Passive and Active Membrane Properties of Isolated Rat Intracardiac Neurons: Regulation by H- and M-Currents
J. Cuevas,
A. A. Harper,
C. Trequattrini, and
D. J. Adams
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101; and Department of Physiology and Pharmacology, University of Queensland, Brisbane, Queensland 4072, Australia
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ABSTRACT |
Cuevas, J., A. A. Harper, C. Trequattrini, and D. J. Adams. Passive and active membrane properties of isolated rat intracardiac neurons: regulation by H- and M-currents. J. Neurophysiol. 78: 1890-1902, 1997. The electrical characteristics of isolated neonatal rat intracardiac neurons were examined at 22 and 37°C using the perforated-patch whole cell recording technique. The mean resting membrane potential was
52.0 mV at 37°C and exhibited no temperature dependence. Lowering the temperature from 37 to 22°C decreased the mean input resistance from 854 to 345 M
, respectively, and reduced the membrane time constant approximately threefold yielding a Q10 of 2.1. Hyperpolarizing current pulses induced time-dependent rectification of the voltage response in all neurons at both temperatures. This behavior was previously not observed in dialyzed neurons and was reversibly blocked by external Cs+ (2 mM) but not Ba2+ (1 mM). Voltage-clamp studies of isolated neurons revealed a hyperpolarization-activated inward current. This inwardly rectifying conductance was isolated from other membrane currents using external Cs+. The time and voltage dependence of this current is consistent with Ih and contributes to the passive electrical properties of rat intracardiac neurons. In >90% of the neurons studied, depolarizing currents evoked firing of multiple, adapting, action potentials at 22°C. The number of action potentials increased with current strength producing a mean discharge of 5.1 (+100 pA, 1 s pulse), which was attenuated at 37°C to a mean of 1.4. The amplitude and kinetics of the slow, muscarine-sensitive inward and outward currents (IM) were highly temperature dependent. Lowering the temperature from 37 to 22°C reduced the steady-state current amplitude by approximately one-third and the rate of deactivation of IM by six- to ninefold at all voltages examined. The average Q10 for the time constant of deactivation of IM was 3.7 ± 0.3 (mean ± SE). Acetylcholine (ACh) induced tonic discharges in response to depolarizing currents (+100 pA, 1 s pulse) at both temperatures. This effect of ACh was inhibited by the muscarinic receptor antagonists, pirenzepine (100 nM), and mL-toxin (60 nM). At 37°C, a mean discharge of 1.5 was increased to 23.5 in the presence of ACh. A similar switch from phasic to tonic discharge was also produced by the potassium channel inhibitors, Ba2+ (1 mM) and uridine-5
-triphosphate (UTP; 100 µM), whereas cadmium, 4-aminopyridine, apamin, charybdotoxin, and dendrotoxin did not alter discharge activity. The pharmacological sensitivity profile and temperature dependence of the active membrane properties are consistent with the muscarine-sensitive potassium current (IM) regulating the discharge activity in rat intracardiac neurons.
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INTRODUCTION |
Postganglionic neurons of the mammalian intracardiac ganglia form an elaborate neural network with anatomic and neurochemical complexity, which suggests a functional sophistication beyond that of a simple relay for the vagus nerve. Mammalian intracardiac ganglia have been shown to function independently of the CNS (see Ardell 1994
), and intracardiac neurons have been shown to form functional afferent, efferent, and local circuits within the plexus (Hardwick et al. 1995
; Moravec and Moravec 1989
). Furthermore, several recent studies have indicated that intrinsic cardiac ganglia contain a heterogenous population of neurons, which have been identified on the basis of their electrical properties (Allen and Burnstock 1987
; Edwards et al. 1995
; Selyanko 1992
; Xi et al. 1994
).
Differences in the resting and firing characteristics of intracardiac neurons have been observed between conventional intracellular microelectrode recordings of isolated neurons from adult rat hearts at 36°C (Selyanko 1992
; Xi-Moy and Dun 1995
) and dialyzed whole cell patch-clamp recording in neonatal rat intracardiac neurons at 22°C (Xu and Adams 1992a
). For example, depolarizing current pulses evoked a single action potential in 85% of dialyzed neonatal rat intracardiac neurons, whereas trains of action potentials were observed in the majority of adult rat intracardiac neurons (Selyanko 1992
). Furthermore, adult rat and guinea pig intracardiac neurons investigated at 34-35°C using intracellular microelectrode recording exhibit a hyperpolarizationactivated inwardly rectifying current (Edwards et al. 1995
;Xi-Moy and Dun 1995
), which was not observed in dialyzed rat intracardiac neurons (Xu and Adams 1992a
). The discrepancy in these results may reflect the limitations of the techniques used and may not accurately reflect the function of cardiac neurons in vivo. For example, the requirement of a diffusible cytosolic second messenger for a functional response may underlie the absence of repetitive firing in dialyzed intracardiac neurons. The perforated-patch recording configuration, however, maintains cytosolic integrity and functional responses dependent on intracellular second messengers (Horn and Marty 1988
), which contribute to the electrical properties of neurons in vivo.
Tonic firing in adult rat intracardiac neurons in situ has been shown to be regulated by a transient outward K+current (IA) and a muscarine-sensitive K+ current (IM)(Xi-Moy and Dun 1995
), whereas a hyperpolarization-activated, nonselective cation current (Ih) has been proposed to promote rhythmic discharges in cardiac neurons of guinea pigs in situ (Edwards et al. 1995
). Cell dialysis-evoked changes in the properties and tonic regulation of these currents may account for the observed lack of repetitive firing in neonatal rat intracardiac neurons (Xu and Adams 1992a
). Application of the whole cell perforated-patch technique, however, has demonstrated the presence of IM and its modulation by muscarinic receptor activation in isolated intracardiac neurons of neonatal rats (Cuevas and Adams 1994
). In the present study, the passive and active electrical properties of isolated rat intracardiac neurons have been reexamined using the perforated-patch technique to determine the source of the reported differences in the firing properties of mammalian intracardiac neurons. Preliminary reports of some aspects of this work have been published (Cuevas et al. 1993
; Harper et al. 1995
).
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METHODS |
Preparation
The isolation and culture of parasympathetic neurons from neonatal rat intracardiac ganglia has been described previously (Xu and Adams 1992a
). Briefly, neonatal rats, 2-7 days old, were killed by decapitation, and the hearts were excised and placed in a saline solution containing (in mM) 140 NaCl, 3 KCl, 2.5 CaCl2, 0.6 MgCl2, 7.7 glucose, and 10 histidine (pH 7.2). The atria were separated and the medial region containing the pulmonary veins and superior vena cava was identified, isolated, and incubated in saline solution containing collagenase (1 mg/ml, Worthington-Biomedical Type 2, specific activity ~200 units/mg) at 37°C for 60 min. Clusters of ganglia were dissected from the epicardial ganglion plexus and dispersed by trituration in a high glucose culture medium (Dulbecco's Modified Eagle media), 10% fetal calfserum, 100 units/ml penicillin and 0.1 mg/ml streptomycin. The dissociated neurons were plated onto glass coverslips coated with laminin and incubated at 37°C under a 95% air: 5% CO2 atmosphere for 36-72 h.
Electrophysiological recording
Neurons plated on glass coverslips were transferred to a recording chamber (volume 0.5 ml) mounted on an inverted phase contrast microscope (×400 magnification) allowing isolated neurons to be identified. Membrane voltage responses and currents in intracardiac neurons (20-30 µm diam) were studied under current-clamp and voltage-clamp mode, respectively, using the whole cell patch-clamp technique. Electrical access was achieved through the use of the perforated-patch method (Horn and Marty 1988
; Rae et al. 1991
), which maintains the intracellular integrity and prevents the loss of cytoplasmic components and subsequent alteration of the functional response of these neurons.
Patch electrodes were pulled from thick-walled borosilicate glass (GC150F; Clark Electromedical Instruments, Reading, UK) and had resistances of 1-3 M
. The electrode was connected to a patch-clamp amplifier (List L/M-EPC 7) by an Ag-AgCl wire with an Ag-AgCl/0.15 M KCl-agar bridge in the recording chamber providing the earth reference. The patch electrode was back-filled with an intracellular solution containing 50 µg/ml nystatin and pluronic in 0.4% dimethyl sulfoxide (DMSO) or 360 µg/ml amphotericin B in 0.6% DMSO. To prevent nystatin- or amphotericin-induced seal disruption, the tips of the electrodes were first filled with antibiotic-free pipette solution and then back-filled with nystatin- or amphotericin-containing solution. Since no evidence of differences in the passive or active properties were noted using nystatin as compared with amphotericin-containing pipette solutions, the results from the two methods of patch perforation are presented together. Following gigaseal formation, the neurons were maintained under current-clamp conditions. Antibiotic incorporation into the membrane revealed the resting membrane potential (Em) and under voltage-clamp conditions a slow capacitive transient and a decrease in the series resistance (Rs) were apparent. Experiments were continued only if Em was greater than or equal to
40 mV, and Rs was reduced to
10 M
within 10 min of seal formation. Rupture of the membrane under the pipette tip was indicated by a rapid change in Em or the holding current, at which time the experiment was terminated. Cell input resistance (Ri) and membrane time constant (
) were measured from the voltage response to hyperpolarizing current pulses less than or equal to
50 pA. Membrane potentials were not corrected for liquid junction potentials, which by theoretical calculations (Barry 1994
) have values of 8.1 and 8.5 mV at 22 and 37°C, respectively.
Membrane potential and current records were recorded on video tape through an A/D recorder adaptor (PCM 4, Medical Systems, Greenvale, NY). Taped signals were subsequently filtered (4-pole Bessel filter, Frequency Devices 902) and transferred to a PC 80486/50 MHz computer by means of an A-D/D-A interface (Tecmar TL1-125 DMA). For computer analysis, voltage and current responses were filtered at 1-5 kHz (
3 dB), digitized and analyzed using Axotape and pClamp programs (Axon Instruments, Foster City, CA).
Solutions
The control external solution for perforated-patch whole cell recordings was physiological saline solution (PSS) containing (in mM) 140 NaCl, 3 KCl, 2.5 CaCl2, 1.2 MgCl2, 7.7 glucose and 10 N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid (HEPES)-NaOH buffered to pH 7.2 at the target temperature. The pipette "intracellular" solution contained (in mM) 75 K2SO4, 55 KCl, 5 MgSO4, and 10 HEPES (titrated to pH 7.2 with N-methyl-D-glucamine). Alterations in extracellular K+ concentration were made by equimolar substitution of K+ for Na+. The osmolality of the extra- and intracellular solutions was monitored with a vapor pressure osmometer (Wescor 5500, Logan, UT) and were in the range of 285-290 mmol/kg. The recording chamber was continuously superfused (2 ml/min) with indicated solutions at either room temperature (22°C) or 37°C, with a maximum deviation of 1°C in any individual procedure. The temperature of the superfusing solutions was controlled by Peltier thermoelectric elements (Melcor Electronics, Trenton, NJ) and monitored by an independent thermistor probe in the recording chamber (Yellow Springs Instruments, Yellow Springs, OH). During whole cell recordings, pharmacological agents were bath applied and/or focally applied using a pressure ejection device (68-101 kPa, Picospritzer II, General Valve Corporation, NY), and the pressure ejection pipette was positioned
50 µm from the neuronal soma to maximize the response to agonist application.
Temperature coefficients (Q10) were calculated using the van't Hoff equation
where k1 and k2 are the rates obtained at the lower (T1) and higher (T2) temperatures, respectively.
Statistical analysis
Data are presented as the means ± SE of the number of observations indicated, unless otherwise stated, and were compared using paired or unpaired t-tests. The Mann-Whitney rank-sum test or nonparametric Wilcoxon signed-rank test was used where the groups of data were not normally distributed and the t-test was inappropriate.
Reagents
All chemicals used were of analytic grade. The following drugs were used: ACh, 4-aminopyridine (4-AP), amphotericin B, apamin, mecamylamine chloride, (±)muscarine chloride, nystatin and uridine-5
-triphosphate (UTP), were purchased from Sigma Chemical (St. Louis, MO). Charybdotoxin (CTX) was obtained from Peptides International (Louisville, KY), dendrotoxin (DTX) from L. C. Laboratories (Woburn, MA), pirenzepine dihydrochloride from Research Biochemicals International (Natick, MA), and pluronic F-127 from Molecular Probes (Eugene, OR). m1-toxin was generously supplied by Dr. L. T. Potter (University of Miami School of Medicine, Miami, FL).
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RESULTS |
Results presented were obtained from isolated rat intracardiac neurons that exhibited stable resting membrane potentials greater than or equal to
40 mV and an overshooting action potential(s) evoked by depolarizing current pulses. All neurons examined were quiescent, with spontaneous discharges not being observed under control conditions at either 22 or 37°C (n > 160).
Effect of temperature on the passive and active membrane properties
Passive and active properties of cardiac ganglion neurons were examined at 22 and 37°C under current-clamp conditions using the perforated-patch technique. The mean resting membrane potential under control conditions was
50.0 ± 0.7 mV (n = 30) and
52.0 ± 0.6 mV (n = 16) at room temperature (22°C) and 37°C, respectively. This difference was not statistically significant (P = 0.217). In 10 neurons, increasing the bath temperature from 22 to 37°C had no significant effect on the resting membrane potential but significantly reduced both the input resistance (Ri) and the membrane time constant (
m; see Table 1). The Q10 obtained for the change in
m between 22 and 37°C was 2.1. No apparent difference in the results was obtained when the temperature was either increased or decreased.
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TABLE 1.
Passive electrical properties and discharge activity to depolarizing current pulses in isolated parasympathetic neurons of rat intracardiac ganglia
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The resting membrane potential was measured as a function of the extracellular K+ concentration (1, 3, 10, 20, 50, 100, and 140 mM) both at 22 and 37°C, and the data were fitted with the Goldman-Hodgkin-Katz (GHK) constant field equation (see Hille 1992
). The relative permeability to Na+ (PNa/PK) calculated from the GHK equation was 0.11 ± 0.01 (n = 4) at both 22 and 37°C, consistent with that reported by Xu and Adams (1992a)
using the dialyzed whole cell recording configuration.
Typical voltage responses to hyperpolarizing and depolarizing current pulses are shown in Fig. 1. Hyperpolarizing current pulses (
50 to
200 pA) evoked a time-dependent rectification of the voltage response at both temperatures in all neurons examined (Fig. 1, A and B). Suprathreshold depolarizing current steps (greater than or equal to +50 pA) at 22°C produced trains of action potentials that exhibit adaptation (Fig. 1A). In contrast, similar depolarizing current pulses evoked only single action potentials at 37°C (Fig. 1B). Current-voltage (I-V) relationships obtained for both peak and steady-state voltage responses to current pulses of varying intensity at 22 and 37°C are shown in Fig. 1, C and D, respectively. Whole cell I-V relationships obtained at both temperatures were outwardly rectifying. A linear regression fit of the I-V curve at hyperpolarized membrane potentials (
50 to
120 mV) yielded a slope conductance of 1.7 nS for the peak and 2.5 nS for the steady-state voltage response at 22°C. Raising the bath temperature to 37°C increased the slope conductance approximately twofold, to 3.4 and 4.1 nS for steady-state and peak voltage responses, respectively.

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| FIG. 1.
Voltage responses to depolarizing (+100 pA) and hyperpolarizing ( 50, 100, 150, 200 pA) current pulses (1 s) recorded from an isolated rat intracardiac neuron at 22°C (A) and 37°C (B). Resting membrane potential: 45 mV (22°C), 48 mV (37°C). The corresponding current-voltage (I-V) relationships obtained at 22°C (C) and 37°C (D) are plotted for peak ( ) and steady-state ( ) voltage responses.
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Effects of extracellular Ba2+ and Cs+
The K+ channel blockers, Ba2+ and Cs+, were used to investigate the ionic conductances underlying action-potential adaptation and time-dependent rectification in these neurons. Figure 2 shows representative voltage responses to current pulses obtained in the absence (Control) and presence of either extracellular Ba2+ (1 mM) or 1 mM Ba2+ + 2 mM Cs+ at 22 and 37°C. Superfusion of Ba2+-containing PSS depolarized the neurons by 4.3 ± 0.6 mV (n = 4) at 22°C and by 8.0 ± 1.3 mV (n = 3) at 37°C. The time-dependent rectification observed in response to hyperpolarizing current pulses was not appreciably altered by extracellular Ba2+ at either temperature. However, the amplitude of both the peak and steady-state voltage response to hyperpolarizing currents was increased in the presence of extracellular Ba2+ (Fig. 2, A and B). Bath perfusion with a solution containing both Ba2+ (1 mM) and Cs+ (2 mM) abolished the time-dependent rectification of the voltage response observed under control conditions and in the presence of Ba2+ alone (Fig. 2, A and B, bottom traces).

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| FIG. 2.
Effect of extracellular Ba2+ and Ba2+ plus Cs+ on voltage responses to depolarizing and hyperpolarizing current pulses at 22°C (A; ±50 pA) and 37°C (B; ±100 pA). A and B: records are shown for control conditions, in the presence of 1 mM Ba2+, and in the presence of 1 mM Ba2+ plus 2 mM Cs+. Resting membrane potentials: 56 mV (A) and 52 mV (B). C:I-V relation for each maneuver at 22°C ( and ) and 37°C ( and ) is plotted for the peak voltage response ( and ), and the steady-state response measured at the end of the current pulse ( and ).
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The firing frequency observed in response to depolarizing current pulses and on anode break was increased in the presence of extracellular Ba2+ (Fig. 2, A and B). Bariumincreased the number of action potentials discharged inresponse to depolarizing current pulses (+100 pA, 1 s), with the mean discharge rising from 4.5 to 11.3 at 22°C and 1.0 to 3.7 at 37°C (n = 4). In the presence of extracellular Cs+ and Ba2+, the action potential discharge observed in response to depolarizing current pulses was similar to that observed in the presence of Ba2+ alone.
The I-V relations for peak and steady-state voltage responses at both temperatures obtained in the absence (control) and presence of extracellular Ba2+ and Ba2+ + Cs+ are shown in Fig. 2C. Extracellular Ba2+ reduced the slope conductance determined from steady-state voltage responses at hyperpolarized membrane potentials (
60 to
150 mV) by approximately one-third, from 1.1 to 0.7 nS at 22°C and from 2.3 to 1.5 nS at 37°C. The slope conductance was further decreased to 0.5 nS (22°C) and 0.9 nS (37°C) by the subsequent addition of external Cs+. In the presence of external Cs+, the difference between the slope conductances calculated for peak and steady-state voltage responses to hyperpolarizing current pulses was abolished at both temperatures.
Voltage-clamp experiments were carried out to characterize the ionic current(s) underlying the time-dependent rectification observed in response to hyperpolarizing current pulses. Figure 3A shows typical membrane currents evoked by hyperpolarizing voltage steps (
65 to
125 mV) obtained in the absence and presence of external Ba2+ and Ba2+ + Cs+ at 22°C. Extracellular Ba2+ (1 mM) attenuated the steady-state inward current amplitude relative to control (Fig. 3Aii). Coapplication of Cs+ (2 mM) and Ba2+ (1 mM) further reduced the steady-state current amplitude by ~46% (n = 6), from
552 ± 68 pA (Ba2+) to
315 ± 62 pA (Ba2+ + Cs+; Fig. 3Aiii). The Cs+-sensitive current was determined by subtracting the current obtained in the presence of Ba2+ + Cs+ from that obtained in the presence of Ba2+ alone (Fig. 3Aiv). The time constant of activation of the Cs+-sensitive current was 827 ± 50 ms at
85 mV and decreased to 679 ± 53 ms at
95 mV (n = 6). Raising the temperature from 22 to 37°C in one cell increased the hyperpolarization-activated inward current amplitude and reduced the activation time constant fivefold at
95 mV (135 ms), with corresponding Q10s of 2.2 and 3.0, respectively. A plot of the I-V relationship for the Cs+-sensitive inward current measured in six neurons is shown in Fig. 3B. A fit of the data with a linear regression (solid line) gives a slope conductance of 4 nS and a null (zero-current) potential of
69 mV.

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| FIG. 3.
Hyperpolarization-activated inwardly rectifying current (Ih) at 22°C. A: membrane currents evoked by hyperpolarizing steps to test potentials between 65 and -125 mV from a holding potential of 55 mV in normal physiological saline solution (PSS; i), in PSS containing 1 mM Ba2+ (ii), and in PSS containing 1 mM Ba2+ and 2 mM Cs+ (iii). The Cs+-sensitive, Ba+-insensitive current (iv) was obtained by subtracting the currents recorded in the presence of Ba2+ from those recorded in the presence of Ba2+ plus Cs+. B: steady-state I-V relation of the Cs+-sensitive, Ba+-insensitive current (n = 6).
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Discharge characteristics of the soma membrane
The discharge characteristics of neurons electrically accessed using the perforated-patch method were studied under current clamp at 22 and at 37°C. Typical responses of a neuron challenged with depolarizing current pulses at 22°C (A) and 37°C (B) are shown in Fig. 4. Increasing suprathreshold depolarizing current amplitude at 22°C produced a growing train of action potentials that became sustained throughout the current pulse. The discharge frequency increased as a function of current strength but declined as a function of time (Fig. 4C). The action-potential overshoot frequently decreased during the course of depolarization. Depolarizing current pulses evoked multiple, adaptive firing in >90% of the neurons examined at 22°C (n > 30). Peak discharge rate, measured at the onset of discharge, was17 ± 1.6 Hz (n = 4) for +100-pA current pulses. In some neurons (10 of 13), however, the number of action potentials declined when challenged with current pulses
250 pA, suggesting faster adaptation to stronger depolarization (data not shown).

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| FIG. 4.
Neuronal discharge characteristics as a function of temperature and current strength. Action potential discharges evoked by 1-s depolarizing current pulses obtained at 22°C (A) and 37°C (B). Frequency-time plots of the discharge in response to depolarizing currents as a function of the elapsed time from the start of the current pulse at 22°C (C) and 37°C (D).
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In contrast, most neurons at 37°C respond to depolarizing current steps with high-frequency phasic bursts of action potentials (Fig. 4B). Two distinct patterns of adaptation were observed: in 85% of the neurons examined (17 of 20), the somata fired only a single action potential in response to depolarizing current pulses, even at current magnitudes significantly greater than threshold (
200 pA). The remaining 15% of neurons fired short bursts of action potentials (5.3 ± 0.6) in response to +200-pA depolarizing current pulses. A peak firing frequency of 33.6 ± 1.9 Hz (n = 3) was obtained for a +100-pA current pulse. The discharge characteristics of neurons recorded at both temperatures are given in Table 1. Afterhyperpolarizations were observed after each action potential and on termination of the depolarizing pulse.
Effects of muscarinic receptor activation on the action potential and discharge characteristics
The Ba2+-induced increase in repetitive firing suggests the involvement of the Ba2+-sensitive M-current (IM), which is inhibited by muscarinic receptor activation (Adams et al. 1982
), in the adaptation response of these neurons. The effect of muscarinic receptor activation on the discharge characteristics was investigated to determine whether IM underlies the adaptation of action potential firing in rat intracardiac neurons at 22°C. ACh (100 µM) and the ganglionic nicotinic receptor antagonist, mecamylamine (3 µM), were coapplied focally to selectively activate muscarinic receptors. Application of ACh depolarized neurons by 5.9 ± 1.2 mV (n = 9), which was associated with an ~35% increase in input resistance. In all neurons studied (n > 30), brief current pulses (+140 pA, 20 ms) evoked a single action potential under control conditions at 22°C (Fig. 5Ai), whereas after focal application of ACh, current pulses of similar amplitude evoked repetitive firing (7 ± 1.0, n = 3; Fig. 5Aii). Similar changes in discharge activity were observed with focally applied muscarine (5 µM; data not shown). ACh modified the action potential waveform, reducing the overshoot from +53 ± 1 mV to +43 ± 1 mV(n = 3) but not appreciably altering the duration (Fig. 5B). However, if the membrane potential was current clamped at
60 mV in the presence of ACh, the action potential overshoot was unchanged (+54 mV). Figure 5C shows representative voltage responses to 1-s depolarizing current pulses (+50 pA) in the absence (i) and presence of 100 µM ACh (ii) and 100 µM ACh + 100 nM pirenzepine (iii). ACh increased the number of action potentials evoked approximately threefold, from 4.3 ± 0.2 to 13.5 ± 0.4 (n = 6), which was statistically significant (P < 0.01). Bath application of the M1 muscarinic receptor antagonist pirenzepine (100 nM) antagonized the ACh-induced increase in action-potential firing (Fig. 5C), reducing the number of action potentials evoked in response to depolarizing current pulses to 6.3 ± 0.2 (n = 3). Neither mecamylamine nor pirenzepine alone altered the resting membrane potential or firing characteristics.

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| FIG. 5.
Acetylcholine (ACh) modulation of discharge activity is mediated by muscarinic receptor activation. A: action potentials recorded in response to brief current pulses (+140 pA, 10 ms) in control, PSS solution (i) and in the presence of focally applied 100 µM ACh coapplied with 3 µM mecamylamine (ii). B: superimposed traces of action potentials, shown on expanded time scale, obtained in the absence (A) and presence of ACh (B). In the absence (control) and presence of ACh (+ACh), the resting membrane potentials were 54 and 42 mV, respectively. C: discharge activity recorded in response to a depolarizing current pulse (+50 pA, 1 s) in the absence (i) and presence of 100 µM ACh (ii) and after coapplication of ACh with muscarinic receptor antagonist, pirenzepine (100 nM; iii). Temperature 22°C.
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Temperature dependence of muscarine-sensitive K+ current (Im) amplitude and kinetics
The ACh-sensitive current mediating the firing adaptation was studied using the whole cell perforated-patch configuration to determine and compare its voltage- and time-dependent properties to those previously reported for IM. Application of 100 µM ACh in the presence of mecamylamine (3 µM) to the cell soma at 22°C evoked a slow decrease in the holding current at the resting membrane potential (
50 mV) and inhibited the outward current evoked in response to a slow voltage ramp (
60 mV to +50 mV, 50 mV/s; data not shown). Muscarinic receptor activation inhibited outward currents in >90% (23 of 25) of the neurons examined, and this inhibition was antagonized by either 100 nM pirenzepine (n = 7) or 60 nM m1-toxin, a specific M1 muscarinic receptor antagonist (n = 3) (Max et al. 1993
).
To examine the kinetics and temperature dependence of the M-current, neurons were voltage clamped at
30 mV and hyperpolarized by voltage jumps (500 ms duration) in the presence of 3 mM Cs+, 1 mM 4-AP and 300 nM tetrodotoxin. Figure 6A shows typical membrane currents evoked at 22°C (a) and 37°C (b) when the membrane was stepped from
30 to
110 mV in 10-mV increments. An instantaneous inward current was observed at the onset of membrane hyperpolarization followed by a much slower inward relaxation. On return to the holding potential (
30 mV), the instantaneous current was reduced and followed by a slow outward relaxation. I-V relations obtained for the instantaneous and steady-state currents at 22 and 37°C are shown in Fig. 6B. The steady-state current amplitude was reduced at all voltages by at least one-third on lowering the temperature from 37 to 22°C. The inward current reversed at approximately
85 mV, which is close to that predicted by the Nernst equation for K+-selective electrode. The muscarine-sensitive component of the inward and outward current relaxations obtained at 22 and 37°C is demonstrated in Fig. 6C. The slow inward and outward relaxations are reversibly inhibited in the presence of 20 µM muscarine. These slow, muscarine-sensitive inward and outward relaxations are characteristic of the deactivation and activation of the noninactivating, time- and voltage-dependent K+ current, IM(Adams et al. 1982
).

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| FIG. 6.
Voltage dependence of M-current amplitude in rat intracardiac neurons at 22 and 37°C. A: membrane currents recorded in response to step hyperpolarizations in 10-mV increments from 30 to 110 mV at 22°C (a) and 37°C (b). Bath solution contained 3 mM Cs+, 1 mM4-aminopyridine (4-AP) and 300 nM tetrodotoxin (TTX). B: I-V relations obtained for instantaneous ( and ) and steady-state ( and ) currents at 22 ( and ) and 37°C ( and ). Lines were best fit to data using a 2nd-order regression and the reversal potentials obtained for IM were 83 mV (22°C) and 75 mV (37°C). C: superimposed traces of membrane currents evoked from the same neuron by step hyperpolarizations from 30 to 50 mV in the absence and presence of 20 µM muscarine at 22and 37°C.
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Figure 7A shows superimposed traces of IM at 22 and 37°C obtained in response to hyperpolarizing voltage steps from
30 to
40 mV (a),
50 mV (b), and
60 mV (c). The deactivation kinetics of IM at both 22 and 37°C were best fit by a single exponential function. At
60 mV, the mean time constant for deactivation was increased 8.8-fold from 12.0 ± 5.0 ms at 37°C to 106.0 ± 21.0 ms at 22°C(n = 6). The logarithm of the time constants for deactivation of IM at 22 and 37°C are plotted as a function of membrane potential in Fig. 7B. The linear relationship between ln
and membrane potential yields an e-fold change in
for a 32- and 19-mV change in membrane potential at 22 and 37°C, respectively. The Q10 of
varied as a function of voltage with values of 3.23, 3.51, and 4.27 obtained at
40,
50, and
60 mV, respectively, yielding a mean Q10 of3.7 ± 0.3 over this voltage range.

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| FIG. 7.
Voltage and temperature dependence of M-current kinetics. A: superimposed membrane currents evoked by step hyperpolarizations from 30 mV to the indicated test potentials (a-c) at 22°C (top traces) and 37°C (bottom traces). Current relaxations were best fit by single exponential functions(- - -) with the following time constants ( ): a, 202.0 ms (22°C) and 33.7 ms (37°C); b, 126.4 ms (22°C) and 16.4 ms (37°C); and c, 120.5 ms (22°C) and 8.5 ms (37°C). B: logarithm of time constants for IM ( , ms) plotted as a function of test potential. Lines represent linear regressions to the data points (mean ± SE, n = 6) with correlation coefficients of 0.932 and 0.995, and slopes of 0.031 and 0.052 at 22 and 37°C, respectively.
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To determine whether IM contributes to the temperature-dependent adaptation of action-potential firing, ACh was applied to the soma under current-clamp conditions at 37°C. Figure 8 shows representative voltage responses to depolarizing current pulses (+100 pA, 1 s), recorded from the same neuron in the absence and presence of 100 µM ACh at 22°C (A) and 37°C (B). At 37°C, ACh depolarized the neuron by 5.5 ± 0.7 mV (n = 4), and the number of action potentials evoked by depolarizing current steps (+100 pA, 1 s) was increased from 2.3 to 36.5. In all neurons studied, ACh (100 µM) evoked a greater increase in the firing frequency at 37°C than at 22°C. Current clamping the membrane potential at
60 mV in both the absence and presence of ACh reduced the number of action potentials evoked by superimposed depolarizing current pulses, but action-potential firing in the presence of ACh remained substantially elevated above control (data not shown).

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| FIG. 8.
Conversion from phasic to tonic discharge in rat intracardiac neurons by ACh. Voltage responses to depolarizing current pulses (+100 pA, 1 s) in normal PSS and during focal application of 100 µM ACh at 22°C (A) and 37°C (B). ACh (100 µM) depolarized the neuron from 54 to 46 mV.
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Effects of K+ channel inhibitors on action-potential discharge characteristics
Several K+ channel currents, including the transientA-current (IA), the slowly inactivating D-current (ID), the Ca2+-dependent K+ current (IK,Ca), and a muscarine-sensitive K+ current (IM), have been reported to regulate repetitive firing and adaptation in autonomic ganglion neurons (see Adams and Harper 1995
). The effects of specific K+ channel inhibitors on the discharge activity and adaptation were examined in rat intracardiac neurons at 22 and 37°C.
The involvement of the transient outward K+ currents, IA or ID, were investigated using 4-AP and the peptide toxin, DTX, respectively. Neither 4-AP (10 µM to 1 mM; n = 4), which inhibits the delayed rectifier, IA and ID in hippocampal neurons (Storm 1988
; Wu and Barish 1992
), nor DTX(10-300 nM; n = 6), which blocks ID in primary afferent and hippocampal neurons (Stansfeld and Feltz 1988
; Wu and Barish 1992
), altered the discharge activity in response to depolarizing currents at either temperature (Fig. 9, A and B). The effects of charybdotoxin and apamin, which selectively block the large and small conductance Ca2+-dependent K+ channels (see Dreyer 1990
), respectively, were also examined. Charybdotoxin at a concentration of 100 nM, which has been previously shown to completely inhibit Ca2+-activated K+ currents in rat intracardiac neurons (Xu and Adams 1992a
), failed to affect the discharge activity in response to depolarizing currents (n = 4, Fig. 9C). Similarly, apamin (30-100 nM) did not appreciably change the discharge pattern in rat intracardiac neurons (n = 3). In the presence of 100 µM Cd2+ externally, which completely inhibits depolarization-activated Ca2+ currents in rat intracardiac neurons (Xu and Adams 1992b
), neither the resting membrane potential nor action potential firing behavior observed at 37°C was affected (n = 4, data not shown). In contrast, superfusion of UTP (100 µM), which selectively inhibits IM in bullfrog sympathetic neurons (Adams et al. 1982
), substantially increased action potential firing inresponse to step depolarizing currents (Fig. 9D). In four neurons studied, the number of action potentials evoked by a 1-s, +100-pA current pulse was increased approximately fourfold in the presence of 100 µM UTP at 37°C.

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| FIG. 9.
E f f e c t s o f p h a r m a c o l o g i c a lblockers of K+ channels and uridine-5 -triphosphate (UTP) on the discharge activity evoked by depolarizing current pulses in rat intracardiac neurons. A-D: representative traces of the discharge activity observed in response to a depolarizing pulse (+100 pA, 1 s) in the absence (top traces) and presence (bottom traces) of inhibitors of the transient K+ A-channel, 4-AP (1 mM 4-AP, A), the D-channel, dendrotoxin (10 nM DTX, B), the Ca2+-activated K+ channel, charybdotoxin (100 nM ChTX, C), and an inhibitor of M-current, UTP (10 µM UTP, D). Temperature 37°C.
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DISCUSSION |
Regulation of passive membrane properties by Ih
The passive and active membrane properties of isolated neonatal rat intracardiac neurons studied using the whole cell perforated-patch recording configuration are qualitatively distinct from those observed under dialyzing conditions and displayed marked temperature sensitivity. There was no significant difference between the resting membrane potential or input resistance (Ri) measured in neurons at 22°C using the perforated-patch and dialyzed whole cell recording configurations (Table 1). Raising the bath temperature from 22 to 37°C failed to significantly alter the resting membrane potential, but Ri was reduced by >60% and
m threefold. The temperature dependence of
m corresponded to a Q10 of 2.1, and the temperature sensitivity of Ri was similar to that reported in hippocampal CA1 neurons (Thompson et al. 1985
).
The voltage response to hyperpolarizing current pulses exhibited time-dependent rectification, which was not observed in dialyzed neurons (Xu and Adams 1992a
). The ionic conductances underlying time-dependent rectification were identified using the K+ channel blockers, Ba2+ and Cs+. Extracellular Ba2+ (1 mM) depolarized the neuron by 4.3 mV at 22°C using the perforated-patch configuration, compared with 13.0 mV in dialyzed rat intracardiac neurons (Xu and Adams 1992a
). At 37°C, the Ba2+-induced depolarization was 8.0 mV, which is consistent with the depolarization reported in adult rat and guinea pig intracardiac neurons (Edwards et al. 1995
; Xi-Moy and Dun 1995
). Hyperpolarization-activated time-dependent rectification was still observed in the presence of extracellular Ba2+; however, the input resistance was increased approximately one-third at both 22 and 37°C. The addition of external Cs+ further increased input resistance and abolished the time-dependent rectification. Under voltage clamp, membrane hyperpolarizations greater than or equal to
60 mV activated an inward current that was attenuated by extracellular Cs+ but not Ba2+, consistent with Ih. This hyperpolarization-activated inward current exhibited a voltage-dependent activation time constant (0.14 s at
95 mV) similar to that reported in guinea pig intracardiac neurons (Edwards et al. 1995
). Recently, Ih has been shown to be regulated by intracellular cyclic nucleotides in mammalian central and peripheral neurons (see Pape 1996
). The wash out of cyclic nucleotides following cell dialysis may account for the absence of Ih-mediated time-dependent rectification in dialyzed rat intracardiac neurons (Xu and Adams 1992a
). The amplitude and time constant of Ih were temperature sensitive, with Q10s of 2.2 and 3.0, respectively. A Cs+-sensitive, hyperpolarization-activated inward current has also been observed in adult rat and guinea pig intracardiac neurons (Edwards et al. 1995
; Xi-Moy and Dun 1995
) and has been proposed to contribute to spontaneous activity in P-cells (Edwards et al. 1995
).
Regulation of active membrane properties by Im
The firing characteristics in response to depolarizing current pulses were markedly different in neurons examined using the perforated-patch and dialyzed recording configurations. In all neurons studied at 22°C using the perforated-patch technique, suprathreshold depolarizing current pulses evoked trains of action potentials that exhibited adaptation. In contrast, at 37°C, only a single action potential was elicited in the majority of neurons by depolarizing current pulses. Similarly, 65-75% of cultured neurons of guinea pig cardiac ganglia (Allen and Burnstock 1987
), >90% of in vitro intrinsic cardiac neurons of the pig (Smith et al. 1992
), and 65% of canine cardiac ganglion neurons in situ (Xi et al. 1994
) fired only a single action potential at the onset of the depolarizing pulse at 34-37°C. Intrinsic cardiac neurons have been classified on the basis of their responses to depolarizing pulses. With the use of this criteria, three distinct cell types have been identified at 34-37°C in the rat [type I (Ib and Im) and type II neurons] (Selyanko 1992
), canine (S-, R-, and N-cells) (Xi et al. 1994
), and guinea pig (AHs, AHm, and M cells, Allen and Burnstock 1987
; S-, SAH-, and P- cells, Edwards et al. 1995
). In response to depolarizing current pulses Type II neurons, S-cells and AHs cells fire single action potentials; whereas type Ib neurons, R-cells, and AHm cells fire short bursts of action potentials. M-cells and type Im neurons produce nonadapting trains of action potentials and often exhibit spontaneous discharges. N-cells fail to produce action potentials even with large depolarizations. It appears that the neurons of the present investigation are similar in firing characteristics to the S- and R-, AH, and type Ib and II cells. M-type cells were of smaller diameter than AH types (<10 µm) (Allen and Burnstock 1987
), and such neurons were not investigated in the present study.
The K+ channel blocker, Ba2+ (1 mM), increased the action potential firing frequency at 22°C and initiated repetitive discharge in neurons at 37°C. External Ba2+ has also been reported to induce repetitive discharges in adult rat intracardiac neurons at 34°C (Xi-Moy and Dun 1995
). Action-potential firing in response to depolarizing current pulses was also promoted at 22 and 37°C by focal application of 100 µM ACh in the presence of the ganglionic nicotinic receptor antagonist, mecamylamine. Furthermore, ACh had a greater effect on the firing frequency at 37°C than at 22°C. Although ACh depolarized the neurons, the repetitive firing was still apparent when the resting membrane potential was held at
60 mV before application of the depolarizing current pulse. The effect of ACh on action potential firing at 37°C was mimicked by muscarine and reversibly inhibited by the muscarinic receptor antagonist, pirenzepine (100 nM). Muscarinic receptor activation has also been shown to induce tonic firing in isolated guinea pig intracardiac neurons (Allen and Burnstock 1990
) and canine intracardiac neurons in situ (Xi-Moy et al. 1993
).
In voltage-clamped neurons, muscarinic receptor activation by ACh evoked a slow decrease in the holding current, which was associated with an increase in input resistance. The inhibition by ACh of outward currents obtained in response to slow voltage ramps at potentials positive to
60 mV was antagonized by the M1-muscarinic receptor antagonists, pirenzepine and m1-toxin (Cuevas and Adams 1994
). The voltage and time dependence of muscarine-sensitive K+ currents in neonatal rat intracardiac neurons exhibit properties characteristic of IM (see Brown 1988
) and consistent with those recently described in adult rat intracardiac neurons (Xi-Moy and Dun 1995
). The time constant of deactivation of IM (
) was voltage dependent, changing e-fold for a 19- and 32-mV change in membrane potential at 37 and 22°C, respectively. The amplitude and kinetics of IM in rat intracardiac neurons were highly temperature dependent. Lowering the temperature from 37 to 22°C reduced the steady-state current amplitude by approximately one-third and the rate of deactivation of IM by six- to ninefold at all voltages examined. The average Q10 for the time constant of deactivation of IM was 3.7 ± 0.3, which indicates that the kinetic transitions in M-channel gating are associated with a high activation energy of ~100 kJ/mol. The high temperature dependence of IM kinetics is similar to that observed for the activation kinetics of K+ channels in rat skeletal muscle (Q10 of 6 below 10°C) (Beam and Donaldson 1983
), minK channels cloned from rat kidney and expressed in Xenopus oocytes (Q10 of 4.0 at + 20 mV) (Busch and Lang 1993
), and high-threshold, voltage-activated Ca2+ currents in chick sensory neurons (Q10 of 5 between 17 and 37°C) (Nobile et al. 1990
). The high temperature sensitivity of IM kinetics is likely to account for the temperature dependence of the discharge characteristics observed in rat intracardiac neurons.
Further evidence that the muscarine-sensitive K+ current, IM, regulates discharge activity in neonatal rat intracardiac neurons was provided by the observation that the specific K+ channel inhibitors including, 4-AP, charybdotoxin, dendrotoxin, apamin, and tetraethylammonium (TEA), failed to promote repetitive firing in response to depolarizing current pulses at 22 and 37°C. In contrast, UTP induced repetitive firing in rat intracardiac neurons at 37°C. Both UTP and Ba2+, which facilitate multiple firing, have been shown to depress IM (Adams et al. 1982
), whereas charybdotoxin and apamin, inhibitors of Ca2+-activated K+ currents, had no effect on firing frequency. Furthermore, inhibition of depolarization-activated Ca2+ currents and Ca2+-activated K+ currents by extracellular Cd2+ (Xu and Adams 1992a
,b
) did not significantly increase the number of action potentials evoked by depolarizing current pulses in isolated rat intracardiac neurons. In contrast, the addition of 100 µM Cd2+ has been reported to increase excitability and evoke multiple action potentials in response to small depolarizing current steps in sympathetic neurons of rat superior cervical ganglion(Davies et al. 1996
). Muscarine has previously been suggested to promote multiple firing predominantly by inhibiting a Ca2+-activated K+ conductance in guinea pig intracardiac neurons (Allen and Burnstock 1990
); however, in rat intracardiac neurons, IM and not a Ca2+-activated K+ conductance, appears to mediate muscarinic-induced repetitive firing. In contrast to neonatal rats, rhythmic discharges were induced by superfusion of TEA (10 mM) and 4-AP (1 mM) in addition to Ba2+ in adult rat intracardiac neurons (Xi-Moy and Dun 1995
), suggesting that both IA and IM may regulate tonic firing. Repetitive firing in rat intracardiac neurons was not suppressed by external Cs+, suggesting that Ih is not involved in the regulation of tonic firing. However, in guinea pig intracardiac P-cells, spontaneous discharges have been shown to be inhibited by external Cs+ (Edwards et al. 1995
).
In summary, IM appears to regulate repetitive firing in neonatal rat intracardiac neurons. IM has previously been shown to be responsible for adaptation of firing in rat sympathetic neurons, shortening action-potential trains evoked by prolonged depolarization (Brown et al. 1982
). Muscarinic receptor-mediated inhibition of IM and the discharge characteristics of these neurons is more pronounced at 37°C than at 22°C. This temperature sensitivity is consistent with the finding that the amplitude and rates of activation and deactivation of IM is increased at higher temperature in rat intracardiac neurons similar to that observed in rat sympathetic and guinea pig hippocampal neurons (Brown 1988
).
Functional significance
Autonomic ganglion neurons can be differentiated on the basis of the response of the soma membrane to depolarizing current pulses and are typically classified as being either tonic (slowly adapting) or phasic (rapidly adapting) (see Adams and Harper 1995
). Differences in potassium channel expression in pre- and paravertebral rat sympathetic neurons have recently been shown to make significant contributions to their firing properties (Wang and McKinnon 1995
). The anatomic distribution of the functional types of neurons within mammalian sympathetic ganglia has been documented and suggested to be related to the efferent activity of the tissue innervated by the neuron (McLachlan 1987
). Although at least three morphologically and functionally distinct types of intracardiac neuron have been identified, collateral information for mammalian epicardial and intraseptal ganglia is at present lacking. Intrinsic cardiac neurons of the mammalian heart are likely to serve not only an integrative function, facilitating local feedback and autoregulation, but may also exert local regulation over cardiac performance.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood Institute Grant HL-35422, National Health and Medical Research Council of Australia Grant 961138 to D. J. Adams, Training Grant HL-07188 to J. Cuevas, and a Travel Research Award from the Wellcome Trust and the Dale Fund of the Physiological Society to A. A. Harper.
Present addresses: J. Cuevas, Dept. of Biology, University of California, San Diego, La Jolla, CA 92093; D. J. Adams, Dept. of Physiology and Pharmacology, University of Queensland, Brisbane, QLD 4072, Australia; A. A. Harper, Dept. of Anatomy and Physiology, University of Dundee, Dundee DD1 4HN, Scotland, UK; C. Trequattrini, Dept. of Cellular and Molecular Biology, University of Perugia, 06100 Perugia, Italy.
 |
FOOTNOTES |
Address for reprint requests: D. J. Adams, Dept. of Physiology and Pharmacology, University of Queensland, Brisbane, QLD 4072, Australia.
E-mail: dadams{at}plpk.uq.edu.au
Received 14 January 1997; accepted in final form 3 June 1997 .
 |
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