Alrich L. Gray et al- Parasympathetic control of the heart. II. A novel interganglionic intrinsic cardiac circuit mediates neural control of heart ratediac innervation of the ventricles

8/3/2019 Alrich L. Gray et al- Parasympathetic control of the heart. II. A novel interganglionic intrinsic cardiac circuit mediates

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doi:10.1152/japplphysiol.00616.200396:2273-2278, 2004. First published 20 February 2004;J Appl Physiol

Alrich L. Gray, Tannis A. Johnson, Jeffrey L. Ardell and V. John Massarineural control of heart rateinterganglionic intrinsic cardiac circuit mediatesParasympathetic control of the heart. II. A novel

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Parasympathetic control of the heart. II. A novel interganglionic intrinsic

cardiac circuit mediates neural control of heart rate

Alrich L. Gray,1 Tannis A. Johnson,1 Jeffrey L. Ardell,3 and V. John Massari1,2

1 Department of Pharmacology and2Specialized Neuroscience Research Program, Howard University

College of Medicine, Washington, District of Columbia 20059; and 3Department of Pharmacology, East Tennessee State University, James H. Quillen College of Medicine, Johnson City, Tennessee 37614

Submitted 13 June 2003; accepted in final form 12 September 2003

Gray, Alrich L., Tannis A. Johnson, Jeffrey L. Ardell, and V.John Massari. Parasympathetic control of the heart. II. A novelinterganglionic intrinsic cardiac circuit mediates neural control ofheart rate. J Appl Physiol 96: 22732278, 2004. First publishedFebruary 20, 2004; 10.1152/japplphysiol.00616.2003.Intracardiacpathways mediating the parasympathetic control of various cardiacfunctions are incompletely understood. Several intracardiac gangliahave been demonstrated to potently influence cardiac rate [the sino-atrial (SA) ganglion], atrioventricular (AV) conduction (the AV gan-glion), or left ventricular contractility (the cranioventricular ganglion).However, there are numerous ganglia found throughout the heartwhose functions are poorly characterized. One such ganglion, theposterior atrial (PA) ganglion, is found in a fat pad on the rostraldorsal surface of the right atrium. We have investigated the potentialimpact of this ganglion on cardiac rate and AV conduction. We reportthat microinjections of a ganglionic blocker into the PA ganglionsignificantly attenuates the negative chronotropic effects of vagalstimulation without significantly influencing negative dromotropiceffects. Because prior evidence indicates that the PA ganglion doesnot project to the SA node, we neuroanatomically tested the hypoth-esis that the PA ganglion mediates its effect on cardiac rate through aninterganglionic projection to the SA ganglion. Subsequent to micro-injections of the retrograde tracer fast blue into the SA ganglion,70% of the retrogradely labeled neurons found within five intracar-

diac ganglia throughout the heart were observed in the PA ganglion.The neuroanatomic data further indicate that intraganglionic neuronalcircuits are found within the SA ganglion. The present data supportthe hypothesis that two interacting cardiac centers, i.e., the SA and PAganglia, mediate the peripheral parasympathetic control of cardiacrate. These data further support the emerging concept of an intrinsiccardiac nervous system.

ganglia; neurocardiology; vagus; retrograde transport; atrioventricularconduction; posterior atrial ganglion

IT HAS LONG BEEN KNOWN that parasympathetic influences oncardiac function are mediated via the vagus nerves. Stimulationof the vagi results in multiple cardiac effects, including brady-

cardia, atrioventricular (AV) block, and reduced myocardialcontractility (32). Over the past three decades, the late WalterRandall and colleagues, as well as other investigators, haveshown that postganglionic neurons contained in discrete fatpads on the surface of the heart are responsible for mediatingcertain aspects of cardiac function (3, 11, 14). Randallsfindings have been supported in the dog by independent inves-tigators not associated with their group (15, 17, 36), in primates(9), in humans (13, 30), and in our own laboratories in the cat

(19, 20). For example, ganglion cells found in a fat pad locatedadjacent to the right pulmonary veins near the junction of theright atrium and superior vena cava have been shown tomediate a selective negative chronotropic effect. Thus surgicalexcision of this fat pad or local microinjections of ganglionicblocking agents result in blockade of the negative chronotropiceffects of vagal stimulation. Due to the potent negative chro-notropic nature of the ganglion cells found in this fat pad, wehave referred to the ganglion cells found in this region as the

sinoatrial (SA) ganglion (18, 20, 24, 25). In a similar fashion,ganglion cells found in a fat pad located at the junction of theinferior border of the left atrium and the inferior vena cavahave been shown to mediate a potent reduction in the rate ofAV conduction. Therefore, we have referred to the negativedromotropic ganglion cells found in this region as the AVganglion (20, 23, 24). Furthermore, a fat pad found at thecranial margin of the left ventricle near its junction with theright ventricle has been found to contain ganglion cells thatselectively mediate negative inotropic vagal effects on the leftventricle without influencing cardiac rate or AV conduction(16, 19). We refer to the ganglion cells contained within thisregion as the cranioventricular (CV) ganglion. Different au-thors have referred to the various intracardiac ganglia utilizingdifferent names (1, 8, 14, 28, 29, 35); however, the anatomiclocation of functionally associated intracardiac ganglia acrossdifferent species shows substantial homology [for further de-tails about nomenclature and locations of intracardiac ganglia,see Johnson et al. (21)]. Taken as a whole, the data reviewedabove are consistent with our working hypothesis that there areanatomically separated and functionally independent intracar-diac ganglia found in fat pads on the surface of the heart.

There are numerous ganglia found throughout the heart forwhich little or no functional role(s) have been ascribed (1, 4,12, 21, 37). One such ganglion is found in a fat pad on therostral dorsal surface of the right atrium (26, 31). To maintainconsistency with our nomenclature scheme, we refer to this

ganglion as the posterior atrial (PA) ganglion. Preliminarystudies conducted by David Randall and colleagues utilizing adiscriminative classical Pavlovian conditioning procedure haveprovided evidence suggesting that this ganglion may be in-volved in attenuating sympathetic cardioacceleration in the dog(31); however, little is known about the potential effects of thePA ganglion on cardiac rate or AV conduction. We haveinvestigated these issues in the present report with both phys-iological and neuroanatomic methods.

Address for reprint requests and other correspondence: V. J. Massari, Dept.of Pharmacology, Howard Univ. College of Medicine, 520 W St., N.W.,Washington, DC 20059 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J Appl Physiol 96: 22732278, 2004.First published February 20, 2004; 10.1152/japplphysiol.00616.2003.

8750-7587/04 $5.00 Copyright 2004 the American Physiological Societyhttp://www.jap.org 2273


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MATERIALS AND METHODS

Physiological studies. The Institutional Animal Care and Use

Committee of Howard University reviewed and approved the exper-

imental design of all animal experiments. Acute physiological exper-

iments were performed on five mongrel cats weighing 2.84.0 kg.Baseline vital signs such as temperature, respiration rate, and heart

rate were recorded. Cats were anesthetized with 25 mg/kg iv pento-

barbital sodium. Supplemental doses of 1.251.5 mg/kg iv pentobar-bital sodium were administered as needed. The cats were assessed for

depth of anesthesia by testing for the absence of the palpebral, ocular,

and pedal reflexes both before the beginning of surgery and every halfhour thereafter. Each animals core temperature was maintained at3738C using a servocontrolled heating pad. Cats were prepared forthe experiment by inserting an intravenous catheter into the brachial

vein and intubating with a cuffed endotracheal tube. Limb leads were

attached to appropriate appendages to record lead II of the ECG. All

electrical leads were connected to a Gould TA 6000 thermal array

recorder. A Pulse-Oximeter (Vet-Ox) clamp was placed on the tongue

and was used to monitor blood oxygen concentration throughout the

experiment. Once the surgical plane of anesthesia was reached, the

right cervical vagus was isolated, a 0.5-mm platinum-stimulating

electrode was placed on it, and it was bathed in mineral oil. The leftfemoral artery was also isolated, and a Millar probe (Millar Instru-

ments, Houston, TX) was inserted into it to record blood pressure.

Blood pressure readings served as a very sensitive indicator of depth

of anesthesia. A thoracotomy was performed via the right fifthintercostal space. Cats were artificially respired with 95% O2-5% CO2on a positive-pressure respirator cycling at 12 breaths/min with a tidal

volume setting between 50 and 100 ml. A longitudinal incision was

made in the pericardium, and the heart was removed from the

pericardial sac. A pacing wire was sutured onto the right atrial

appendage. Atrial pacing was performed at a rate 10% greater than

resting sinus rate. The right vagus was stimulated over 20-s intervals

at 20 Hz, 15 V, and 0.1-ms duration. These parameters were found to

result in maximal vagal stimulation. Heart rates were obtained from

the R-R interval. AV conduction measurements were obtained from

the stimulus artifact-R interval during atrial pacing. Heart rate and AVconduction were recorded before and during the stimulation interval.

Heart rate and AV conduction measurements were calculated from

beats that occurred during the middle of the stimulation interval.

Baseline AV conduction and heart rate responses to three consec-

utive vagal stimulations were recorded with and without atrial pacing,

respectively. The PA ganglion was identified by its gross anatomiclocation (21), and 10 l of the ganglionic blocker hexamethonium

(C6) (5 g/l) was injected into it. Five minutes later, the vagus was

stimulated three consecutive times with and without the heart being

paced, and C6-induced changes in heart rate and AV conduction were

recorded. Thirty minutes was allowed for the ganglion to recover from

the effects of the ganglionic blocker. After this time, the right vagus

nerve was again stimulated three consecutive times with and without

the heart being paced, and effects on heart rate and AV conductionwere recorded again. If the condition of the cat permitted, the process

was repeated.

At the end of the experimental protocol, a series of controls was

performed. Because in the experimental animals the heart was re-

moved from the pericardial sac, potential leakage of the ganglionic

blocker from the injection site would result in its deposition in the

chest cavity. To control for potential effects produced by such leak-

age, 10 l of C6 (5 g/ul) was injected into the chest cavity of all fiveexperimental animals, and the effects of vagal stimulation on heart

rate and AV conduction before and after C6 injection were compared.

Hemodynamic data analysis. The heart rates obtained before vagal

stimulation and during the peak response to vagal stimulation (max-

imum R-R interval during the stimulation period) each were averagedover three consecutive trials to provide a mean heart rate before andduring vagal stimulation. Mean AV intervals were calculated in ananalogous fashion (maximal stimulus-R interval during the stimula-tion period) during the trials while the heart was being paced. Theseresults were obtained before blockade of the PA ganglion (baseline),during blockade of the PA ganglion, and after recovery of the PAganglion from that blockade. The mean peak response to vagalstimulation was normalized to percent change from the mean pre-stimulation levels. The percent change in heart rate and AV conduc-tion during right cervical vagal stimulation before pharmacologicalblockade and during pharmacological blockade of the PA ganglionwas analyzed with a paired t-test. A P value of0.05 was consideredstatistically significant.

Retrograde tracing studies. Experiments were performed on eightmongrel cats weighing 2.84.0 kg. Baseline vital signs of temperature,respiration rate, and heart rate were recorded. Cats were pretreatedwith 0.05 mg/kg of atropine to reduce secretions followed by 22mg/kg of ketamine and 0.2 mg/kg of acepromazine for induction ofanesthesia. Cats were prepared for surgery by inserting an intravenouscatheter into the brachial vein and intubating with a cuffed endotra-cheal tube. Isoflurane gas anesthesia was then used to bring the animal

to a surgical plane of anesthesia. Cardiac rate and blood oxygenconcentration were monitored as described above. A thoracotomy wasperformed via the right fifth intercostal space. At this point, the catwas artificially respired on a positive-pressure respirator with a tidalvolume setting between 50 and 100 ml and cycling at 12 breaths/min.An incision was made into the pericardium that was large enough toexpose the heart and allow for neuroanatomic identification andaccess to the SA ganglion (21). One microliter of a 2% solution of theretrograde tracer fast blue (27, 33) was injected into the SA ganglionin five cats and into the pericardial sac in an additional three cats.After injections were made, the pericardium was closed, the musclesand skin were sutured in layers, spontaneous respiration was reestab-lished, fluids along with the potent analgesic butorphanol (0.2 mg/kg)and the antibiotic penicillin procaine G (30,000 IU/kg) were admin-istered, and the animal was awakened from anesthesia. Four days after

the injection, the cats were killed under deep barbiturate anesthesia(50 mg/kg ip pentobarbital sodium) by intravascular perfusion with aphosphate-buffered solution containing fixatives as previously de-scribed in detail (21). The hearts were removed and then postfixed inthe same solution for 2 h. Hearts were then cryoprotected by placingthem into a 20% sucrose phosphate-buffered solution for 3 daysfollowed by a 30% sucrose phosphate-buffered solution for 4 days.After cryoprotection, hearts were flash frozen, and transverse serialsections 50 m thick were cut as previously described (21). Everythird section was examined via fluorescence microscopy using UVoptics. All intrinsic cardiac ganglia were inspected for the presence ofretrogradely labeled neurons. Neuronal counts that are reported wereadjusted by multiplication by a factor of three.

RESULTS

Physiological studies. Stimulation of the right cervical va-gus, before pharmacological blockade of the PA ganglion,resulted in a decrease in heart rate from a baseline of 183 6to 110 12 beats/min, a 40 5% change (P 0.001; n 5)(Figs. 1 and 2). After hexamethonium was injected into the PAganglion, the vagally induced negative chronotropic effectswere attenuated, and stimulation of the right cervical vagusresulted in a decrease in heart rate from 186 12 to 144 14beats/min, a 23 4% change (P 0.001; n 5) (Figs. 1 and2). After time for drug-induced ganglionic blockade was al-lowed to wear off, recovery of function was observed (Fig. 1).

2274 PARASYMPATHETIC CONTROL OF CARDIAC RATE

J Appl Physiol VOL 96 JUNE 2004 www.jap.org


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During atrial pacing, vagal stimulation before blockade ofthe PA ganglion prolonged the rate of AV conduction from abaseline of 111 6 to 149 14 ms, a 35 10% change (P 0.01; n 5) (Fig. 2). During blockade of the PA ganglion,stimulation of the right cervical vagus did not significantlychange the effects of vagal stimulation on AV conduction time

(P 0.05) (Fig. 2). Injecting C6 at the same dose into the chestcavity or intravenously had no effect on vagally inducedchanges in heart rate or AV conduction.

Retrograde tracing studies. We inspected six intrinsic car-diac ganglia for the presence of neurons retrogradely labeledfrom the microinjection of fast blue into the SA ganglion.Three of these ganglia were associated with the atria, i.e., thePA ganglion, the SA ganglion, and the AV ganglion. The otherthree ganglia were distributed on or within the ventricles, i.e.,the CV ganglion on the surface of the left ventricle, the rightventricular (RV) ganglion on the surface of the right ventricle,and the interventriculoseptal (IVS) ganglion within the inter-ventricular septum (for details, see Ref. 21).

This study involved the injection of 1 l of fast blue

fluorescent tracer into two different sites. In the experimentalgroup of animals, the tracer was injected directly into the SAganglion (Fig. 3), whereas in the control group of animals, thetracer was injected into the pericardial sac. This control groupwas exceptionally conservative insofar as it presumes that asmuch as 100% of the tracer could potentially leak from theinjection site in the SA ganglion onto surrounding structures inthe heart.

Four days after the injection offluorescent tracer into the SAganglion, the extent of diffusion of the tracer was limited to theSA fat pad. A total of 78.6 2.7% of the neurons in the SAganglion, which were identifiable, were labeled; however, not

all labeled neurons could be unequivocally identified due to theintense fluorescence that was seen at the center of the injectionsite (Fig. 3A). While many of the neurons of the SA ganglionappeared to be nonspecifically labeled by their close appositionto high concentrations of the tracer near the center of theinjection site (Fig. 3A), multiple neurons within the SA gan-glion were clearly labeled but were not surrounded by detect-

able levels of fast blue (Fig. 3B) in each of the animalsexamined.

Retrogradely labeled neurons containing the tracer were alsoreadily identifiable within intracardiac ganglia outside of theSA ganglion (Fig. 4). Statistical analysis of the percentage oflabeled cells found within each ganglion in the experimentalgroup utilizing a one-way ANOVA test (Fig. 5) showed thatthere was a statistically significant difference in the number oflabeled neurons found within different intrinsic cardiac ganglia[F(4,15) 45.62 (P 0.001)]. A Dunnetts T3 post hoc testshowed that the percentage of labeled cells found in the PAganglion was significantly larger than that found in all otherganglia, with all P values of0.01. All other permutations ofcomparisons between ganglia, however, were statistically non-significant. Furthermore, when tested with an independenttwo-sample t-test assuming unequal variances, there was asignificant difference in the mean percentage of labeled cellsfound in the PA ganglion in the experimental group of animals(71.4 6.8%) compared with that found in the PA ganglion inthe control group of animals (26.8 4.2%) (P 0.001) (Fig.5). Additionally, the pattern of labeling seen in the experimen-tal group of animals (Fig. 5) was significantly different fromthe pattern of labeling seen in the control group of animals(Fig. 5) when tested with a two-factorial ANOVA [F(1,25) 5.415, P 0.05].

In one of the five animals utilized in this study, the injectionof the fluorescent tracer missed the SA ganglion by 2 mm. In

this animal, only 29.2% of the retrogradely labeled neuronswere found in the PA ganglion, which is similar to that foundin the experimental group. The results from this animal wereexcluded from analysis but served as a negative control. It was

Fig. 2. Bar graph illustrating the percent decrease in heart rate and atrioven-tricular (AV) conduction caused by vagal stimulation before ( filled bars) andafter (open bars) pharmacological blockade of the PA ganglion (n 5). Therewas a statistically significant 39 9% attenuation of vagal effects on cardiacrate (*P 0.02) caused by microinjections of hexamethonium into the PAganglion. However, pharmacological blockade of the PA ganglion did notsignificantly alter vagal effects on AV conduction (P 0.05).

Fig. 1. Three different ECG tracings taken before (Control; A), during(Blocked; B), and after (Recovery; C) pharmacological blockade of nicotinicsynaptic transmission subsequent to local microinjections of hexamethonium(C6) into the posterior atrial (PA) ganglion. A: unilateral electrical stimulationof the right vagus nerve (stimulation onset indicated by arrow) leads to apronounced bradycardia. B: subsequent to microinjections of hexamethoniuminto the PA ganglion, vagal stimulation (arrow) no longer elicits a pronouncedbradycardia. Cis taken 30 min after the microinjection of hexamethonium intothe PA ganglion, and partial recovery of function after vagal stimulation(arrow) is demonstrated.

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not possible to identify the exact location of the axonal path-way interconnecting the PA and SA ganglia in the tissuesexamined using fast blue as a tracer, due both to low levels ofthe tracer in axons and problems with fluorescence quenchingduring microscopic observation of the heart.

DISCUSSION

In the classical tradition of Langley (22), neurons within theheart have been considered to be exclusively postganglionicparasympathetic efferents. This concept of the neuroanatomicorganization of the heart has persisted for almost a century. Inmore recent times, however, an increasing body of primarilyphysiological data has been consistent with the hypothesis that

neurons within intracardiac ganglia do not serve as simplerelays between the central nervous system and their effectors(2, 4, 6, 7, 31, 34). Rather, it has been postulated that individualcardiac ganglia may serve as complex integrative centerswithin which considerable processing of autonomic signals

may occur. These physiological data have resulted in theproposal of several relatively elaborate schematic descriptionsof the organization of what has been referred to as an intrinsiccardiac nervous system (1, 5). It has been suggested that theintrinsic cardiac nervous system is composed of a hierarchy ofnested feedback control loops that include parasympatheticefferent neurons, afferent neurons, local circuit intraganglionicneurons, interganglionic neurons, sympathetic postganglionicneurons, and the terminals of cardiac neurons projecting fromhigher centers (1, 4, 5).

While the available physiological data have provoked thisradical reinterpretation of the autonomic innervation of theheart, morphological evidence in support of many proposed

physiological hypotheses has been absent. Numerous physio-logical studies have shown that the SA ganglion is responsiblein large part for mediating parasympathetic effects on cardiacrate. Ablation of this ganglion, either by surgical excision ortopical application of phenol, results in complete inhibition of

Fig. 3. A: intense fast blue fluorescence wasobserved in the right atrium in close conti-

guity to a cluster of sinoatrial (SA) ganglionneurons (arrows) that are presumptively non-specifically labeled. B: some fluorescent neu-rons were also found in loci within the SAganglion that were not surrounded by detect-able fast blue fluorescent labeling. Theseneurons were presumptively retrogradely la-beled from adjacent areas within the injec-tion site.

Fig. 4. A: arrows identify 4 of the multipleneurons in the PA ganglion that were retro-gradely labeled after microinjections of fastblue into the SA ganglion using a fast bluefilter set. B: the same section utilizing anFITC filter set. Note that fast blue-labeledneurons in A comprise a subset of the totalnumber of neurons contained within the PAganglion as illustrated in B.




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vagally induced bradycardia (3). Similar but reversible physi-ological effects result from the injection of ganglionic blockingagents (20, 35). Furthermore, injecting fluorescent tracers into

the SA nodal region results in retrogradely labeling neurons inthe SA ganglion (26). This evidence clearly indicates that theSA ganglion mediates its negative chronotropic effect bymodulating SA nodal activity.

On the other hand, little has been known about the functionalproperties and intracardiac projections of the PA ganglion.Previous studies have indicated a potential role for the PAganglion in mediating in part sympathetic-parasympatheticinteractions for control of chronotropic function (31). In thepresent report, we have performed a coordinated series ofrelated physiological and neuroanatomic studies. We havedemonstrated that microinjections of a ganglionic blockingdrug into the PA ganglion attenuates the negative chronotropiceffects of vagal stimulation on the heart without significantly

effecting vagal effects on AV conduction. Therefore, these dataand other physiological data indicate that there are at least twointracardiac ganglia that mediate the vagal control of cardiacrate, i.e., the SA ganglion and the PA ganglion.

The SA ganglion controls cardiac rate via parasympatheticpostganglionic axons that project to the pacemaker cells of theSA node (26). However, neuroanatomic data clearly indicatethat neurons in the PA ganglion do not project to the SA node(26). We hypothesized, therefore, that the negative chrono-tropic effects mediated by the PA ganglion might be due to aninterganglionic projection to the SA ganglion. We now reportthat subsequent to microinjections of a retrograde neuronal

tracer into the SA ganglion, the vast majority of retrogradely

labeled neurons found throughout the heart were found in the

PA ganglion. Several lines of evidence indicate that failure to

find retrogradely labeled neurons within other intracardiacganglia was not due to an inadequate survival time or relative

distance from the injection site. Thus, for example, the PA and

AV ganglia are approximately equidistant from the SA gan-

glion (21); however, retrograde labeling in these two ganglia

was quite different. Furthermore, the 4-day survival time that

was used in the present report has also been demonstrated to

provide ample staining of cardioinhibitory vagal preganglionic

neurons in the nucleus ambiguus of the brain stem after

microinjections into the SA or AV ganglia (10), a distance that

is substantially greater than that separating the SA ganglion

from other intracardiac ganglia. These neuroanatomic data are

therefore consistent with the hypothesis that the PA ganglion

may affect the parasympathetic control of cardiac rate through

an intracardiac neuronal circuit, which thereby modulates

the actions of the SA ganglion on the SA node. The present

data are, to our knowledge, the first neuroanatomic demon-

stration of an interganglionic circuit between any two intra-cardiac ganglia.

Microinjections of a retrograde neuronal tracer into the SA

ganglion labeled 80% of the neurons in this ganglion. Al-

though many of these neurons are probably nonspecificallylabeled due to their contiguity to high concentrations of the

tracer, in all animals multiple neurons also appear to be labeled

by retrograde transport within the SA ganglion because the

tissues surrounding these neurons did not contain detectable

levels of tracer. This morphological conclusion is consistent

with recent physiological data that indicate that there is a

functional interdependence of spontaneous and reflex-evokedactivity between neurons within the SA ganglion much of the

time (34). These morphological and physiological data suggestthat intraganglionic neuronal circuits may be found in the SA

ganglion; however, an ultrastructural analysis is urgently

needed to confirm this hypothesis.In summary, we have presented both physiological and

neuroanatomic data to support the hypothesis that the periph-

eral autonomic control of cardiac rate is controlled by two

interacting cardiac centers, i.e., the SA and PA ganglia. An

interganglionic neuronal circuit connects the PA ganglion to

the SA ganglion. The SA ganglion then projects to the SA node

to mediate parasympathetic effects on cardiac rate. Our neu-

roanatomic data indicate that intraganglionic neuronal circuits

are also found within the SA ganglion. These circuits may also

function in a coordinated fashion to modulate cardiac ratewithin the feline heart. Taken as a whole, the present data

support the emerging concept of an intrinsic cardiac nervous

system (1, 32, 34).

GRANTS

This research was supported in part by grants from the National Heart,

Lung, and Blood Institute (NHLBI) (RO1-HL-51917) and American Heart

Association to V. J. Massari and from the NHLBI (R01-HL-58140) to J. L.

Ardell. Additional funding was provided by the Gustavus and Louise Pfeiffer

Research Foundation to A. L. Gray and the Specialized Neuroscience Research

Program (1U54-NS-39407, M. A. Haxhiu, Principal Investigator).

Fig. 5. Percentage of the total number of labeled cells found within eachganglion after injecting fast blue into the SA ganglion. A significantly largernumber of retrogradely labeled neurons was found in the PA ganglion than inany other intracardiac ganglion (**P 0.01). Furthermore, the total number oflabeled cells found in the PA ganglion in the experimental group (n 4) wassignificantly different than that found in the control group (n 3; *P 0.001).PA, posterial atrial ganglion; RV, right ventricular ganglion; CV, cranioven-tricular ganglion; AV, atrioventricular ganglion; IVS, interventriculo-septalganglion.

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Alrich L. Gray et al- Parasympathetic control of the heart. II. A novel interganglionic intrinsic cardiac circuit mediates neural control of heart ratediac innervation of the ventricles