J. Leger et al- Regional Distribution and Extrinsic Innervation of Intrinsic Cardiac Neurons in the Guinea Pig

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Regional Distribution and ExtrinsicInnervation of Intrinsic Cardiac Neurons

in the Guinea Pig

J. LEGER, 1 R.P. CROLL, 2 AND F.M. SMITH 1*1Depart ment ofAnat omy and Neurobiology, Dalhousie Un iversity,

Halifax, NS Cana da B3H 4H72Department of Physiology and Biophysics, Dalhousie University,

Halifax, NS Cana da B3H 4H7

ABSTRACTMammalian intrinsic cardiac neurons subserve different functions in different cardiac

regions, but the regional anatomical organisation of the intracardiac nervous system is notwell understood. We investigated t he qua ntit ative an d qua litative distribut ion of cholinergicand adrenerg ic e lements , and the in t raca rd iac pa thways of ext r ins ic ca rd iac ne rves , inwhole-mount preparations of guinea pig atria. Protein gene product 9.5 immunoreactivity(PGP 9.5-IR)m arked intracardiac neuronal elements; immunoreactions for choline a cetyltrans-ferase (ChAT-IR) and tyrosine hydroxylase (TH-IR) distinguished cholinergic and adrenergiccomponents, respectively. Catecholamine-containing components were identied by aldehyde-induced uorescence histochemistry. Mean total number of atrial neurons was 1510 25 1(SE); 85% of these occurred in ganglia of 20 neur ons. All neur onal somata expressing PGP9.5-IR also expressed ChAT-IR, suggesting th at these neur ons were cholinergic. Right (RA)and left (LA) atr ia had s ta t is t ical ly s imilar neuronal densi t ies (6.4 1.2 and 2.4 0.7neurons/mm 2 , respectively; analysis of variance, P 0.05). Neur ons in RA were concentr at edintercaval ly; LA neurons were concentrated near pulmonary vein ost ia . Greatest densi tyoccurred in the interatrial septum (16.3 4.0 neurons/mm 2). No neuronal somat a expressed

TH-IR or contained detectable am ines but these element s were expressed by somat a of smallcells (mean t otal 124 33) throughout the atria, primarily associated with ganglia. Amine-and TH- containing var icosities were also present in gan glia, repr esenting potentia l sites foradren ergic modulation of ganglionic neu rotran smission. Bran ches of extrinsic cardiopulmo-nary and vagus nerves were dis t r ibuted to a l l par ts of both atr ia . The organisat ion of theintracardiac nervous system revealed in this s tudy wil l faci li ta te fur th er invest igat ions of regional autonomic control of the heart. J. Comp. Neurol. 407:303–317, 1999.

1999 Wiley-Liss, Inc.

Indexing te rms: autonomic ne rvous s ystem; cardiac ganglia; vagus; sympathetic; parasympathetic

Neurons with their somata located in the mammalianintrin sic car diac nervous system are capable of modifyingpacemaker activity, force of contr action and conduction.Classically, these neur ons have been regarded a s para sym-path etic postganglionic efferent neur ons, and t heir a ctiva-t ion has been considered to inhibi t cardiac funct ions.However, recent physiological s tudies have shown thatact ivat ion of some intracardiac n eurons by electr ical orchemical stimu lation can a ugment cardiodynamics (Butleret al., 1990; Armour et al., 1993; Huang et al., 1993a,b,c),suggest ing that some cardiac sympathomimetic effectsmay be mediated by neur ons with th eir somat a intr insic tothe hear t . Evidence is a lso accumulat ing to suggest thepresence of neurons of other functional types within the

hear t, including afferent and ‘‘local-circuit’’ neurons orintern eurons (Ardell, 1994; Steele et a l., 1994; Edwards eta l ., 1995). I t has been p roposed th a t neurons of t hesediverse types constitute a distributed network with connec-

Grant sponsor : Medica l Research Council of Canada; Grant number :MT-11622; Grant sponsor: Heart and Stroke Foundation of Canada; Gran tsponsor: Natural Sciences and Engineering Research Council of Canada;Grant number: OGP0038863.

*Correspondence to: Dr. Frank M. Smith, Department of Anatomy andNeurobiology, Faculty of Medicine, Dalhousie U niversity, Halifax, NSCana da B3H 4H 7. E-mail: [email protected]

Received 25 June 1997; Revised 12 August 1998; Accepted 4 December1998

THE JOURNAL OF COMPARATIVE NE UROLOGY 40 7:303 –317 ( 19 99 )

1999 WILEY-LISS, INC.



t ions to a l l regions of the hear t . This network, possiblyoperat ing with the aid of local feedback on a beat-by-beatbasis, could poten tia lly provide great exibility in modulat -ing regional and global myocardia l function to help mat chcardiac output to the ow requirements of the systemicand pulmonary a rter ial circulat ions (Armour, 1994;Ardell,1994). However, details of the regional patterns of innerva-tion of int racardia c neurons by extrin sic nerves, the qua n-t i ty and dis t r ibut ion of neurons within the hear t , an d therelative abun dan ce of different neur onal phenotypes havenot been established in the mammalian heart.

I t h as been r eported in anat omical s tudies of the mam-malian heart that ganglia containing the somata of intrin-s ic cardiac neurons are associated with an intracardiacnerve plexus extending throughout both atria and into theventr ic les (King and Coakley, 1958; Calaresu and St .Louis, 1967;Ander son, 1972; Ellison an d Hibbs, 1976; Tayet a l., 1984; Ja nes et al. , 1986; Pardini et al. , 1987; Yuan etal. , 1994), but the anatomical arrangement of this systemis not well understood. Cont rol of the hea rt by intra cardiacneurons has been s tu died in in vivo and in vi t ro prepara-tions in a n umber of mamma lian species, including canin e,

porcine, and rodent, but these studies have been hinderedby a l ack of un derstand ing o f the organ isa t ion of th eintracardiac nervous system. The overall objective of thepresent s tudy, t herefore , was to invest igate the regionalorganisat ion of the a tr ia l port ion of the intracardiac ner-vous system in t he hear t of a small mam mal, the guineapig. A major aim was t o determ ine the genera l distributionand phenotype o f neurons th roughou t the a t r i a of theguinea pig heart, their total number and their location byregion. The somata of neurons were ident ied by theirimmunoreactivity to a general vertebra te neuronal mark er,protein gene pr oduct 9.5 (PGP 9.5-IR). This procedure a lsolabelled axons in nerves associated with the heart, facilitat-ing the analysis of regional patterns of innervation of theatr ial ganglionat ed plexus by the extrinsic cardia c nerves.

In st udies of the ph enotype of intracardia c neurons, histo-chemica l su rveys of t he mammal ian hea r t have shownthat many nerve bres , terminals , and neuronal somataexhibit stainin g for acetylcholinestera se (AChE), an en-zyme involved in me tabol ism of the pa rasympathe t icneurotransmitter acetylcholine (ACh; James and Spence,1966; Ja cobowitz, 1967; Ehinger e t a l ., 1968; Bojsen-Moller an d Tran um-J ensen, 1971; Anderson, 1972; Ellisonand Hibbs, 1976; Ha ncock et a l ., 1987; Roberts e t a l .,1989). However AChE may not be the mos t su it ab leindicat or for cholinergic neuronal ph enotype, because th isenzyme is also found in non-neuronal elements (Fibiger,1982). The presence of choline acetyltran sferase (ChAT),an enzyme involved in acetylcholine synthesis, is consid-ered the most reliable indicator of cholinergic phenotype in

neurons of t he central nervous system (Fibiger, 1982;Satoh et a l., 1983). This enzyme can be detected immun o-histochemically in both t he centra l and peripher al nervoussystems, but in only one previous study ha s this t echniqueb ee n u s ed a s a p r ob e for s u r ve yin g t h e i nt r a ca r d ia cnervous system. Mawe et al. (1996) used ChAT immunore-activity (ChAT-IR) to characterise neur onal ph enotype inse lected pa r t s of th e gu inea p ig hea r t , r epor t ing tha tneur ons in the a tria l intercaval region, the ba sal portion of the in tera t r i a l sep tum and the ca rd iac s inus region ap-peared to be ChAT-IR. However, nei ther this s tudy norearlier histochemical analyses of AChE location coveredthe en tire extent of the a tria , so the overall distribution of

atr ial neu rons exhibiting cholinergic phenotype is still notknown. The present s tu dy th erefore a imed to provide anestimate of the total number and distribution of ChAT-IRneurons in the guinea pig atria.

Evidence that augmentation of cardiac function can beevoked by focal activation of the somata of some intr insiccardiac neurons has led to the suggest ion that the somata

of adrenergic neurons may be present in the heart (Butleret al. , 1990; Armour et al. , 1993; Hu ang et al. , 1993a,b,c;Armour, 1994). Neur ons potentia lly involved in such aug-mentatory responses might be expected to display one ormore aspects of adrenergic phenotype, including expres-sion of tyrosine hydroxylase (TH, the rate-limiting enzymein n orepinephrine synthesis), or upt ake and s torage of catecholamines. This study aimed to establish the patternof TH occurrence within the atria of the guinea pig heartby using immunohistochemical detection of this enzyme,and to compare this with the intra-atr ia l dis t r ibut ion of catecholamines as indicated by an aldehyde histouores-cence reaction.

This study shows that t he somata of identiable neuronsin the atria display aspects of cholinergic phenotype and

are dis t r ibuted throughout the atr ia with concentrat ionsnear the sinoat rial node, the roots of the pu lmonary veins,and in the interatrial septum. Cells which phenotypicallyand morphologically resemble th e sma ll, intensely uores-cent cells found th roughout th e au tonomic nervous systemare a lso distributed in t he at ria, largely in association withintracardiac gangl ia . The regional intracardiac dis t r ibu-t ion of b ranches of the ext r ins ic ca rd iac ne rves i s de -scr ibed. The detai ls of t he regional organisat ion of th eintracardiac nervous system and the phenotypes of i tselements provided by th is s tudy establish an anat omicalsubstrate for guiding further functional investigations of the roles of intracardiac neurons in the control of cardiody-namics.

MATERIALS AND METHODSExperiments were conducted on Hart ley guinea pigs

(Charles River, Quebec City, Que., Canada) of both sexes,weighing 150–300 g and r anging in age from 3 t o 7 weeks.Experimental protocols were approved by the DalhousieUniversi ty Animal Use Commit tee and conformed withanimal use guidelines established by the Canadian Coun-cil on Anima l Car e.

ImmunohistochemistryThir teen animals were u sed for immunohistochemical

studies of the intracardiac nervous system. Animals wereki l led by a blow to the head, their hear ts were quicklyremoved through a midline sternotomy and washed in 0.1

M phosphate-buffered sal ine (0.9% Na Cl, pH 7.4). Theexternal walls of the atria and the interatrial septum wereseparated from the ventr ic les , the septum was t r immedfree of the a tr ial walls, and both pieces of at rial tissue werepinned at in a dissect ing dish. Tissue was xed for 18hours at 4°C in 4% paraformaldehyde dissolved in 0.1 Mphosphate buffered saline, rinsed, dehydrated in a gradedser ies of e tha nol solut ions and cleared in xylene. Thetissue was then r ehydrat ed and incubated in a 4% solutionof Triton-X 100 in phosphate-buffered saline for 48 hours.Tissue was th en removed from t he dish an d incubated asfree-oatin g whole-mounts in blocking solutions of 2.5%normal goat , donkey, or rabbi t serum as appropriate for

304 J. LEGER ET AL.



the antibody used, to reduce nonspecic staining (detailsand sources of p r imary and secondary an t ibod ies anddilutions u sed in t his stu dy are given in Table 1). Double-label immunocytochemistr y was performed with an initialincubat ion (48 hours) in a pr imary a nt iserum directedagainst either ChAT or TH , followed by a second incuba-tion (48 hours) in primar y antiseru m directed against PGP9.5 (Tab le 1 ). Afte r t r ea tment with p r imary an t i sera ,t issue was incubated with secondary ant ibodies conju-gated with rh odamine or uorescein-isothiocyana te (FITC),as shown in Table 1. For viewing and photography, whole-mounts were made by hea t -dry ing the t i ssue on g lasss lides ; covers lips were then app li ed over a mount ingmedium consisting of a 1:1 solution of glycerol an d phos-pha te-buffered sa line in which gelat in was d issolved (1 g of gelatin per 12 ml of solution). Alternatively, some tissuewas rapidly dehydrated in a lcohol and cleared in xylenebefore being mounted in Accumount (Baxter Scient icProducts , McGraw Park, IL). Both mounting t echniquesyielded tissue suitable for analysis of uorescent labels.These labels were visual ised with a s tandard compoundmicroscope equipped for epiuorescence (Arist oplan, Leica

Canada, Ltd., Willowdale, Onta r io, Cana da) t hat usedappropriate lter cubes (Type N21 for rhodamine and TypeL3 for FITC). The number of labelled cells in all regions of the at ria and septum was count ed in each specimen. Atria ltissue was ph otographed (Kodak T-Max 100 lm) th roughthe microscope on a grid pat tern of overlapping rectan gu-lar elds (1.5- 2.5-mm eld size). These photographswere digitised with a desktop scanner (Deskjet, Hewlett-Packard, Palo Alto, CA) and stored on disk; the resultingimage les were then assembled into montages by usingimage processing softwar e (Photoshop, Adobe Systems,Inc., Mountain View, CA) to produce maps of the distribu-t ion of labelled nerve bres and intracardiac neurons.Selected ar eas of some heart s were also examined with theaid of the optical sectioning capability of a confocal micro-

scope (Axiovert 100 microscope equipped with LSM 410confocal system; Carl Zeiss, In c., N orth York, Ont ar io,Canada) to de termine de ta il s of ce llu la r and t e rmina lmorphology within intracardiac ganglia. Ventricular tis-sue was not analyzed in this study, because it was gener-ally too thick to be processed for visualization of neuralelements in whole-mount prepar at ions.

Both positive and negative controls were used to estab-lish t he specicity of the immun ohistochemical protocolsused in this study. To verify ChAT- and TH-IR labelling,samples of spinal cord and s te l la te gangl ia were takenfrom the same animals yielding cardiac t issue and werexed and sectioned (40 µm) before being processed with

the same immunohistochemical protocols as the cardiactissue. Motor neur ons in the ventr al horn of the spinal cordof th e guinea pig have been r eported t o display ChAT-IR inprevious studies (Eckenstein and Sofroniew, 1983), a ndneur ons in the stellat e ganglia have been shown to displaystrong TH-IR (Bowden and Gibbins, 1992; Heym et al. ,1993). In the present s tudy, ChAT-IR neurons were ob-served in the dorsal h orn, in the intermediolateral cel lcolumn and in th e ventra l horn of spinal cord sections; allof these neurons were also immunoreactive for PGP 9.5.Stellate ganglion neu rons were strongly TH-IR, and th eseneur ons were a lso double-labelled with ant ibodies againstPGP 9.5. Nonspecic background labelling in all t issuesprocessed for PGP 9.5-, ChAT-, and TH-IR wa s low, as wastissue a utouorescence at t he wavelengths u sed for visual-ising the uorophores.

To establish negative controls, the atria were removedfrom a separa te group of four an imals an d processed withthe same protocol used for t issues in t he experimentalgroup, with one of th e following chan ges: eith er pr ima ry orsecondary ant ibodies were omit ted from the incubat ingsolut ion; or pr imary ant ibodies were preabsorbed withtheir a ppropriate an tigens (100 times the concentra tion of th e an tibodies) for 24–48 hour s before being us ed for t issueincubation. Cont rol cardia c tissue exposed t o primar y butnot to seconda ry an tibodies, and vice versa, did not cont ainany label, nor did tissue in which primary antibodies hadbeen prea bsorbed.

Aldehyde histouoresce nceThe presence of catecholamines in whole-mounts of

freshly dissected atrial tissue in six animals was detectedby using a technique modied from that of Furness et al.(1977) and Molist et al. (1993). Briey, cardiac tissue wasobtained as descr ibed above, pinned at and xed in asolut ion of 4% para forma ldehyde, 0.5% glutar aldehyde,and 35% glucose in phosphate-buffered saline at 4°C for24–48 hours . The t i ssue was then removed from thexative, placed onto a glass slide, desiccated in a light-proof box for 48 hours, cleared in xylene, and mounted inAccumount. Tissue was examined and photographed un-der ultraviolet epi-illumination (Leitz D lter cube, excita-t ion bandwidth 355–425 nm, 460 nm long-pass bar r ierlter) . With this technique cells , nerve bres a nd termi-nals containing catecholamines uoresced br ight blue-green. Tissue which was xed with paraformaldehydeonly, th en desiccat ed an d m ounted for viewing served a s anegat ive control for background autouorescence; thistissue displayed either very faint or no uorescence underultra violet illuminat ion.

Data analysesCounts of ChAT- and TH-IR cell bodies were made over

t h e e n t ir e e xt e n t of b ot h a t r ia a n d i n t h e i nt e r a t ri a lseptum. Statistically signicant regional differences in cellcounts and other var iables among the atr ia l areas wereanalyzed by analysis of variance (single f-factor). Wheresignicant f-values were obtained, pairwise comparisonsof means were done by using Tukey’s mult iple meanscomparison test (Zar, 1984); P 0 .05 was t aken as thelimit of signicance. Numerical values are expressed asmean 1 s tandar d error.

TABLE 1. Sources and Dilut ion of Primar y and Secondary AntibodiesUsed In This Study

Ant igen Host Su pplier Dilu t ion

Primary antibodiesPr ot ein gen e pr odu ct 9.5 Ra bbit Ult ra clon e 1 1:400–800Cholin e acetylt ran sfer ase Goat Ch emicon 2 1:250Tyrosine hydroxylase Mou se Incst a r 3 1:500

Secondar y an tibodies

Rabbit IgG Rh odamine Goat J ackson 4 1:50Goa t IgG FITC Don key J ackson 4 1:50Mouse IgG FITC Sh eep J ackson 4 1:50

1Ultraclone Limited, Isle of Wight, E ngland.2Chemicon Inter nat ional, Inc., Temecula, CA.3Incstar Corporat ion, Stillwater, MN.4Ja ckson Immu noresearch Laborat ories, Inc., West Grove, PA.

DISTRIBUTION OF INTRINSIC CARDIAC NEURONS 305



Fig. 1 . Confocal photomicrographs of a t r ia l ganglion neuronsdouble label led with ant ibodies against choline acetyl t ransferase(ChAT), tyrosine hydr oxylase (TH), and pr otein gene product 9.5 (PGP9.5). A–C: ChAT-immu noreactivit y (IR) indicated by red label (second-ary antibody conjugated with rhodamine) and PGP 9.5-IR indicated bygreen label (secondar y ant ibody conjugat ed with uorescein isothiocya-nate [FITC]) in r ight a t r ium. A: Rhodamine-lter exposure showingChAT-IR neuronal somata and n erve bres. B: FITC-lter exposure of same gangl ion as A, showing PGP 9.5-IR elements . C: Combinedexposure showing same gangl ion a s i n A and B , t oge ther w ithsurr ounding tissue. In this example, and th roughout the atr ia, all PGP

9.5-IR neur ons were also ChAT-IR. D: Left atrial tissue double labelledwith an tibodies against PGP 9.5 (green; FITC secondary) and TH (red;rhodamine secondary). No colocalization of these labels was observedin somata in this example or anywhere else in the atria. PGP 9.5-IRidentied neuronal somata and axons; TH-IR identied cells proposedto be sm all, intensely uorescent (SIF) cells (see text). Note t he sh ortprocess extending from one of the TH-IR cel ls (arrowhead withasterisk). Some TH-IR varicosities a re visible within t he ganglion(arrowheads). The arr ows indicate large processes emer ging from oneend of the spheroid neuronal somata . Scale bars 50 µm in C (appliesto A–C) and D.



RESULTSDistribution of cholinergic and adrene rgic

components of the intracardiacnervous system

ChAT-IR components . ChAT-IR occurren ce over-lapped completely with immun oreactivity for the n euronalmarker PGP 9.5 in the somata of intra cardiac neurons inall eight hearts processed for double labelling with these

markers, a typical example of which is shown in Figure 1(A-C). Based on th i s nding, i t was assumed for thepurposes of this s tudy that a l l neurons marked by PGP9.5-IR in the heart were cholinergic.

The somata of individual neurons in guinea pig atr iawere predominant ly prolate spheroid in shape (Fig. 1).Estimates of mean neuronal dimensions were obtained bymeasu ring, with an ocular micrometer, the long and sh ortaxes of 60 ChAT-IR neurons ra ndomly sampled from vehear ts. Mean length of th e long axis was 27 1 µm (range,20–45µm), whereas the mean length of the short axis was21 0 .5 µm (range, 15–30µm). When v iewed in theconfocal microscope to clarify cellular details, most neu-

rons appeared to have on ly one l a rge p rocess whichemerged from one end of t he oval soma (e .g ., F ig. 1D,arrows).

Some isolated neurons were observed throughout theatr ia, but the vast majority were cluster ed into ganglia , asshown in Figures 1–3. Gangl ia ranged in s ize f rom 2 tomore than 100 neurons and were a r ranged as a t t enedmasses, usu ally one cell-layer th ick, oriented to lie in t heplane of the atr ia l wal l. Within t he external a t r ia l wal ls ,

gangl ia were loca ted between the ep ica rd ium and themyocardium. They were covered supercially by the meso-thel ium, a subepicardial layer of connect ive t issue, andw er e s it u a t ed e xt e r n a l t o t h e t h r ee or fou r l a ye r s of myocardial cells constituting the bulk of the atrial walls.In the in tera t r i a l sep tum, where the myocard ium wasman y cell layers th ick, numer ous ganglia were distr ibutedamong the layers of myocytes . In a l l areas of the atr ia ,ganglia were locat ed either at the junctions of two or m orelarge intracardiac plexus nerves, or were s i tuated to oneside of a nerve, as shown in Figure 1.

Gangl ia were a lways connected to the in t raca rd iacplexus by more than one bre bundle; in some cases up to

Fig. 2. Composite photomicrograph of whole-mount pr eparat ion of the external wal ls of the lef t and r ight a t r ia af ter processing forprotein gene product 9.5 immunoreactivity. A line running betweenthe tips of the large arrows (top and bottom of the image) delineatesthe junct ion of the interatr ia l septum with the external a t r ia l wal l .Right atrium is to the right of this line; left atrium is on the left side.With the exception of the areas labelled PV (pulmonary veins), IVC(inferior vena cava), and SVC (superior vena cava), the edges of thetissue represent the atrioventricular (AV) borders. The approximate

location of the sinoatrial node is outlined by the dashed oval in ther ight a t r ium. Neuronal somata, most ly occurr ing in gangl ia , weredistributed throughout both atria; examples of ve large ganglia areindicated by the small arrows. Apexes of white arrowheads indicatecut ends of branches of right (RCPN) and left (LCPN) cardiopulmo-nary nerves and the cardiac branches of the right (RVN) and left (LVN)vagal nerves. LAA, RAA: left and right atr ial appendages, r espec-tively. Scale ba r 2.5 mm.




10 nerves of varying sizes were associated with in dividualganglia.

The mea n tota l num ber ofCh AT-positive neurons count edin t he a t r i a and the quan t i t a t ive d i st r ibu t ion of theseneurons by region are presented in Table 2. The standarderror of the mean total neuron count was relatively large(17% of the m ean value), indicatin g substa ntia l variabilityin total num ber of neur ons among the h eart s in this study.This high var iabi l i ty was reected in the range of totalcounts (from 717 to 2,542 atrial neurons per heart) in theeight hear ts sa mpled. The distribut ion of intr insic cardiacganglia, and th e microan atomy of the extr insic nerves an dth e ganglionated plexus is shown in t he composite photomi-crographs of the external atrial walls and septum (Figs. 2and 3 , r e spect ive ly ) in a r ep resen ta t ive hea r t . For theanalysis of the quantitative regional distribution of atrialneurons shown in Table 2, the atria were divided into the

three regions i l lustra ted in Figures 2 and 3. The regiondesignated as right atrium (RA) consisted of the externalatr ia l wal l extending from i ts junct ion with the septum(along a line r unn ing between th e large ar rows in Fig. 2) tothe r ight ventr icular border. The lef t a t r ia l (LA) regionconsis ted of the external a t r ia l wal l extending from i ts junction with t he septu m t o the border of the left vent ricle.The left a t r ia l appendage contained n o neurons and wasremoved to prevent folds in the tissue during the prepara-t ion of whole-mounts . The smaller r ight appendage alsowas devoid of neurons but was left att ached because it didnot hinder whole-mount preparat ion. The region desig-na ted sep tum in Tab le 2 cons i s t ed o f the sep ta l t i s sue

dividing the r ight an d left a tria l cham bers. The borders of a l l three regions were readi ly dis t inguishable in whole-mount specimens, thus ensuring consistent sampling pro-cedures a mong heart s in this study.

To develop a quan tita tive index of the regional distribu-t ion of a t r i a l n eurons , the a rea and number o f neuronspresen t in the designa ted regions of each hea r t wereobtained. A value for th e density of neuron occurrence ineach atr ia l region was t hen der ived from the quot ient of mea n n eur on num ber an d mea n ar ea (‘‘densit y’’in Table 2).The mean area of the RA was s ignicant ly greater thanthat of the LA, but the number of neurons in the RA wasnot signicant ly different from t he n umber in th e LA, andour analysis shows that the corresponding density valuesfor RA and LA were n ot signicantly different . H owever,during th e counting process, it became a pparen t th at t here

were differences in the patterns of overall distribution of neurons within these regions. The majority of neurons inthe RA was concentra ted near t he SA node and t he ost iumof the inferior vena cava (IVC; Fig. 2, ganglia indicated bysmall arr ows in RA). In contr ast, neur ons in th e LAt endedto be distributed in a broad band between the roots of thepulmonary veins a nd the atr ioventr icular border (Fig. 2 ,small ar rows in LA), as well a s being more widely scat-tered over the rest of th e myocardium in t his chamber.

The mean area of the septum was s ignicant ly smallerthan ei ther RA or LA areas (Table 2); the septum repre-s en t e d on l y 5 % o f m e a n t ot a l a t r ia l a r e a . T h e m ea nnum ber of neur ons contained in t he septu m (21% of meantotal number) was also signicantly less than the numberin t he LA (but was n ot s ignicant ly different from th e

number in the RA), yet the value of neuron density in thesep tum was s ign ican t ly g rea te r than the densi ty o f neurons in e ithe r the LA (septa l neurons 2 .5 t imes asdense) or the RA (more than s ix t imes as dense) . Theseresults show that the greatest concentration of neurons inthe atr ia was in the septum.

To de termine whethe r the re were d iffe rences in therelative sizes of ganglia in different parts of the atria thenumber of neurons per ganglion was counted in all eighth e a r t s s t u d ie d, a n d fr e qu e n cy h i st og r a m s w er e con -structed. The frequency of occurrence of ganglia of differ-ent sizes was analyzed by categorising ganglia into 11 sizeran ges. The sma llest cat egory in t his ra nge included single

Fig. 3. Composite photomicrograph of a whole-mount of the int er-atr ia l septum ta ken from th e same hear t as th e external a t r ia l wallsshown in Figure 2. The tissue is oriented with the dorsal edge of theseptum to the r igh t of t he f r ame and the mos t cr an ia l por t ion i suppermost. This gure is shown at a higher ma gnication than Figure2 to illustrat e more clearly th e ganglia and nerves. Scale bar 1 mm.

TABLE 2. Mean Number and Proportional Distribution of CholineAcetyltransferase-Immunoreactive (ChAT-IR) Neurons (n 8)

and Tyrosine Hydroxylase (TH)-IR Cells (n 5) in Guinea-Pig Atria,and Regional Density of Neuron Occurrence (neurons/mm 2 )1

Lefta t r ium

Righta t r ium

Intera t r ia lseptum

Meantotal

C hAT-I R n eu r on s 7 14 127 485 124 312 2 56 1510 251% of t ot al neu rons 47% 32% 21%Area (mm 2 ) 129 3 6 210 9 19 4 1 359 14% of t ot al a rea 36% 59% 5%Densit y 6.4 1.2 2.4 0.7 16.3 5 4.0 5.2 0.7

TH-IR cells 46 11 60 27 18 6 124 33% of t ot al cells 37% 48% 14%Area (mm 2 ) 120 3 12 193 13 22 4 1 372 24% of t ot al a rea 32% 52% 6%Densit y 0.4 0.1 0.3 0.1 0.8 0.3 0.3 0.1

1Values are expressed as mean 1 SE.2Mean n umber of ChAT-IR neurons in region of interatr ia l septum signicant ly lessthan in t he left a t r ium.3Area of left atrium signicantly smaller than right atrium.4Area of interatr ia l septum signicant ly smaller than areas of lef t a t r ium and r ightatr ium.5Density of neurons in interatrial septum signicantly greater than in right atrium andleft atrium.

308 J. LEGER ET AL.



neur ons together with ganglia conta ining from t wo to fourcells.

Cat egory size increas ed by incremen ts of four cells to thelargest category, which included ganglia with 41 or moreneurons. Although a few gangl ia containing up to 100neurons were found , gangl ia con ta in ing more than 40neurons were relat ively rare . An analysis of the overal lfrequency-to-ganglion size relat ionship for all ganglia re-gardless of atrial location was rst done, as shown in theupper left panel of Figure 4. This ana lysis showed that themajori ty of a t r ia l neurons occurred in re la t ively small

ganglia, with 85% of the n eurons being locat ed in gangliaof fewer than 20 neurons . Subsequen t b reakdown of frequency vs. ganglion size by at rial r egion, illustra ted inthe other pa nels of Figure 4, showed that the t rend in eachregion reected the overal l pat tern. To summar ise thesedata , there was no trend t oward a preponderan ce of largergangl ia in any a t r i a l r eg ion , even t hough a few la rgeganglia were observed in all regions.

Adrenerg ic comp onen t s . TH-IR elements. Fibres andcell bodies within the a tria ofve heart s were immun oreac-tive for TH, a s sh own in t he confocal photomicrograph of Figure 1D (in red). Individual bres were a ssociated withmyocytes or were bundled together in ner ve trun ks within

the intracardiac plexus. TH-IR bres were also presentwithin int racar diac ganglia; moreover, examination in t heconfocal microscope sh owed t hat some of these bres hadvaricosities near PGP 9.5-IR ganglion neurons (Fig. 1D,arrowheads). No TH-IR cell bodies were observed to bedouble-labelled with immun oreactivity for PGP 9.5; in-s tead, TH-IR was found only in small , spheroid cel ls .These cells had a mean long axis dimension of 10 0.4 µm(range, 9–12µm) and a m ean short a xis dimension of 7.40 .3 µm (range, 5–10 µ m; da ta from 21 ce ll s r a ndomlysampled from thr ee hear ts). The lar gest of these cells was

smaller than the smallest neurons label led with ei therChAT- or PGP 9.5-IR, an d st at istical compar isons (t- test,adjusted for unequal n , P 0.05) conrmed signicantdifferences in dimensions between the two cell types.Confocal microscopy showed th at TH-IR cells had one ortwo short (length, 5–30 µm) processes; an example of sucha pr ocess can be seen in Figure 1D (small ar rowhead withasterisk).

TH-IR cells occurred singly or were grouped together inclusters with the majority (more than 90%) located eitherwithin or closely associated with intracardiac gangl ia .F igure 1D shows a typica l example o f the ana tomica lrelationship of TH-IR cells to pr incipal ganglion neur ons.

Fig. 4. Bar graph of proport ional dis t r ibut ion of a t r ia l neurons,expressed as mean frequency of occurrence of ganglia of different sizecategories (num ber of neur ons in each ganglion category indicated

under the bars) for both atr ia combined (ALL REGIONS, top leftpanel) and by region, in e ight hear ts . Ganglion s ize increases by 4

neurons per s ize category unt i l the largest category (41 ), wh ichrepresents the frequency of gangl ia of 41 neurons or greater. Themajority (85%) of neurons were contained in ganglia of fewer th an 20

neurons. Error bars 1 SE.




Only a few TH-IR cells were locat ed in area s rem ote fromnerve trunks or ganglia; these cells occurred singly or insmall clusters of fewer than 10 cells. Eighty-ve percent of TH-IR cells was found in the right and left atrial regions(Table 2), with no signicant difference between the rightand left atria in the number or the density of TH-IR cells.The mean num ber of TH-IR cells in t he septu m (represent-ing 15% of mean total n umber) was signicant ly less th anthe numbers of cells in either the right or left atrium, but

cell density in the septum was not signicantly differentthan in the external a t r ia l wal ls . Therefore , TH-IR cellsdid not appear to congregate preferentially in any region of the a t r i a . At r i a l TH-IR ce ll s were l es s numerous thanatrial neurons; the mean total number of TH-IR cells wasapproximately 12% of the mean total nu mber of neurons(Table 2).

An an alysis of the frequen cy of occur ren ce of TH-IR cellsin clusters of different sizes over the whole extent of theatr ia (upper left panel , Fig. 5) showed a qual i ta t ivelysimilar pa tt ern to t hat for ChAT-positive neur ons (cf. Fig.4). Overall, 85% of TH-positive cells occurred singly orwere contained in clusters of up to eight cell bodies. The

remaining 15% were distributed in a few clusters rangingin size from 9 to 40 cells, and this overall patt ern wa s alsoreected in the regional distributions shown in Figure 5.TH-IR cel ls in the atr ia thus appear, l ike ChAT-IR neu-rons, to be preferent ially distribut ed in clusters containingrela tively few cells.

A number of sma ll TH-IR varicosi t ies on individualprocesses occurred within ganglia observed under theconfocal m icroscope, severa l exam ples of which ar e sh own

in Figure 1D (arr owheads). It was not clear whet her t hesevaricosities were a ssociat ed with sympath etic postgan gli-onic a xons of extracardiac or igin or were processes of int ra cardia c TH-IR cells. To clarify th is, th e optical section-ing capa bility of the confocal microscope was used to followth e pat hs of some of th e processes emer ging from a nu mberof TH-IR cel ls l ike that shown in Figure 1D (arrowheadwith aster isk) to determine whether these processes h advaricosities. None were observed in the specimens exam-ined. However, the processes of some TH-IR cells wereobserved to a pproach t he somata of other n earby TH-IRcells (dat a n ot shown), ra ising th e possibility of cell-to-cellcommu nication with in th is population.

Fig. 5. Bar graph of proportional distribution of tyrosine hydroxy-lase immunoreact ive (TH-IR) cells in the atr ia , expressed as meanfrequency of clusters of different size categories (number of cells ineach category indicated under each bar) for both atria combined and

by region in ve heart s, presented in a format similar to th at of Figure4. Eighty-ve percent of TH-IR cells were found in clusters of eightcells or less. Error bars 1 SE.

310 J. LEGER ET AL.



Biogenic amines. Tyrosine hydroxylase is involved inthe synthesis of norepinephrine, so to determine whetherthe morphology of atrial TH-IR cells an d cells conta iningcatecholamines were similar, aldehyde-induced u ores-cence was used in this study to histochemically identifycatecholamine occurrence. With this technique, elementscontaining catecholamines uoresce a bright blue-green

which is ea sily distinguishable from the faint backgroundau touorescence (Fig. 6) . This me th od was not compa tiblewith t he imm unohistochemical visualizat ion of TH, so thetwo procedures could not be combined for direct compari-son. Catecholamine-containing cel ls , nerve bres , andtheir associated varicosities (Fig. 6) were similar in size,dist r ibut ion, and morphology to a t r ia l cel ls and bresexpressing TH immu nohistochemistr y (Fig. 1D). Fur th er-more, the relationship of catecholamine-containing cellsand var icosi t ies t o nearby s t ru ctures was very s imilar tothat of TH-IR elements . The s imilar i t ies in morphologyand distribution of catecholamine-containing and TH-IRin t raca rd iac cel ls and bres sugges t s tha t in t raa t r i a le lements which contain catecholamines may also be ca-pable ofsynth esising th ese compounds. No atr ial cells with

somatic dimensions s imilar to t he neurons displayingChAT- or PGP 9.5-IR were found to contain catechol-amines.

One of the most striking ndings of th is part of the studywas that large numbers of amine-containing varicositieswere observed not only in association with cardiac musclecells but also within a tr ial ganglia (Fig. 6B,C) where t heyformed basket-l ike networks closely apposed to someprincipal gangl ion neurons. Although both TH-IR andaldehyde uorescence t echniques labelled intr agan glionicvar icos it i es in the p resen t s tudy, these s t ructu res ap -peared to be more intensely labelled after processing forcatecholamine histochemistr y th an for TH-IR (cf. Fig. 1Dand Fig. 6C). Aldehyde his tochemistry thus appears toafford a better view of the intraganglionic distribution of these str uctures th an does TH immu nohistochemistr y.

Ext rinsic innervation of int racardiac plex usF ig ur e s 2 a n d 3 s h ow t h e r st -d egr e e i nt r a ca r d ia c

ramications ofextrinsiccardiacsympatheticand parasym-pathetic nerves. Parasympathetic innervation of the heartor igina ted as ne rve b ranches f rom the l eft and r igh tthorac ic vaga l t runks , and the con t inua t ions of thesebranches within the cardiac plexus are represented by the

Fig. 6. Composite photomicrographs of whole-mount prepara tionsof two different left atr ial ganglia after histochemical processing foraldehyde-induced uorescence, showing catecholamin e-containin g cells

(arrows), terminals (arrowheads), and nerve bres .A:

Low-powerphotomicrograph showing several cluster s of sma ll, intensely uores-cent (SIF) cel ls (arrows) associated with gangl ia . The somata of ganglion neur ons appear faintly au touorescent; no neuronal somatain th i s example or e lsewhere in the a t r i a d isp layed the b r igh tuorescence indicative of the presence of catecholamines. B: Higher-magnication view of a different ganglion showing a cluster of SIFcells (arrow) and ne networks of catecholamine-containing bres andvaricosities (arrowheads). One of these networks is shown enlarged(area indicated by corner brackets) in C. C: Enlarged portion of B toillustrate network of catecholamine-containing intraganglionic vari-cosities in close apposition to ganglion neurons. The cluster of brightlyuorescing cells in th e upper left par t of the pan el (arrow) is th e samecluster appearing in B but is out of the focal plane. Scale bars 100µm in A, 50 µm in B, 25 µm in C.




large nerve bundles labelled LVN a nd RVN, r espectively,in Figure 2.

Both LVN and RVN entered the left atrium superior tothe roots of the pulmonary veins . From t hese entry s i tesextensive ramicat ions contr ibuted to the nerve plexussupplying the atr ia l myocardium. From LVN numeroussmall nerve bundles could be traced: (1) laterally towardand along the border between the left a t r ium and the leftventr ic le ; (2) to more anter ior par t s of the atr ium in th edirect ion of the left a t r ia l appendage and the junct ion of the ca rd iopu lmonary ne rves wi th the a t r ium; and (3 )toward th e midline of the hear t wher e some bundles mixedwith those or iginat ing from the r ight vagus. From RVNnerve bundles were dis t r ibuted to: (1) the medial r ightatr ium; (2) a long th e border of the r ight a t r ium with theright ventricle (seen in the lower right portion of Fig. 2);and (3) toward the area of the s inoatr ia l node, locatedapproximately as indicated by the da shed oval in F igure 2.

Sympathe t i c bres coursed to the hea r t by means o f cardiopulmonary nerves ar is ing from the left and r ights t e ll a t e gangl ia , the ansae subclavia , and the midd lecervical gan glia. The intra cardiac routes of branches of the

left and right cardiopulmonary nerves are represented bythe lar ge nerve bundles labelled LCPN an d RCPN, respec-tively, as illustra ted in Figure 2.

On t he left side, major cardiopulmonary n erve bran chesfol lowed t he left superior intercostal vein toward theatr ium, then coursed dorso- and ventrolateral ly aroundthe opening of the left a t r ia l appendage, innervat ing thea t r i a l myocard ium dorsa l , l a t e ra l, and ven t ra l to t h i sopening. In some hear ts , a few of these branches weretraced across the atrioventricular border and into the leftvent ricle (not shown in F ig. 2). Sma ller ner ves arisin g fromthe sympathet ic branches around the dorsal a spect of theopening of the left a t r ia l appendage cont inued dorsal lyinto the area la teral to the pulmonary veins; this area wasalso innervated by la t eral branches of the LVN. Several

bran ches of the LCPN ram ied in a dorsomedial directiontoward th e intera trial septu m (see Fig. 3 and description of septal innerva tion below) and the ar ea just superior to theroots of the pulmonar y veins wher e th ere was a lso a densevagal innervation. The RCPN entered the myocardium of the r ight a t r ium m ore ventral ly and medially than did th eLCPN. In addi t ion, th e dis t r ibut ion of RCPN within themyocardium was more diffuse compared with the patternon the left. It was expected t hat the majority of car diopul-monary nerve bran ches into the plexus of the r ight atr iumwould innervate the area of the sinoatrial node, atrioven-tr icular node, or both, yet in the hear ts examined in thisstudy only one or two small nerve bundles ran directly tonodal regions (Fig. 2). The m ajority of nerve bra nches ra n(1 ) in to and a long the dorsa l and l a t e ra l wa l l s o f the

superior vena cava; (2) medially and vent rally toward an dacross the junct ion of the septum with the external a t r ia lwal l, cont inuing into t he left a t r ium; a nd (3) toward thein fe r ior vena cava and in to an ad jacen t r egion of themyocar dium which was also heavily inn ervat ed by bran chesof the right vagus. In addition, some small nerve bundlescould be traced from the inferior vena cava laterally intothe cr is ta terminal is and into the wal l of the r ight a t r ia lappendage.

Due to separation of the septum from the atrial walls tofacilitate penetration of antibodies and subsequent tissueprocessing, specic pathways of vagal and sympathet icnerve branches into the septum could not be identied in

this st udy. Nerve bre bundles in th e septum were concen-trated primarily in the cranial portion (Fig. 3), with bothlarge and small bundles present. Typically, one large andseveral smaller bundles ran from the cranial aspect to-ward the atr ioventr icular valves ( in the direct ion of thebottom edge of Fig. 4). Nerve bres in the cranial part of the septum were concentrated in an area corresponding

with the location of the densely innervated region of theexternal a t r ia l myocardium near the septum-atr ia l wal l junct ion noted above. The nerve plexus of the externalatr ial wall was contiguous with the septa l innervation.

DISCUSSIONPhenotype of intrinsic cardiac neurons

The resul ts of the phenotype analysis showed that a l lneurons in t he a t r i a of the gu inea p ig hea r t express ingimmunoreact ivi ty for the general neuronal marker PGP9.5 were also immunoreactive for the enzyme ChAT, therat e-limiting enzyme in ACh synt hesis. This suggests th atthese neurons were chol inergic. Our resul ts conrm andextend th e ndings of previous st udies which showed th atthe majori ty of neurons in the hear ts of guinea pigs andother mamma lian species display chol inergic a t t r ibutes(Ja cobowitz, 1967; Eh inger et a l., 1968; Bojsen-Moller a ndTranum-Jensen, 1971; Hancock et al., 1987; Roberts et al.,1989; Seabrook et a l ., 1990; Steele a nd Choate , 1994;Steele e t a l ., 1994; Edwards et a l ., 1995; Mawe et a l .,1996).

Histochemical reactions for AChE (Koelle, 1963) ha vebeen widely used to investigate t he distribution ofcholiner-gic neur ons in th e periphera l nervous system (reviewed byKuhar, 1976), and most previous invest igat ions of t hephenotype of in t raca rd iac neurons have re li ed on th i stechnique. However, some noncholinergic neurons andsome non-neuronal elements associated with the nervoussystem also express AChE (Fibiger, 1982; Sat oh et a l . ,

1983), leading to concern a bout the usefulness of AChE asa rel iable indicator of chol inergic neurons in the hear t .Specic antibodies against ChAT have been used to mapcholinergic neur ons in the centr al ner vous system, but th istechnique ha s only recently been applied to the periph eralnervous system (Keast et al. , 1995; Schema nn et a l., 1995;Wan g et al., 1995; Talm age et al., 1996; Dey et a l., 1996). Inthe guinea pig heart , a recent su rvey by Mawe et a l. (1996)found that a l l neurons in the atr ia l intercaval region, thebasa l por t ion of the in tera t r i a l sep ta l r egion , and thecardiac sinus r egion which expressed immun oreactivityfor MAP-2, a microtubu le-associated protein wh ich hasbeen used as a general neuronal marker, were also ChAT-IR. However, the s tudy of Mawe et a l . (1996) excludedmajor areas of both atria. The results of the present study

not only conr m t he pr esence of lar ge nu mber s of ChAT-IRneurons in the regions analyzed by Mawe et al. (1996), butfor the rst t ime demonstrat e tha t ChAT-IR neurons ar edist r ibuted t hroughout both a tr ia . In par t icular, neuronsare present in high concentrat ions within the interatr ia lseptum and in t he region a round the pu lmonary veins,areas which were not included in the study of Mawe et al.(1996). Control experiments in the present study, designedto test the specicity of the ChAT antibody, showed thatneuronal somata in extracardiac t issues , including t hedorsal and ventral horns and the intermediolateral cel lcolumn of the spinal cord, processed with th e sam e proto-col as the experimental tissue, were ChAT-IR.

312 J. LEGER ET AL.



The nding in the present s tu dy that a l l neurons in th eatr ia are chol inergic could be taken to support the ideathat these neurons are all parasympathetic postganglionicefferent neurons, as predicted by t he classical model of neur al contr ol of the hea rt. In this m odel, intr insic cardiacneurons are considered to be all postganglionic cholinergicneurons, act ing as s imple relays conveying parasympa-

thet ic cardiomotor dr ive to the myocardium. However,intrinsic cardiac neurons are neurochemically very com-plex, expressing a num ber of neuropeptides in addition toACh (Steele e t a l ., 1994, 1996) in a mann er s imilar tocholinergic neur ons elsewhere in t he peripher al au tonomicnervous system. Janig and McLachlan (1992) have pro-posed t hat the variety of peptide neur omodulat ors colocal-ized with classic neurotransmitters in specic subpopula-tions of autonomic neurons chemically code these neuronalpopulations to subserve specic functions. In atrial neu-rons of the guinea pig mu ltiple peptides have been sh ownto be coexpressed in distinct subpopulations (Steele et al. ,1994, 1996), and it is likely that, even though all of theseneurons can synthesise ACh, the colocalization of specicgroups of peptides in different subpopulations indicates

specic functional roles for neurons in these subpopula-tions. Physiological evidence in several mammalian spe-cies has been obtained for the existence of several types of neurons, including a fferent neurons, interneurons, an dlocal c ircuit neurons in addi t ion to efferent neurons ca-pable of generat ing cardioaugmen tat ion a s well as cardio-inhibition (summarised in Armour, 1994). Therefore, al-though the p resen t s tudy has shown ana tomica l ly tha tatr ia l neurons share a common cholinergic phenotype,fur th er s tudies of t he funct ional propert ies of specicclasses of neurons must be combined with techniques t odetermine which neuromodulators may be colocalized inthese neur ons.

Number and distributionof intracardiac neurons

The mean total nu mber of neur ons found in adult guineapig atr ia in the present s tudy (1,510, Table 2) was 50%higher than the mean number r epor ted by Mawe e t a l .(1996) in the atr ia of adul ts of the same species , l ikelyreect ing the larger area analyzed in the present s tudy.The number of neurons in the present s tudy was estab-l ished by u sing ant ibodies a gainst th e general ver tebrateneur onal mar ker P GP 9.5 (Thompson et al. , 1983); immu-noreaction for this marker has previously been shown to bere li able in de termin ing t he pa thways and exten t of themyocardial innervation and the locations of intracardiacneur onal somat a in a ran ge of mamma lian species, includ-ing hu man s (Gulbenkian et al. , 1987, 1993, 1994; Chow etal., 1993; Gordon et al., 1993; Crick et al., 1994; Fu et al.,

1994; Marron et al. , 1994). In control experiments in thepresent study, PGP 9.5-IR labelled neur ons in sections of the sp ina l co rd and the s t e l l a t e gang l ion ; thus , we a rereasonab ly conden t tha t th i s n eurona l marker gave areliable picture of the distribution of neurons intrinsic tothe guinea pig atr ia .

Neurons were found throughout a l l a t r ia l r egions. Theq u a nt i t at i ve d a t a p r es en t e d in Ta b le 2 s h ow a l a rg evariat ion in total number of neurons per hear t , and in t henumbers of neurons in the different atrial regions, amongthe specimens sampled. However, one consistent patternof neuronal dis t r ibut ion in the r ight a t r ium of a l l speci-mens was a c luster ing of gangl ia in the intercaval area

close to the SA node and near the ost ium of the IVC. Asimilar pat tern was also reported in the guinea pig hear tby Mawe et al. (1996), and t his pat tern has been observedin othe r species a s wel l. Anothe r locus which had aconsistently high concentration of neurons in the presents t u dy w a s i n t h e l eft a t r iu m a r ou n d t h e b a se of t h epulmonary veins . This nding is s imilar to the s i tuat ion

reported in rat (Burk holder et a l., 1992), can ine (Yua n etal ., 1994), and human (Ja nes et a l ., 1986) hear ts but hasnot been previously reported in the guinea pig heart. Theinteratr ia l septum had the smallest area but contained adisproportionat ely large num ber of neurons, i .e., one-fthof the total number was located here, giving this area thehighest density (Table 2). This nding provides quantita-tive conrmation of previous qualitative reports of highconcentrat ions of neurons in the mammalian interatr ia lseptum (King an d Coakley, 1958; Ehinger e t a l ., 1968;Ellison and Hibbs, 1976). In guinea pigs, the septal areawas also r ichly innervated by nerves and s ingle bres ,primarily in the cranial portion (Fig. 3). These nerves werecont iguous with those in t he heavi ly innervated areas of the exte rna l a t r i a l wa ll s a t the septum-wall junct ion .

In t rasepta l ne rves runn ing in the d irect ion of the ven-tr icles, would th us, appear to represent a major pathwayfor the innerva t ion of t hese chambers . Neurons in theinteratr ia l septum are in c lose proximity to the cardiacvalves as well as to pacemaker and conductive tissues, sowould be well posi t ioned to modify chronotropic anddromotropic funct ions and possibly valve operat ion, asproposed by Moravec a nd Moravec (1987).

Despi te the lef t a t r ium having a s ignicant ly smallerarea than the right, there was no signicant difference innumbers of neurons associated with these two regions(Table 2) . Furt hermore, a comparison of th e density of neur ons in th ese two regions showed no signican t differ-ence. However, there was a qualitative difference in thepatt ern of distribut ion of neur ons within the left and r ightatr ia . With the except ion of a concentra t ion of neur onsnear the pulmonary veins, ganglia in the left atrium weremore widely dis t r ibuted than those in the r ight a t r ium,where the majori ty of neurons was concentra ted n ear theSA node. This difference ma y be relat ed to th e functions of n e u ron s i n t h e t w o ch a m be r s. I n t h e r igh t a t r iu m , acondensed organisation m ay be more effective in promot-ing local n etwork int eractions am ong neurons contr ollingthe complex behaviour of nearby pacemaker and conduc-tion tissues. Given t his ra tionale, it m ight also be expectedtha t right a trial neu rons would be preferentia lly cluster edinto larger ganglia to facilita te int erneur onal communica-t ion. This was, however, not the case: the analysis pre-sented in Figure 4 shows that neurons were consis tent lycluster ed in relatively small ganglia in this ar ea as well asin the res t o f the a t r i a l r eg ions . In the l e f t a t r ium, theneur on populat ion size was similar to that in t he right but ,in contra st to the dis t r ibut ion pat t ern in th e r ight a t r ium,left a trial n eurons were more widespread. Such a distribu-t ion pa t t e rn may reect the pa r t i cipa t ion of l e ft a t r i a lneurons in multiple local feedback loops involved in re-gional control of myocyte contractile properties through-out the cham ber.

Adrenergic elements in the atriaNo TH-IR neur onal somata were observed in th is study,

as judged from the lack of colocalization of TH- an d P GP9.5-IR. These ndings concur with those of Baluk and




Gabella (1990), Steele et al. (1994, 1996), and Mawe et al.(1996), who reported a n absence of TH-IR in int racar diacneur ons in th e guinea pig hear t. Furt hermore, a review byAllen et a l . (1994) summar ised reports that guinea piga t r i a l n eurons in p r imary cu l tu re a l so l ack TH-IR. Incontrast to these reports , Dalsgaard et a l . (1986) foundTH-IR in a small group of neur ons in the guinea pig heartand Horackova e t a l . (1996) r epor ted tha t gu inea p igintracardiac neurons in culture can express TH-IR; someof the n eurons in th e latter study even coexpressed TH andChAT-IR. In the rat hear t, t here is also some evidence forTH-IR in a t r ia l and ventr icular neurons (Moravec andMoravec, 1989; Moravec et a l ., 1990; Slavikova et a l .,1993) . The reasons for these disparate ndings are notobvious but could be due to interspecic var ia t ion ordifferences in the immun ohistochemical procedures used.With respect to t he la tt er possibility, our pr ocedures weresensitive enough to detect TH within primary neurons of the s t e ll a t e gangl ia and , the re fore , shou ld have beencapable of detecting the presence of TH in atrial neurons.As a furt her pr ecau tion, in some experiments we increasedthe concentr at ions of the pr imary an tibody against TH (upto 10 t imes th e concentra t ion necessary to label s te lla teganglion neurons) and prolonged incubat ion t imes, butstill failed to label intra cardiac neur ons.

The lack of TH-IR in intr insic cardiac neurons in theguinea pig heart has important implications for the studyof intr insic neural mechanisms contr olling cardioaugmen -tat ion. In vivo funct ional s tudies in the hear ts of othermam malian species such as t he dog have shown tha t someintrin sic cardiac neur ons are capable ofevoking cardioaug-mentation when activated (Butler et al. , 1990; Armour etal., 1993; Huang et al., 1993a,b,c), and it was proposed ont h e b a si s of s u ch fu n ct ion a l e vi de n ce t h a t t h e r e a r eneur ons in th e canine h eart wh ich ar e capable of synthesis-ing and s tor ing monoamines for re lease when these neu-rons are activated. The presence of TH-IR in neurons is apr imary indicator of the capabi li ty for catecholaminesynthesis , but no evidence h as so far been presented forTH-IR neuronal somata in the dog hear t . This s i tuat ionhas a pa ra l l e l in the gu inea p ig hea r t , i n tha t we havefound in t r ia l experiments t hat s t imulat ion of some neu-rons in the gu inea p ig r igh t a t r ium in v i t ro can causecardioacceleration (Leger a nd Smith, in prepar at ion).

Al though no in t raca rd iac neurons were found in thepresent study to conta in det ecta ble levels of TH, cardioac-celeration evoked by activation of intrinsic cardiac neu-rons could be driven by other mechanisms, such a s releaseof catecholamines from neurons which can take up ands tore bu t not syn thes ise these compounds . A po in t infavour of th is idea is that some intracar diac neurons whichlack the synthet ic enzymes for biogenic amines h ave beenshown to accumulat e th ese compounds in culture (Allen etal., 1994). However, the results of the present study do notsupport th is because n o neuronal somat a were observed toconta in catecholamines in a tria l tissue pr ocessed for a lde-hyde h is touorescence (Fig. 6). In this sense, then, ourr e su lt s con rm a n d e xt e n d t h e r e su lt s of B a lu k a n dGabella (1990) in the guinea pig.

Release of catecholamines from n on-neur onal st ores inthe hear t may be an al ternat ive explanat ion for intr insi-cally mediated cardioaugmen ta tion. Immun oreactivity forTH was found in high concentration in a class of cells withsomata which were smaller than , an d morphologically

distinct from, intracardiac neurons (Fig. 1D). The resultsof a compar ison of morphologic char acteristics of the twocel l types showed that there was no overlap in somaticdimensions between the smallest neurons and the largestTH-IR cells. This nding, in combinat ion with t he lack of PGP 9.5-IR in cells expressing TH-IR, argues a gainst theidea tha t th ese cells are simply small neur ons. Instea d, themorphology of atrial TH-IR cells in our study was similarto that of sma ll, in tensely uorescent cel ls (SIF cel ls)obse rved th roughou t the v isce ra and in the pe r iphera lautonomic nervous system af ter s ta ining for catechol-amines using a n aldehyde uorescence reaction (Ja cobow-itz, 1967; Biscoe, 1971; Burn stock an d Costa , 1975; Dail eta l ., 1975; Dai l, 1976; Howe et a l ., 1978; de Groat andBooth, 1980; Wurst er et al. , 1990; Kriebel et al. , 1991).Specically, SIF cel ls in the hear t have been found inprevious s tudies to contain TH in high concentra t ions(Heym et a l ., 1994; Mawe et a l ., 1996). In the presentstudy, to determine which cells contained catecholaminesand to see whether there were any morphologic s imilar i -ties between sma ll atrial TH -IR cells and t hose containingcatecholamines, we used aldehyde his touorescence to

localise these compounds. Brightly uorescent catechol-amine-containing cells with a morphology very similar tothat of small TH-IR cel ls were found most ly in or nearganglia (Fig. 6), with some cells scattered in the myocar-dium. The protocols for determining catecholamine con-tent and TH-IR were not compatible for double-labellingbut based on the morphologic similarities of cells visual-ised by these techniques, we hypothesise that cells contain-ing catecholamines a nd those immun oreact ive for THcomprise t he sa me populat ion of intra cardiac SIF cells.

The mean total number of these cel ls in the atr ia wasabout 12% of the mean total nu mber of atr ial neurons, butun l ike neurons , the densi ty o f S IF ce ll s was un i formamong the different atrial regions. At least 90% of thesecells were associated with ganglia. The quant itat ive analy-

sis of the t endency of these cells to group t ogether (Fig. 5)showed th at they preferent ia l ly occurred in re la t ivelysmall c lusters throughout the atr ia with l i t t le regionalvar ia t ion in this pat tern. Such a c luster ing pat tern wasalso similar t o that displayed by intra cardiac neurons. Theclose approximation of the majority of SIF cells to ganglia,and the s imi la r i ty in cluste r ing pa t t e rn of the two ce lltypes, suggests th at SIF cells may be involved in neuralcontr ol of the h eart .

The funct ional role of SIF cells in the hear t is not yetclear, but these cel ls are posi t ioned to play potent ia l lyimportant roles in cardioaugment ation and in intr agan gli-onic neurotra nsmission. Release of cat echolamines st oredin SIF cells of pelvic gan glia can be in duced by activa tion of cholinergic receptors on t he membra nes of these cells (de

Groat and Booth, 1980). If, in the heart, a subpopulation of intr insic chol inergic neurons projects to SIF cel ls , thenactivation of neurons in this pathway may evoke SIF cellcatecholamine release. Such a release mechanism, if underlocal neuronal control, may provide an alternative explana-t ion for cardioaugmentat ion mediated by intra cardiacneu rons. Additiona lly, th e pr ocesses of some SI F cells wereobserved to approach nearby SIF cell somata, suggestingsome form ofcell-to-cell commun ication with in t his popula -tion.

A large num ber of catecholamine-conta ining var icoseterminals was observed both in the myocardium and inganglia in the present study (Fig. 6). It was not, however,

314 J. LEGER ET AL.



possible to determine whether these terminals (1) wereassociated with t he a xons of intrin sic car diac neurons; (2)were of extracardiac or igin (i .e ., axons of sympathet icpos tgang lion ic n eurons with the ir somata in thorac icganglia); or (3) were associated with the processes of SIFcells. Regardin g th e lat ter possibility, no SIF cell processesin tissue which had been prepared for amine histouores-

cence could, in the standard uorescence microscope, bet raced fa r enough from the ce ll bod ies to de terminewheth er th ey included am ine-contain ing varicosities. Theseobservations were not, however, conclusive because tissuesamples were t oo thick to visualise cell processes well inthis microscope at high magnicat ion. To a ddress t hisissue, the opt ical sect ioning capabi li ty of the confocalmicroscope was used on tissue containing TH-IR. Underthe assumpt ion tha t TH-IR ce ll s and ca techolamine-containing cel ls comprised th e same populat ion, a n at-tempt was made to determine whether processes of intra-cardiac TH-IR cells conta ined varicosities. As with SIFcells, no var icosities were found along th e processes of TH-IR cells as far as t hese could be t raced (up t o 30 µmdistal to the cel l bodies). I t i s , therefore , possible that

catecholamine-containing varicosities and TH-IR varicosi-ties were associated with axons. Regardless of origin, th epresence of t his type of var icosi ty within intracardiacganglia suggests th at release of biogenic amines may h avepowerful m odulating effects on ganglionic neu rotran smis-s ion, as proposed by Smith et a l . (1992) on the basis of physiological evidence.

Extrinsic innervationof t he at rial myocardium

Isolated an d in vitro prepara tions of mam malian h eart s,including those from rodents, have been used extensivelyfor investigating functional aspects of neural control of theheart, yet the details of anatomical pathways of extrinsiccardiac nerves within the heart have not been established

in a ny of these species. In neonata l ra ts , Seabrook et a l .(1990) invest igated the parasympathet ic innervat ion of intracardiac gangl ia in a small area of the dorsal a t r ia lwa ll nea r the a t t achment of the in tera t r i a l sep tum, bu tthese a uth ors did not explore other cardia c regions. On th esympath etic side, no description of the r elationship of thecardiopulmonary nerves to the intracardiac plexus existsfor t he rodent hear t . In th e present s tudy we ha ve showntha t both vaga l and ca rd iopu lmonary ne rves r amiedextensively into the cardiac plexus in the general patternillustr ated in Figure 2.

Major bran ches of th e sympat hetic and para sympath eticnerves entered t he plexus in regional patt erns which wereconsistent among the specimens, but these branches werenot na rrowly targeted on specic cardiac regions. Inst ead,

these n erves ram ied widely within th eir regional t errito-ries. In particular it was surprising, given the concentra-t ion of n e u ron s n e a r t h e s in oa t r ia l n od e i n t h e r ig htatr ium, t hat the branches of extr insic nerves innervat ingthis chamber were broadly dis t r ibuted over the chamberwall and not targeted pr imari ly on the node and nearbyneurons.

A m a jor fe a t u r e o f a l l t h e h e a rt s s t u di ed w a s t h a tpart icular at rial regions such as the left atr ium around th ebase of the pu lmonary veins, th e area of the a tt achment of the septu m to the externa l atria l wall, and the r egion of theSA node , were dua l ly innerva ted by b ranches of thesympathet ic and parasympathet ic nerves . Another major

nding was that the cardiac branches of the r ight vagusenter the heart in association with the left atrium near the junct ion of the external a t r ia l wal l with the interatr ia lseptum. One importan t consequence of this anat omicalfea tu re i s th a t in v it ro p repara t ions for s tudying r igh tvagal inuences on th e hear t mu st include a portion of th el eft a t r iu m n e a r t h e s ep t u m t o e n s ur e t h a t a l l v a ga l

pathways to the r ight a t r ium are present in the prepara-tion. The resu lts of this pa rt of the st udy have esta blishedthe genera l ana tomica l pa thways for r egiona l ca rd iacinnervat ion by extr insic autonomic nerves, thu s providingan organisat ional substrate on which fur ther funct ionalstudies of the aut onomic control of the myocardium can bebased.

CONCLUSIONSAll neurons found in the guinea pig atria were coreactive

for the neuronal marker PGP 9.5 and ChAT, suggest ingthat these neurons are chol inergic . Greater numbers of a t r i a l neurons were found in th i s s tudy than have beenreported in previous s tudies. The regional densit ies of neurons in t he external wal ls of the RA and LA were n ots ignicant ly different , but density in the interatr ia l sep-tum was s ignicant ly greater than in e i ther of the otheratr ia l r egions. Neurons throughout the a tr ia tended to beclustered in relatively small ganglia, with the majority inganglia of less than 20 cells. Neurons were more widelydist r ibuted in the left a t r ium th an in t he r ight , where themajority was located in the intercaval region near the SAnode. This difference in regional distr ibution is proposed toreect the roles of these neur ons in contr olling inotropic,chronotropic, and dromotropic aspects of cardiac function.No cells identied as neurons contained either TH-IR ordetectable levels of catecholamines. However, both TH-IRand catecholamines were detected in high concentrationsin sma ll atria l cells which r esembled the SIF cells reportedby othe r s to be p resen t in the hea r t and th roughou t theperipheral autonomic nervous system. Catecholamine-containing varicosities were observed within intracardiacganglia a nd were associated with the atr ia l myocardium,as were TH-IR varicosities. This study provides, for therst time, detailed anatomical data on the regional organ-isat ion of the intracardiac nervous system in t he guineapig hear t . This information is essent ia l to guide fur therphysiological studies of the roles of intr insic cardiac neu -rons con t ro ll ing the ra t e of d ischarge of s inoa t r i a l oratr ioventr icular pacemaker cel ls and regional myocytecontr actile pr opert ies.

ACKNOWLEDGMENTSThe authors thank Helen Sauveur and Jeanet te Nason

for technical assistance. J.L. was supported by a scholar-ship from Dalhousie University during part of this study.Some of the dat a ha ve been presented at t he Experimenta lBiology 1996 ann ua l meetin g, Wash ington, DC, USA, April14–17, 1996 (Smith FM, Leger J , Croll R. 1996. Regionaldistribution of ChAT- an d TH -immu noreactive neur onalelements in guinea pig atr ia . FASEB J 10:A337). Thecomments of Pr of. David A. Hopkins on a draf t of thisma nu script a re gra tefully acknowledged. F.M.S. received aresearch scholarship from the Heart and Stroke Founda-tion of Canada. R.P.C. received an operating grant fromthe N atu ral Sciences and E ngineering Research Council of Canada .




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J. Leger et al- Regional Distribution and Extrinsic Innervation of Intrinsic Cardiac Neurons in the Guinea Pig