THE JOURNAL OF COMPARATIVE NEUROLOGY 407:303317 (1999)

Regional Distribution and Extrinsic Innervation of Intrinsic Cardiac Neurons in the Guinea Pig
1Department

J. LEGER,1 R.P. CROLL,2 AND F.M. SMITH1* of Anatomy and Neurobiology, Dalhousie University, Halifax, NS Canada B3H 4H7 2Department of Physiology and Biophysics, Dalhousie University, Halifax, NS Canada B3H 4H7

ABSTRACT Mammalian intrinsic cardiac neurons subserve different functions in different cardiac regions, but the regional anatomical organisation of the intracardiac nervous system is not well understood. We investigated the quantitative and qualitative distribution of cholinergic and adrenergic elements, and the intracardiac pathways of extrinsic cardiac nerves, in whole-mount preparations of guinea pig atria. Protein gene product 9.5 immunoreactivity (PGP 9.5-IR) marked intracardiac neuronal elements; immunoreactions for choline acetyltransferase (ChAT-IR) and tyrosine hydroxylase (TH-IR) distinguished cholinergic and adrenergic components, respectively. Catecholamine-containing components were identied by aldehydeinduced uorescence histochemistry. Mean total number of atrial neurons was 1510 251 (SE); 85% of these occurred in ganglia of 20 neurons. All neuronal somata expressing PGP 9.5-IR also expressed ChAT-IR, suggesting that these neurons were cholinergic. Right (RA) and left (LA) atria had statistically similar neuronal densities (6.4 1.2 and 2.4 0.7 neurons/mm2, respectively; analysis of variance, P 0.05). Neurons in RA were concentrated intercavally; LA neurons were concentrated near pulmonary vein ostia. Greatest density occurred in the interatrial septum (16.3 4.0 neurons/mm2). No neuronal somata expressed TH-IR or contained detectable amines but these elements were expressed by somata of small cells (mean total 124 33) throughout the atria, primarily associated with ganglia. Amineand TH- containing varicosities were also present in ganglia, representing potential sites for adrenergic modulation of ganglionic neurotransmission. Branches of extrinsic cardiopulmonary and vagus nerves were distributed to all parts of both atria. The organisation of the intracardiac nervous system revealed in this study will facilitate further investigations of regional autonomic control of the heart. J. Comp. Neurol. 407:303317, 1999.
1999 Wiley-Liss, Inc.

Indexing terms: autonomic nervous system; cardiac ganglia; vagus; sympathetic; parasympathetic

Neurons with their somata located in the mammalian intrinsic cardiac nervous system are capable of modifying pacemaker activity, force of contraction and conduction. Classically, these neurons have been regarded as parasympathetic postganglionic efferent neurons, and their activation has been considered to inhibit cardiac functions. However, recent physiological studies have shown that activation of some intracardiac neurons by electrical or chemical stimulation can augment cardiodynamics (Butler et al., 1990; Armour et al., 1993; Huang et al., 1993a,b,c), suggesting that some cardiac sympathomimetic effects may be mediated by neurons with their somata intrinsic to the heart. Evidence is also accumulating to suggest the presence of neurons of other functional types within the 1999 WILEY-LISS, INC.

heart, including afferent and local-circuit neurons or interneurons (Ardell, 1994; Steele et al., 1994; Edwards et al., 1995). It has been proposed that neurons of these diverse types constitute a distributed network with connec-

Grant sponsor: Medical Research Council of Canada; Grant number: MT-11622; Grant sponsor: Heart and Stroke Foundation of Canada; Grant sponsor: Natural Sciences and Engineering Research Council of Canada; Grant number: OGP0038863. *Correspondence to: Dr. Frank M. Smith, Department of Anatomy and Neurobiology, Faculty of Medicine, Dalhousie University, Halifax, NS Canada B3H 4H7. E-mail: fsmith@is.dal.ca Received 25 June 1997; Revised 12 August 1998; Accepted 4 December 1998

304 tions to all regions of the heart. This network, possibly operating with the aid of local feedback on a beat-by-beat basis, could potentially provide great exibility in modulating regional and global myocardial function to help match cardiac output to the ow requirements of the systemic and pulmonary arterial circulations (Armour, 1994; Ardell, 1994). However, details of the regional patterns of innervation of intracardiac neurons by extrinsic nerves, the quantity and distribution of neurons within the heart, and the relative abundance of different neuronal phenotypes have not been established in the mammalian heart. It has been reported in anatomical studies of the mammalian heart that ganglia containing the somata of intrinsic cardiac neurons are associated with an intracardiac nerve plexus extending throughout both atria and into the ventricles (King and Coakley, 1958; Calaresu and St. Louis, 1967; Anderson, 1972; Ellison and Hibbs, 1976; Tay et al., 1984; Janes et al., 1986; Pardini et al., 1987; Yuan et al., 1994), but the anatomical arrangement of this system is not well understood. Control of the heart by intracardiac neurons has been studied in in vivo and in vitro preparations in a number of mammalian species, including canine, porcine, and rodent, but these studies have been hindered by a lack of understanding of the organisation of the intracardiac nervous system. The overall objective of the present study, therefore, was to investigate the regional organisation of the atrial portion of the intracardiac nervous system in the heart of a small mammal, the guinea pig. A major aim was to determine the general distribution and phenotype of neurons throughout the atria of the guinea pig heart, their total number and their location by region. The somata of neurons were identied by their immunoreactivity to a general vertebrate neuronal marker, protein gene product 9.5 (PGP 9.5-IR). This procedure also labelled axons in nerves associated with the heart, facilitating the analysis of regional patterns of innervation of the atrial ganglionated plexus by the extrinsic cardiac nerves. In studies of the phenotype of intracardiac neurons, histochemical surveys of the mammalian heart have shown that many nerve bres, terminals, and neuronal somata exhibit staining for acetylcholinesterase (AChE), an enzyme involved in metabolism of the parasympathetic neurotransmitter acetylcholine (ACh; James and Spence, 1966; Jacobowitz, 1967; Ehinger et al., 1968; BojsenMoller and Tranum-Jensen, 1971; Anderson, 1972; Ellison and Hibbs, 1976; Hancock et al., 1987; Roberts et al., 1989). However AChE may not be the most suitable indicator for cholinergic neuronal phenotype, because this enzyme is also found in non-neuronal elements (Fibiger, 1982). The presence of choline acetyltransferase (ChAT), an enzyme involved in acetylcholine synthesis, is considered the most reliable indicator of cholinergic phenotype in neurons of the central nervous system (Fibiger, 1982; Satoh et al., 1983). This enzyme can be detected immunohistochemically in both the central and peripheral nervous systems, but in only one previous study has this technique been used as a probe for surveying the intracardiac nervous system. Mawe et al. (1996) used ChAT immunoreactivity (ChAT-IR) to characterise neuronal phenotype in selected parts of the guinea pig heart, reporting that neurons in the atrial intercaval region, the basal portion of the interatrial septum and the cardiac sinus region appeared to be ChAT-IR. However, neither this study nor earlier histochemical analyses of AChE location covered the entire extent of the atria, so the overall distribution of

J. LEGER ET AL. atrial neurons exhibiting cholinergic phenotype is still not known. The present study therefore aimed to provide an estimate of the total number and distribution of ChAT-IR neurons in the guinea pig atria. Evidence that augmentation of cardiac function can be evoked by focal activation of the somata of some intrinsic cardiac neurons has led to the suggestion that the somata of adrenergic neurons may be present in the heart (Butler et al., 1990; Armour et al., 1993; Huang et al., 1993a,b,c; Armour, 1994). Neurons potentially involved in such augmentatory responses might be expected to display one or more aspects of adrenergic phenotype, including expression of tyrosine hydroxylase (TH, the rate-limiting enzyme in norepinephrine synthesis), or uptake and storage of catecholamines. This study aimed to establish the pattern of TH occurrence within the atria of the guinea pig heart by using immunohistochemical detection of this enzyme, and to compare this with the intra-atrial distribution of catecholamines as indicated by an aldehyde histouorescence reaction. This study shows that the somata of identiable neurons in the atria display aspects of cholinergic phenotype and are distributed throughout the atria with concentrations near the sinoatrial node, the roots of the pulmonary veins, and in the interatrial septum. Cells which phenotypically and morphologically resemble the small, intensely uorescent cells found throughout the autonomic nervous system are also distributed in the atria, largely in association with intracardiac ganglia. The regional intracardiac distribution of branches of the extrinsic cardiac nerves is described. The details of the regional organisation of the intracardiac nervous system and the phenotypes of its elements provided by this study establish an anatomical substrate for guiding further functional investigations of the roles of intracardiac neurons in the control of cardiodynamics.

MATERIALS AND METHODS

Experiments were conducted on Hartley guinea pigs (Charles River, Quebec City, Que., Canada) of both sexes, weighing 150300 g and ranging in age from 3 to 7 weeks. Experimental protocols were approved by the Dalhousie University Animal Use Committee and conformed with animal use guidelines established by the Canadian Council on Animal Care.

Immunohistochemistry
Thirteen animals were used for immunohistochemical studies of the intracardiac nervous system. Animals were killed by a blow to the head, their hearts were quickly removed through a midline sternotomy and washed in 0.1 M phosphate-buffered saline (0.9% NaCl, pH 7.4). The external walls of the atria and the interatrial septum were separated from the ventricles, the septum was trimmed free of the atrial walls, and both pieces of atrial tissue were pinned at in a dissecting dish. Tissue was xed for 18 hours at 4C in 4% paraformaldehyde dissolved in 0.1 M phosphate buffered saline, rinsed, dehydrated in a graded series of ethanol solutions and cleared in xylene. The tissue was then rehydrated and incubated in a 4% solution of Triton-X 100 in phosphate-buffered saline for 48 hours. Tissue was then removed from the dish and incubated as free-oating whole-mounts in blocking solutions of 2.5% normal goat, donkey, or rabbit serum as appropriate for

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TABLE 1. Sources and Dilution of Primary and Secondary Antibodies Used In This Study Antigen
Primary antibodies Protein gene product 9.5 Choline acetyltransferase Tyrosine hydroxylase Secondary antibodies Rabbit IgG Goat IgG Mouse IgG
1Ultraclone 2Chemicon 3Incstar

305 the same immunohistochemical protocols as the cardiac tissue. Motor neurons in the ventral horn of the spinal cord of the guinea pig have been reported to display ChAT-IR in previous studies (Eckenstein and Sofroniew, 1983), and neurons in the stellate ganglia have been shown to display strong TH-IR (Bowden and Gibbins, 1992; Heym et al., 1993). In the present study, ChAT-IR neurons were observed in the dorsal horn, in the intermediolateral cell column and in the ventral horn of spinal cord sections; all of these neurons were also immunoreactive for PGP 9.5. Stellate ganglion neurons were strongly TH-IR, and these neurons were also double-labelled with antibodies against PGP 9.5. Nonspecic background labelling in all tissues processed for PGP 9.5-, ChAT-, and TH-IR was low, as was tissue autouorescence at the wavelengths used for visualising the uorophores. To establish negative controls, the atria were removed from a separate group of four animals and processed with the same protocol used for tissues in the experimental group, with one of the following changes: either primary or secondary antibodies were omitted from the incubating solution; or primary antibodies were preabsorbed with their appropriate antigens (100 times the concentration of the antibodies) for 2448 hours before being used for tissue incubation. Control cardiac tissue exposed to primary but not to secondary antibodies, and vice versa, did not contain any label, nor did tissue in which primary antibodies had been preabsorbed.

Host
Rabbit Goat Mouse Rhodamine FITC FITC Goat Donkey Sheep

Supplier
Ultraclone1 Chemicon2 Incstar3 Jackson4 Jackson4 Jackson4

Dilution
1:400800 1:250 1:500 1:50 1:50 1:50

Limited, Isle of Wight, England. International, Inc., Temecula, CA. Corporation, Stillwater, MN. 4Jackson Immunoresearch Laboratories, Inc., West Grove, PA.

the antibody used, to reduce nonspecic staining (details and sources of primary and secondary antibodies and dilutions used in this study are given in Table 1). Doublelabel immunocytochemistry was performed with an initial incubation (48 hours) in a primary antiserum directed against either ChAT or TH , followed by a second incubation (48 hours) in primary antiserum directed against PGP 9.5 (Table 1). After treatment with primary antisera, tissue was incubated with secondary antibodies conjugated with rhodamine or uorescein-isothiocyanate (FITC), as shown in Table 1. For viewing and photography, wholemounts were made by heat-drying the tissue on glass slides; coverslips were then applied over a mounting medium consisting of a 1:1 solution of glycerol and phosphate-buffered saline in which gelatin was dissolved (1 g of gelatin per 12 ml of solution). Alternatively, some tissue was rapidly dehydrated in alcohol and cleared in xylene before being mounted in Accumount (Baxter Scientic Products, McGraw Park, IL). Both mounting techniques yielded tissue suitable for analysis of uorescent labels. These labels were visualised with a standard compound microscope equipped for epiuorescence (Aristoplan, Leica Canada, Ltd., Willowdale, Ontario, Canada) that used appropriate lter cubes (Type N21 for rhodamine and Type L3 for FITC). The number of labelled cells in all regions of the atria and septum was counted in each specimen. Atrial tissue was photographed (Kodak T-Max 100 lm) through the microscope on a grid pattern of overlapping rectangular elds (1.52.5-mm eld size). These photographs were digitised with a desktop scanner (Deskjet, HewlettPackard, Palo Alto, CA) and stored on disk; the resulting image les were then assembled into montages by using image processing software (Photoshop, Adobe Systems, Inc., Mountain View, CA) to produce maps of the distribution of labelled nerve bres and intracardiac neurons. Selected areas of some hearts were also examined with the aid of the optical sectioning capability of a confocal microscope (Axiovert 100 microscope equipped with LSM 410 confocal system; Carl Zeiss, Inc., North York, Ontario, Canada) to determine details of cellular and terminal morphology within intracardiac ganglia. Ventricular tissue was not analyzed in this study, because it was generally too thick to be processed for visualization of neural elements in whole-mount preparations. Both positive and negative controls were used to establish the specicity of the immunohistochemical protocols used in this study. To verify ChAT- and TH-IR labelling, samples of spinal cord and stellate ganglia were taken from the same animals yielding cardiac tissue and were xed and sectioned (40 m) before being processed with

Aldehyde histouorescence
The presence of catecholamines in whole-mounts of freshly dissected atrial tissue in six animals was detected by using a technique modied from that of Furness et al. (1977) and Molist et al. (1993). Briey, cardiac tissue was obtained as described above, pinned at and xed in a solution of 4% paraformaldehyde, 0.5% glutaraldehyde, and 35% glucose in phosphate-buffered saline at 4C for 2448 hours. The tissue was then removed from the xative, placed onto a glass slide, desiccated in a lightproof box for 48 hours, cleared in xylene, and mounted in Accumount. Tissue was examined and photographed under ultraviolet epi-illumination (Leitz D lter cube, excitation bandwidth 355425 nm, 460 nm long-pass barrier lter). With this technique cells, nerve bres and terminals containing catecholamines uoresced bright bluegreen. Tissue which was xed with paraformaldehyde only, then desiccated and mounted for viewing served as a negative control for background autouorescence; this tissue displayed either very faint or no uorescence under ultraviolet illumination.

Data analyses
Counts of ChAT- and TH-IR cell bodies were made over the entire extent of both atria and in the interatrial septum. Statistically signicant regional differences in cell counts and other variables among the atrial areas were analyzed by analysis of variance (single f-factor). Where signicant f-values were obtained, pairwise comparisons of means were done by using Tukeys multiple means comparison test (Zar, 1984); P 0.05 was taken as the limit of signicance. Numerical values are expressed as mean 1 standard error.

Fig. 1. Confocal photomicrographs of atrial ganglion neurons double labelled with antibodies against choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and protein gene product 9.5 (PGP 9.5). AC: ChAT-immunoreactivity (IR) indicated by red label (secondary antibody conjugated with rhodamine) and PGP 9.5-IR indicated by green label (secondary antibody conjugated with uorescein isothiocyanate [FITC]) in right atrium. A: Rhodamine-lter exposure showing ChAT-IR neuronal somata and nerve bres. B: FITC-lter exposure of same ganglion as A, showing PGP 9.5-IR elements. C: Combined exposure showing same ganglion as in A and B, together with surrounding tissue. In this example, and throughout the atria, all PGP

9.5-IR neurons were also ChAT-IR. D: Left atrial tissue double labelled with antibodies against PGP 9.5 (green; FITC secondary) and TH (red; rhodamine secondary). No colocalization of these labels was observed in somata in this example or anywhere else in the atria. PGP 9.5-IR identied neuronal somata and axons; TH-IR identied cells proposed to be small, intensely uorescent (SIF) cells (see text). Note the short process extending from one of the TH-IR cells (arrowhead with asterisk). Some TH-IR varicosities are visible within the ganglion (arrowheads). The arrows indicate large processes emerging from one end of the spheroid neuronal somata. Scale bars 50 m in C (applies to AC) and D.

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Fig. 2. Composite photomicrograph of whole-mount preparation of the external walls of the left and right atria after processing for protein gene product 9.5 immunoreactivity. A line running between the tips of the large arrows (top and bottom of the image) delineates the junction of the interatrial septum with the external atrial wall. 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 the tissue represent the atrioventricular (AV) borders. The approximate

location of the sinoatrial node is outlined by the dashed oval in the right atrium. Neuronal somata, mostly occurring in ganglia, were distributed throughout both atria; examples of ve large ganglia are indicated by the small arrows. Apexes of white arrowheads indicate cut ends of branches of right (RCPN) and left (LCPN) cardiopulmonary nerves and the cardiac branches of the right (RVN) and left (LVN) vagal nerves. LAA, RAA: left and right atrial appendages, respectively. Scale bar 2.5 mm.

RESULTS Distribution of cholinergic and adrenergic components of the intracardiac nervous system
ChAT-IR components. ChAT-IR occurrence overlapped completely with immunoreactivity for the neuronal marker PGP 9.5 in the somata of intracardiac neurons in all eight hearts processed for double labelling with these markers, a typical example of which is shown in Figure 1 (A-C). Based on this nding, it was assumed for the purposes of this study that all neurons marked by PGP 9.5-IR in the heart were cholinergic. The somata of individual neurons in guinea pig atria were predominantly prolate spheroid in shape (Fig. 1). Estimates of mean neuronal dimensions were obtained by measuring, with an ocular micrometer, the long and short axes of 60 ChAT-IR neurons randomly sampled from ve hearts. Mean length of the long axis was 27 1 m (range, 2045m), whereas the mean length of the short axis was 21 0.5 m (range, 1530m). When viewed in the confocal microscope to clarify cellular details, most neu-

rons appeared to have only one large process which emerged from one end of the oval soma (e.g., Fig. 1D, arrows). Some isolated neurons were observed throughout the atria, but the vast majority were clustered into ganglia , as shown in Figures 13. Ganglia ranged in size from 2 to more than 100 neurons and were arranged as attened masses, usually one cell-layer thick, oriented to lie in the plane of the atrial wall. Within the external atrial walls, ganglia were located between the epicardium and the myocardium. They were covered supercially by the mesothelium, a subepicardial layer of connective tissue, and were situated external to the three or four layers of myocardial cells constituting the bulk of the atrial walls. In the interatrial septum, where the myocardium was many cell layers thick, numerous ganglia were distributed among the layers of myocytes. In all areas of the atria, ganglia were located either at the junctions of two or more large intracardiac plexus nerves, or were situated to one side of a nerve, as shown in Figure 1. Ganglia were always connected to the intracardiac plexus by more than one bre bundle; in some cases up to

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TABLE 2. Mean Number and Proportional Distribution of Choline Acetyltransferase-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/mm2 )1 Left atrium
ChAT-IR neurons % of total neurons Area (mm2 ) % of total area Density TH-IR cells % of total cells Area (mm2 ) % of total area Density
1Values 2Mean

Right atrium
485 124 32% 210 9 59% 2.4 0.7 60 27 48% 193 13 52% 0.3 0.1

Interatrial septum
3122 56 21% 194 1 5% 16.35 4.0 18 6 14% 224 1 6% 0.8 0.3

Mean total
1510 359 5.2 124 372 0.3 251 14 0.7 33 24 0.1

714 127 47% 1293 6 36% 6.4 1.2 46 11 37% 1203 12 32% 0.4 0.1

are expressed as mean 1 SE. number of ChAT-IR neurons in region of interatrial septum signicantly less than in the left atrium. 3Area of left atrium signicantly smaller than right atrium. 4Area of interatrial septum signicantly smaller than areas of left atrium and right atrium. 5Density of neurons in interatrial septum signicantly greater than in right atrium and left atrium.

Fig. 3. Composite photomicrograph of a whole-mount of the interatrial septum taken from the same heart as the external atrial walls shown in Figure 2. The tissue is oriented with the dorsal edge of the septum to the right of the frame and the most cranial portion is uppermost. This gure is shown at a higher magnication than Figure 2 to illustrate more clearly the ganglia and nerves. Scale bar 1 mm.

10 nerves of varying sizes were associated with individual ganglia. The mean total number of ChAT-positive neurons counted in the atria and the quantitative distribution of these neurons by region are presented in Table 2. The standard error of the mean total neuron count was relatively large (17% of the mean value), indicating substantial variability in total number of neurons among the hearts in this study. This high variability was reected in the range of total counts (from 717 to 2,542 atrial neurons per heart) in the eight hearts sampled. The distribution of intrinsic cardiac ganglia, and the microanatomy of the extrinsic nerves and the ganglionated plexus is shown in the composite photomicrographs of the external atrial walls and septum (Figs. 2 and 3, respectively) in a representative heart. For the analysis of the quantitative regional distribution of atrial neurons shown in Table 2, the atria were divided into the three regions illustrated in Figures 2 and 3. The region designated as right atrium (RA) consisted of the external atrial wall extending from its junction with the septum (along a line running between the large arrows in Fig. 2) to the right ventricular border. The left atrial (LA) region consisted of the external atrial wall extending from its junction with the septum to the border of the left ventricle. The left atrial appendage contained no neurons and was removed to prevent folds in the tissue during the preparation of whole-mounts. The smaller right appendage also was devoid of neurons but was left attached because it did not hinder whole-mount preparation. The region designated septum in Table 2 consisted of the septal tissue

dividing the right and left atrial chambers. The borders of all three regions were readily distinguishable in wholemount specimens, thus ensuring consistent sampling procedures among hearts in this study. To develop a quantitative index of the regional distribution of atrial neurons, the area and number of neurons present in the designated regions of each heart were obtained. A value for the density of neuron occurrence in each atrial region was then derived from the quotient of mean neuron number and mean area (density in Table 2). The mean area of the RA was signicantly greater than that of the LA, but the number of neurons in the RA was not signicantly different from the number in the LA, and our analysis shows that the corresponding density values for RA and LA were not signicantly different. However, during the counting process, it became apparent that there were differences in the patterns of overall distribution of neurons within these regions. The majority of neurons in the RA was concentrated near the SA node and the ostium of the inferior vena cava (IVC; Fig. 2, ganglia indicated by small arrows in RA). In contrast, neurons in the LA tended to be distributed in a broad band between the roots of the pulmonary veins and the atrioventricular border (Fig. 2, small arrows in LA), as well as being more widely scattered over the rest of the myocardium in this chamber. The mean area of the septum was signicantly smaller than either RA or LA areas (Table 2); the septum represented only 5% of mean total atrial area. The mean number of neurons contained in the septum (21% of mean total number) was also signicantly less than the number in the LA (but was not signicantly different from the number in the RA), yet the value of neuron density in the septum was signicantly greater than the density of neurons in either the LA (septal neurons 2.5 times as dense) or the RA (more than six times as dense). These results show that the greatest concentration of neurons in the atria was in the septum. To determine whether there were differences in the relative sizes of ganglia in different parts of the atria the number of neurons per ganglion was counted in all eight hearts studied, and frequency histograms were constructed. The frequency of occurrence of ganglia of different sizes was analyzed by categorising ganglia into 11 size ranges. The smallest category in this range included single

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Fig. 4. Bar graph of proportional distribution of atrial neurons, expressed as mean frequency of occurrence of ganglia of different size categories (number of neurons in each ganglion category indicated under the bars) for both atria combined (ALL REGIONS, top left panel) and by region, in eight hearts. Ganglion size increases by 4

neurons per size category until the largest category (41 ), which represents the frequency of ganglia of 41 neurons or greater. The majority (85%) of neurons were contained in ganglia of fewer than 20 neurons. Error bars 1 SE.

neurons together with ganglia containing from two to four cells. Category size increased by increments of four cells to the largest category, which included ganglia with 41 or more neurons. Although a few ganglia containing up to 100 neurons were found, ganglia containing more than 40 neurons were relatively rare. An analysis of the overall frequency-to-ganglion size relationship for all ganglia regardless of atrial location was rst done, as shown in the upper left panel of Figure 4. This analysis showed that the majority of atrial neurons occurred in relatively small ganglia, with 85% of the neurons being located in ganglia of fewer than 20 neurons. Subsequent breakdown of frequency vs. ganglion size by atrial region, illustrated in the other panels of Figure 4, showed that the trend in each region reected the overall pattern. To summarise these data, there was no trend toward a preponderance of larger ganglia in any atrial region, even though a few large ganglia were observed in all regions. Adrenergic components. TH-IR elements. Fibres and cell bodies within the atria of ve hearts were immunoreactive for TH, as shown in the confocal photomicrograph of Figure 1D (in red). Individual bres were associated with myocytes or were bundled together in nerve trunks within

the intracardiac plexus. TH-IR bres were also present within intracardiac ganglia; moreover, examination in the confocal microscope showed that some of these bres had varicosities near PGP 9.5-IR ganglion neurons (Fig. 1D, arrowheads). No TH-IR cell bodies were observed to be double-labelled with immunoreactivity for PGP 9.5; instead, TH-IR was found only in small, spheroid cells. These cells had a mean long axis dimension of 10 0.4 m (range, 912m) and a mean short axis dimension of 7.4 0.3 m (range, 510 m; data from 21 cells randomly sampled from three hearts). The largest of these cells was smaller than the smallest neurons labelled with either ChAT- or PGP 9.5-IR, and statistical comparisons (t- test, adjusted for unequal n, P 0.05) conrmed signicant differences in dimensions between the two cell types. Confocal microscopy showed that TH-IR cells had one or two short (length, 530 m) processes; an example of such a process can be seen in Figure 1D (small arrowhead with asterisk). TH-IR cells occurred singly or were grouped together in clusters with the majority (more than 90%) located either within or closely associated with intracardiac ganglia. Figure 1D shows a typical example of the anatomical relationship of TH-IR cells to principal ganglion neurons.

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Fig. 5. Bar graph of proportional distribution of tyrosine hydroxylase immunoreactive (TH-IR) cells in the atria, expressed as mean frequency of clusters of different size categories (number of cells in each category indicated under each bar) for both atria combined and

by region in ve hearts, presented in a format similar to that of Figure 4. Eighty-ve percent of TH-IR cells were found in clusters of eight cells or less. Error bars 1 SE.

Only a few TH-IR cells were located in areas remote from nerve trunks or ganglia; these cells occurred singly or in small 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 right and left atria in the number or the density of TH-IR cells. The mean number of TH-IR cells in the septum (representing 15% of mean total number) was signicantly less than the numbers of cells in either the right or left atrium, but cell density in the septum was not signicantly different than in the external atrial walls. Therefore, TH-IR cells did not appear to congregate preferentially in any region of the atria. Atrial TH-IR cells were less numerous than atrial neurons; the mean total number of TH-IR cells was approximately 12% of the mean total number of neurons (Table 2). An analysis of the frequency of occurrence of TH-IR cells in clusters of different sizes over the whole extent of the atria (upper left panel, Fig. 5) showed a qualitatively similar pattern to that for ChAT-positive neurons (cf. Fig. 4). Overall, 85% of TH-positive cells occurred singly or were contained in clusters of up to eight cell bodies. The

remaining 15% were distributed in a few clusters ranging in size from 9 to 40 cells, and this overall pattern was also reected in the regional distributions shown in Figure 5. TH-IR cells in the atria thus appear, like ChAT-IR neurons, to be preferentially distributed in clusters containing relatively few cells. A number of small TH-IR varicosities on individual processes occurred within ganglia observed under the confocal microscope, several examples of which are shown in Figure 1D (arrowheads). It was not clear whether these varicosities were associated with sympathetic postganglionic axons of extracardiac origin or were processes of intracardiac TH-IR cells. To clarify this, the optical sectioning capability of the confocal microscope was used to follow the paths of some of the processes emerging from a number of TH-IR cells like that shown in Figure 1D (arrowhead with asterisk) to determine whether these processes had varicosities. None were observed in the specimens examined. However, the processes of some TH-IR cells were observed to approach the somata of other nearby TH-IR cells (data not shown), raising the possibility of cell-to-cell communication within this population.

DISTRIBUTION OF INTRINSIC CARDIAC NEURONS Biogenic amines. Tyrosine hydroxylase is involved in the synthesis of norepinephrine, so to determine whether the morphology of atrial TH-IR cells and cells containing catecholamines were similar, aldehyde-induced uorescence was used in this study to histochemically identify catecholamine occurrence. With this technique, elements containing catecholamines uoresce a bright blue-green which is easily distinguishable from the faint background autouorescence (Fig. 6) . This method was not compatible with the immunohistochemical visualization of TH, so the two procedures could not be combined for direct comparison. Catecholamine-containing cells, nerve bres, and their associated varicosities (Fig. 6) were similar in size, distribution, and morphology to atrial cells and bres expressing TH immunohistochemistry (Fig. 1D). Furthermore, the relationship of catecholamine-containing cells and varicosities to nearby structures was very similar to that of TH-IR elements. The similarities in morphology and distribution of catecholamine-containing and TH-IR intracardiac cells and bres suggests that intraatrial elements which contain catecholamines may also be capable of synthesising these compounds. No atrial cells with somatic dimensions similar to the neurons displaying ChAT- or PGP 9.5-IR were found to contain catecholamines. One of the most striking ndings of this part of the study was that large numbers of amine-containing varicosities were observed not only in association with cardiac muscle cells but also within atrial ganglia (Fig. 6B,C) where they formed basket-like networks closely apposed to some principal ganglion neurons. Although both TH-IR and aldehyde uorescence techniques labelled intraganglionic varicosities in the present study, these structures appeared to be more intensely labelled after processing for catecholamine histochemistry than for TH-IR (cf. Fig. 1D and Fig. 6C). Aldehyde histochemistry thus appears to afford a better view of the intraganglionic distribution of these structures than does TH immunohistochemistry.

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Extrinsic innervation of intracardiac plexus

Figures 2 and 3 show the rst-degree intracardiac ramications of extrinsic cardiac sympathetic and parasympathetic nerves. Parasympathetic innervation of the heart originated as nerve branches from the left and right thoracic vagal trunks, and the continuations of these branches within the cardiac plexus are represented by the

Fig. 6. Composite photomicrographs of whole-mount preparations of two different left atrial ganglia after histochemical processing for aldehyde-induced uorescence, showing catecholamine-containing cells (arrows), terminals (arrowheads), and nerve bres. A: Low-power photomicrograph showing several clusters of small, intensely uorescent (SIF) cells (arrows) associated with ganglia. The somata of ganglion neurons appear faintly autouorescent; no neuronal somata in this example or elsewhere in the atria displayed the bright uorescence indicative of the presence of catecholamines. B: Highermagnication view of a different ganglion showing a cluster of SIF cells (arrow) and ne networks of catecholamine-containing bres and varicosities (arrowheads). One of these networks is shown enlarged (area indicated by corner brackets) in C. C: Enlarged portion of B to illustrate network of catecholamine-containing intraganglionic varicosities in close apposition to ganglion neurons. The cluster of brightly uorescing cells in the upper left part of the panel (arrow) is the same cluster appearing in B but is out of the focal plane. Scale bars 100 m in A, 50 m in B, 25 m in C.

312 large nerve bundles labelled LVN and RVN, respectively, in Figure 2. Both LVN and RVN entered the left atrium superior to the roots of the pulmonary veins. From these entry sites extensive ramications contributed to the nerve plexus supplying the atrial myocardium. From LVN numerous small nerve bundles could be traced: (1) laterally toward and along the border between the left atrium and the left ventricle; (2) to more anterior parts of the atrium in the direction of the left atrial appendage and the junction of the cardiopulmonary nerves with the atrium; and (3) toward the midline of the heart where some bundles mixed with those originating from the right vagus. From RVN nerve bundles were distributed to: (1) the medial right atrium; (2) along the border of the right atrium with the right ventricle (seen in the lower right portion of Fig. 2); and (3) toward the area of the sinoatrial node, located approximately as indicated by the dashed oval in Figure 2. Sympathetic bres coursed to the heart by means of cardiopulmonary nerves arising from the left and right stellate ganglia, the ansae subclavia, and the middle cervical ganglia. The intracardiac routes of branches of the left and right cardiopulmonary nerves are represented by the large nerve bundles labelled LCPN and RCPN, respectively, as illustrated in Figure 2. On the left side, major cardiopulmonary nerve branches followed the left superior intercostal vein toward the atrium, then coursed dorso- and ventrolaterally around the opening of the left atrial appendage, innervating the atrial myocardium dorsal, lateral, and ventral to this opening. In some hearts, a few of these branches were traced across the atrioventricular border and into the left ventricle (not shown in Fig. 2). Smaller nerves arising from the sympathetic branches around the dorsal aspect of the opening of the left atrial appendage continued dorsally into the area lateral to the pulmonary veins; this area was also innervated by lateral branches of the LVN. Several branches of the LCPN ramied in a dorsomedial direction toward the interatrial septum (see Fig. 3 and description of septal innervation below) and the area just superior to the roots of the pulmonary veins where there was also a dense vagal innervation. The RCPN entered the myocardium of the right atrium more ventrally and medially than did the LCPN. In addition, the distribution of RCPN within the myocardium was more diffuse compared with the pattern on the left. It was expected that the majority of cardiopulmonary nerve branches into the plexus of the right atrium would innervate the area of the sinoatrial node, atrioventricular node, or both, yet in the hearts examined in this study only one or two small nerve bundles ran directly to nodal regions (Fig. 2). The majority of nerve branches ran (1) into and along the dorsal and lateral walls of the superior vena cava; (2) medially and ventrally toward and across the junction of the septum with the external atrial wall, continuing into the left atrium; and (3) toward the inferior vena cava and into an adjacent region of the myocardium which was also heavily innervated by branches of the right vagus. In addition, some small nerve bundles could be traced from the inferior vena cava laterally into the crista terminalis and into the wall of the right atrial appendage. Due to separation of the septum from the atrial walls to facilitate penetration of antibodies and subsequent tissue processing, specic pathways of vagal and sympathetic nerve branches into the septum could not be identied in

J. LEGER ET AL. this study. Nerve bre bundles in the septum were concentrated primarily in the cranial portion (Fig. 3), with both large and small bundles present. Typically, one large and several smaller bundles ran from the cranial aspect toward the atrioventricular valves (in the direction of the bottom 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 the external atrial myocardium near the septum-atrial wall junction noted above. The nerve plexus of the external atrial wall was contiguous with the septal innervation.

DISCUSSION Phenotype of intrinsic cardiac neurons

The results of the phenotype analysis showed that all neurons in the atria of the guinea pig heart expressing immunoreactivity for the general neuronal marker PGP 9.5 were also immunoreactive for the enzyme ChAT, the rate-limiting enzyme in ACh synthesis. This suggests that these neurons were cholinergic. Our results conrm and extend the ndings of previous studies which showed that the majority of neurons in the hearts of guinea pigs and other mammalian species display cholinergic attributes (Jacobowitz, 1967; Ehinger et al., 1968; Bojsen-Moller and Tranum-Jensen, 1971; Hancock et al., 1987; Roberts et al., 1989; Seabrook et al., 1990; Steele and Choate, 1994; Steele et al., 1994; Edwards et al., 1995; Mawe et al., 1996). Histochemical reactions for AChE (Koelle, 1963) have been widely used to investigate the distribution of cholinergic neurons in the peripheral nervous system (reviewed by Kuhar, 1976), and most previous investigations of the phenotype of intracardiac neurons have relied on this technique. However, some noncholinergic neurons and some non-neuronal elements associated with the nervous system also express AChE (Fibiger, 1982; Satoh et al., 1983), leading to concern about the usefulness of AChE as a reliable indicator of cholinergic neurons in the heart. Specic antibodies against ChAT have been used to map cholinergic neurons in the central nervous system, but this technique has only recently been applied to the peripheral nervous system (Keast et al., 1995; Schemann et al., 1995; Wang et al., 1995; Talmage et al., 1996; Dey et al., 1996). In the guinea pig heart, a recent survey by Mawe et al. (1996) found that all neurons in the atrial intercaval region, the basal portion of the interatrial septal region, and the cardiac sinus region which expressed immunoreactivity for MAP-2, a microtubule-associated protein which has been used as a general neuronal marker, were also ChATIR. However, the study of Mawe et al. (1996) excluded major areas of both atria. The results of the present study not only conrm the presence of large numbers of ChAT-IR neurons in the regions analyzed by Mawe et al. (1996), but for the rst time demonstrate that ChAT-IR neurons are distributed throughout both atria. In particular, neurons are present in high concentrations within the interatrial septum and in the region around the pulmonary veins, areas which were not included in the study of Mawe et al. (1996). Control experiments in the present study, designed to test the specicity of the ChAT antibody, showed that neuronal somata in extracardiac tissues, including the dorsal and ventral horns and the intermediolateral cell column of the spinal cord, processed with the same protocol as the experimental tissue, were ChAT-IR.

DISTRIBUTION OF INTRINSIC CARDIAC NEURONS The nding in the present study that all neurons in the atria are cholinergic could be taken to support the idea that these neurons are all parasympathetic postganglionic efferent neurons, as predicted by the classical model of neural control of the heart. In this model, intrinsic cardiac neurons are considered to be all postganglionic cholinergic neurons, acting as simple relays conveying parasympathetic cardiomotor drive to the myocardium. However, intrinsic cardiac neurons are neurochemically very complex, expressing a number of neuropeptides in addition to ACh (Steele et al., 1994, 1996) in a manner similar to cholinergic neurons elsewhere in the peripheral autonomic nervous system. Janig and McLachlan (1992) have proposed that the variety of peptide neuromodulators colocalized with classic neurotransmitters in specic subpopulations of autonomic neurons chemically code these neuronal populations to subserve specic functions. In atrial neurons of the guinea pig multiple peptides have been shown to be coexpressed in distinct subpopulations (Steele et al., 1994, 1996), and it is likely that, even though all of these neurons can synthesise ACh, the colocalization of specic groups of peptides in different subpopulations indicates specic functional roles for neurons in these subpopulations. Physiological evidence in several mammalian species has been obtained for the existence of several types of neurons, including afferent neurons, interneurons, and local circuit neurons in addition to efferent neurons capable of generating cardioaugmentation as well as cardioinhibition (summarised in Armour, 1994). Therefore, although the present study has shown anatomically that atrial neurons share a common cholinergic phenotype, further studies of the functional properties of specic classes of neurons must be combined with techniques to determine which neuromodulators may be colocalized in these neurons.

313 close to the SA node and near the ostium of the IVC. A similar pattern was also reported in the guinea pig heart by Mawe et al. (1996), and this pattern has been observed in other species as well. Another locus which had a consistently high concentration of neurons in the present study was in the left atrium around the base of the pulmonary veins. This nding is similar to the situation reported in rat (Burkholder et al., 1992), canine (Yuan et al., 1994), and human (Janes et al., 1986) hearts but has not been previously reported in the guinea pig heart. The interatrial septum had the smallest area but contained a disproportionately large number of neurons, i.e., one-fth of the total number was located here, giving this area the highest density (Table 2). This nding provides quantitative conrmation of previous qualitative reports of high concentrations of neurons in the mammalian interatrial septum (King and Coakley, 1958; Ehinger et al., 1968; Ellison and Hibbs, 1976). In guinea pigs, the septal area was also richly innervated by nerves and single bres, primarily in the cranial portion (Fig. 3). These nerves were contiguous with those in the heavily innervated areas of the external atrial walls at the septum-wall junction. Intraseptal nerves running in the direction of the ventricles, would thus, appear to represent a major pathway for the innervation of these chambers. Neurons in the interatrial septum are in close proximity to the cardiac valves as well as to pacemaker and conductive tissues, so would be well positioned to modify chronotropic and dromotropic functions and possibly valve operation, as proposed by Moravec and Moravec (1987). Despite the left atrium having a signicantly smaller area than the right, there was no signicant difference in numbers of neurons associated with these two regions (Table 2). Furthermore, a comparison of the density of neurons in these two regions showed no signicant difference. However, there was a qualitative difference in the pattern of distribution of neurons within the left and right atria. With the exception of a concentration of neurons near the pulmonary veins, ganglia in the left atrium were more widely distributed than those in the right atrium, where the majority of neurons was concentrated near the SA node. This difference may be related to the functions of neurons in the two chambers. In the right atrium, a condensed organisation may be more effective in promoting local network interactions among neurons controlling the complex behaviour of nearby pacemaker and conduction tissues. Given this rationale, it might also be expected that right atrial neurons would be preferentially clustered into larger ganglia to facilitate interneuronal communication. This was, however, not the case: the analysis presented in Figure 4 shows that neurons were consistently clustered in relatively small ganglia in this area as well as in the rest of the atrial regions. In the left atrium, the neuron population size was similar to that in the right but, in contrast to the distribution pattern in the right atrium, left atrial neurons were more widespread. Such a distribution pattern may reect the participation of left atrial neurons in multiple local feedback loops involved in regional control of myocyte contractile properties throughout the chamber.

Number and distribution of intracardiac neurons

The mean total number of neurons found in adult guinea pig atria in the present study (1,510, Table 2) was 50% higher than the mean number reported by Mawe et al. (1996) in the atria of adults of the same species, likely reecting the larger area analyzed in the present study. The number of neurons in the present study was established by using antibodies against the general vertebrate neuronal marker PGP 9.5 (Thompson et al., 1983); immunoreaction for this marker has previously been shown to be reliable in determining the pathways and extent of the myocardial innervation and the locations of intracardiac neuronal somata in a range of mammalian species, including humans (Gulbenkian et al., 1987, 1993, 1994; Chow et al., 1993; Gordon et al., 1993; Crick et al., 1994; Fu et al., 1994; Marron et al., 1994). In control experiments in the present study, PGP 9.5-IR labelled neurons in sections of the spinal cord and the stellate ganglion; thus, we are reasonably condent that this neuronal marker gave a reliable picture of the distribution of neurons intrinsic to the guinea pig atria. Neurons were found throughout all atrial regions. The quantitative data presented in Table 2 show a large variation in total number of neurons per heart, and in the numbers of neurons in the different atrial regions, among the specimens sampled. However, one consistent pattern of neuronal distribution in the right atrium of all specimens was a clustering of ganglia in the intercaval area

Adrenergic elements in the atria

No TH-IR neuronal somata were observed in this study, as judged from the lack of colocalization of TH- and PGP 9.5-IR. These ndings concur with those of Baluk and

314 Gabella (1990), Steele et al. (1994, 1996), and Mawe et al. (1996), who reported an absence of TH-IR in intracardiac neurons in the guinea pig heart. Furthermore, a review by Allen et al. (1994) summarised reports that guinea pig atrial neurons in primary culture also lack TH-IR. In contrast to these reports, Dalsgaard et al. (1986) found TH-IR in a small group of neurons in the guinea pig heart and Horackova et al. (1996) reported that guinea pig intracardiac neurons in culture can express TH-IR; some of the neurons in the latter study even coexpressed TH and ChAT-IR. In the rat heart, there is also some evidence for TH-IR in atrial and ventricular neurons (Moravec and Moravec, 1989; Moravec et al., 1990; Slavikova et al., 1993). The reasons for these disparate ndings are not obvious but could be due to interspecic variation or differences in the immunohistochemical procedures used. With respect to the latter possibility, our procedures were sensitive enough to detect TH within primary neurons of the stellate ganglia and, therefore, should have been capable of detecting the presence of TH in atrial neurons. As a further precaution, in some experiments we increased the concentrations of the primary antibody against TH (up to 10 times the concentration necessary to label stellate ganglion neurons) and prolonged incubation times, but still failed to label intracardiac neurons. The lack of TH-IR in intrinsic cardiac neurons in the guinea pig heart has important implications for the study of intrinsic neural mechanisms controlling cardioaugmentation. In vivo functional studies in the hearts of other mammalian species such as the dog have shown that some intrinsic cardiac neurons are capable of evoking cardioaugmentation when activated (Butler et al., 1990; Armour et al., 1993; Huang et al., 1993a,b,c), and it was proposed on the basis of such functional evidence that there are neurons in the canine heart which are capable of synthesising and storing monoamines for release when these neurons are activated. The presence of TH-IR in neurons is a primary indicator of the capability for catecholamine synthesis, but no evidence has so far been presented for TH-IR neuronal somata in the dog heart. This situation has a parallel in the guinea pig heart, in that we have found in trial experiments that stimulation of some neurons in the guinea pig right atrium in vitro can cause cardioacceleration (Leger and Smith, in preparation). Although no intracardiac neurons were found in the present study to contain detectable levels of TH, cardioacceleration evoked by activation of intrinsic cardiac neurons could be driven by other mechanisms, such as release of catecholamines from neurons which can take up and store but not synthesise these compounds. A point in favour of this idea is that some intracardiac neurons which lack the synthetic enzymes for biogenic amines have been shown to accumulate these compounds in culture (Allen et al., 1994). However, the results of the present study do not support this because no neuronal somata were observed to contain catecholamines in atrial tissue processed for aldehyde histouorescence (Fig. 6). In this sense, then, our results conrm and extend the results of Baluk and Gabella (1990) in the guinea pig. Release of catecholamines from non-neuronal stores in the heart may be an alternative explanation for intrinsically mediated cardioaugmentation. Immunoreactivity for TH was found in high concentration in a class of cells with somata which were smaller than, and morphologically

J. LEGER ET AL. distinct from, intracardiac neurons (Fig. 1D). The results of a comparison of morphologic characteristics of the two cell types showed that there was no overlap in somatic dimensions between the smallest neurons and the largest TH-IR cells. This nding, in combination with the lack of PGP 9.5-IR in cells expressing TH-IR, argues against the idea that these cells are simply small neurons. Instead, the morphology of atrial TH-IR cells in our study was similar to that of small, intensely uorescent cells (SIF cells) observed throughout the viscera and in the peripheral autonomic nervous system after staining for catecholamines using an aldehyde uorescence reaction (Jacobowitz, 1967; Biscoe, 1971; Burnstock and Costa, 1975; Dail et al., 1975; Dail, 1976; Howe et al., 1978; de Groat and Booth, 1980; Wurster et al., 1990; Kriebel et al., 1991). Specically, SIF cells in the heart have been found in previous studies to contain TH in high concentrations (Heym et al., 1994; Mawe et al., 1996). In the present study, to determine which cells contained catecholamines and to see whether there were any morphologic similarities between small atrial TH-IR cells and those containing catecholamines, we used aldehyde histouorescence to localise these compounds. Brightly uorescent catecholamine-containing cells with a morphology very similar to that of small TH-IR cells were found mostly in or near ganglia (Fig. 6), with some cells scattered in the myocardium. The protocols for determining catecholamine content and TH-IR were not compatible for double-labelling but based on the morphologic similarities of cells visualised by these techniques, we hypothesise that cells containing catecholamines and those immunoreactive for TH comprise the same population of intracardiac SIF cells. The mean total number of these cells in the atria was about 12% of the mean total number of atrial neurons, but unlike neurons, the density of SIF cells was uniform among the different atrial regions. At least 90% of these cells were associated with ganglia. The quantitative analysis of the tendency of these cells to group together (Fig. 5) showed that they preferentially occurred in relatively small clusters throughout the atria with little regional variation in this pattern. Such a clustering pattern was also similar to that displayed by intracardiac neurons. The close approximation of the majority of SIF cells to ganglia, and the similarity in clustering pattern of the two cell types, suggests that SIF cells may be involved in neural control of the heart. The functional role of SIF cells in the heart is not yet clear, but these cells are positioned to play potentially important roles in cardioaugmentation and in intraganglionic neurotransmission. Release of catecholamines stored in SIF cells of pelvic ganglia can be induced by activation of cholinergic receptors on the membranes of these cells (de Groat and Booth, 1980). If, in the heart, a subpopulation of intrinsic cholinergic neurons projects to SIF cells, then activation of neurons in this pathway may evoke SIF cell catecholamine release. Such a release mechanism, if under local neuronal control, may provide an alternative explanation for cardioaugmentation mediated by intracardiac neurons. Additionally, the processes of some SIF cells were observed to approach nearby SIF cell somata, suggesting some form of cell-to-cell communication within this population. A large number of catecholamine-containing varicose terminals was observed both in the myocardium and in ganglia in the present study (Fig. 6). It was not, however,

DISTRIBUTION OF INTRINSIC CARDIAC NEURONS possible to determine whether these terminals (1) were associated with the axons of intrinsic cardiac neurons; (2) were of extracardiac origin (i.e., axons of sympathetic postganglionic neurons with their somata in thoracic ganglia); or (3) were associated with the processes of SIF cells. Regarding the latter possibility, no SIF cell processes in tissue which had been prepared for amine histouorescence could, in the standard uorescence microscope, be traced far enough from the cell bodies to determine whether they included amine-containing varicosities. These observations were not, however, conclusive because tissue samples were too thick to visualise cell processes well in this microscope at high magnication. To address this issue, the optical sectioning capability of the confocal microscope was used on tissue containing TH-IR. Under the assumption that TH-IR cells and catecholaminecontaining cells comprised the same population, an attempt was made to determine whether processes of intracardiac TH-IR cells contained varicosities. As with SIF cells, no varicosities were found along the processes of TH-IR cells as far as these could be traced (up to 30 m distal to the cell bodies). It is, therefore, possible that catecholamine-containing varicosities and TH-IR varicosities were associated with axons. Regardless of origin, the presence of this type of varicosity within intracardiac ganglia suggests that release of biogenic amines may have powerful modulating effects on ganglionic neurotransmission, as proposed by Smith et al. (1992) on the basis of physiological evidence.

315 nding was that the cardiac branches of the right vagus enter the heart in association with the left atrium near the junction of the external atrial wall with the interatrial septum. One important consequence of this anatomical feature is that in vitro preparations for studying right vagal inuences on the heart must include a portion of the left atrium near the septum to ensure that all vagal pathways to the right atrium are present in the preparation. The results of this part of the study have established the general anatomical pathways for regional cardiac innervation by extrinsic autonomic nerves, thus providing an organisational substrate on which further functional studies of the autonomic control of the myocardium can be based.

CONCLUSIONS
All neurons found in the guinea pig atria were coreactive for the neuronal marker PGP 9.5 and ChAT, suggesting that these neurons are cholinergic. Greater numbers of atrial neurons were found in this study than have been reported in previous studies. The regional densities of neurons in the external walls of the RA and LA were not signicantly different, but density in the interatrial septum was signicantly greater than in either of the other atrial regions. Neurons throughout the atria tended to be clustered in relatively small ganglia, with the majority in ganglia of less than 20 cells. Neurons were more widely distributed in the left atrium than in the right, where the majority was located in the intercaval region near the SA node. This difference in regional distribution is proposed to reect the roles of these neurons in controlling inotropic, chronotropic, and dromotropic aspects of cardiac function. No cells identied as neurons contained either TH-IR or detectable levels of catecholamines. However, both TH-IR and catecholamines were detected in high concentrations in small atrial cells which resembled the SIF cells reported by others to be present in the heart and throughout the peripheral autonomic nervous system. Catecholaminecontaining varicosities were observed within intracardiac ganglia and were associated with the atrial myocardium, as were TH-IR varicosities. This study provides, for the rst time, detailed anatomical data on the regional organisation of the intracardiac nervous system in the guinea pig heart. This information is essential to guide further physiological studies of the roles of intrinsic cardiac neurons controlling the rate of discharge of sinoatrial or atrioventricular pacemaker cells and regional myocyte contractile properties.

Extrinsic innervation of the atrial myocardium

Isolated and in vitro preparations of mammalian hearts, including those from rodents, have been used extensively for investigating functional aspects of neural control of the heart, yet the details of anatomical pathways of extrinsic cardiac nerves within the heart have not been established in any of these species. In neonatal rats, Seabrook et al. (1990) investigated the parasympathetic innervation of intracardiac ganglia in a small area of the dorsal atrial wall near the attachment of the interatrial septum, but these authors did not explore other cardiac regions. On the sympathetic side, no description of the relationship of the cardiopulmonary nerves to the intracardiac plexus exists for the rodent heart. In the present study we have shown that both vagal and cardiopulmonary nerves ramied extensively into the cardiac plexus in the general pattern illustrated in Figure 2. Major branches of the sympathetic and parasympathetic nerves entered the plexus in regional patterns which were consistent among the specimens, but these branches were not narrowly targeted on specic cardiac regions. Instead, these nerves ramied widely within their regional territories. In particular it was surprising, given the concentration of neurons near the sinoatrial node in the right atrium, that the branches of extrinsic nerves innervating this chamber were broadly distributed over the chamber wall and not targeted primarily on the node and nearby neurons. A major feature of all the hearts studied was that particular atrial regions such as the left atrium around the base of the pulmonary veins, the area of the attachment of the septum to the external atrial wall, and the region of the SA node, were dually innervated by branches of the sympathetic and parasympathetic nerves. Another major

ACKNOWLEDGMENTS
The authors thank Helen Sauveur and Jeanette Nason for technical assistance. J.L. was supported by a scholarship from Dalhousie University during part of this study. Some of the data have been presented at the Experimental Biology 1996 annual meeting, Washington, DC, USA, April 1417, 1996 (Smith FM, Leger J, Croll R. 1996. Regional distribution of ChAT- and TH-immunoreactive neuronal elements in guinea pig atria. FASEB J 10:A337). The comments of Prof. David A. Hopkins on a draft of this manuscript are gratefully acknowledged. F.M.S. received a research scholarship from the Heart and Stroke Foundation of Canada. R.P.C. received an operating grant from the Natural Sciences and Engineering Research Council of Canada.

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