studies have suggested that the attenuation of these …

Neural control hierarchy of the heart has not evolved to deal with myocardial ischemia

The consequences of myocardial ischemia are examined from the standpoint of the neural control system of the heart, a hierarchy of three neuronal centers residing in central command, intrathoracic ganglia, and intrinsic cardiac ganglia. The basis of the investigation is the premise that while this hierarchical control system has evolved to deal with "normal" physiological circumstances, its response in the event of myocardial ischemia is unpredictable because the singular circumstances of this event are as yet not part of its evolutionary repertoire. The results indicate that the harmonious relationship between the three levels of control breaks down, because of a conflict between the priorities that they have evolved to deal with. Essentially, while the main priority in central command is blood demand, the priority at the intrathoracic and cardiac levels is heart rate. As a result of this breakdown, heart rate becomes less predictable and therefore less reliable as a diagnostic guide as to the traumatic state of the heart, which it is commonly used as such following an ischemic event. On the basis of these results it is proposed that under the singular conditions of myocardial ischemia a determination of neural control indexes in addition to cardiovascular indexes has the potential of enhancing clinical outcome.

Vesicular glutamate transporter 1‐immunoreactive sensory neurons in the rat intrinsic cardiac ganglia

Sensory neurons that reside in intrinsic cardiac ganglia (ICG) have an important role in monitoring the cardiac milieu for regional cardiac dynamic control. We recently described sensory neurons in ICG that project to the cardiac ventricular wall and currently are evaluating their neurochemical phenotype. Since many primary sensory neurons are glutamatergic, we hypothesized that ICG sensory neurons are glutamatergic, utilizing vesicular glutamate transporter 1 (VGLUT1) to package glutamate into synaptic vesicles. In the present study, the rat ventricle wall was injected (retro‐diaphragmatic, 8μl) with the retrograde tracer wheat germ agglutinin‐horseradish peroxidase (WGA‐HRP). After 72h, rats were euthanized and transcardially perfused with fixative. ICG were processed for immunohistochemistry studies. Our results demonstrated that: (1) VGLUT1‐immunoreactive (ir) neurons were present in ICG; (2) Some VGLUT1‐ir neurons were labeled from the ventricles with WGA‐HRP. Our previous studies indicated that sensory ICG neurons contain glutaminase, the synthetic enzyme for glutamate. These and the current results indicate that ICG sensory neurons are glutamatergic in neurochemical phenotype. Further investigation of VGLUTs during cardiac physiological and pathological conditions will help in deciphering the function of glutamate as a neurotransmitter in the ICG. Supported by NIH AR47410 (KEM).Grant Funding Source: NIH AR47410

Complexity of the frog intracardiac neurons. Intracellular injection study

The goal of this study was to determine the structure of intracardiac neurons in the frog Rana temporaria . Fifty-six intracardiac neurons from 8 animals were labelled ionophoretically by the intracellular markers AlexaFluor 586 and Lucifer Yellow CH. Among the labelled neurons, we found the cells of unipolar, bipolar, multipolar and pseudounipolar types. Multiple neuronal processes originated from the soma, the axon hillock and the initial segment of axon. With respect to the soma, the neuron contained (Mean ± SE) 3.5 ± 0.3 long and 5.5 ± 0.6 short processes. Most neurons had the spine, the bubble or the flake like extensions on their soma surface and were classified as Golgi I typeneurons. Few Golgi II type neurons, the presumptive interneurons, were also found. Our findings contradict to a general view that the frog intracardiac ganglia contain only the adendritic neurons of the unipolar type. Our findings demonstrate that the frog intracardiac neurons are structurally complex and diverse. This diversity may account for the complicated integrative functions of the frog intrinsic cardiac ganglia.

The Heart of the Matter

b l PRESENTATION In this case, a man with multiple cardiac symptoms suffers cardiac arrest during his medical evaluation, is revived, and is then treated for a very unusual tumor. The patient was a 46-year-old, previously healthy man who presented with complaints of shortness of breath and chest heaviness. Further inquiry revealed generalized fatigue and numerous episodes of profuse sweating over the preceding 2 months. A review of systems was otherwise negative.

Foundations of Neurocardiology: Neural control of the normal and diseased heart

Neurons in the intrinsic cardiac and intrathoracic extracardiac networks of the cardiac neuronal hierarchy form distinct nested feedback loops that act in concert with central feedback loops in the spinal cord, medulla and higher centers to coordinate regional cardiac function. Intrathoracic ganglia contain afferent, local circuit (interneurons) and sympathetic efferent postganglionic neurons. Cardiac‐related parasympathetic efferent postganglionic neurons are confined to intrinsic cardiac ganglia. The peripheral aspects of this hierarchy are, in turn, modulated by central neurons and circulating neurohumoral agents such as Ang II and catecholamines. Imbalances in cardiac neurohumoral control, especially those leading to excessive efferent neuronal activation, can lead to adverse short‐ or long‐term alterations in cardiac function. These involve structural and functional changes within the intrinsic cardiac nervous system, as well as cardiac arrhythmia induction and pump failure. We have proposed that stabilization of imbalances within select elements of the cardiac neuronal hierarchy can i) reduce the arrhythmogenic potential of cardiac tissues and ii) act to maintain myocyte viability in the presence of regional ischemia. Thus, the cardiac neuronal hierarchy has emerged as a novel therapeutic target for managing cardiac disease via pharmacological, physical or electrical means. HL71830

Distribution, structure and projections of the frog intracardiac neurons

Histochemistry for acetylcholinesterase was used to determine the distribution of intracardiac neurons in the frog Rana temporaria. Seventy-nine intracardiac neurons from 13 frogs were labelled iontophoretically by the intracellular markers Alexa Fluor 568 and Lucifer Yellow CH to determine their structure and projections. Total neuronal number per frog heart was (Mean ± SE) 1374 ± 56. Largest collections of neurons were found in the interatrial septum (46%), atrioventricular junction (25%) and venal sinus (12%). Among the intracellularly labelled neurons, we found the cells of unipolar (71%), multipolar (20%) and bipolar (9%) types. Multiple processes originated from the neuron soma, hillock and proximal axon. These processes projected onto adjacent neuron somata and cardiac muscle fibers within the interatrial septum. Average total length of the processes from proximal axon was 348 ± 50 μm. Average total length of processes from soma and hillock was less, 118 ± 27 μm and 109 ± 24 μm, respectively. The somata of 59% of neurons had bubble- or flake-shaped extensions. Most neurons from the major nerves in the interatrial septum sent their axons towards the ventricle. In contrast, most neurons from the ventral part of the interatrial septum sent their axons towards the atria. Our findings contradict to a view that the frog intracardiac ganglia contain only non-dendritic neurons of the unipolar type. We conclude that the frog intracardiac neurons are structurally complex and diverse. This diversity may account for the complicated integrative functions of the frog intrinsic cardiac ganglia.

Sinoatrial Node Activity is Modulated by Nerves Emerging From the Intrinsic Cardiac Ganglia of the Pulmonary Veins in the Mouse Heart

Sinoatrial Node Activity is Modulated by Nerves Emerging From the Intrinsic Cardiac Ganglia of the Pulmonary Veins in the Mouse Heart

Neural mechanisms of atrial arrhythmias

The past 5 years have seen great advances in the knowledge of neural mechanisms of atrial arrhythmogenesis. Direct autonomic nerve recordings demonstrate that simultaneous sympathovagal discharges and intrinsic cardiac nerve activities are common triggers of paroxysmal atrial tachycardia and atrial fibrillation. While activity of the autonomous nervous system (ANS) is crucial in triggering paroxysmal atrial fibrillation, a high incidence of sympathovagal co-activation at baseline is associated with a high vulnerability to pacing-induced sustained atrial fibrillation, suggesting that ANS has a role in the development of persistent atrial fibrillation. Modulation of ANS activity may constitute an important therapeutic strategy for the management of atrial tachyarrhythmias. Specifically, continuous, low-level stimulation of the left cervical vagus nerve effectively suppresses atrial tachyarrhythmias by reducing the nerve activity of the stellate ganglion. Clinically, compared with pulmonary vein isolation alone, the addition of ablation of intrinsic cardiac ganglia may confer better outcomes for patients with paroxysmal atrial fibrillation. These findings suggest that further investigation of the neural mechanisms of atrial arrhythmias might lead to better management of patients with atrial arrhythmias. In this article, we review the role of the ANS in the induction and maintenance of atrial arrhythmias and the role of neural modulation as a treatment strategy for atrial arrhythmias.

Neural control of heart rate: The role of neuronal networking

Neural control of heart rate, particularly its sympathetic component, is generally thought to reside primarily in the central nervous system, though accumulating evidence suggests that intrathoracic extracardiac and intrinsic cardiac ganglia are also involved. We propose an integrated model in which the control of heart rate is achieved via three neuronal “levels” representing three control centers instead of the conventional one. Most importantly, in this model control is effected through networking between neuronal populations within and among these layers. The results obtained indicate that networking serves to process demands for systemic blood flow before transducing them to cardiac motor neurons. This provides the heart with a measure of protection against the possibility of “overdrive” implied by the currently held centrally driven system. The results also show that localized networking instabilities can lead to sporadic low frequency oscillations that have the characteristics of the well-known Mayer waves. The sporadic nature of Mayer waves has been unexplained so far and is of particular interest in clinical diagnosis.

The cardiac neuronal hierarchy and susceptibility to arrhythmias

The cardiac neuronal hierarchy and susceptibility to arrhythmias

Immunohistochemistric study of the intra-cardiac ganglia of the pig heart

Heart contains a variety of neurons known to influence cardiac functions. Little is known about the neurochemistry of these neurons. In this study, neurochemistry of intra cardiac neurons of the intrinsic cardiac ganglia was investigated. The technique used allows studying the morphology of the ganglionated nerve plexus found within the atria and great cardiac vessels as well as of individual neurons. The epicardial surface of pig hearts obtained postmortem using immunohistochemically staining in conjunction with confocal microscopy neurons in the cardiac intrinsic ganglia immunoreactive for PGP 9.5 identified in the epicardial fat pad around the aortic artery and pulmonary vein. Immunocytochemistry with the marker Protein gene product PGP9.5 was used to study the presence of neurons within clusters of intrinsic cardiac ganglia. Briefly, Tissues were fixed with neutral buffered formalin. Then, they were dissected, dehydrated, cleared, washed and incubated overnight at room temperature with PGP 9.5 (1/1000 in phosphate buffered saline with 0.2 Triton X-100). The slides were washed in PBS and incubated with a mixture of Cy3 labeled Donkey anti-rabbit antibody IgG for 2 hour at room temperature. The slides were evaluated and photographed with a confocal microscope. The results of this study indicate a moderate level of chemical diversity within the intra cardiac neurons of the pig such chemical diversity may reflect functional specialization of neurons in the intra cardiac ganglia. A distinct population of ganglia was seem to arise from in the endocardial plexus of the atria and was predominantly protein gene product 9.5 positive. The small black depressions on the neurons represented satellite cells. Neurons of the intrinsic cardiac ganglia are distributed more widely than has been described in experimented animals. Intra cardiac ganglia formed a complex plexus around the origin of pulmonary veins and were connected by nerve fibers. All neurons included in intrinsic cardiac ganglia contained immunoreactivity to PGP 9.5. Neurons express PGP 9.5 immunoreactivity, suggesting that catecolamine may be present in autonomic nerves in the heart.