Ca2+ and
K+ currents regulate accommodation
and firing frequency in guinea pig bronchial ganglion neurons
Allen C.
Myers
The Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland
21224-6821
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ABSTRACT |
Intracellular microelectrode recordings were
obtained from neurons located in adult guinea pig bronchial
parasympathetic ganglia in situ to determine the calcium and potassium
currents regulating repetitive action potential activity and firing
rates by these neurons. Neurons in these ganglia respond to prolonged
suprathreshold depolarizing current steps with either a burst of action
potentials at the onset of the stimulus (accommodating or phasic
neurons) or repetitive action potentials throughout the stimulus
(nonaccommodating or tonic neurons). Instantaneous and adapted firing
rates during prolonged threshold and suprathreshold stimuli were lower
in tonic than in phasic neurons, indicating a longer interspike
interval between repetitive action potentials in tonic neurons. In
tonic neurons, blockade of A-type current with 4-aminopyridine
increased accommodation; 4-aminopyridine or apamin decreased the
interspike interval in tonic neurons. Calcium-free buffer, cadmium
ions, or
-conotoxin GVIA also increased accommodation in tonic
neurons but did not affect the interspike interval; nifedipine or
verapamil did not affect the tonic firing pattern. Accommodation in
phasic neurons could be decreased by a conditioning hyperpolarization step of the resting potential, which could be subsequently blocked by
4-aminopyridine or calcium-free buffer. Accommodation in phasic neurons
could also be decreased by apamin or barium ions: the repetitive action
potentials observed during these treatments could be reversed by
cadmium ions or calcium-free buffer. These results indicate that tonic
and phasic neurons in guinea pig bronchial parasympathetic ganglia have
similar types of calcium currents, but potassium channels may
ultimately regulate the accommodation pattern, the firing rate, and,
consequently, the output from these neurons.
parasympathetic; tonic neurons; phasic neurons; trachea; bronchoconstriction; calcium channels; potassium channels; N-type
channel;
-conotoxin GVIA; apamin; 4-aminopyridine
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INTRODUCTION |
TWO DISTINCT ACTION POTENTIAL PATTERNS have been
described by intracellular studies of neurons in a number of different
autonomic ganglia (10, 15, 28, 32). When stimulated with a
long suprathreshold constant-current depolarizing step, one population of neurons responds with a brief burst of action potentials at the
onset of the depolarization, accommodating to the stimulus, and the
other population responds with repetitive action potentials persisting
throughout the duration of the stimulus, not accommodating to the
stimulus. Neurons displaying these different discharge characteristics
have been designated "phasic" and "tonic," respectively (14, 15). In most autonomic ganglia, the two types of neurons appear to
have different active and passive membrane properties (10, 15) and may
have different anatomic characteristics as well (6). Tonic neurons
generally exhibit a prominent potassium current referred to as A-type
current, which increases the interspike interval between action
potentials, whereas accommodation to the stimulus in most phasic
neurons is due to activation of calcium-activated potassium current(s)
and/or the M-type current (1, 10, 16, 31).
Parasympathetic neurons in guinea pig bronchial ganglia display tonic
or phasic action potential patterns but have similar neurophysiological
and anatomic characteristics (28). Accommodative properties have also
been determined for intrinsic ganglion neurons located in guinea pig
(19) and rat (4) tracheae, and a preliminary study (23) identified
tonic and phasic firing patterns by neurons in human bronchial
parasympathetic ganglia; other studies on ferret (5, 8) or cat (22)
tracheal neurons did not address these properties. In guinea pig
bronchial ganglia, most phasic neurons respond to a suprathreshold
current stimulus, with action potentials decreasing in amplitude during
the burst similar to those of guinea pig parasympathetic phasic neurons
in urinary bladder ganglia (16) or sympathetic neurons in celiac
ganglia (31). By contrast, phasic neurons in sympathetic (32), vagal
sensory (11), airway parasympathetic (7), and vesical pelvic
parasympathetic (15) ganglia generally respond with a burst of spikes
of an amplitude similar to that observed at the onset of the stimulus.
With very prolonged (5-s) stimuli, most neurons in rat tracheal ganglia respond with rhythmic, high-frequency (50- to 90-Hz) bursts of action
potentials of equal amplitude throughout the stimulus and display a
long (3-s) calcium-dependent afterhyperpolarization (4). Neurons in
guinea pig bronchial ganglia do not display a prolonged (>500-ms)
calcium-dependent spike afterhyperpolarization, and only subtle
differences in active and passive membrane properties between guinea
pig bronchial tonic and phasic parasympathetic neurons were observed
(28); consequently, the membrane properties responsible for tonic and
phasic action potential patterns and firing frequency in this
preparation are unknown.
Accommodative properties of neurons in bronchial parasympathetic
ganglia may greatly affect their ability to relay preganglionic stimuli
(24, 28) and, consequently, parasympathetic tone in the airway (24).
Accommodation in guinea pig bronchial neurons is altered by immediate
hypersensitivity (allergic) reactions; specific antigen challenge in
vitro causes neurons that normally respond with a phasic action
potential pattern to respond with a tonic pattern (29). Our
understanding of the mechanism by which inflammatory mediators affect
accommodation is limited by the lack of information regarding the ionic
currents that govern the repetitive action potential activity. Although
there have been several voltage-clamp studies (2, 4) characterizing the
different currents in airway parasympathetic neurons, none have
determined the role these currents play in regulating action potential accommodation or firing frequency. The present study further
characterizes differences in active membrane properties of tonic and
phasic neurons in guinea pig bronchial parasympathetic ganglia. The
role voltage- and ionic-dependent currents have in regulating the
accommodation properties and frequency of action potentials by
these neurons is also examined.
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MATERIALS AND METHODS |
Male albino guinea pigs (180-300 g) were asphyxiated with carbon
dioxide and exsanguinated; the bronchus was isolated, cut longitudinally along the ventral surface, opened, and then tightly pinned as a sheet to the floor of a Sylgard-coated recording chamber (0.2-ml volume) with Z-shaped pins. Ganglia were located in the extrachondral plexus near the peribronchial nerves and were visualized, without staining, after removal of the overlying connective tissue by
fine dissection. The tissue was equilibrated with flowing (5-8 ml/min) Krebs bicarbonate buffer at 36°C for at least 1 h in the recording chamber before experimentation. The composition of the Krebs
buffer was (in mM) 118 NaCl, 5.4 KCl, 1 NaH2PO4,
1.2 MgSO4, 1.9 CaCl2, 25 NaHCO3, and 11.1 dextrose and was
bubbled with 95% oxygen-5% carbon dioxide at a pH of 7.4. To avoid
precipitation of cadmium ions, the tissue was isolated and equilibrated
as above, but, for the cadmium experiments, the tissue was temporarily
superfused with a solution containing the same sodium, potassium,
magnesium, calcium, and chloride ion and dextrose concentrations and
was buffered with HEPES (30 mM), pH 7.4, before the addition of cadmium to this superfusate.
Micropipettes were fabricated from thick-walled capillary stock (0.5-mm
ID, 1.0-mm OD; World Precision Instruments, Sarasota, FL) by a
Brown-Flaming microelectrode puller (model P-87, Sutter Instruments,
San Rafael, CA). Electrodes were filled with 3.0 M potassium chloride
(pH 7.4), and the electrolyte in the micropipette was connected by a
chloridized silver wire in an electrode holder (Axon Instruments,
Foster City, CA) by a headstage to an electrometer (Axoclamp 2A, Axon
Instruments). A silver-silver chloride pellet in the bath was connected
to a headstage ground. The electrode DC resistance in Krebs solution
ranged between 60 and 70 M
. Intracellular data (voltage and current)
were displayed on-line on an oscilloscope and a chart recorder and
stored on digital audiotape. Data epochs were digitized by a Macintosh
computer equipped with a data-translation interface and displayed and
analyzed off-line with an oscilloscope simulation-and-analysis program
(AxoData and AxoGraph, Axon Instruments). The delivery of
constant-current pulses through the microelectrode was also controlled
by the computer. Intracellular recordings were performed with the
electrometer in either discontinuous current clamp (3.0- to 4.0-kHz
sampling rate) or active bridge mode. Quantitative voltage clamp was
not used because of the location of the ganglia deep within the tissue
and bath and because of space-clamp distortion from the nonisopotential
spread of voltage over the neuronal soma, dendrites, and axon(s) of the
in situ guinea pig airway neuron (19, 28).
Drug and ionic substitutions were utilized as follows.
CaCl2 was substituted for with
MgCl2 on an equimolar basis to
produce a nominally "calcium-free" Krebs solution. The following
compounds were added to the control Krebs solution (final
concentration): apamin (1 µM), 4-aminopyridine (0.1 mM),
-conotoxin GVIA (1 µM), nifedipine (10 µM), tetrodotoxin (TTX; 1 µM), and verapamil (50 µM). Cadmium chloride (0.1 mM) was added to
the HEPES solution as described above. All reagents used to prepare the
Krebs and HEPES solutions were purchased from J. T. Baker Chemical
(Phillipsburg, NJ).
-Conotoxin GVIA was purchased from Research
Biochemicals International (Natick, MA). All other reagents were
purchased from Sigma (St. Louis, MO).
Results are presented as means ± SE. Means were compared as
unpaired samples (see RESULTS), with
Student's t-test statistics for two
means; means were considered to differ significantly if P was <0.05. These results, as well
as the slope of the change in rate of action potential frequency, were
analyzed by the Statview statistics program (Abacus Concepts, Berkeley,
CA).
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RESULTS |
Repetitive firing characteristics.
Intracellular recordings were made from 152 neurons in parasympathetic
ganglia located on the primary (predominantly right side) bronchi from
guinea pigs; of these, 145 were used in these studies. The
classification of bronchial neurons as either tonic or phasic was
similar to that previously described (28): in response to threshold
(
0.5-nA) rectangular anodal constant-current steps, 100-500 ms
in duration, tonic neurons (52 of 145 neurons) responded with either
one or several action potentials of equal size at the onset of the
stimulus (Fig.
1A) or
action potentials throughout the step. Tonic neurons responded to
suprathreshold (1- to 3-nA) current steps with action potentials
throughout the depolarization (Fig.
1B). Phasic neurons responded to
threshold (
0.5-nA) rectangular anodal constant-current steps,
100-500 ms in duration, with one to several action potentials at
the onset of the stimulus (Fig. 1C);
2-10 times threshold stimuli (1-5 nA) elicited either one or
a burst of action potentials in the initial 50 ms of the stimulus
followed by accommodation (91 of 145 neurons). In phasic neurons,
action potentials decreased in amplitude during the burst until no
further regenerative spikes were generated (Fig.
1D). On occasion (7 of 152 neurons),
clear differentiation of tonic and phasic action potential patterns was
not evident; these neurons were not included in these studies. The mean
resting membrane potentials for tonic and phasic neurons used in the
study were
53 ± 3 and
50 ± 2 mV, respectively.
The mean resting input resistances for tonic and phasic neurons used in
these experiments were 52 ± 7 and 38 ± 4 M
,
respectively (P < 0.05). In recording sessions that
involved characterization of >1 neuron/ganglion, both tonic and
phasic neurons were recorded in 15 of 27 ganglia.

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Fig. 1.
Accommodating and nonaccommodating responses to threshold and
suprathreshold current steps by bronchial ganglion neurons.
A: response by a tonic-type neuron to
threshold (1.0-nA) rectangular anodal current step, 500 ms in duration,
which elicits several action potentials at onset of stimulus.
B: 2 times threshold current steps
(2.0 nA) evoke action potentials throughout current step.
C: phasic neuron response to threshold
stimuli (0.5 nA) with either 1 (data not shown) or a burst of action
potentials at onset of stimulus. D: 4 times threshold stimuli (2.0 nA) elicit either 1 (data not shown) or a
burst of action potentials followed by accommodation (note action
potentials decrease in amplitude during burst until no further
regenerative spikes were generated).
Bottom traces, duration and amplitude
of current step stimuli.
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The interval between the first two action potentials (measured peak to
peak) was used to compare the instantaneous firing rate of tonic
neurons and phasic neurons that responded with two or more action
potentials at a threshold (0.5-nA), 500-ms current step; the interspike
interval between the next five spikes at 0.5, 1.0, 1.5, and 2.0 nA
during prolonged (500-ms) current steps was measured in six bursting
phasic neurons and six tonic neurons to determine the steady-state
(adapted) firing rate. The instantaneous firing rate between tonic and
phasic neurons was different at threshold (0.5-nA) stimuli where the
interspike intervals were 23 ± 3 and 16 ± 1 ms, respectively
(P < 0.05;
n = 10 for both cell types; Fig.
2A),
with a similar observation at four times threshold (2.0 nA; Fig.
2A). The interspike interval between
successive action potentials (this "adapted" firing rate was
calculated from intervals between peaks of spikes
2-5) decreased
with increasing current amplitude (0.5-2.0 nA): from 41.6 to 14.0 ms for tonic neurons and from 22.2 to 10.9 ms for phasic neurons (Fig.
2B). Adapted firing rates were
different at each current intensity studied (0.5, 1.0, 1.5, and 2.0 nA), with phasic neurons firing at higher frequencies than tonic
neurons (P < 0.05; Fig.
2B, Table 1); the slope of the change in adapted rate
for the four stimulus intensities was similar for tonic and phasic
neurons (R = 0.968 and 0.966, respectively).

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Fig. 2.
Instantaneous (A) and adapted
(B) firing rate of tonic and phasic
neurons were determined from interval between 1st 2 and subsequent
action potentials, respectively, during threshold and suprathreshold
prolonged current steps. A:
instantaneous firing rates at threshold and 2-4 times threshold
currents (0.5-2.0 nA) ranged between 43 and 82 spikes/s for tonic
neurons (n = 10; ) and between 62 and 89 spikes/s for phasic neurons (n = 10; ). B: frequency of successive
action potentials (between spikes 2 and 5) increased with increasing
current from 24 to 71 spikes/s for tonic neurons ( ) and from 45 to
91 spikes/s for phasic neurons ( ).
* P < 0.05 for differences
between tonic and phasic neurons at respective current steps.
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Table 1.
Adapted interspike intervals for tonic and phasic neurons: effects of
apamin, 4-aminopyridine, -conotoxin GVIA and barium
chloride
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Effects of blocking agents and hyperpolarization on
accommodation and action potential frequency. The role
of sodium and calcium conductance in action potential accommodation was
determined in tonic and phasic neurons. The sodium channel-blocker TTX
was bath applied after the neurons were typed as either tonic or phasic with 1- to 3-nA, 100-ms depolarizing steps (Fig.
3). TTX (1 µM) reduced the amplitude and
frequency of repetitive spikes in tonic neurons
(n = 4; Fig.
3A,
middle trace) during the
depolarization. The amplitude of the remaining action potentials could
be further reduced by calcium-free buffer
(n = 4 neurons; Fig.
3A,
right trace) and/or cadmium
chloride (0.1 mM; n = 3 neurons; data
not shown). TTX (1 µM) blocked action potentials in the burst of
spikes in phasic neurons, leaving only one or two nonregenerative
spikes at the onset of the depolarizing step
(n = 4; Fig.
3B,
middle trace). This remaining spike
could be reduced in amplitude by calcium-free buffer
(n = 4 neurons; Fig.
3B,
right
trace).

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Fig. 3.
Effects of blocking sodium and calcium conductances on action potential
accommodation in tonic and phasic neurons.
A:
left, repetitive action potentials are
evoked in a control tonic neuron with a 2.0-nA, 100-ms depolarizing
step; middle, tetrodotoxin (TTX)
reduced amplitude of repetitive spikes in this cell, leaving broad,
lower-frequency spikes during depolarization
(n = 4 neurons);
right, amplitude of remaining spike
could be further reduced by calcium-free buffer
(n = 4 neurons) and/or cadmium
chloride (0.1 mM; n = 4 neurons; data
not shown). B:
left, burst of spikes is evoked in a
control phasic neuron with a 2.0-nA, 100-ms depolarizing step;
middle, TTX blocked action potentials
in burst of spikes, leaving only 1 nonregenerative spike at onset of
depolarizing step (n = 4 neurons);
right, remaining spike could be
reduced in amplitude by calcium-free buffer
(n = 4 neurons). Scale bars apply to
respective tonic and phasic traces to
right.
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Reducing calcium currents increases accommodation in tonic neurons, but
reducing potassium currents affected both the firing rate and
accommodation in these cells. With a 500-ms, 1.0-nA depolarizing constant-current step, accommodation increased in the presence of
calcium-free buffer in all tonic neurons tested, leaving action potentials of equal amplitude at the onset of the stimulus
(n = 8 neurons; Fig.
4A). A
similar increase in accommodation was observed during superfusion with
cadmium chloride (0.1 mM; n = 10 neurons; data not shown).
-Conotoxin GVIA (1.0 µM;
n = 6 neurons) blocked repetitive
action potentials, leaving action potentials during the beginning of
the stimulus (Fig. 4B). The adapted
firing rate of the remaining action potentials during prolonged
depolarization steps, as described above, was unaffected by
calcium-free buffer (n = 8 neurons),
cadmium chloride (0.1 mM; n = 8 neurons), or
-conotoxin GVIA (n = 4 neurons; Fig. 4, Table 1). Bath-applied nifedipine (10 µM;
n = 4 neurons) decreased spike
frequency in one of four neurons but did not block the repetitive
firing pattern; verapamil (50 µM; n = 3) had a similar effect (data not shown). The potassium-channel
blocker 4-aminopyridine (0.1 mM) increased accommodation in tonic
neurons (n = 6; Fig.
4C,
right trace, 2-nA stimulus); in the
presence of this compound, the adapted interspike interval between the
remaining action potentials in the burst was 8.0 ± 1 ms
(P < 0.05 compared with control
neurons above; Table 1). An inhibitor of calcium-activated potassium
currents, apamin (1 µM), decreased the adapted interspike interval of
repetitive action potentials during a 500-ms, 2-nA current step to 11.5 ± 2 ms in four tonic neurons (P < 0.05 compared with control frequencies; Table 1). Bath application
of barium chloride (1.0 mM) had little effect on the adapted interspike
interval (12.2 ± 3 ms) of repetitive action potentials during a
500-ms, 2-nA current step in tonic neurons
(P < 0.5;
n = 3; Table 1).

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Fig. 4.
Blocking calcium conductance or decreasing A-type current increases
accommodation in tonic neurons. A:
repetitive action potentials by a control tonic neuron
(left) elicited by a 500-ms, 1.5-nA
depolarizing current (current trace not shown) was changed to a
phasiclike pattern (right) in
presence of calcium-free buffer (n = 8 neurons) or with 0.1 mM cadmium chloride (data not shown).
B: a similar change in accommodation
(evoked by a 500-ms, 2.0-nA current stimulus not shown) was also
induced by -conotoxin GVIA
(right) in 6 tonic neurons.
Bath-applied nifedipine (10 µM) decreased spike frequency in 4 tonic
neurons but did not block repetitive firing pattern; verapamil (50 µM; n = 2 neurons) had a similar
effect (data not shown). C:
4-aminopyridine (right) also
increased accommodation (n = 6 neurons; 500-ms, 2.0-nA current stimulus not shown).
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After an initial hyperpolarizing step and return to the resting
potential, application of a depolarization stimulus resulted in a
decrease in accommodation in phasic neurons. Current clamping the
resting membrane potential (
50 mV) to a range of
90 to
100 mV for 1 or more seconds changed the action potential
pattern of five of seven bronchial phasic neurons; after the
hyperpolarizing step and return to the resting potential, the
depolarization stimuli (500 ms, 0.5 nA) now elicited repetitive action
potentials (Fig. 5A)
similar to those observed in tonic neurons. The induction of the
tonic-type firing pattern was transient; within 10-15 s, the
firing pattern returned to the normal phasic pattern (data not shown).
These changes in spike accommodation could be entirely blocked by bath
application of 4-aminopyridine (0.1 mM;
n = 4 neurons; Fig.
5B) or with bath application of
cadmium chloride (0.1 mM; n = 3 neurons; data not shown).

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Fig. 5.
Effects of membrane potential hyperpolarization on accommodating action
potential pattern by bronchial phasic neurons.
A: after neuron was typed as phasic
(500-ms, 0.5-nA current trace;
bottom), resting potential was
clamped to 90 mV for 5 s (B;
full time not shown). C: after return
to preclamp resting potential, typing the same neuron results in a
tonic firing pattern. D: the same
neuron as in A was superfused with
4-aminopyridine (0.1 mM) and was clamped to 90 mV for 5 s
(E; full time not shown) and returned
to preclamp potential. F: typing the
same neuron results in no change in accommodation
(n = 4 neurons). Similar results were
observed in presence of cadmium chloride (0.1 mM in HEPES buffer; data
not shown).
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Bath-applied barium chloride (1 mM; n = 6 neurons; Fig.
6A,
right trace) or apamin (1.0 µM;
n = 4 neurons; Fig.
6B,
right trace) also decreased spike
accommodation in phasic neurons evoked by a 500-ms depolarizing current
step (2.0 nA), and this effect could be reversed by adding cadmium
chloride (0.1 mM; n = 4 neurons) for
both treatments (data not shown). Apamin (1.0 µM) had little effect
on the adapted firing rate as measured above (Table 1) but did decrease
the duration of the afterhyperpolarization that followed four
consecutive action potentials (elicited by 20-Hz, 3.0-nA, 2-ms steps)
from a control duration of 175 ± 35 to 65 ± 30 ms
(P < 0.05); apamin (1 µM;
n = 4 neurons) had no effect on the
amplitude of the afterhyperpolarization (data not shown).
-Conotoxin
GVIA (1.0 µM; n = 4 neurons),
calcium-free buffer (n = 8 neurons)
and cadmium chloride (0.1 mM; n = 4 neurons) reduced the number of action potentials in the burst of action
potentials at the onset of the stimulus in phasic neurons (data not
shown). The adapted firing rate of the remaining action potentials
during prolonged depolarization steps, as described above, could not be
determined in the presence of calcium-free buffer
(n = 8 neurons), cadmium chloride (0.1 mM; n = 4 neurons), or
-conotoxin
GVIA (n = 4 neurons) because too few
action potentials were generated in the presence of these compounds.

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Fig. 6.
Bath-applied barium chloride (A) or
apamin (B) decreases accommodation
in phasic neurons. A: control phasic
neuron (left) fires repetitive
spikes evoked during a 500-ms current step (2.0 nA) in presence of
barium chloride (right).
B: apamin decreased accommodation in
phasic neuron (right).
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DISCUSSION |
In the present study, further differences in active membrane properties
were observed for guinea pig bronchial tonic and phasic parasympathetic
ganglion neurons, and the ionic currents regulating accommodation and
action potential frequency were determined. With the use of 100- to
500-ms depolarizing steps, the interspike interval between the first
two action potentials was greater (lower instantaneous firing rate) in
tonic neurons than in phasic neurons at threshold and four times
threshold (2.0-nA) stimulus intensities (Fig.
2A). For subsequent action
potentials, tonic neurons had a more prolonged interspike interval than
phasic neurons (lower adapted firing rate) at all depolarizing currents
studied (Fig. 2B). These
instantaneous and adapted patterns by bronchial phasic and tonic
neurons were similar to guinea pig inferior mesenteric ganglion phasic
type I and tonic type III and IV neurons, respectively (32). Although
phasic neurons fire a burst of action potentials at a higher frequency
than tonic neurons, the greater interspike interval in tonic neurons
may be necessary for the maintenance of the repetitive action potential
pattern observed in these cells (28). Blocking sodium conductance with
TTX reduced, but did not entirely eliminate, action potentials in tonic
and phasic neurons; evidence for a role of calcium channels in the
remaining action potentials came from the elimination of the remaining
spikes with calcium-free buffer (Fig. 3) or cadmium ions.
If the repetitive activity observed in control tonic neurons is calcium
dependent, then decreasing the calcium conductance should affect
repetitive action potentials in these neurons. By reducing the
extracellular calcium concentration with nominally calcium-free buffer,
the number of action potentials during the prolonged depolarizing
stimulus decreased in tonic neurons; this indicates that calcium
channels are the major charge carriers during the repetitive action
potential activity that follows the initial burst of action potentials
(Fig. 4). Reducing the calcium conductance, however, had no effect on
the adapted firing rate of the remaining action potentials (Table 1).
Similarly, cadmium ions, which are more efficacious at blocking T-
and N-type calcium channels (17), decreased repetitive spikes by tonic
neurons. Thus it may be inferred that the calcium-channel subtype
active during the repetitive action potentials is the N-type channel because
-conotoxin GVIA (relatively specific for the N-type calcium channel) blocked repetitive spikes, whereas nifedipine, relatively specific for the L-type calcium channel (reviewed in Refs. 17, 20), was
without overt effect. The presence of both high-threshold (L-type) and
-conotoxin-sensitive (N-type) calcium currents have been identified
in dissociated neurons from rat tracheal (2) and cardiac (18)
parasympathetic ganglia; N-type calcium channels have been identified
in amphibian cardiac parasympathetic ganglion neurons as well (21). The
characterization of calcium currents and their effects on repetitive
action potential pattern is a relatively unexplored area of
parasympathetic neurophysiology (reviewed in Ref. 20).
The differences in accommodation patterns by tonic and phasic neurons
is likely due to different expression and/or activation of
potassium channels. This study provides evidence that a potassium current may be responsible for the increase in interspike interval in
tonic neurons. The A-type current blocker 4-aminopyridine reduced the
number of action potentials evoked by a prolonged depolarizing stimulus, making them fire with an action potential pattern similar to
that of phasic neurons (Fig. 3). These results indicate that activation
of the A-type current contributes to the increased interspike interval
observed in guinea pig bronchial tonic neurons because 4-aminopyridine
decreased the interspike interval in tonic neurons to a level similar
to that in phasic neurons (Table 1). The A-type current has also been
identified in rat tracheal parasympathetic neurons (2) and is a very
prominent current in tonic neurons located in guinea pig sympathetic
ganglia (10, 31). Furthermore, the accommodation to the stimulus by
phasic neurons could be reversed after the resting potential was
current clamped to more negative levels that could be blocked by
4-aminopyridine (Fig. 5), indicating activation of the A-type current
during the hyperpolarization (17). It is unlikely the
hyperpolarization-activated h-type current plays a role in action potential accommodation because it is
activated only at potentials more negative than
90mV, a potential that is not reached during action potential
afterhyperpolarization in bronchial phasic cells (Fig. 3) (28), and,
furthermore, it is not blocked by barium (17; see below).
Further evidence for the role of potassium currents in accommodation is
based on the decrease in accommodation in phasic neurons by apamin or
barium ions, indicating the presence of a calcium-activated potassium
channel. Although it was originally suggested that the decrease in
accommodation induced by barium is due to inhibition of the M-type
current (12), a similar effect by apamin on bronchial ganglion neurons
indicates that perhaps barium decreases accommodation by inhibiting
calcium-activated potassium currents and perhaps by "uncovering"
or increasing current carried by calcium channels (reviewed in Ref.
17). Furthermore, Myers and Undem previously demonstrated
that muscarinic- (26) or neurokinin-receptor (25) agonists have no
effect on accommodation in phasic neurons, eliminating any role of
M-type current associated with these agonists in accommodation, unlike
that reported for rat tracheal (4) or guinea pig intracardiac (3)
parasympathetic ganglion neurons.
That similar calcium and potassium currents are present in tonic and
phasic neurons indicates that these cells may not represent distinct
neuronal populations as has been suggested for tonic and phasic neurons
in sympathetic ganglia (6, 10, 31) but represent a single population
with different states of potassium-channel activation or inactivation.
Phasic neurons may have calcium channels similar to tonic neurons; the
decrease in accommodation in guinea pig bronchial phasic neurons
induced by hyperpolarization, barium, or apamin in all instances could
be reversed by blocking the calcium current. Such results indicate the
presence of calcium current in phasic neurons, which may be shunted by
an outward current with similar temporal kinetics. Thus blocking the
antagonistic outward potassium conductance with apamin or barium may
unmask or amplify the calcium currents necessary for repetitive action potential activity. However, calcium-activated potassium currents may
also be active but have a different role in tonic neurons, i.e.,
regulating the frequency of repetitive action potentials, as apamin
increased the firing rate in these neurons (Table 1). As mentioned
above, guinea pig bronchial ganglion neurons have similar anatomic
characteristics (28); furthermore, chemical coding for
neurotransmitters in guinea pig bronchial ganglion neurons is similar
as well: all neurons in the guinea pig bronchial ganglia synthesize
acetylcholine, i.e., are choline acetyltransferase positive (9), and do
not synthesize nitric oxide (13).
The results from these studies may explain the mechanism for the
decrease in accommodation in bronchial neurons observed after antigen
challenge (29): inflammatory mediators such as prostaglandin D2 may directly or indirectly
affect conductance through N-type calcium channels or decrease an
opposing conductance, e.g., through calcium-activated potassium
channels, as has been reported for guinea pig vagal sensory neurons
(30). Thus activation or inhibition of these channels by inflammatory
mediators (29) or neurotransmitters (27, 28) released near a ganglion
neuron may ultimately affect the ability of that neuron to relay
excitatory stimuli from the central nervous system and, consequently,
regulate airway parasympathetic tone. Results from the present study
suggest that regulation of accommodation properties may be on the level
of a single neuron and not on the entire population of neurons within
the ganglion or bronchus because recordings were made from both tonic
and phasic neurons in single ganglia.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grant HL-48198.
 |
FOOTNOTES |
Address for reprint requests: A. C. Myers, The Johns Hopkins Asthma and
Allergy Center, 5501 Hopkins Bayview Circle 3A62, Baltimore, MD
21224-6821.
Received 13 November 1997; accepted in final form 24 April 1998.
 |
REFERENCES |
1.
Adams, P. R.,
and
M. Galvan.
Voltage-dependent currents of vertebrate neurons and their role in membrane excitability.
In: Advances in Neurology. New York: Raven, 1986, p. 137-170.
2.
Aibara, K.,
S. Ebihara,
and
N. Akaike.
Voltage-dependent ionic currents in dissociated paratracheal ganglion cells of the rat.
J. Physiol. (Lond.)
457:
591-610,
1992[Abstract].
3.
Allen, T. G. J.,
and
G. Burnstock.
M1 and M2 muscarinic receptors mediate excitation and inhibition of guinea-pig intracardiac neurones in culture.
J. Physiol. (Lond.)
422:
463-480,
1990[Abstract].
4.
Allen, T. G. J.,
and
G. Burnstock.
A voltage clamp study of the electrophysiological characteristics of the intramural neurones of the rat trachea.
J. Physiol. (Lond.)
423:
593-614,
1990[Abstract].
5.
Baker, D. G.,
C. B. Basbaum,
D. A. Herbert,
and
R. A. Mitchell.
Transmission in airway ganglia of ferrets: inhibition by norepinephrine.
Neurosci. Lett.
41:
139-143,
1983[Medline].
6.
Boyd, H. D.,
E. M. McLachlan,
J. R. Keast,
and
H. Inokuchi.
Three electrophysiological classes of guinea pig sympathetic postganglionic neurons have distinct morphologies.
J. Comp. Neurol.
369:
372-387,
1996[Medline].
7.
Burnstock, G.,
T. G. J. Allen,
and
C. J. S. Hassell.
The electrophysiological and neurochemical properties of paratracheal neurons in situ and in dissociated cell culture.
Am. Rev. Respir. Dis.
136:
S23-S26,
1987[Medline].
8.
Cameron, A. R.,
and
R. F. Coburn.
Electrical and anatomic characterization of cells of ferret paratracheal ganglion.
Am. J. Physiol.
246 (Cell Physiol. 15):
C450-C458,
1984[Abstract].
9.
Canning, B. J.,
and
A. Fischer.
Localization of cholinergic nerves in lower airways of guinea pigs using antisera to choline acetyltransferase.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L731-L738,
1997[Abstract/Free Full Text].
10.
Cassell, J. F.,
A. L. Clark,
and
E. M. McLachlan.
Characteristics of phasic and tonic sympathetic ganglion cells of the guinea-pig.
J. Physiol. (Lond.)
372:
457-483,
1986[Abstract].
11.
Christian, E. P.,
J. Togo,
and
K. E. Naper.
Guinea pig visceral C-fiber neurons are diverse with respect to the K+ currents involved in action-potential repolarization.
J. Neurophysiol.
71:
561-674,
1994[Abstract/Free Full Text].
12.
Constanti, A.,
and
D. A. Brown.
M-currents in voltage-clamped mammalian sympathetic neurones.
Neurosci. Lett.
24:
289-294,
1981[Medline].
13.
Fischer, A.,
P. Mundel,
U. Preissler,
B. Philippin,
and
W. Kummer.
Nitric oxide synthase in guinea pig lower airway innervation.
Neurosci. Lett.
149:
157-160,
1993[Medline].
14.
Granit, R.,
D. Kernell,
and
R. S. Smith.
Delayed depolarization and the repetitive response to intracellular stimulation of mammalian motoneurones.
J. Physiol. (Lond.)
168:
890-910,
1961.
15.
Griffith, W. H., III,
J. P. Gallagher,
and
J. P. Shinnick-Gallagher.
An intracellular investigation of cat vesical pelvic ganglia.
J. Neurophysiol.
43:
343-354,
1980[Abstract/Free Full Text].
16.
Hanani, M.,
and
N. Maudlej.
Intracellular recordings from intramural neurons in the guinea pig urinary bladder.
J. Neurophysiol.
74:
2358-2365,
1995[Abstract/Free Full Text].
17.
Hille, B.
Ionic Channels of Excitable Membranes (2nd ed.). Sunderland, MA: Sinauer, 1992.
18.
Jeong, S.-W.,
and
R. D. Wurster.
Calcium channel currents in acutely dissociated intracardiac neurons from adult rats.
J. Neurophysiol.
77:
1769-1778,
1997[Abstract/Free Full Text].
19.
Lees, G. M.,
E. G. Pacitti,
and
G. M. MacKenzie.
Morphology and electrophysiology of guinea pig paratracheal neurons.
Anat. Rec.
247:
261-270,
1997[Medline].
20.
Lundy, P. M.,
and
R. Frew.
Review: Ca2+ channel sub-types in peripheral efferent autonomic nerves.
J. Auton. Pharmacol.
16:
229-241,
1996[Medline].
21.
Merriam, L. A.,
and
R. L. Parsons.
Neuropeptide galanin inhibits omega-conotoxin GVIA-sensitive calcium channels in parasympathetic neurons.
J. Neurophysiol.
73:
1374-1382,
1995[Abstract/Free Full Text].
22.
Mitchell, R. A.,
D. A. Herbert,
and
C. A. Richardson.
Neurohumoral regulation of airway smooth muscles role of tracheal ganglia.
In: Chemoreceptors and Reflexes in Breathing: Cellular and Molecular Aspects, edited by S. Lahiri,
R. E. Forster,
R. O. Davies,
and A. I. Pack. New York: Oxford University Press, 1989, p. 299-309.
23.
Myers, A. C.
Evidence for neural integration by human bronchial parasympathetic ganglia neurons (Abstract).
Am. J. Respir. Crit Care Med.
155:
A575,
1997.
24.
Myers, A. C.,
and
B. J. Undem.
Analysis of preganglionic nerve evoked cholinergic contractions of the guinea pig bronchus.
J. Auton. Nerv. Syst.
35:
175-184,
1991[Medline].
25.
Myers, A. C.,
and
B. J. Undem.
Electrophysiological effects of tachykinins and capsaicin on guinea-pig bronchial parasympathetic ganglion neurones.
J. Physiol. (Lond.)
470:
665-679,
1993[Abstract].
26.
Myers, A. C.,
and
B. J. Undem.
Muscarinic receptor regulation of synaptic transmission in airway parasympathetic ganglia.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L630-L636,
1996[Abstract/Free Full Text].
27.
Myers, A. C.,
B. J. Undem,
and
W. Kummer.
Anatomical and electrophysiological comparison of the sensory innervation of bronchial and tracheal parasympathetic ganglion neurons.
J. Auton. Nerv. Syst.
61:
162-168,
1996[Medline].
28.
Myers, A. C.,
B. J. Undem,
and
D. Weinreich.
Electrophysiological properties of neurons in guinea pig bronchial parasympathetic ganglia.
Am. J. Physiol.
259 (Lung Cell. Mol. Physiol. 3):
L403-L409,
1990[Abstract/Free Full Text].
29.
Myers, A. C.,
B. J. Undem,
and
D. Weinreich.
Influence of antigen on membrane properties of guinea pig bronchial ganglion neurons.
J. Appl. Physiol.
71:
970-976,
1991[Abstract/Free Full Text].
30.
Undem, B. J.,
W. Hubbard,
and
D. Weinreich.
Immunologically induced neuromodulation of guinea pig nodose ganglion neurons.
J. Auton. Nerv. Syst.
44:
35-44,
1993[Medline].
31.
Vanner, S.,
R. J. Evans,
S. G. Matsumoto,
and
A. Surprenant.
Potassium currents and their modulation by muscarine and substance P in neuronal cultures from adult guinea pig celiac ganglia.
J. Neurophysiol.
69:
1632-1614,
1993[Abstract/Free Full Text].
32.
Weems, W. A.,
and
J. H. Szurszewski.
An intracellular analysis of some intrinsic factors controlling neural output from inferior mesenteric ganglion of guinea pig.
J. Neurophysiol.
41:
305-321,
1978[Abstract/Free Full Text].
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