Stochastic behavior of atrial and ventricular intrinsic cardiac neurons

M. Waldmann, G. W. Thompson, G. C. Kember, J. L. Ardell and J. A. Armour

You might find this additional info useful... This article cites 32 articles, 18 of which can be accessed free at: http://jap.physiology.org/content/101/2/413.full.html#ref-list-1 This article has been cited by 2 other HighWire hosted articles -Adrenoceptor blockade modifies neurally induced atrial arrhythmias Louis-Philippe Richer, Alain Vinet, Teresa Kus, Ren Cardinal, Jeffrey L. Ardell and John Andrew Armour Am J Physiol Regul Integr Comp Physiol, October 1, 2008; 295 (4): R1175-R1180. [Abstract] [Full Text] [PDF] Potential clinical relevance of the 'little brain' on the mammalian heart J. A. Armour Exp Physiol, February 1, 2008; 93 (2): 165-176. [Abstract] [Full Text] [PDF] Updated information and services including high resolution figures, can be found at: http://jap.physiology.org/content/101/2/413.full.html Additional material and information about Journal of Applied Physiology can be found at: http://www.the-aps.org/publications/jappl J Appl Physiol 101:413-419, 2006. First published 27 April 2006; doi:10.1152/japplphysiol.01346.2005

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Journal of Applied Physiology publishes original papers that deal with diverse areas of research in applied physiology, especially those papers emphasizing adaptive and integrative mechanisms. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2006 by the American Physiological Society. ISSN: 0363-6143, ESSN: 1522-1563. Visit our website at http://www.the-aps.org/.

J Appl Physiol 101: 413 419, 2006. First published April 27, 2006; doi:10.1152/japplphysiol.01346.2005.

Stochastic behavior of atrial and ventricular intrinsic cardiac neurons

M. Waldmann,1 G. W. Thompson,2 G. C. Kember,3 J. L. Ardell,4 and J. A. Armour5
Department of Cardiology, Technical University RWTH, Aachen, Germany; Departments of 2Physiology and Biophysics and 3Physics, Dalhousie University, Halifax, Nova Scotia, Canada; 4Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee; and 5 Department of Pharmacology, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada
Submitted 21 October 2005; accepted in nal form 15 February 2006
1

Waldmann, M., G. W. Thompson, G. C. Kember, J. L. Ardell, and J. A. Armour. Stochastic behavior of atrial and ventricular intrinsic cardiac neurons. J Appl Physiol 101: 413 419, 2006. First published April 27, 2006; doi:10.1152/japplphysiol.01346.2005.To quantify the concurrent transduction capabilities of spatially distributed intrinsic cardiac neurons, the activities generated by atrial vs. ventricular intrinsic cardiac neurons were recorded simultaneously in 12 anesthetized dogs at baseline and during alterations in the cardiac milieu. Few (3%) identied atrial and ventricular neurons (2 of 72 characterized neurons) responded solely to regional mechanical deformation, doing so in a tightly coupled fashion (cross-correlation coefcient r 0.63). The remaining (97%) atrial and ventricular neurons transduced multimodal stimuli to display stochastic behavior. Specically, ventricular chemosensory inputs modied these populations such that they generated no short-term coherence among their activities (cross-correlation coefcient r 0.21 0.07). Regional ventricular ischemia activated most atrial and ventricular neurons in a noncoupled fashion. Nicotinic activation of atrial neurons enhanced ventricular neuronal activity. Acute decentralization of the intrinsic cardiac nervous system obtunded its neuron responsiveness to cardiac sensory stimuli. Most atrial and ventricular intrinsic cardiac neurons generate concurrent stochastic activity that is predicated primarily upon their cardiac chemotransduction. As a consequence, they display relative independent short-term (beat-to-beat) control over regional cardiac indexes. Over longer time scales, their functional interdependence is manifest as the result of interganglionic interconnections and descending inputs. atrial neuron; intrinsic cardiac nervous system; myocardial ischemia; stochastic control; ventricular neuron

single, intrinsic cardiac ganglionated plexus display interdependent behavior over short time scales (32). Their local interactions likely contribute to coordination of efferent outputs to specic cardiac indexes, as with the integrated neuromodulation of the sinoatrial (SA) nodal complex (6, 10, 25). There is functional and anatomical evidence to support the concept of local circuit neuron-dependent interactions occurring among various intrinsic cardiac ganglionated plexuses. For example, Gray et al. (15) demonstrated the existence of anatomical interconnections among neurons within the right atrial and the posterior atrial ganglionated plexuses, both of which have been associated with reex control of SA nodal function (3, 24, 27, 28). Data likewise indicate that coordination of SA and atrioventricular nodal function may reect interconnections within the intrinsic cardiac nervous system and/or common shared inputs from the extracardiac sources (21, 26). It remains to be established how neurons in atrial and ventricular ganglionated plexuses interact on an ongoing basis in the short-term control of disparate cardiac regions. Given the fact that, as yet, we do not understand how atrial and ventricular neurons interact on a short-term basis, the coherence of their activities was determined to elucidate how they concurrently transduce physiological as well as pathological states. This is particularly relevant when considering targeting select intrinsic cardiac neurons therapeutically to stabilize control over regional cardiac electrical or mechanical indexes in the presence of cardiac pathology (7, 19, 31).
METHODS

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the intrinsic cardiac nervous system acts as a local distributive processor, given its capacity to differentially transduce ascending sensory and descending efferent neuronal inputs in the coordination of postganglionic efferent neuronal regulation of different cardiac regions (5). Atrial and ventricular ganglionated plexuses contain heterogeneous populations of neurons (11, 18, 20) that are known to exert control over broad and overlapping cardiac regions (12, 33, 34). It has been proposed that regional cardiac control depends in large part on the multiple functional interactions that exist among neurons in major intrinsic cardiac ganglionated plexuses (2, 13, 24, 28). Neurons in intrinsic cardiac vs. intrathoracic extracardiac ganglia are known to display noncoherent behavior (5). As a result, if a pathological process compromises one level within this neuronal hierarchy, other elements can compensate to ensure adequate regional cardiac control (2, 5). It is also known that neurons in a
IT HAS BEEN PROPOSED THAT

Animal preparation. Adult mongrel dogs (n 12) of either sex, weighing between 15 and 22 kg, were employed in this study. All experiments were performed in accordance with the guidelines for animal experimentation described in the Guiding principles for research involving animals and human beings (1). The institutional animal care and use committee of Dalhousie University approved these experiments. Canines were tranquilized with pentothal sodium (1520 mg/kg iv) and then anesthetized with pentothal sodium (5 mg/kg iv to effect every 510 min for the duration of the surgical procedures). Thereafter, the animals were intubated, and positive-pressure ventilation was maintained with a Bird Mark 7A ventilator at a respiratory rate of about 30 times/min, using a gas mixture of 95% O2 and 5% CO2. After all the surgery had been completed, anesthesia was changed to -chloralose that was rst administered as a bolus (2550 mg/kg iv) and then as repeat doses (25 mg/kg iv) every hour or less throughout the experiments, as required. Noxious stimuli were applied to a paw
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Address for reprint requests and other correspondence: J. A. Armour, Centre de recherche, Hopital du Sacre-Coeur, 5400 boul. Gouin ouest, Montreal, Quebec, H4J-1C5, Canada (e-mail: JA-Armour@crhsc.rtss.qc.ca). http://www. jap.org

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periodically throughout the experiments to ascertain (limb withdrawal, heart rate changes) adequacy of the anesthesia. Experimental procedures. A bilateral thoracotomy was performed in the fourth intercostal space. Umbilical tape was placed around the inferior vena cava and the descending thoracic aorta so that each of these vessels could be transiently occluded later in the experiments. A 3-0 silk ligature was placed around the ventral descending coronary artery 1 cm from its origin and lead through polyethylene tubing so that this vessel could be transiently occluded later in the experiments. A midline incision was made in the neck to expose the caudal cervical vagosympathetic trunks, and silk threads were placed around them so that they could be severed later in the experiments. Silk ligatures were also placed around the right and left subclavian ansae so that the middle cervical, mediastinal, and intrinsic cardiac ganglia could be decentralized from the central nervous system. A lead II ECG was recorded. Left atrial and left ventricular chamber pressures, as well as aortic pressure, were monitored using Bentley Trantec model 800 transducers connected, respectively, to a PE-50 catheter placed in the left atrial cavity via its appendix, a Cordis no. 7 pig-tail catheter inserted into the left ventricular chamber via the right femoral artery and a Cordis no. 6 catheter inserted into the ascending aorta via the left femoral artery. Miniature solid-state pressure transducers (Konigsberg Instruments, Pasadena, CA, model P190; 5-mm diameter, 1.5-mm thick) were inserted in the right ventricular conus and into the left ventricular ventral wall to record regional intramyocardial pressures in both chambers. These sensing devices were employed because ventricular chamber pressure by itself is inadequate for detecting regional ventricular inotropic alterations induced by activating select populations of cardiac efferent neurons. All data, including intrinsic cardiac neuronal activity (see below), were recorded on an Astro-Med, model MT 9500, eight-channel rectilinear recorder. Data were stored on VHS tape (T120 Scotch, 3M Canada, London, ON, Canada) using a VCR recorder (A. R. Vetter, model 820, Rebersburg, PA) for later analysis. Recording neuronal activity. The activity generated by neurons in a locus of the right atrial ganglionated plexus (atrial neurons) was recorded concurrently with the activity generated by neurons in a locus of the cranial medial ventricular ganglionated plexus (ventricular neurons). The ventral pericardium was incised and retracted laterally to expose fat on the ventral surface of the right atrium that contains the ventral component of the right atrial ganglionated plexus and the fat overlying the ventral interventricular grove that contains the ventral septal component of the cranial medial ventricular ganglionated plexus (35). Separate circular rings of heavy-gauge wire were placed on epicardial fat located on the ventral surface of the right atrium and cranial interventricular groove to minimize epicardial motion. Fatty tissues in these two regions were explored via two separate tungsten microelectrodes mounted on micromanipulators. These recording microelectrodes had 250- m shank diameters, exposed tips of 10 m, and impedances of 9 11 M at 1,000 Hz. Indifferent electrodes for each were attached to the mediastinum. Epicardial fatty tissues were examined from their surfaces to the underlying cardiac musculature. Signals generated by atrial and ventricular neurons were differentially amplied via separate Princeton Applied Research model 113 ampliers that had band-pass lters set at 300 Hz to 10 kHz and amplication ranges of 100 500. The outputs of these devices were further amplied ( 50 200) and ltered (bandwidth 100 Hz to 2 kHz) by two optically isolated ampliers (Applied Microelectronics Institute, Halifax, NS, Canada). The activity generated by individual neurons, as identied by the amplitude and shape of recorded action potentials with signal-to-noise ratios greater than 3:1, were recorded. Using these techniques and criteria, action potentials generated by cell bodies and/or dendrites rather than axons of passage can be identied (32). Interventions. Loci on the epicardium of the left atrium, the right ventricular conus or sinus, as well as the ventral or lateral surfaces of the
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cranial left ventricle, are known to contain sensory inputs to the intrinsic cardiac nervous system (5). These regions were touched sequentially with a saline-soaked cotton swab. Right atrial tissues were not disturbed in order not to disturb the recording electrode in right atrial fat. Thereafter, chemicals known to activate cardiac sensory neurites (5, 32) were applied (60 100 s) individually to ventricular epicardial loci that responded to mechanical stimuli via 1-cm 1-cm gauze squares soaked with 0.5 ml of a chemical. After removing a square containing a chemical, each site was washed with normal saline ( 2 ml/s) for 30 s; each response terminated on average within 1 min of chemical removal. Gauze squares soaked with room-temperature normal saline were also applied to identied ventricular epicardial sensory elds to determine whether neuronal responses elicited by epicardial chemical application were due to vehicle effects or the mechanical effects elicited by gauze squares. The following chemicals were applied individually to ventricular epicardial sensory elds: adenosine (1 M); angiotensin II (1 M); bradykinin (1 M); the -adrenoceptor agonist phenylephrine (1 M); the 2-adrenoceptor agonist clonidine (1 M); the 1-adrenoceptor agonist dobutamine (1 M); the 2-adrenoceptor agonist terbutaline (1 M); the nitric oxide donor S-nitroso-N-acetyl-penicillamine (SNAP; 10 M); and the sodium channel modier veratridine (5 10 6 g). The order of chemical application varied among experimental animals. Active chemicals were reapplied at least twice to the same epicardial locus to verify response reproducibility. To discretely activate one neuronal population while recording activity change generated by both, nicotine was administered to right atrial neurons via their local arterial blood supply. Nicotine is known to activate intrinsic cardiac neurons in a consistent manner when administered into their local arterial blood supply (23). Nicotine (0.1 ml bolus of a 100 g/ml solution) was administered locally via a 24-French catheter placed in the right coronary artery. That cannula was threaded retrograde to ow so that its tip lay 1 cm proximal to the arterial branch that supplied blood to the right atrial ganglionated plexus without obstructing blood ow in either artery. The cannula was xed in place with 0.5 ml of super-glue adhesive. PE-15 tubing was inserted into the hub of this catheter with a stopcock in order that nicotine could be administered repeatedly into the local arterial blood supply of ventral, right atrial neurons. Monitored cardiac indexes were unaffected by cannula placement. Postmortem examination of appropriate catheter placement was conrmed by injecting methylene blue dye through this catheter. By this method, nicotine could be delivered into the regional arterial blood supply of right atrial neurons and other adjacent tissues while leaving the ow of blood in the right coronary artery patent. The arteries supplying blood to the cranial medial ventricular ganglionated plexus arise along the proximal left anterior descending coronary artery (22); thus neurons in this ganglionated plexus were not in the direct perfusion path of such right-sided intracoronary administered nicotine. To control for potential systemic effects elicited by local nicotine administration or epicardial chemical application, each agent was administered into descending aorta blood in the same doses. To determine whether altered cardiovascular mechanical status affected the activity generated by either population of neurons, the inferior vena cava and then the descending thoracic aorta were partially occluded for 35 s. Thereafter, the ventral descending coronary artery was occluded for 30 s. Once all of the interventions had been completed, the cervical vagosympathetic complexes were severed, as were all connections between the stellate ganglia and spinal cord. Following decentralizing intrathoracic autonomic ganglia from the central nervous system, interventions that had previously induced responses were repeated. At least 5 min were allowed to elapse between interventions to ensure preparation stabilization. Data analysis. Spontaneous cardiodynamic uctuations were minimal during control periods: heart rate varying less than 5 beats/min, and systolic pressure uctuating less than 5 mmHg. Thresholds for classifying induced cardiovascular changes were chosen to be greater than these ranges. Action potentials recorded simultaneously from the right atrial and ventral ventricular ganglionated plexuses were counted
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415

for 30-s periods to establish average activity immediately before and during maximal responses elicited by each intervention. Fluctuations in the amplitude of action potentials generated by a unit varied by 50 V over several minutes, with action potentials retaining the same congurations over time. Action potentials recorded from each locus with the same conguration and amplitude ( 50 V) were considered to be generated by a single unit. Recorded action potentials with signal-to-noise ratios greater than 3:1 were analyzed. Changes in neuronal activity and monitored cardiac indexes induced by each intervention were evaluated by comparing data generated immediately before each intervention with data obtained at the point of maximum change during that intervention. Data were expressed as means SD. One-way ANOVA and paired t-test with Bonferroni correction for multiple tests were used for statistical comparisons. A signicance value of P 0.05 was used for these determinations. The coupling of activities generated by atrial and ventricular neurons was determined by continuous cross-covariance analysis (32). To accomplish this, the activity generated by atrial and ventricular neurons was digitized ofine at sampling frequencies of 2,000 Hz. This was done by window discriminating the action potentials recorded at each site, i.e., values below a threshold were zeroed, and those above the assigned window value were truncated to have unit value. In that manner, the activity recorded from neurons in the two ganglionated plexuses could be computed simultaneously so that the activities generated by identied atrial and ventricular neurons could be cross-correlated over time. This was done by using a sliding window of data lasting 10 s (32). This approach permitted the determination of the ongoing cross-covariance function between identied atrial and ventricular neurons in basal states and in response to chemical or ischemic activation of local cardiac sensory neurites. Continuous analysis of heart rate and cardiac inotropic function was performed over the same time periods to compare alterations in neuronal activity with concomitant changes in the monitored cardiac indexes.
RESULTS

Overview. Action potentials generated concurrently by one to three neurons in the right atrial and ventral ventricular ganglionated plexuses, as characterized by their specic action potential congurations, were identied in each animal. During control states, active sites generated 9 26 impulses/min. Both populations were modied when mechanical (Table 1) and/or Table 1. Mechanical stimuli and ischemia
Interventions HR, beats/min LAP, mmHg RV IMP, mmHg

chemical (Table 2) stimuli were applied to the right ventricular conus ( 80% of active sites) or the cranial medial surface of the left ventricle ( 20% of active sites). Solely mechanosensory transducing neurons. The activity generated by one identied atrial and one identied ventricular neuron generated activity that was simultaneously related to regional cardiac dynamics, thereby displaying cardiac cyclerelated activity. The activities generated by both neurons occurred concomitantly during isovolumetric contraction for two to ve consecutive cardiac cycles, with cardiac cycles in between in which no activity was identied (Fig. 1). The activity of both of these neurons increased when focal mechanical stimuli were applied repetitively to the outow tract of the right ventricle. Their activities increased immediately upon application of these epicardial mechanical stimuli and terminated immediately upon removal of such stimuli. The activity generated by either neuron was not affected when mechanical stimuli were applied to other epicardial regions. They did not respond when vertridine was applied to that same locus or other cardiac epicardial loci. In control states, these two neurons displayed short-term coherence of their activities (crosscorrelation coefcient of their activities was 0.63). Their tightly coupled behavior persisted during focal mechanical stimuli of the right ventricle outow tract, a stress that enhanced both of their activities. Following characterization of this neuron pair, the activities of additional atrial and ventricular neurons were investigated in this animal by repositioning the recording electrodes. All subsequent neuron pairs so identied exhibited multimodal transduction characteristics. Multimodal sensory neurons. In every animal, atrial and ventricular neurons were concurrently identied that transduced mechanical and chemical stimuli. Both populations generated irregular activity patterns with no xed temporal relationship to atrial or ventricular dynamics. On average, a cross-correlation coefcient of 0.21 0.07 was exhibited between their respective activities during basal states, as well as during enhancement of their ventricular chemosensory inputs. The right atrial neurons so identied responded preferentially to ventricular mechanosensory inputs, while those

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LV IMP, mmHg

LVP, mmHg

Atrial Neuronal Activity, ipm

Ventricular Neuronal Activity, ipm

Control Touch LA Control Touch RV Control Touch LV Control IVC occlusion Control Aortic occlusion Control Nicotine (cor art) Control Coronary art occ

123 123 128 129 128 129 120 125 120 112 120 119 119 119

21 21 24 24 24 24 24 31 24 42 31 24 24 24

10 10 9 9 9 9 10 7 10 10 10 9 10 10

3 3 3 3 3 3 3 3 3 3 3 3 3 3

29 29 29 30 29 30 28 24 28 26 28 26 28 29

10 10 7 7 7 7 7 10 7 10 7 7 7 10

95 98 99 101 93 97 97 95 98 128 99 99 96 94

21 24 24 21 14 21 17 21 21 38* 17 21 17 21

128 129 124 124 104 104 123 118 121 143 124 124 129 129

17 24 21 21 21 21 21 17 24 38* 21 21 21 21

14.9 15.5 12.0 24.3 11.7 22.0 14.0 8.4 7.9 15.6 8.6 18.3 7.0 17.7

10.7 14.9 13.5 17.3* 12.5 19.1* 11.1 10.4* 5.2 22.2* 5.2 22.2* 7.3 10.7*

9.5 21.8 26.4 19.2 20.2 15.6 15.3 17.9 14.3 17.1 14.0 28.2 9.1 16.5

10.4 18.7* 17.3 14.9 17.0 16.6 11.1 17.0 12.1 11.1 10.4 31.2* 11.8 14.5*

Values are means SD; n 12 dogs. In all 12 dogs, activity changes were initiated in either population of neurons by the mechanical interventions, regional arterial nicotine administration, or transient coronary artery occlusion. Mechanical stimuli applied to the left atrium (LA), right ventricle (RV), or left ventricle (LV) activated neurons in all animals, as did inferior vena cava (IVC) or aortic occlusion. Nicotine administered locally to right atrial neurons activated both populations; so did transient coronary artery occlusion. Heart rate (HR), LA systolic pressure (LAP), RV and LV intramyocardial systolic pressures (IMP), and LV chamber systolic pressure (LVP) data are tabulated. ipm, Impulses per minute. *P 0.01 control vs. intervention. J Appl Physiol VOL
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Table 2. Cardiovascular variables recorded along with atrial and ventricular neuronal activities before and after epicardial application of various chemicals
Epicardial Chemical HR, beats/min LAP, mmHg RV IMP, mmHg LV IMP, mmHg LVP, mmHg Atrial Neuronal Activity, ipm Ventricular Neuronal Activity, ipm

Control Adenosine Control Angiotensin II Control Bradykinin Control Clonidine Control (n 7) Phenylephrine Control Dobutamine Control (n 11) Terbutaline Control Veratridine Control SNAP Decentralized SNAP decentralized

122 122 117 119 124 123 119 121 122 122 119 120 122 122 118 120 121 122 105 105

24 24 24 24 21 21 24 21 13 13 24 24 17 17 21 28 21 24 17 17

9 9 10 10 10 10 10 10 10 10 10 10 10 10 9 9 9 9 10 11

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

27 27 27 27 29 29 28 28 29 29 28 29 29 29 26 28 27 29 25 25

7 7 10 10 10 10 10 7 8 8 14 10 10 10 10 10 7 7 7 7

96 96 87 87 96 97 88 89 86 85 89 91 86 89 95 97 94 87 91 91

17 21 24 24 21 21 24 24 19 16 21 24 23 23 21 24 17 21 17 28

127 128 125 127 128 128 129 127 128 127 127 129 128 129 121 121 127 130 118 120

17 17 28 24 17 17 21 21 13 16 17 21 17 20 31 21 17 17 24 21

9.5 13.7 17.7 53.0 5.7 31.7 9.3 18.4 20.2 12.2 12.5 24.7 24.6 43.5 10.2 29.0 7.4 20.3 9.4 10.9

11.8 22.2* 13.9 55.8* 6.6 28.8* 10.7 19.4* 20.6 14.3 11.8 32.6* 25.9 33.2* 10.0 19.4* 5.9 8.7* 13.9 12.1

24.8 20.1 16.2 30.1 19.0 26.5 10.6 19.0 22.3 11.8 19.2 29.6 24.9 36.5 17.8 23.0 16.0 21.4 11.2 11.7

23.2 15.2 15.6 25.6* 12.1 22.9 12.1 18.7* 10.6 5.6* 14.9 20.1* 14.6 17.2 12.8 20.4 8.0 14.2* 9.4 10.0

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Values are means SD; n 12 dogs. Adenosine, angiotensin II, bradykinin, clonidine, dobutamine, veratridine, and the nitric oxide donor nitroprusside S-nitroso-N-acetyl-penicillamine (SNAP) modied one or both populations in all animals. Following acute decentralization, epicardial SNAP no longer activated neurons. *P 0.01 control vs. intervention.

ventricular ones responded preferentially to left atrial mechanosensory inputs (Table 1). Transient occlusion ( 5 s) of the inferior vena cava reduced the activity generated by atrial neurons, but not ventricular neurons. That intervention was restricted to short enough periods of time such that monitored cardiac indexes did not change overall (Table 1). Transient occlusion ( 5 s) of the descending aorta increased left ventricular chamber systolic pressure concomitant with right atrial neuronal activation; ventricular neurons did not respond overall to that intervention (Table 1). Following decentralization of the intrinsic cardiac nervous system later in the experiment, fewer neurons responded to repeat mechanical stimuli (data not shown). All multimodal neurons responded to more than one of the chemicals applied to ventricular epicardial loci. Both populations were activated when angiotensin II, clonidine, dobutamine, or the nitric oxide donor SNAP were applied individually to the right ventricular outow tract (Table 2). Fewer responded when chemicals where applied to the ventral cranial, left ventricular epicardium. Right atrial neurons were also activated consistently by ventricular application of adenosine, bradykinin, terbutaline, and veratridine; phenylephrine suppressed neuronal activity. These responses took time to develop (Fig. 2) and lasted for 1 min after chemical removal and epicardial rinsing. Reapplication of each chemical to previously identied epicardial sites induced similar neuronal responses. Cardiovascular variables were unaffected by epicardial chemical application, presumably due to the small quantities of chemicals tested. Neuronal activity and monitored cardiac variables were unaffected by epicardial application of gauze squares soaked with room-temperature normal saline. Systemic administration of these chemicals in the doses studied did not modify recorded variables. Administration of nicotine to right atrial neurons via their regional arterial blood supply activated both populations (TaJ Appl Physiol VOL

ble 1). Monitored cardiac indexes were unaffected overall by that intervention. Administration of the same dose of nicotine into the aortic bloodstream did not affect monitored neuronal activities or cardiovascular variables. Neuronal activity increased in a transient fashion immediately following acute decentralization of the intrathoracic nervous system (atrial neurons: 7.5 8.7 to 31.5 45.7 impulses/ min; ventricular neurons: 11.2 10.4 to 24.5 21.8 impulses/ min; P 0.01). Within 5 min of acute decentralization, the activities of both populations returned to baseline values. Thereafter, reapplication of the same chemicals to previously responsive epicardial sites affected fewer neurons, such that neuronal activity did not change overall. As an example, local epicardial application of SNAP increased the activity generated by both populations in the intact, but not acutely decentralized, state (Table 2). Regional ventricular ischemia. In the intact state, transient occlusion (30 s) of the left ventral descending coronary artery distal to the site of origin of the small arteries supplying blood to the ventral ventricular ganglionated plexus enhanced the activity generated by both neuronal populations (Fig. 3; Table 1). Recorded cardiovascular variables were unaffected overall by these brief occlusions. Following acute decentralization of the intrinsic cardiac nervous system, coronary artery occlusion no longer generated signicant activity changes.
DISCUSSION

In contradistinction to the tightly coupled behavior displayed by most neurons in one intrinsic cardiac ganglionated plexus that control select cardiac indexes (32), the results of the present investigation indicate that the vast majority of spatially distributed atrial and ventricular neurons display independent activity patterns. As their sensory inputs are primarily related to a relatively slowly varying cardiac chemical milieu (5), the
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Fig. 1. Individual atrial and ventricular neurons displaying coupled behavior. Top to bottom: lead II ECG, left ventricular (LV) ventral wall intramyocardial pressure (IMP), and concurrent atrial and ventricular neuronal activity (the activity of each major unit was discriminated as unit activity and displayed below their raw activity data). The activity generated by these two neurons occurred during isovolumetric contraction episodically for 25 consecutive cardiac cycles. The correlation coefcient of their activities (r) was computed to be 0.63. The activity generated by both neurons increased when a locus on the right ventricular outow tract epicardium was touched; coherence of their activities was maintained in that state (data not shown).

activity displayed by most is stochastic in nature. Specically, the irregular nature of their activities apparently involves transduction of the cardiac chemical milieu without a major inuence being imposed by phasic mechanosensory inputs derived from local mechanical events. As a consequence, short-term coordination of activities is not to be expected among the vast majority of atrial and ventricular neurons. It is now recognized that the intrinsic cardiac nervous system does not act as a simple monosynaptic relay station for central efferent neuronal control of regional cardiac function (5, 30). As demonstrated in this study, this target organ nervous system processes sensory information arising from various regions of the heart (Tables 1 and 2). As a consequence of their multiple multimodal inputs, the activity generated by most atrial and ventricular neurons is irregular with respect to time. Given their capacity to transduce multiple chemicals, including those known to be liberated by the ischemic myocardium, such as adenosine (29) and bradykinin (17), most intrinsic cardiac neurons respond to regional ventricular ischemia. Transduction
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of myocardial ischemia involves mechanosensory and sensory inputs, with chemosensitive being the predominant signal (5a). Previous data indicate that the majority of neurons whose activity can be recorded by the techniques employed in this study represent local circuit ones (2, 4). That is because of their predominance numerically as well as the relatively large size of their somata with respect to other intrinsic cardiac neuronal populations (2, 4). Presumably, most neurons identied in this study belonged to that category. Limited subpopulations of intrinsic cardiac neurons display cardiac phase-related activity, reective of primary mechanosensory inputs arising from restricted cardiac regions (5, 32). Presumably, such inputs accounted for the fact that a small ( 3%) population of solely mechanotransducing neurons was identied in both ganglionated plexuses studied. Because of the similar nature of their inputs, they displayed tightly coupled behavior (Fig. 1). Thus commonality in mechanosensitive sensory inputs may impose a degree of coordination among disparate intrinsic cardiac neuronal populations. The inherent stochastic nature of cardiac chemosensory inputs minimizes the potential for short-term coordination between disparate atrial and ventricular populations during myocardial ischemia or during the subsequent reperfusion. Yet, because of the underlying neuronal substrate of local circuitdependent interganglionic interactions, separate elements of the intrinsic cardiac nervous system do have the capacity to exert longer term coordination of disparate efferent outows. Coordination among and between different populations of intrinsic cardiac neurons amplies control over select cardiac indexes (8, 9, 12, 14). For example, control of SA nodal function resides primarily with neurons in the right atrial and posterior atrial ganglionated plexuses (13, 24, 28). Gray et al. (15) have recently demonstrated the anatomical substrate for interganglionic coordination between these two ganglionated plexuses. The present study enlarges upon such a concept. Specically, administering nicotine to one population (right atrial neurons) activated not only that population but another (ventricular) as well (Table 1). As no vascular connections exist between these populations, these data support the concept of functional interconnectivity existing among atrial and ventricular neurons. Future studies should be directed at identifying the anatomical substrate for such atrial/ventricular neural interactions. The common, shared inputs from higher centers can also impose coordination of activities among spatially discrete populations of intrinsic cardiac neurons. In fact, both independent and shared preganglionic inputs to separate intrinsic cardiac ganglionic plexuses (right atrial and posterior atrial ones) originate from neurons in the ventral lateral nucleus ambiguous (16). In agreement with that concept, in the present study, the activity generated by both right atrial and cranial medial ventricular neurons transiently increased and then adapted at lower activity levels following their acute decentralization. Moreover, acute decentralization also obtunded the responsiveness of intrinsic cardiac neurons to cardiac milieu alterations (Table 2). Limitations. There are multiple factors that may impact on the data generated in the present study. 1) Anesthesia and the surgery necessitated to record the activity generated by these neurons may impact on their basal activity, as well as reexes generated within the cardiac nervous system. 2) Coordination
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Fig. 2. Effects on cardiovascular variables and the activity generated by atrial and ventricular neurons (two bottom channels) of applying bradykinin (beginning at arrow below) to a LV ventral epicardial locus lying over the regional LV IMP sensor. This intervention enhanced the activity generated by both neuronal populations, while minimally affecting LV dynamics. LVP, LV chamber pressure.

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Fig. 3. Effects of briey occluding the left anterior descending coronary artery on concomitantly recorded atrial and ventricular neuronal activity. This intervention increased the activity generated by both neuronal populations (onset of the occlusion is indicated by the arrow below). J Appl Physiol VOL

among peripheral neuronal populations may be inuenced by the neuronal subtype identied. For this study, the predominant neuronal subtype identied was likely local circuit neurons. 3) Coordination among neuron population may be inuenced by multiple functional factors. For example, neurons involved in SA nodal function are tightly linked (15, 27, 28), whereas those involved in control of global electrical or mechanical function may not be, as indicated in this study. Finally, conclusions based on acute decentralization of the intrinsic cardiac nervous system may underestimate the potential for interganglionic interconnections due to the profound suppressor effects that acute decentralization exerts on autonomic ganglia. Perspectives. In the context of previous studies, these data imply the existence of 1) overlapping cardiac sensory inputs to atrial and ventricular neurons, and 2) functional connectivity among such populations that subserve longer term interactions. Moreover, these data indicate that cardiac phase-related activity generated by sympathetic and parasympathetic preganglionic inputs to the intrinsic cardiac nervous system is not reected in similar activity proles of its local circuit neurons. That most atrial and ventricular neurons display no short-term interactive behavior appears to be predicated upon their stochastic chemosensory inputs. As a consequence, they display relatively independent beat-to-beat reex control of regional cardiac indexes, responding rapidly and discretely to localized cardiac stress. Their functional interdependence, manifest by interganglionic interconnections and varied descending inputs, presumably acts to maintain overall balance of efferent neuronal control over longer time scales. Such an anatomical funcwww.jap.org

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tion arrangement may provide a substrate that compensates for altered function of select populations. Much more research is required to elucidate the varied linkages within the intrinsic cardiac nervous system to determine how its redundancy affects cardiac electrical and mechanical control in the presence of emergent pathology.
ACKNOWLEDGMENTS The authors gratefully acknowledge the technical assistance of Richard Livingston. GRANTS This study was supported by the Canada Institutes of Health Research, the National Heart, Lung, and Blood Institute (HL-58140 and HL-71830), and the American Heart Association. REFERENCES 1. [Anon]. Guiding principles for research involving animals and human beings. Am J Physiol Regul Integr Comp Physiol 283: R281R283, 2002. 2. Ardell JL. Intrathoracic neuronal regulation of cardiac function. In: Basic and Clinical Neurocardiology, edited by Armour JA and Ardell JL. New York: Oxford University Press, 2004, p. 118 152. 3. Ardell JL and Randall WC. Selective vagal innervation of sinoatrial and atrioventricular nodes in canine heart. Am J Physiol Heart Circ Physiol 251: H764 H773, 1986. 4. Armour JA. Cardiac neuronal hierarchy in health and disease. Am J Physiol Regul Integr Comp Physiol 287: R262R271, 2004. 5. Armour JA, Collier K, Kember G, and Ardell JL. Differential selectivity of cardiac neurons in separate intrathoracic autonomic ganglia. Am J Physiol Regul Integr Comp Physiol 274: R939 R949, 1998. 5a.Armour JA and Kember G. Cardiac sensory neurons. In: Basic and Clinical Neurocardiology, edited by Armour JA and Ardell JL. New York: Oxford University Press, 2004, p. 79 117. 6. Armour JA, Richer LP, Page PL, Vinet A, Kus T, Vermeulen M, Nadeau R, and Cardinal R. Origin and pharmacological response of atrial tachyarrhythmias induced by activation of mediastinal nerves in canines. Auton Neurosci 118: 68 78, 2005. 7. Arora RC, Cardinal R, Smith FM, Ardell JL, DellItalia LJ, and Armour JA. Intrinsic cardiac nervous system in tachycardia induced heart failure. Am J Physiol Regul Integr Comp Physiol 285: R1212R1223, 2003. 8. Blinder KJ, Johnson TA, and Massari VJ. Negative inotropic vagal preganglionic neurons in the nucleus ambiguus of the cat: neuroanatomical comparison with negative chronotropic neurons utilizing dual retrograde tracers. Brain Res 804: 325330, 1998. 9. Blomquist TM, Priola DV, and Romero AM. Source of intrinsic innervation of canine ventricles: a functional study. Am J Physiol Heart Circ Physiol 252: H638 H644, 1987. 10. Cardinal R and Page PL. Neuronal modulation of atrial and ventricular electrical properties. In: Basic and Clinical Neurocardiology, edited by Armour JA and Ardell JL. New York: Oxford University Press, 2004, p. 315339. 11. Cheng Z, Powley TL, Schwaber JS, and Doyle FJ. Vagal afferent innervation of the atria of the rat heart reconstructed with confocal microscopy. J Comp Neurol 381: 117, 1997. 12. Dickerson L, Rodak D, Fleming T, Gatti PJ, Massari VJ, McKenzie J, and Gillis R. Parasympathetic neurons in the cranial medial ventricular fat pad on the dog heart selectively decrease ventricular contractility. J Auton Nerv Syst 28: 129 141, 1998. 13. Furukawa Y, Hoyano Y, and Chiba S. Parasympathetic inhibition of sympathetic effects on sinus rate in anesthetized dogs. Am J Physiol Heart Circ Physiol 271: H44 H50, 1996. 14. Gatti PJ, Johnson TA, Phan P, Jordan KI III, Coleman W, and Massari VJ. The physiological and anatomical demonstration of functionally selective parasympathetic ganglia in discrete fat pads on the feline myocardium. J Auton Nerv Syst 51: 255259, 1995. 15. Gray AL, Johnson CI, Ardell JL, and Massari VJ. Parasympathetic control of the heart. A novel interganglionic intrinsic cardiac circuit mediates neural control of the heart. J Appl Physiol 96: 22732278, 2004.

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