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Extrinsic inputs to intrinsic neurons in the porcine heart in vitro

F. M. Smith

Am J Physiol Regul Integr Comp Physiol 276:R455-R467, 1999.

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Extrinsic inputs to intrinsic neurons in the porcine heart in vitro

F. M. SMITH Department of Anatomy and Neurobiology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7

Smith, F. M. Extrinsic inputs to intrinsic neurons in the porcine heart in vitro. Am. J. Physiol. 276 (Regulatory Integrative Comp. Physiol. 45): R455–R467, 1999.—Conver- gence of inputs from extrinsic cardiac nerves [vagus and cardiopulmonary (CPN)] on intrinsic cardiac neurons was investigated in the pig (Sus scrofa). A segment of the right atrial wall containing epicardial neurons along with attached stumps of the right vagus nerve and CPN was maintained in vitro; intracellular recordings were made from 57 neurons. Three types of neuron were identified by their responses to long intracellular depolarizing current pulses: phasic [dis- charged 1 action potential (AP); 40%]; accommodating (dis- charged multiple APs decrementing in frequency during pulse; 33%); and tonic (discharged multiple APs at a high frequency; 27%). Sixty-six percent of the neurons responded with excitatory postsynaptic potentials (EPSP) to vagal nerve stimulation; two-thirds of these cells fired APs when EPSP amplitude exceeded threshold level. Postsynaptic responses to vagal nerve stimulation were mediated by nicotinic ion channels; responses were eliminated by hexamethonium. CPN stimulation produced EPSPs but no APs in 17% of the neurons.All neurons responding with postsynaptic depolariza- tions to CPN stimulation also received vagal inputs. Com- bined stimulation of the vagus nerve and CPN produced APs in all but one of these neurons. Timolol eliminated postsynap- tic responses from CPN stimulation, indicating that these responses involved -adrenergic receptors and likely resulted from activation of sympathetic postganglionic terminals. These results show that some intrinsic cardiac neurons receive convergent inputs from the CPN and vagus nerve. It is suggested that such neurons represent intraganglionic sites for sympathetic-parasympathetic interactions in neural control of the heart.

intrinsic cardiac ganglia; sympathetic innervation; vagus nerve; cardiopulmonary nerve; intracellular recording

THE MAMMALIAN HEART is dually innervated by the sympathetic and parasympathetic limbs of the auto- nomic nervous system. The cardiac branches of the vagus nerves convey parasympathetic efferent pregan- glionic axons to the heart (29, 30) where some of these terminate on postganglionic neurons in the intrinsic cardiac ganglia. The majority of sympathetic postgangli- onic axons innervating the heart course in cardiopulmo- nary nerves originating from the middle cervical, stel- late, and, in some species, upper thoracic ganglia of the paravertebral ganglion chain (30, 31, 40). The anatomic separation of these autonomic pathways has informed analyses of their functions in cardiac regulation, and the sympathetic and parasympathetic limbs are tradi- tionally considered to control the heart in a reciprocal fashion. That is, when vagal cardiodepressant activity is low, sympathetic cardioaugmentatory activity is high, and vice versa (14). However, studies in which activity

of vagal and sympathetic cardiac nerves was recorded simultaneously have shown that these inputs to the heart are coactivated under a wide range of physiologi- cal conditions, for example during periods of increased atrial filling (36). When parasympathetic and sympathetic inputs to the heart are activated together, significant interac- tions between these inputs occur, and the primary sites for these interactions are considered to be the auto- nomic neuroeffector junctions at the myocardium (24, 38, 39, 46, 47, 53). At these sites, interactions occur through sympathetic modulation of vagal function or vagal modification of sympathetic function, depending on the prevailing physiological circumstances (58). These interactions are believed to be mediated by the combined influences of transmitter and modulator sub- stances released by sympathetic and parasympathetic terminals on the myocardium (39), as well as by prejunctional modulatory mechanisms on neuroeffector terminals (38, 47). However, another potential site for sympathetic-parasympathetic interactions is within the intracardiac ganglia at the level of single neurons. These neurons are organized into ganglionated plex- uses, which are connected into a network by intergangli- onic nerves (5, 9, 10, 12, 13, 22, 51). Classically, intracardiac neurons have been viewed as simple re- lays between parasympathetic preganglionic axons and the myocardium. However, recent anatomic reports indicate that sympathetic terminals exist within intra- cardiac ganglia (22, 43, 44), and it is therefore possible that sympathetic and parasympathetic inputs may converge on single intracardiac neurons. The main objective of this study was to investigate the intracellular responses of single intrinsic cardiac neurons of the pig right atrium in vitro to stimulation of attached cardiac vagal and cardiopulmonary nerve stumps to identify neurons potentially capable of inte- grating information from both efferent limbs of the autonomic nervous system. Recordings made from intra- cardiac neurons in this study show that some of these neurons receive both parasympathetic and sympa- thetic inputs and that interactions between these dual inputs can modify neuronal firing properties.


Experiments were done on 22 pigs of both sexes, 20–26 days old and with a mean body mass of 8.5 0.3 kg (mean SE). Pilot studies were conducted on a range of species (dog, pig, and guinea pig) to determine the best preparation for recording intracellularly from intracardiac neurons with ex- trinsic inputs. The pig heart was chosen because, in this species, a population of intracardiac neurons with responses to extrinsic nerve stimulation was located in the right atrial

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wall close to the entry point of the extrinsic nerves. The overall size of this segment of atrial wall and attached nerve stumps was small enough to be maintained in vitro, and intracardiac ganglia receiving extrinsic inputs could be ac- cessed easily for recording. Animals were obtained from a local livestock supplier and were maintained in the Univer- sity Animal Care Facility for up to 3 days at 20°C under a 12:12-h light-dark cycle before the experiments. Animals were treated in conformance with the guidelines of the Canadian Council on Animal Care; the protocol for these experiments was approved by the Dalhousie University Com- mittee on Animal Care. Animals were killed by an overdose of pentobarbital sodium (1,000 mg) administered intraperitone- ally. The sternum was opened in the midline, and the heart with attached stumps of right vagal and cardiopulmonary nerves was quickly removed and further dissected in a dish containing flowing oxygenated Tyrode solution at room tem- perature. This solution contained (in mM) 120 NaCl, 4 KCl, 1.2 KH 2 PO 4 , 1.2 MgSO 4 , 11 glucose, 10 HEPES, and 1.9 CaCl 2 ; pH was adjusted to 7.4 before use. All chemicals were obtained from Sigma (St. Louis, MO). The solution was saturated with 100% O 2 using a gas disperser. A block of tissue including the intercaval right atrial wall along with attached nerves was dissected free of the heart for in vitro recording from intracardiac neurons. To ensure inclusion of all right cardiac vagal branches, segments of this nerve extending cranially from the heart to the level of the middle cervical ganglion near the thoracic inlet and caudally from the heart to the diaphragm were included. The attached sympathetic cardiac nerves included all cardiopulmonary branches running to the heart from the right stellate and middle cervical ganglia and ansae subclavia. The tissue block was pinned to the silicone rubber-covered bottom of a 30-ml recording chamber and perfused with oxygenated Tyrode solution maintained at 36°C by a thermostatically controlled heater. The stumps of the vagal and cardiopulmonary nerves were gathered into bipolar suction electrodes filled with perfusate for stimulation with rectangular current pulses (0.5-ms dura- tion). These electrodes were driven by a stimulator (S88, Grass Instrument, Quincy, MA) through constant-current stimulus isolation units. Epicardial ganglia were exposed by blunt dissection with the aid of a dissecting microscope. In this area of the atrium, ganglia were embedded in epicardial fat and were connected together by small nerves in a ganglion- ated plexus. A ganglion selected for recording was lightly supported on a metal platform (0.2 mm wide, 0.6 mm long) attached to a micropositioner. With this platform the ganglion was held slightly away from the underlying tissue to provide mechanical stabilization, thus facilitating penetration of the tough ganglion sheath by the recording electrode (50). Gan- glion cells and connecting nerves were not damaged by this procedure. Ganglia were explored with a glass micropipette electrode filled with 3 M KCl (electrode resistance 50–120

M ), mounted on a three-axis mechanical micromanipulator.

The electrode was connected to an intracellular amplifier equipped with current injection and bridge-balancing cir- cuitry (model 1600, A-M Systems, Everett, WA) for nulling the electrode resistance. Before neurons were impaled, elec- trode potential was nulled with reference to the bath poten- tial, and this null was confirmed after withdrawing the electrode from the neuron at the end of a recording session. The difference between intracellular and bath potentials was taken as the transmembrane potential. The bath reference electrode consisted of a silver wire, coated with silver chlo- ride, inserted into a capillary tube filled with 1% agar dissolved in the same solution used to fill the recording

electrode, with the tip of the capillary tube immersed in the bath. To determine threshold and maximal responses to nerve stimulation, stimulus current was increased by increments until the first response appeared (threshold response), then further increased until the response reached peak amplitude (maximal response). Transmembrane potential, intracellular stimulus waveforms, and responses to nerve stimulation were monitored on an oscilloscope during the experiments

and stored on videotape through a digital data converter

(model PCM4, Medical Systems, Greenvale, NY) connected to

a videocassette recorder for later analysis. Selected responses were played back from the tape into a data-acquisition board attached to a personal computer and analyzed with pCLAMP software (Axon Instruments, Foster City, CA).

Data Analysis

Whole cell input resistance was estimated from the slope of

a line plotting membrane potential displacements from rest-

ing potential against the amplitudes of a series of hyperpolar- izing current pulses (200- to 400-ms duration) delivered through the recording electrode to clamp membrane current at preset levels. Some neurons displayed time-dependent voltage changes at the start of hyperpolarizing pulses, so only

the last 50 ms of responses, when potentials had reached

steady-state values, were sampled. Plots of current-voltage

relationships were linear over a range of voltages up to 35–40

mV more negative than the resting potential. All neurons

displayed marked nonlinearity in the current-voltage relation-


when depolarizing pulses subthreshold for action poten-


(AP) generation were injected, so these responses were

not included in estimates of input resistance. Membrane time constant was estimated from the time course of responses to

small (0.1 nA) hyperpolarizing currents. A curve-fitting pro- gram (CLAMPAN in the suite of pCLAMP programs) indi-

cated that these responses followed a single-exponential time course, as reported for other autonomic neurons (28), so time constant for each cell was calculated as the time for mem- brane potential to change by 1 1/e of the final steady-state value during hyperpolarization. The amplitude of depolariza-

tion to threshold for AP generation was taken as the differ-

ence between resting membrane potential and the voltage at

which regenerative changes were initiated. AP duration was measured at one-half peak amplitude. Afterhyperpolariza-

tion (AHP) amplitude was taken as the difference between

resting membrane potential and peak amplitude of the hyperpolarization following the AP. For estimating AHP duration, the time was measured from the point at which the repolarizing waveform crossed the level of the resting mem- brane potential, to the point at which the potential had returned to one-half the peak amplitude of the AHP. Differences among mean values of intracellular variables were analyzed by ANOVA (P 0.05). Where f values were significant, comparisons between means were done using Scheffe´’s multiple means comparison test (57). Experimental design and statistical analysis of the data were carried out in accordance with guidelines suggested by Wallenstein et al. (52), Glantz (25), and Denenberg (20).


Membrane and Firing Properties of Intracardiac Neurons

Neurons with resting membrane potentials that did not settle to values more negative than 40 mV within 5 min of impalement were excluded from this study. Mean values of resting membrane potential, whole cell

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Table 1. Membrane properties and characteristics of action potentials for neurons grouped by firing pattern

Neuron Type


Phasic (40%)


Tonic (27%)

(n 23)

(n 19)

(n 15)

Resting membrane potential, mV Whole cell input


49 4 (23)

47 5


49 5


resistance, M

72 8* (23)

33 7


39 7


Whole cell time constant, ms Action potential characteristics Threshold depo-

5.8 0.7 (8)

7.2 1.1 (9)

6.1 1.2 (11)

larization, mV

19 4† (23)

27 2


25 2


Total amplitude,

73 5


75 4


72 5


Overshoot, mV

24 4


27 4


24 2


Duration, ms

1.5 0.4 (23)

1.9 0.3 (18)

1.6 0.2 (15)

After hyperpolar-

ization charac-


Amplitude, mV

8 3


13 3


10 2 (15)

Duration, ms

48 4


39 5


130 11‡ (15)

Values are expressed as means SE. No. of measurements contributing to each mean is shown in parentheses beside each value. Characteristics of action potentials were evoked by intracellular depolarizing currents delivered through the recording electrode. Significantly different from: *accommodating neurons and tonic neurons; †accommodating neurons but not tonic neurons; ‡phasic neurons and accommodating neurons.

input resistance, and whole cell time constant, along with neuronal responses to depolarization, are summa- rized in Table 1. All neurons sampled in this study responded with depolarizations to inward current pulses of 50- to 60-ms duration delivered through the record- ing electrode, and generated APs when stimulus cur- rent reached threshold (Fig. 1). Three types of response to prolonged (400- to 600-ms duration) depolarizing current pulses permitted classification of intracardiac neurons into three groups: 1) 40% were phasic (dis- charged 1 AP at the start of the stimulus pulse, Fig. 2A); 2) 33% were accommodating (discharged multiple APs that decreased in frequency as the stimu-

multiple APs that decreased in frequency as the stimu- Fig. 1. Response of membrane potential (

Fig. 1. Response of membrane potential (top trace) of an intracardiac neuron to a depolarizing current pulse (0.2 nA, 60-ms duration; bottom trace) delivered through the intracellular recording electrode. Stimulus current intensity was at threshold for evoking an action potential (AP) in this cell. Resting membrane potential before current injection was 48 mV; peak of AP overshoots 0 V (indicated at left). Vertical calibration bar: 20 mV, 1 nA. Horizontal calibration bar:

20 ms.

lus continued, Fig. 2B); and 3) 27% were tonic (dis- charged multiple APs with little or no frequency decre- ment throughout the stimulus pulse, Fig. 2C). There were no statistically significant differences in mean resting membrane potential or mean whole cell time constant among the three types of neuron, but whole cell input resistance was significantly higher in phasic than in accommodating or tonic neurons. Phasic neu-

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Fig. 2. Examples of responses of 3 types of intracardiac neurons to long intracellular depolarizing current pulses. AC: top main trace represents transmembrane potential (0 V indicated at left), and bottom main trace represents stimulus current. Smaller traces (insets) above main voltage traces show AP and afterhyperpolariza- tion (AHP) in response to a brief intracellular depolarizing pulse (5-ms duration; current artifact precedes AP). A: phasic neuron; 1.2-nA, 520-ms duration current pulse evoked 1 AP. A, inset: AHP duration was 35 ms. Resting membrane potential before stimulation was 67 mV. B: accommodating neuron; 0.7-nA, 540-ms-duration current pulse elicited multiple APs, which decreased in frequency during the depolarization pulse. B, inset: AHP duration was 29 ms. Resting membrane potential was 42 mV. C: tonic neuron; 0.6-nA, 440-ms-duration current pulse produced multiple APs that did not decrement in frequency over duration of pulse. C, inset: AHP duration was 118 ms. Resting membrane potential was 57 mV. Tonic neuron (C) had longest-duration AHP despite displaying highest firing frequency. Vertical calibration bar: 30 mV for inset traces; 20 mV, 1 nA for main traces. Horizontal calibration bar: 150 ms for inset traces, 60 ms for main traces.



Table 2. Patterns of extrinsic input to physiologically identified intracardiac neurons

Response to

Extrinsic Nerve


Neuron Type





No extrinsic input







of total





Vagal input only








of total





Vagal and CPN input








of total





CPN, cardiopulmonary nerve; n, no. of neurons of each type within each category of response to extrinsic nerve stimulation.

rons also had a significantly lower depolarization thresh- old for firing APs than did the other two cell types. The APs of all neurons in this study overshot 0 V, and AP duration was not significantly different among the groups. AHPs were of similar amplitude in all cell types, but the duration of AHP in tonic neurons was significantly longer than in phasic or accommodating neurons (Table 1; examples shown in Fig. 2, insets).

Responses to Extrinsic Nerve Stimulation

All intracardiac neurons in this study were tested for the presence of inputs from extrinsic cardiac nerves. Intracellular responses evoked by single-pulse stimula- tion of vagus and cardiopulmonary nerves in the three classes of neurons are summarized by neuron type in Table 2. All phasic neurons except one received input from the vagus nerve, and a subset of these neurons also responded to cardiopulmonary nerve stimulation. Many accommodating neurons also received vagal in- puts, and one of these cells responded to cardiopulmo- nary nerve stimulation as well. Only one tonic neuron responded to vagal nerve stimulation, and no tonic cells received inputs from cardiopulmonary nerves. All intra- cellular responses to nerve stimulation were confirmed as orthodromic by their elimination in a modified perfusate containing 0 Ca 2 and high Mg 2 (10 mM) (see Figs. 3B, 4B, and 5B). No antidromic responses to extrinsic nerve stimulation were recorded in this study. All postsynaptic responses were depolarizing; no hyper- polarizing responses were observed. Responses to va- gus and cardiopulmonary nerve stimulation are de- scribed in more detail in the following sections. Vagus nerve stimulation. In this study, 66% of neu- rons sampled responded to vagus nerve stimulation (Table 2; Figs. 3 and 4). Threshold neuronal responses were without exception marked by the appearance of small excitatory postsynaptic potentials (EPSP) at la- tencies of up to 40 ms (Figs. 3 and 4); as stimulus current intensity was increased, multiple EPSPs were evoked (Figs. 3 and 4). Graded increases in stimulation produced corresponding increases in the amplitude of the EPSPs (Figs. 3 and 4). In some neurons additional EPSPs frequently occurred at latencies of 70 to 100 ms after the stimulus pulse (e.g., Fig. 4A, 2.1-mA trace

from top). One-third of the neurons responding to vagal stimulation generated EPSPs that did not reach thresh-

old for AP initiation (Fig. 3) even when the intensity of

nerve stimulation was maximal. Some of these EPSPs were followed by small AHPs (e.g., Fig. 4A, 2.5-mA trace). The duration of EPSPs evoked by vagal nerve

stimulation was similar in all three cell types, so data from all groups have been combined; the mean value of EPSP duration was 15 2 ms. In eight phasic cells and five accommodating cells responsive to vagal nerve stimulation, EPSPs reached threshold for AP genera- tion. The mean amplitude of EPSPs reaching threshold for AP generation was 17 3 mV in phasic and 23 4

mV in accommodating neurons; these values are consis-

tent with threshold values for intracellularly evoked

APs in these two groups (Table 1). All orthodromically mediated APs overshot 0 V, and, for phasic and accom- modating neurons, peak AP amplitude was within a

few millivolts of the values shown in Table 1 for directly

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Fig. 3. Excitatory response of an intracardiac neuron to single-pulse stimulation (0.5-ms duration) of cardiac branches of right vagus nerve (stimulus indicated by artifacts at start of traces). Resting membrane potential of cell was 49 mV. A: responses to intensity- graded stimulation (current increasing from top to bottom traces; intensity indicated at left of each trace). Note multiple excitatory postsynaptic potentials (EPSP) and a reduction of latency to onset of response at greatest current intensity. No AP could be evoked by nerve stimulation in this neuron. B: response to nerve stimulation was eliminated by 5-min exposure to modified, Ca 2 -free perfusate containing 10 mM Mg 2 . C: exposure to hexamethonium (HEX; 100 µM for 6 min in perfusate) also eliminated response to nerve stimulation, demonstrating that this response was mediated by nicotinic channels in postsynaptic membrane. Stimulus intensity in B and C was 5 mA. All stimulus artifacts have been truncated for clarity. Vertical calibration bar: 10 mV. Horizontal calibration bar:

10 ms.



EXTRINSIC INPUTS TO INTRACARDIAC NEURONS R459 Fig. 4. Intracardiac neuron generating EPSPs and AP in response

Fig. 4. Intracardiac neuron generating EPSPs and AP in response to vagus nerve stimulation (experimental protocol similar to that in Fig. 3). Resting membrane potential was 51 mV. A:

intensity-graded single-pulse nerve stimulation evoked multiple EPSPs that summed to exceed threshold for AP generation at the highest inten- sity (bottom trace; 0-V level indicated at left). B and C: at same stimulus intensity as in bottom trace of A, neuronal responses to nerve stimula- tion were blocked in 0-Ca 2 , high-Mg 2 (lo Ca 2 , hi Mg 2 ) perfusate and in 100 µM bath-applied hexamethonium, respectively (both 5-min expo- sure). As in Fig. 3, neuronal responses to nerve stimulation were mediated by nicotinic postsyn- aptic receptors. D: timolol (TIM; 10 µM, 5 min in perfusate) had no effect on membrane potential or responses to vagus nerve stimulation. AP was truncated for clarity. Vertical calibration bar: 10 mV. Horizontal calibration bar: 20 ms.

evoked APs. Within the group of neurons responding to vagus nerve stimulation, mean AHP amplitude and duration were, respectively, 9 2 mV and 54 6 ms for phasic and 15 4 mV and 47 11 ms for accommodat- ing neurons. In the only tonic neuron responding to vagal nerve stimulation, the EPSP did not reach thresh- old for AP generation. Cardiopulmonary nerve stimulation. Graded cardio- pulmonary nerve stimulation evoked EPSPs with corre- spondingly graded amplitudes in 17% of the neurons in this study, at latencies up to 15 ms after the stimulus pulse (Fig. 5). Nine of these cells were phasic and one was accommodating (Table 2). These neurons consti- tuted a subset of those responsive to vagal stimulation;

no neurons impaled in this study responded only to cardiopulmonary nerve stimulation. As shown in the example in Fig. 5, maximal cardiopulmonary nerve stimulation was not, in itself, sufficient to evoke an AP in any of these neurons. EPSPs evoked in accommodat- ing and phasic neurons by cardiopulmonary nerve stimulation had similar amplitudes and durations, so EPSP data from all neurons were combined for compari- son with the properties of EPSPs evoked in these cells by vagus nerve stimulation, as shown in Table 3. EPSPs evoked by cardiopulmonary nerve stimulation were significantly smaller in amplitude and signifi- cantly shorter in duration than those evoked by vagal nerve stimulation in the same neurons. In none of these

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R460 EXTRINSIC INPUTS TO INTRACARDIAC NEURONS Fig. 5. Effects of cardiopulmonary nerve stimulation on an intracar-

Fig. 5. Effects of cardiopulmonary nerve stimulation on an intracar- diac neuron. Protocol was similar to that of Figs. 3 and 4. Resting membrane potential was 46 mV. A: intensity-graded nerve stimula- tion evoked an EPSP but no AP in this neuron; this was true for all neurons receiving inputs from the cardiopulmonary nerve. B and C: blockade of postsynaptic responses by lo Ca 2 , hi Mg 2 perfusate and by timolol (10 µM in bath) showed that these responses were orthodromic and involved -adrenergic receptors. D: postsynaptic responses were unaffected by 100 µM hexamethonium in the bath. Vertical calibration bar: 10 mV. Horizontal calibration bar: 10 ms.

dually innervated neurons did vagal nerve stimulation alone evoke EPSPs above threshold for AP generation. Costimulation of vagus and cardiopulmonary nerves. There were two types of response to combined vagal and sympathetic nerve stimulation. Two cells, both phasic, displayed responses of the type designated type 1, illustrated in Fig. 6. These neurons produced EPSPs and APs in response to maximal vagal nerve stimula- tion alone (Fig. 6A) but did not show any postsynaptic responses to cardiopulmonary nerve stimulation alone (Fig. 6B). However, simultaneous stimulation of vagal and cardiopulmonary nerves produced an AP of longer duration and an AHP with a slightly greater amplitude

Table 3. Comparison of properties of excitatory postsynaptic potentials evoked in same neurons by stimulation of vagus and cardiopulmonary nerves

Vagus Nerve



Nerve Stimulation

Amplitude, mV

19 3

7 1*

Duration, ms

17 2

11 2*

Values are expressed as means SE; n 10 neurons. *Significant difference from value for vagus nerve stimulation (P 0.05; t-test).

and of longer duration (Fig. 6C) compared with the AP and AHP generated by vagal nerve stimulation alone. These differences are readily apparent in Fig. 6C, where the AP resulting from vagus nerve stimulation has been juxtaposed with the response to combined

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Fig. 6. Example of type 1 response of an intracardiac neuron to vagus nerve and cardiopulmonary nerve (CPN) stimulation. Resting mem- brane potential was 43 mV. A: AP produced by maximal (5.0 mA) single-pulse stimulation of vagus nerve (0-V level indicated at left of trace). B: CPN stimulation produced no direct postsynaptic response, even at highest intensity available (10 mA shown). C: costimulation of vagus nerve and CPN with same stimulus parameters as in A and B produced prolonged AP and AHP duration. In C, a portion of trace A has been inset at right of main trace for comparison. In both, dashed lines indicate level of resting membrane potential, and arrows indicate half-amplitude of each AP (for duration comparison). Verti- cal calibration bar: 10 mV. Horizontal calibration bar: 20 ms.



EXTRINSIC INPUTS TO INTRACARDIAC NEURONS R461 Fig. 7. Example of type 2 response of an intracardiac

Fig. 7. Example of type 2 response of an intracardiac neuron to vagus nerve and CPN stimulation. A: maximal vagus nerve stimulation (6.8 mA) elicited an EPSP but no AP. B: maximal stimulation of CPN (9.0 mA) also generated an EPSP but no AP. C: costimulation of vagus nerve and CPN, using the same stimulus parameters as in A and B, produced EPSPs that summed to exceed firing threshold. Resting membrane potential in this cell was 41 mV before stimulation. Vertical calibration bar: 10 mV. Horizontal calibration bar: 10 ms.

nerve stimulation. As only two neurons displaying a type 1 response were found, no statistical analysis of the effects of combined nerve stimulation was possible. The second type of response, designated type 2, was observed in nine neurons: eight phasic and one accom- modating. In these cells, independent vagal and cardio- pulmonary nerve stimulation evoked EPSPs (Fig. 7, A and B, respectively; Fig. 8A), but neither input alone was strong enough at maximal stimulus intensity to generate an AP. When both nerves were costimulated at their respective maximal stimulus intensities, the resulting EPSPs summed and reached threshold for AP generation (Fig. 7C; Fig. 8A, bottom). One phasic neuron in which separate stimulation of the cardiopulmonary and vagus nerves evoked small- amplitude EPSPs showed no augmentation of postsyn- aptic effects in response to combined nerve stimulation, so fit neither of the above categories and was not included in the analysis. Effects of cholinergic and -adrenergic blockade. Of the 38 neurons having vagal but no cardiopulmonary inputs, a group of six neurons was tested to determine whether nicotinic neurotransmission was involved. In this group of cells, the nicotinic channel blocker hexame- thonium [100 µM in the perfusate for at least 5 min; this dose was found to be effective in pig heart in a previous study (50)] eliminated all postsynaptic depolar- izing potentials evoked by nerve stimulation (Fig. 3C). In another group of six neurons that were dually innervated by the vagus and cardiopulmonary nerves, the postsynaptic responses to vagus nerve stimulation

(Fig. 4A; Fig. 8A, top) were also eliminated by hexame- thonium (Fig. 4C; Fig.8B, top). The responses to cardio- pulmonary nerve stimulation were unchanged after hexamethonium application (Fig. 5D; Fig. 8B, middle), and combined stimulation of the vagus and cardiopul-

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Fig. 8. Nicotinic and -adrenergic receptors are involved in type 2 responses of intracardiac neuron to vagus nerve and CPN stimula- tion. A: control responses to separate stimulation of vagus nerve (7.0 mA) and CPN (9.0 mA) and AP generated by combined nerve stimulation (vagus CPN). Stimulus parameters were unchanged throughout this experiment. B: hexamethonium (100 µM for 5 min in perfusate) eliminated the vagally generated EPSP. EPSP resulting from CPN stimulation was unaffected by hexamethonium but neuron did not fire an AP in response to costimulation. C: timolol (100 µM in perfusate) administered for 5 min in the continued presence of hexamethonium eliminated the EPSP due to CPN stimulation. In this condition, combined nerve stimulation had no postsynaptic effect. Resting membrane potential was 57 mV. Vertical calibration bar: 20 mV. Horizontal calibration bar: 10 ms.



monary nerves then produced the same effect (Fig. 8B, bottom) as did cardiopulmonary nerve stimulation alone. To address the possibility that this concentration of hexamethonium was insufficient to block potential nicotinic neurotransmission through cardiopulmonary inputs, the antagonist dose was increased in two trials to 200 µM, but this did not alter the response to cardiopulmonary nerve stimulation (data not shown). To determine the involvement of -adrenergic recep- tors in responses to nerve stimulation, the -adrenergic antagonist timolol (10 µM) was applied in the perfus- ate. This agent was used because it is a nonselective -antagonist and does not have membrane-stabilizing effects (6). The dose used in this study was within the range found effective in previous studies (6) and was chosen for the present study on the basis of a series of preliminary experiments to determine optimum concen- tration in atrial tissue. Two protocols were followed in these experiments. In the first, timolol was applied during vagus nerve stimulation in four experiments to determine if -adrenergic receptors were involved in these responses. -Blockade had no effect on the post- synaptic response to vagal nerve stimulation in these trials, an example of which is shown in Fig. 4D. In the second protocol (6 experiments), timolol was adminis- tered during vagus and cardiopulmonary nerve stimu- lation in the continued presence of hexamethonium. As shown in Fig. 8C, this protocol eliminated the EPSP due to cardiopulmonary nerve stimulation (Fig. 8C, middle); combined cardiopulmonary and vagus nerve stimulation then had no effect (Fig. 8C, bottom).


Three patterns of innervation of right atrial neurons by axons in extrinsic cardiac nerves have been revealed in this study. One-third of the neurons sampled re- ceived no synaptic inputs of extracardiac origin, whereas the remaining two-thirds were innervated by axons running in the vagus nerve. None of the neurons sampled in this study received inputs solely from cardiopulmonary nerves; instead, inputs from cardiopul- monary axons converged on a subset of vagally inner- vated neurons. Intracardiac neurons could be classified into three physiological types based on their responses to depolarizing test current pulses, and there appeared to be a relationship between the physiological class of a neuron and the probability of that neuron receiving extrinsic inputs. The properties of extrinsic inputs to intracardiac neurons, the relationships between neu- ron type and specificity of input, the integrative capabili- ties conferred by these input patterns and by intrinsic membrane and active properties, and the functional implications of the results are discussed in the follow- ing sections.

Extrinsic Innervation of Atrial Neurons

Sixty-six percent of the cells sampled in this study responded with depolarizing postsynaptic potentials to right vagus nerve stimulation, but in some neurons these depolarizations did not reach threshold for AP

generation. None of the neurons capable of generating APs in response to vagus nerve stimulation exhibited strong postsynaptic activation (i.e., a unitary EPSP large enough to exceed threshold for AP generation). Instead, multiple EPSPs were summed to generate an AP. Neurons generating vagally mediated EPSPs there- fore must integrate synaptic inputs from multiple preganglionic axons to produce a regenerative output, a result that concords with the observations of Seabrook et al. (49) on vagal innervation of neurons in the neonatal rat heart. Those neurons responding with APs to vagal stimulation in the present study may in fact represent classical efferent parasympathetic neurons innervating the myocardium. If this is the case, it is interesting to note that these neurons did not represent the majority of intracardiac neurons sampled. Perfusion with a modified Tyrode solution containing reduced calcium and high magnesium levels (blocking release of neurotransmitter at nerve terminals) or exposure to the nicotinic channel blocker hexametho- nium eliminated the postsynaptic effects of vagal nerve stimulation. These results demonstrate that the effects of nerve stimulation were mediated synaptically and imply that such stimulus-elicited depolarizations were produced by acetylcholine released from vagal pregan- glionic terminals and acting at nicotinic postsynaptic receptors. The latter finding agrees with that of Ed- wards et al. (21), who reported that the nicotinic receptor antagonist D-tubocurarine blocked vagal trans- mission to intracardiac neurons in the guinea pig atrium. Interestingly, Seabrook et al. (49), using the nicotinic receptor antagonist mecamylamine to block cholinergic neurotransmission, found that some intra- cardiac neurons had a residual postsynaptic response to vagal nerve stimulation and suggested that a noncho- linergic neurotransmitter was coreleased from vagal preganglionic terminals in intracardiac ganglia. How- ever, no evidence for this effect was found in the present study. Seventeen percent of intracardiac neurons in this study were dually innervated by axons in the right vagus and cardiopulmonary nerves; none of the neu- rons sampled received inputs exclusively from cardio- pulmonary nerves. Two types of responses were re- corded in these neurons. In type 1 responses, illustrated in Fig. 6, vagus nerve stimulation alone evoked EPSPs and an AP whereas cardiopulmonary nerve stimulation alone had no postsynaptic effect. Stimulation of cardio- pulmonary and vagal nerves together broadened the duration of both the AP and the AHP compared with that produced by vagal stimulation alone. Because there was no direct postsynaptic effect of cardiopulmo- nary nerve stimulation, one possible explanation for this augmentation is that a neuroactive agent released from intraganglionic sympathetic terminals (possibly norepinephrine or a substance coreleased with this transmitter) could have acted presynaptically on vagal terminals to potentiate the release of acetylcholine. Higher acetylcholine concentrations within the synap- tic cleft could then have been responsible for prolonging postsynaptic channel-opening times, thus leading to

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increased AP and AHP durations. The receptor types involved in this response remain unknown. In this study, type 1 responses were in the minority and were found in only two neurons. The majority of neurons influenced by axons in cardiopulmonary nerves displayed type 2 responses:

concomitant stimulation of cardiopulmonary nerves enabled firing of APs during vagal stimulation (Figs. 7 and 8). In these neurons, vagus nerve stimulation alone evoked depolarizations that were orthodromically medi- ated (eliminated by low-calcium, high-magnesium per- fusate), were blocked by hexamethonium, and had electrophysiological properties (mean EPSP amplitude and duration, Table 3) similar to those produced by nerve stimulation in neurons innervated only by the vagus. EPSPs produced by cardiopulmonary nerve stimulation in dually innervated neurons with type 2 responses were significantly smaller in amplitude and shorter in duration than those produced in the same neurons by vagal inputs, as summarized in Table 3. Nicotinic receptors were not involved in responses to cardiopulmonary nerve stimulation, as determined by stimulating these nerves during exposure to hexametho- nium; this antagonist had no effect on the response to cardiopulmonary nerve stimulation either at a concen- tration that blocked all vagally mediated responses (100 µM; Fig. 8) or at double this dose. However, the -antagonist timolol blocked postsynaptic responses to stimulation of cardiopulmonary nerves (Fig. 5), and exposure to hexamethonium and timolol together com- pletely eliminated postsynaptic responses of these neu- rons to combined nerve stimulation (Fig. 8). Given the differences in the properties of EPSPs and the differen- tial effects of cholinergic and adrenergic antagonists on the postsynaptic responses to vagus and cardiopulmo- nary nerve stimulation, it is clear that different synap- tic mechanisms were activated by these inputs. Acetyl- choline, released from vagal preganglionic terminals and acting at nicotinic postsynaptic receptors, likely mediated the effects produced by vagus nerve stimula- tion, whereas the adrenergic neurotransmitter norepi- nephrine, most likely released from postganglionic sympathetic terminals and acting at -adrenergic recep- tors, was involved in the effects of cardiopulmonary nerve stimulation. There have been several anatomic reports demon- strating the presence of sympathetic postganglionic nerve terminals in mammalian intracardiac ganglia (22, 43, 44), and the results of the present study constitute functional evidence that these terminals may be capable of modulating ganglionic neurotransmis- sion when activated. Endogenously released or exog- enously applied norepinephrine has been shown to affect ion conductances and thus membrane and firing properties of peripheral autonomic neurons, either facilitating or inhibiting ganglionic neurotransmission (1, 3, 17, 18, 45, 50). In the pig heart, Smith et al. (50) found that exogenously applied norepinephrine facili- tated orthodromically mediated responses of some neu- rons to stimulation of intracardiac plexus nerves. In that study, it was proposed that facilitation could have

resulted either from direct postsynaptic effects of norepi- nephrine or through action at a presynaptic site. The observation of type 1 and 2 responses in the present study provides evidence that both of these mechanisms may be operative in intrinsic cardiac ganglia in the pig heart. Results of the present in vitro study also support the findings of a previous study of the beating canine heart in vivo showing that adrenergic agonists applied locally to intracardiac ganglia enhanced the frequency of neuronal AP discharge (33). Clearly the facilitatory effects of exogenously applied adrenergic agents in the heart in vivo could have been operating by the same mechanism responsible for the excitatory effects of activating sympathetic inputs to intracardiac neurons seen in the present in vitro study. The results of the present experiments showing that the influences of axons in vagus and cardiopulmonary nerves are mediated by cholinergic and adrenergic receptors, respectively, provide an insight into the issue of possible mixing of sympathetic and parasympathetic fibers within individual extrinsic cardiac nerves. Some parasympathetic preganglionic fibers innervating the mammalian heart have been reported to diverge from the vagus nerve in the neck to course with sympathetic nerves, joining the cervical sympathetic trunk, the middle cervical or stellate ganglia, or the ansae subcla- via (29, 31). It is therefore possible that intracardiac neurons activated by stimulation of vagal and cardiopul- monary nerves in the present study may have been responding to parasympathetic preganglionic inputs running to the heart in both nerves. If this were the case, then the postsynaptic effect of activating parasym- pathetic axons in the cardiopulmonary nerves should have been eliminated by nicotinic blockade. However, because hexamethonium did not eliminate responses to cardiopulmonary nerve stimulation, these responses were likely not mediated by displaced vagal axons. There is also anatomic and functional evidence that sympathetic fibers, originating from postganglionic neu- rons in the sympathetic chain, may reach the heart via the cardiac vagus (11, 35, 48) as well as the cardiopulmo- nary nerves. It is thus possible that stimulation of the vagus nerve in the present study could have activated some sympathetic postganglionic as well as parasympa- thetic preganglionic fibers innervating intracardiac neurons. This also does not appear to be the case, since -adrenergic blockade had no effect on the postsynaptic responses of intracardiac neurons to vagus nerve stimu- lation. Although the number of neurons sampled in this study was limited, the data indicate that stimulation of the vagus nerve activated only parasympathetic inputs and cardiopulmonary nerve stimulation activated only sympathetic inputs to these neurons. This may not, however, be true for all intracardiac neurons.

Intrinsic Neuron Properties and Correlation with Extrinsic Input Pattern

Properties. There were no significant differences be- tween mean resting membrane potentials of the three neuron types (Table 1). Values reported in this study were consistent with the mean value of resting poten-

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tial reported in an earlier in vitro study of intracardiac neurons in the pig heart (50), with the value for R-type canine intracardiac neurons (55), and with mean val- ues reported for guinea pig atrial neurons (21, 32). However, mean values of resting potential in the pre- sent study were smaller than those reported for neona- tal rat intracardiac neurons (49), canine S-type intracar- diac neurons (55), and phasic neurons in the guinea pig left atrium (27). The mean values for resting potential in these studies ranged roughly from 50 to 60 mV, and this variation is likely to be due at least partly to interspecies differences and possibly to differences in recording conditions (see Ref. 2 for discussion). Mean whole cell input resistance of phasic neurons in the present study was double that of accommodating and tonic neurons (Table 1), but resistance values for all neurons in this study were within the range of those reported for intracardiac neurons in a previous study of pig atrial neurons (50) and for atrial neurons of other species (21, 27, 49, 55, 56). The higher resistance (or lower membrane conductance) of phasic neurons in the present study implies that a smaller current would be required to depolarize the voltage-gated channels re- sponsible for AP generation to threshold for firing in these neurons, compared with currents needed to depo- larize lower-resistance neurons to firing threshold. This characteristic of phasic neurons, coupled with a threshold depolarization voltage for AP generation that was significantly less than threshold voltages for accom- modating and tonic neurons (Table 1), means that phasic neurons may be more excitable than the other types of intracardiac neuron. Mean time constants for the three cell types in the present study were statisti- cally similar (Table 1) and were also similar to those reported for atrial neurons in other species (21, 27, 49, 50, 55, 56). Properties of the APs induced by intracellular stimu- lation in the three classes of neuron (with the exception of the significantly lower threshold voltage for AP generation in phasic cells mentioned above) were not significantly different. These properties were, more- over, similar to those reported for neurons in porcine (50), rat (49), and dog (55, 56) hearts. All neurons in the present study displayed promi- nent AHPs following single APs (Figs. 4 and 6). When mean amplitudes and durations of AHPs in the three groups of neurons were compared, the only significant difference found was that mean AHP duration in tonic cells was more than twice the duration of AHPs in the other two groups of neurons (Table 1; Fig. 2). A similar difference was reported for AHP duration between S-type (phasically discharging, AHP 50-ms duration) and ‘‘SAH’’ and P-type (repetitively firing, AHP 200 ms) intracardiac neurons in the guinea pig heart (21). Correlations. In the present study there appears to be a correlation between neuron class (based on electro- physiological criteria) and pattern of innervation by extracardiac inputs. Data compiled by neuron class in Table 2 show that extrinsic vagal inputs appear to be routed to phasic and to some accommodating intracar- diac neurons, whereas extrinsic sympathetic input

primarily targeted phasic neurons; most tonic neurons appeared to receive no extrinsic inputs. Several schemes have recently been proposed to classify peripheral autonomic neurons by functional criteria (summarized in Ref. 2), based either on the time course of the AHP following single intracellularly evoked APs (4, 15, 23, 56), firing behavior in response to long depolarizing currents injected through the recording electrode (26, 54), or a combination of both. In the present study, neurons have been divided into three classes based on their firing behavior in response to depolarization, as shown in Fig. 2. This scheme was adopted because of the implications of this intrinsic property for the poten- tial roles of the different neuron types in controlling intracardiac neurotransmission. The existence of correlations between firing proper- ties and patterns of extrinsic innervation of the three classes of neuron found in this study provides some insight into the potential roles of these neuron types in regulating the heart. Implicit in the use of this classifi- cation scheme is the concept that neurons with differ- ent firing behaviors are involved in different intracar- diac functional pathways. In this respect, autonomic neurons with different types of firing behavior have been shown to participate in anatomically and function- ally distinct pathways involved in visceral control (15, 23, 41, 42). Firing behavior is an important physiologi- cal characteristic that can at least partly determine the responses of neurons to ongoing high-frequency synap- tic input sufficient to depolarize the cell membrane to firing threshold. Thus phasic and accommodating neu- rons would act as low-pass filters in the presence of sustained high-frequency synaptic input. Because the majority of intracardiac neurons receiving vagal pregan- glionic inputs in this study were phasic or accommodat- ing, high levels of vagal preganglionic activity would result in the generation of only one or two APs or a brief decrementing burst of APs at the start of depolariza- tion, depending on the neuron type. The output path- ways of these neurons are as yet unknown, but if they mediate parasympathetic control of the myocardium, the rate of transganglionic transmission of impulses of extracardiac origin to effector sites would thus be subject to a low cut-off frequency even in the presence of strong, high-frequency preganglionic activity. This cor- relation is strengthened further when the pattern of innervation by axons in cardiopulmonary nerves is considered: neurons displaying type 2 responses were almost exclusively phasic, so parasympathetic neuro- transmission to these neurons would be modulated by sympathetic inputs. The finding that none of the neurons expressing type 2 responses could be induced to fire an AP through vagal drive alone suggests that these neurons were not in the pathway for direct efferent parasympathetic control of the myocardium. Indeed, inasmuch as these neurons only generated an output in response to the activation of both sympathetic and parasympathetic inputs, they may represent a special category of neuron concerned with intracardiac integration of information from both efferent limbs of the autonomic nervous

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system. On the other hand, some of the accommodating and all but one of the tonically discharging neurons in this study lacked extrinsic inputs, so presumably these cells would be activated primarily by inputs from sources intrinsic to the heart and may therefore be ‘‘local-circuit’’ neurons or interneurons (7, 8) involved in intracardiac reflex loops. If this is the case, the high- frequency repetitive firing properties of these cells may help promote strong activation of such reflex loops by feedback from intracardiac events. In the guinea pig heart, an alternate, sensory role for intracardiac neurons receiving no extrinsic inputs has been proposed (27). The existence of intracardiac sen- sory neurons has also been postulated in the hearts of other mammalian species (see Ref. 8 for review). In the present study, one-third of the neurons sampled were devoid of extrinsic inputs, raising the possibility that these neurons may be sensory in function. However, solid electrophysiological criteria for identifying intra- cardiac sensory neurons have not yet been established at the cellular level. Efferent axons of potential sensory neurons in the heart that might communicate with the central nervous system or intrathoracic ganglia would be expected to run in the extrinsic cardiac nerves and should have been antidromically activated by nerve stimulation, but no antidromically activated neurons were detected in the present study. As a side issue, data on AHP properties of intracar- diac neurons may provide some insight into the relation- ship of AHP characteristics to neuronal firing behavior. It has been proposed that AHP duration may have a causal relationship with repetitive firing behavior in peripheral autonomic neurons (reviewed in Ref. 2). In cells with long-duration AHPs, it would be expected that repetitive firing in response to prolonged depolar- ization would be limited to a low frequency because of the time needed for membrane potential to recover to a value near the resting level. Phasically firing cells would therefore be expected to have longer AHPs than repetitively firing cells. However, this was not the case for phasic neurons either in the present study (Fig. 2, insets) or for phasic (S-type) cells in the guinea pig heart (21). It is thus possible that AHP duration per se may not be directly related to repetitive firing behavior. In response to intracellular depolarizing current in the tonic neuron illustrated in Fig. 2C, the initial inter- spike interval was 25 ms, yet the AHP duration of this neuron was close to the mean value of 130 ms for tonic cells (Table 1). In contrast, phasically discharging neurons in this study had a significantly shorter mean AHP duration of 48 ms. The characteristics of the AHP and the capability for repetitive firing of a particular neuron type are set by the complement of ionic conduc- tances present in the cell membrane (2, 4), but the results of the present study and those of Edwards et al. (21) indicate that in atrial neurons AHP characteristics and firing behavior may not be closely related. The membrane currents underlying these physiological properties in neurons in the pig heart have not been established.

In conclusion, this study shows that there exists a neuronal substrate within intracardiac ganglia for the convergence of inputs from vagal and cardiopulmonary cardiac nerves onto single intracardiac neurons. Vagal inputs to these neurons appear to be mediated by nicotinic postsynaptic receptors, whereas inputs from cardiopulmonary nerves appear to involve -adrenergic receptors. These convergent inputs can interact to modify the firing properties of intracardiac neurons. Evidence is presented to indicate that there may also be presynaptic modulation of vagal neurotransmission through intracardiac ganglia, mediated by inputs from cardiopulmonary nerves. The results presented here suggest that some interactions between sympathetic and parasympathetic inputs to the heart can occur at a premyocardial site within intracardiac ganglia. Inputs of extracardiac origin targeted phasic and some accom- modating neurons, whereas other accommodating and almost all tonically firing neurons lacked extrinsic inputs. Analysis of the correlations between innerva- tion pattern and electrophysiological properties of the three neuron types found in this study suggests that neurons of each type may play a distinct role in neuronal control of cardiac function.


The major issue addressed in this study was the possibility of vagosympathetic convergence on single intracardiac neurons. The identification of such neu- rons now raises further questions about the implica- tions of this convergence for neural control of the heart. To evoke orthodromically mediated APs in dually inner- vated neurons, activation of both inputs was required because neither input was capable on its own of depolar- izing these cells to threshold for firing. Neurons in this group must therefore integrate signals from both effer- ent limbs of the autonomic nervous system to produce an output. It may be that sympathetic inputs function to ‘‘prime’’ these neurons for firing by increasing their excitability, acting to increase neuronal sensitivity to succeeding vagal inputs. Alternatively, vagal inputs may function to prime sympathetically innervated neu- rons for firing. In any case, either input pathway may function as a ‘‘gate,’’ controlling transmission through the other pathway. In this way, these neurons may represent a major site for vagosympathetic interac- tions, allowing cooperation between these pathways in coordinating the neural regulation of cardiac function. One reflex mechanism that could make use of such an integrative process is the cardiac response to increased atrial filling. In this condition sympathetic activity is reflexly increased to elevate cardiac output by raising stroke volume and heart rate. However, strong sympa- thetic drive can elevate heart rate to a level too high for efficient ventricular ejection and it has been proposed that coactivation of vagal preganglionic cardiomotor axons would ensure that sympathetic overdrive does not result in heart rate exceeding the optimal level for maximizing cardiac output during increased atrial filling (36, 37). Intracardiac neurons that respond to inputs from both limbs of the autonomic nervous sys-

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tem may thus act as dual-input gates capable of balancing sympathetic and parasympathetic drive in control of the heart. A parallel to this type of dual-input integration has been described in prevertebral ganglia that innervate pelvic organs (16, 19, 34), in which parasympathetic and sympathetic inputs interact at the level of postganglionic neurons to control their firing. In this system parasympathetic preganglionic inputs synapse directly on postganglionic neurons inner- vating pelvic end organs, while sympathetic inputs reach the same postganglionic neurons either directly (16) or indirectly via interneurons (18). Sympathetic and parasympathetic convergence within the pelvic ganglia appears to be a key factor in regulating pelvic organ function during changes in relative activity of the two limbs of the autonomic nervous system. Evidence in the present study for a similar type of convergence on intracardiac neurons suggests that sympathetic-para- sympathetic interactions within cardiac ganglia, occur- ring at intracardiac neurons, may help balance the cardiac influences of extrinsic autonomic inputs.

This research was supported by an operating grant from the Medical Research Council of Canada. The author is grateful to the Heart and Stroke Foundation of Canada for a Research Scholarship. Address for reprint requests: F. M. Smith, Dept. of Anatomy and Neurobiology, Faculty of Medicine, Dalhousie Univ., Halifax, Nova Scotia, Canada B3H 4H7.

Received 7 July 1997; accepted in final form 16 October 1998.



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