1The Neuroscience Program and 2Department of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine, Baltimore, Maryland 21201-1559
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ABSTRACT |
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Lancaster, Eric, Eun Joo Oh, and Daniel Weinreich. Vagotomy Decreases Excitability in Primary Vagal Afferent Somata. J. Neurophysiol. 85: 247-253, 2001. Standard patch-clamp and intracellular recording techniques were used to monitor membrane excitability changes in adult inferior vagal ganglion neurons (nodose ganglion neurons, NGNs) 5 days following section of the vagus nerve (vagotomy). NGNs were maintained in vivo for 5 days following vagotomy, and then in vitro for 2-9 h prior to recording. Vagotomy increased action potential (AP) threshold by over 200% (264 ± 19 pA, mean ± SE, n = 66) compared with control values (81 ± 20 pA, n = 68; P < 0.001). The number of APs evoked by a 3 times threshold 750-ms depolarizing current decreased by >70% (from 8.3 to 2.3 APs, P < 0.001) and the number of APs evoked by a standardized series of (0.1-0.9 nA, 750 ms) depolarizing current steps decreased by over 80% (from 16.9 APs to 2.6 APs, P < 0.001) in vagotomized NGNs. Similar decreases in excitability were observed in vagotomized NGNs in intact ganglia in vitro studied with "sharp" microelectrode techniques. Baseline electrophysiological properties and changes following vagotomy were similar in right and left NGNs. A "sham" vagotomy procedure had no effect on NGN properties at 5 days, indicating that changes were due to severing the vagus nerve itself, not surrounding tissue damage. NGNs isolated after being maintained 17 h in vivo following vagotomy revealed no differences in excitability, suggesting that vagotomy-induced changes occur some time from 1-5 days after injury. Decreased excitability was still observed in NGNs isolated after 20-21 days in vivo following vagotomy. These data indicate that, in contrast to many primary sensory neurons that are thought to become hyperexcitable following section of their axons, NGNs undergo a marked decrease in electrical excitability following vagotomy.
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INTRODUCTION |
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The inferior vagal ganglia house
the somata of primary vagal afferent neurons (nodose ganglion neurons,
NGNs), which provide sensory innervation to the lungs, heart, proximal
gastrointestinal tract, aortic baroreceptors, and other thoracic and
abdominal viscera. NGNs play an important role in detecting liquid in
the upper airway, blood pressure, gastric acidity, hepatic glucose levels, and visceral distension, and they contribute to the febrile and
hyperalgesic responses to infection (Mei 1983;
Watkins et al. 1994
). Vagal afferents may also mediate
short-term satiety and satiety-induced analgesia; these functions may
contribute to bulimia nervosa (Raymond et al. 1999
). In
addition, chemical or electrical stimulation of vagal afferents may
either facilitate or inhibit nociception, depending on intensity of
stimulation (Randich and Gebhart 1992
).
Many surgical and pharmacological treatments affect vagal sensory
function. Extensive severing of vagal afferents occurs during heart and
lung transplants, and may occur during resections of the stomach,
esophagus, or intestine. Iatrogenic impairment of vagally mediated
airway protection has been reported following the removal of certain
neoplasms of the neck, parapharyngeal region, and infratemporal fossa
(Leonetti et al. 1996). Carotid endarterectomy (Ballota et al. 1999
) and the implantation of electronic
vagal stimulators for epilepsy treatment (Amar et al.
1998
) have also been reported to damage the vagus
nerve. Vagus nerve stimulation with implanted electronic stimulators
has been used to treat refractory epilepsy in over 6,000 patients
(George et al. 2000
), and these devices are currently
being studied as a therapy for drug-resistant depression (Rush
et al. 2000
). The expanding use of these devices increases the
importance of understanding vagus nerve injury.
In the rat, bilateral vagotomy induces increased sensitivity to several
types of injury and inflammation; this effect has been ascribed to a
reduction in the tone of vagally mediated analgesia (Khasar et
al. 1998a,b
; Miao et al. 1997
). Severing the
vagus nerve bilaterally, or just the celiac branches of the vagus
nerve, augments inflammatory and nociceptive responses to bradykinin injection, mechanical pinch, and other noxious stimuli (Janig et
al. 2000
). Either increased pro-nociceptive vagal activity or
decreased anti-nociceptive vagal activity could account for these observations.
Despite the importance of the vagus nerve in conveying visceral
afferent information, the electrophysiological properties of injured
vagal afferents are far less understood than those of injured spinal
afferents (i.e., those from dorsal root ganglion neurons, DRGNs) (for
review see Devor 1994). Axotomy of DRGNs results from
many types of nerve injuries, and chronic pain and allodynia are common
results of such injuries. Increased excitability and ectopic spiking
noted in axotomized DRGNs may be important in pain syndromes
(Devor 1994
; Nordin et al. 1984
), and
blocking ectopic discharge from reaching the CNS may prevent ongoing
pain and allodynia (Gracely et al. 1992
; Sheen
and Chung 1993
).
The purpose of the present work was to test whether similar increases in excitability occur in NGNs. We find that within 5 days of a unilateral vagotomy, vagotomized NGNs become profoundly less excitable. Specifically, the current required to initiate action potential (AP) firing was increased by over 200%, and the numbers of AP fired in response to standardized strong depolarizing stimuli was reduced by over 80%. Thus this population of visceral afferents responds to injury in a fashion that is radically different from that reported for somatic (dorsal root ganglion, DRG) afferents.
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METHODS |
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Vagotomy
Vagus nerve injury, vagotomy, was elicited by unilaterally removing a section of the right or left cervical vagus nerve of adult (200-300 g), male Sprague Dawley rats, as approved by the Institutional Animal Care and Use Committee of the University of Maryland, Baltimore. Under ketamine (50 mg/kg ip)/xylazine (10 mg/kg ip) anesthesia, a 5-mm section of the vagus nerve was removed approximately 1 cm distal to the right or left inferior vagal (nodose) ganglion. This operation severed the afferents processes of approximately 90% of the NGNs on the operated side (with the remainder projecting their afferents via the superior laryngeal nerve, proximal to the nerve injury). We did not observe any gross impairment of respiration or behavior in vagotomized rats, and within 2 days of the operation, rats resumed gaining weight. Vagotomized and control nodose ganglia remained in vivo for some time (usually 5 days) after the operation. Rats were then killed by CO2 inhalation, and nodose ganglia were removed bilaterally. Intact ganglia were used for "sharp" microelectrode recording in vitro, while dissociated NGNs maintained in culture for 2-9 h after plating were used for patch-clamp analysis.
Dissociation
NGNs were dissociated enzymatically as described previously
(Jafri et al. 1997). Briefly, ganglia were rapidly
removed from animals, desheathed, and then incubated in enzyme solution
[10 mg collagenase type 1A (Sigma, St. Louis, MO), 10 mg dispase II (Boehringer Mannheim, Mannheim, Germany), and 10 ml
Ca2+- and Mg2+-free Hank's
Balanced Salt Solution] for 2 h at 37°C. Neurons were
dissociated by trituration, washed by centrifugation (3 times at
700 g for 45 s), suspended in L15 media (GIBCO BRL,
Rockville, MD) containing 10% fetal bovine serum (JRH Biosciences,
Lenexa, KS), and then transferred onto circular 15-mm glass cover slips (Bellco Glass, Vineland, NJ) coated with poly-D-lysine (0.1 mg/ml, Sigma). NGNs adhered to cover slips and were maintained in
culture for 2-9 h after plating at 37°C prior to recording.
Patch-clamp recording
Whole cell patch-clamp techniques (Hamill et al.
1981) were employed with an Axopatch 200B amplifier and PCLAMP
7 software (Axon Instruments, Foster City, CA). Patch pipettes (1-4
M
) were fabricated from glass capillaries (MTW150F-4, World
Precision Instruments, Sarasota, FL). Pipettes were filled with a
variant of a solution described previously (Ikeda et al.
1986
) for rat NGNs, with a composition of (in mM) 140 KCl, 2 MgCl2, 10 N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid] (HEPES), 0.11 ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 10 dextrose;
titrated to pH 7.3 with KOH, 314 mOsm. Except where specified, all
chemicals were from Sigma. Pipette voltage offset was neutralized prior to the formation of a gigaseal. Membrane input resistance
(Rin), series resistance
(Rs), and capacitance
(Cm) were determined from current
transients elicited by 5-mV depolarizing steps from a holding potential
of
60 mV, delivered using the Membrane Test application of PCLAMP7.
Capacitance compensation and 80% Rs
compensation were used. Criteria for cell inclusion in the study were
as follows: Rs
10 M
,
Rin > 100 M
, and stable recording
with 80% series resistance compensation during the entire experiment.
Cover slips were superfused (2-4 ml/min) continuously during recording
with room temperature (22-24°C) Locke solution (composition in mM: 10 dextrose, 136 NaCl, 5.6 KCl, 1.2 MgCl2
· 6 H2O, 2.2 CaCl2
· 2 H2O, 1.2 NaH2PO4, and 14.3 NaHCO3, equilibrated with 95%
O2-5% CO2 pH between 7.3 and 7.5). The recording chamber was grounded via a 3 M KCl agar bridge.
Sharp electrode recording
Nodose ganglia were removed from some animals 5 days following
vagotomy for study with "sharp" microelectrode recording in vitro.
For sharp microelectrode recording, intact ganglia were placed on the
floor of the recording chamber, covered with gauze thread, and
superfused with Locke solution (3-4 ml/min) at room temperature
(22-24°C). Conventional current-clamp recording was performed with
an Axoclamp 2A amplifier (Axon Instruments). Sharp microelectrodes
filled with 3 M KCl (40-100 M) were inserted into NGNs blindly.
Entry into a NGN was confirmed by a resting membrane potential and by
the presence of an action potential in response to depolarizing
current. Rin was measured from the voltage deflection produced by a 100-pA injection of hyperpolarizing current from a potential of
60 mV, and
Em was the membrane potential recorded
with zero current injected (corrected for tip potential). Criteria for
cell acceptance include the following: an action potential overshooting
0 mV and DC membrane input resistance
10 M
(typically
20-100 M
).
Statistical methods
The NGNs associated with a cut vagus (NGNs vagotomized for 5 days) were compared with NGNs from the contralateral (control) ganglia using the statistical software SIGMASTAT (Jandel Scientific, San Rafael, CA). The effects of treatment (vagotomy vs. contralateral control) and intra-animal variation on NGN properties were measured using two-way ANOVA. Vagotomized and contralateral NGNs from animals vagotomized for 17 h were also compared with each other using two-way ANOVA, as were vagotomized and contralateral NGNs from animals vagotomized for 20-21 days, and NGNs from the operated and contralateral ganglia of the "sham" vagotomy animals.
To determine whether the properties of contralateral NGNs were altered following vagotomy, vagotomized NGNs, contralateral NGNs, and NGNs from control animals were compared using one-way ANOVA with Dunn's pairwise comparisons.
The properties of right and left NGNs from control animals were compared using a two-way ANOVA. To determine whether there was a differential effect of right and left vagotomy, a two-way ANOVA examined the effects of vagotomy (vagotomy vs. contralateral control) and side (right vs. left) on NGN properties.
P < 0.05 was considered significant for all tests.
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RESULTS |
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Passive and active membrane properties of vagotomized vagal afferents recorded in isolated NGNs
The vagotomized and contralateral (control) nodose ganglia were removed from 13 animals 5 days after vagotomy and dissociated separately. In eight animals the vagotomy was performed on the right side, and in five animals on the left side. Using the whole cell patch-clamp recording technique, the passive membrane properties, firing properties, and AP characteristics of NGNs from vagotomized ganglia were compared with those of control ganglia. The experimenter was "blind" to cell status (the vagotomized vs. control NGNs). However, the obvious differences in excitability measured in NGNs from each ganglia allowed the experimenter to guess accurately (13 of 13 experiments) which side was vagotomized.
Passive membrane properties (input resistance and capacitance) were
determined in vagotomized and contralateral control NGNs in whole cell
voltage clamp at 60 mV using ±2.5-mV voltage steps. The resting
membrane potential was estimated in current-clamp mode with zero
injected current. The capacitance of vagotomized NGNs (46 ± 1.4 pF, mean ± SE, n = 66) was significantly
(P < 0.001) increased compared with control NGNs
(34 ± 1.4 pF, n = 68). This indicates that
vagotomy may cause an increase in membrane surface area, although the
possibility that there is differential survival of large cells has not
yet been discounted. Membrane input resistance was significantly
(P < 0.01) decreased by vagotomy (284 ± 20 M
, n = 66) relative to control values (371 ± 21 M
, n = 67). The increase in cell membrane
conductance (1/Rin) in vagotomized
NGNs could be satisfactorily accounted for by their increased membrane surface area (approximated by Cm);
conductance per unit surface area,
1/(Rin * Cm), was not significantly different
in control and vagotomized NGNs. The estimated values for membrane
potential in vagotomized NGNs were significantly (P < 0.01) more hyperpolarized (
53 ± 0.8 mV, n = 65)
than in control NGNs (
50 ± 0.8 mV, n = 67). The
population values are tabulated in Table
1.
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To examine the characteristics of individual APs, brief (3-ms)
depolarizing current steps were used to evoke single APs (see Fig.
1). The width (at 0 mV), overshoot (above
0 mV), and most hyperpolarized potential (peak hyperpolarization) of
these APs were quantified, and the values are summarized in Table 1.
Overshoot was significantly decreased by vagotomy (+40 ± 2.2 mV,
n = 64) when compared with control values (+49 ± 1.5 mV, n = 65, P < 0.01), suggesting
that vagotomized NGNs may have decreased sodium current and/or
augmented potassium current. There was also a significant decrease in
peak hyperpolarization magnitude in vagotomized NGNs (68 ± 0.6 mV, n = 64, P < 0.001) versus control
NGNs (
71 ± 0.5, n = 65). AP duration was not
significantly different in the two groups (P > 0.45).
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AP firing patterns were also examined in control and vagotomized NGNs.
For these experiments, a DC current was applied to adjust baseline
membrane potential to between 55 and
60 mV. Subsequently, a series
of 750-ms depolarizing current steps were used to evoke APs. For each
neuron, a threshold current (rheobase) was determined with incremental
(10 pA) 750-ms current steps. Then we applied 750-ms depolarizing
current steps to 1, 2, and 3 times threshold and recorded the number of
APs evoked by each current step. To determine the responsiveness of
NGNs to absolute (as opposed to relative to threshold) depolarizing
stimuli, we injected a series of 750-ms incremental current steps
(0.1-0.9 nA). As illustrated in Fig. 2,
control NGNs typically fired multiple APs in response to maintained
suprathreshold stimuli; most neurons accommodated (ceased firing)
before the end of the 750-ms current injection for stimuli of any
strength. Vagotomized NGNs, by contrast, accommodated much more
rapidly, generally firing only one or two APs in response to any step
injection of depolarizing current. Vagotomy increased rheobase by over
200% (264 ± 19 pA, n = 66) compared with control
values (81 ± 20 pA, n = 68; P < 0.001). The number of APs evoked by a 1, 2, or 3 times threshold
stimulus was significantly decreased, with the decrease exceeding 70%
(from 8.3 to 2.3 APs, P < 0.001) at 3 times threshold.
The number of APs evoked by the standardized series of current steps
decreased by over 80% (from 16.9 APs to 2.6 APs, P < 0.001) in vagotomized NGNs. Population data for these measurements are
shown in Table 1.
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Passive and active membrane properties of vagotomized vagal afferents recorded in the intact nodose ganglion
To test whether the decrease in excitability observed in isolated
vagotomized NGNs may have arisen from neuronal isolation methodology or
from the patch-clamp recording methods, we employed sharp
microelectrode techniques to record electrical properties from NGNs in
the intact nodose ganglia in vitro. Membrane electrical properties from
vagotomized (right) and contralateral control ganglia removed 5 days
following vagotomy from three rats were compared and are summarized in
Table 2. There was a trend
(P = 0.28) toward more negative resting membrane
potentials in vagotomized NGNs (56 ± 2 mV, n = 23) versus controls (
51 ± 2 mV, n = 20) and a
trend (P = 0.09) toward decreased membrane input
resistance in vagotomized NGNs (38 ± 6 M
, n = 21) compared with control NGNs (62 ± 10 M
, n = 19). Like dissociated vagotomized NGNs, vagotomized NGNs in intact
ganglia showed dramatic decreases in action potential discharge in
response to supra-threshold depolarizing current injection (Fig.
3). In all NGNs recorded in vagotomized ganglia, only a single action potential could be evoked by 750-ms current steps to 1, 2, or 3 times threshold (in 23 of 23 cells), or by
currents steps ranging from 1 to 3 nA (in 15 of 16 cells). In contrast,
most (15 of 20) NGNs recorded in contralateral (control) ganglia fired
multiple (>1) action potentials in response to current steps of 2 times threshold or greater. Vagotomized NGNs fired significantly
(P < 0.01) fewer APs in response to 2 times threshold stimuli (1 ± 0 vs. 5 ± 1 for controls) or 3 times threshold
stimuli (1 ± 0 vs. 10 ± 2). Significant decreases in the
numbers of APs discharged by vagotomized NGNs were also seen in
response to 1-, 2-, and 3-nA depolarizing current steps (Table 2).
These results from the intact ganglia clearly demonstrate that
decreased excitability in vagotomized NGNs was not associated with
dissociation procedures, or the whole cell patch-clamp technique.
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Excitability in contralateral nodose ganglia
While differences in the excitability of vagotomized and contralateral control NGNs are most likely due to decreased excitability in vagotomized NGNs, it is also possible that the excitability of contralateral NGNs changed. To examine this possibility, we recorded the passive membrane properties, AP characteristics, and firing properties of the right and left NGNs from four control animals. There were no statistically significant differences between the right and left NGNs of control animals for any of the properties measured, so they were tabulated as a single population in Table 1. Further, properties of NGNs from control rats were similar to the properties of contralateral control NGNs from vagotomized rats; there were no statistically significant difference between the two groups for any variable measured (P > 0.05 for each, 1-way ANOVA). Compared to NGNs from control animals, vagotomized NGNs had significantly (P < 0.05 for each) larger capacitance; lower membrane resistance; higher AP threshold; reduced AP firing at 1, 2, and 3 times threshold, reducing firing in response to standardized stimuli; reduced AP overshoot; and reduced peak hyperpolarization (1-way ANOVA). Thus, each of the differences between vagotomized and contralateral control NGNs is due to changes in the vagotomized NGNs. There was no evidence of any significant change in contralateral NGN electrophysiological properties following vagotomy.
Effects of right versus left vagotomy on excitability changes in NGNs
The right and left vagus nerves do not innervate visceral tissues
in an entirely symmetrical fashion. For example, the liver is
innervated predominately by the left vagus (Carobi
1990), the proximal duodenum by the left vagus, and the distal
duodenum/jejunum by the right (Berthoud et al. 1997
).
These differences raise the possibilities that the baseline
excitability of right and left NGNs may be different, and that right
and left NGNs may respond differently to vagotomy. The data presented
in the previous section demonstrate that baseline excitability is
similar in right and left NGNs. To test for a differential effect of
right and left vagotomy, the effects of treatment (vagotomy vs.
contralateral control) and vagotomy side (right or left) on each of the
variables in Table 1 were measured using a two-way ANOVA. For each
variable, a Student-Newman-Keuls test revealed no significant
interaction between the side of vagotomy (right/left) and treatment
(vagotomy/control). That is, either right or left vagotomy induced
similar decreases in excitability and alterations in membrane and
passive properties in NGNs of the vagotomized side.
Sham vagotomy
Although excitability changes are most likely due to severing of the vagus nerve per se, it is possible that they are triggered by damage to tissues surrounding the vagus caused by the vagotomy procedure. To examine this issue, we performed a sham vagotomy surgery on two animals and examined the excitability of their right (sham, n = 12) and left (control, n = 13) NGNs 5 days later. The sham surgery consisted of exposing the vagus nerve and retracting the surrounding tissues, as during vagotomy, but not touching the nerve itself. The excitability, passive properties, and AP characteristics of the sham and control neurons were similar; no statistically significant differences existed for any of the variables listed in Table 1. Changes in the excitability of NGNs following vagotomy were therefore a result of vagus nerve section, and not damage to surrounding tissues.
Time course
The above data were obtained from NGNs removed 5 days post-vagotomy. To examine the time course of the changes in excitability and passive membrane properties following vagotomy, we repeated similar measurements on NGNs removed 17 h and 20-21 days post-vagotomy.
Properties of vagotomized NGNs (n = 14) and
contralateral control (n = 14) NGNs removed from two
animals 17 h after vagotomy were similar to the values for control
NGNs shown in Table 1. There were no statistically significant
differences between vagotomized and contralateral NGNs for AP
threshold, AP waveform, or the number of APs elicited by the stimulus
protocols (2-way ANOVA for each). The only statistically significant
difference between the two groups was a modest (~20%) increase in
membrane input resistance on the vagotomized side (626 ± 60 M
vs. 503 ± 37 M
for contalateral NGNs). Further, there were no
statistically significant differences between either vagotomized or
contralateral NGNs from these animals and NGNs from control animals for
any of the properties listed in Table 1 (1-way ANOVA).
Significantly decreased neuronal excitability was still apparent in vagotomized NGNs removed from two animals 20-21 days following surgery, indicating that the effects of vagotomy were maintained at least this long. Specifically, vagotomized NGNs (n = 13) had significantly (P < 0.05) higher rheobase (212 ± 46 vs. 56 ± 1 pA), and fired significantly fewer APs in response to 3 times threshold stimuli (6 ± 1.4 vs. 34 ± 4.8 APs) and fewer APs in response to the standardized series of current steps (4.4 ± 1.2 vs. 5.5 ± 1.4 APs) than their contralateral controls (n = 15). Vagotomized NGNs also had decreased AP overshoot (34 ± 3.4 vs. 47 ± 2.4 mV for controls) and slightly increased capacitance (38 ± 2 vs. 31 ± 2 pF for controls). Interestingly, while the differences between vagotomized NGNs and contralateral control NGNs persisted, there was a trend toward increased excitability in both groups relative to the earlier time points. This observation was not pursued in the current work.
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DISCUSSION |
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Vagotomy dramatically decreased the excitability of NGNs,
increasing AP threshold by over 200%, and reducing AP discharge by up
to 80% in response to strong depolarizing stimuli. Despite differences
in the target organs of the right and left vagus nerves, baseline
excitability and changes induced by vagotomy were similar in right and
left NGNs. Sham operations did not duplicate these changes in
excitability, suggesting that local inflammatory reactions near the
vagus nerve do not trigger changes in the neurons. While the mechanism
of vagotomy-induced hypoexcitability in NGNs is unknown, the time
course reported here was similar to that of ionic current changes in
axotomized DRG neurons that appear to be due to the removal of
peripheral trophic factors (Dib-Hajj et al. 1998;
Fjell et al. 1999
). Vagotomy or the disruption of axoplasmic transport has also been shown to change the expression of
mRNA and protein for various neuropeptides in the nodose ganglia (Zhuo et al. 1994
, 1995
), with a time
course similar to that of the excitability changes we report here.
Approximately 90% of NGNs are C-fiber afferents and the remainder
A-afferents (Gallego and Adrover 1990;
Gallego and Eyzaguirre 1978
; Mei 1983
).
Previous investigators (Schild et al. 1994
) have classified isolated NGNs as presumed A- or C-type afferents based on
their electrophysiological characteristics, specifically the presence
of a "hump" on the falling phase of the C-cell action potential.
Because the AP characteristics of NGNs are changed by vagotomy, we
avoided classifying our cells in this regard. Due to the large
differences between control and vagotomized NGN populations, we can
conclude that most C-type NGNs become less excitable following
vagotomy. Additional experiments will be required to definitively
establish whether A-afferents are also affected, and whether specific
populations of C-afferents (heart afferents, airway afferents, etc.)
respond differently to vagotomy.
The concordance of data obtained with patch pipettes in isolated NGNs and sharp microelectrode recording in intact ganglia demonstrated that excitability changes observed were unlikely to have arisen from an artifact of the dissociation process or cell dialysis during patch-clamp recording. The close correspondence of the two data sets (patch and sharp electrode) further validated the use of the isolated soma as a model for studies of excitability of rat nodose ganglion neurons.
The excitability of control NGNs in our study differed from those
described previously by Schild et al. (1994), who noted that most neonatal rat NGNs fire only a single AP in response to
suprathreshold depolarizing current steps. However, many nodose A- and
C-fiber afferents of the adult rabbit and adult cat discharge multiple
action potentials in response to supra-threshold depolarizing stimuli
(Gallego and Eyzaguirre 1978
; Jaffe and Sampson
1976
). This suggests that differences between our observations
and those of Schild et al. (1994)
may reflect a
difference between neonatal and adult afferents, although differences
in tissue culture conditions, external and internal solutions, and
recording protocols may also contribute to the disparate results. Based
on the similarity between accommodative AP properties observed in
intact ganglia with sharp microelectrodes and in acutely dissociated
NGNs with patch pipettes, we believe our results may represent an
accurate description of the response of adult rat NGNs to sustained
depolarizing stimuli.
The somata and the neuroma of some axotomized DRG neurons have been
implicated as regions generating spontaneous AP activity (Wall
and Devor 1983). By contrast, the somata of vagotomized NGNs
were less excitable than control NGN somata by every measure examined,
and less able to generate APs in response to sustained depolarizing
current. Our work did not directly study whether vagal neuromas could
generate spontaneous AP activity. However, the neuromas were attached
to nodose ganglia during our "sharp" microelectrode experiments on
vagotomized NGNs, and we did not observe spontaneous APs in any of the
NGNs during excitability measurements. It is therefore possible that
the vagal neuroma, like the somata of axotomized NGNs, is also
electrically hypoexcitable (relative to those of spinal afferents).
Electrical activity in the somata of NGNs, like those of DRGNs, may not
be necessary for AP conduction to the CNS. Thus, the question arises
whether excitability changes recorded in primary sensory somata
adequately reflect changes in the electrophysiology of the entire
neuron (including central and peripheral axons, growth cone, and
central terminals). Although our work reveals that the somata of
vagotomized NGNs are hypoexcitable, and conduction velocity has been
reported to be decreased in vagotomized vagal C-fiber afferents
(Gallego and Adrover 1990), additional work will be
required to determine whether AP generation is suppressed throughout
the entire vagotomized nodose neuron.
Following vagotomy, rats display enhanced responses to several types of
noxious stimuli (Khasar et al. 1998a,b
; Miao et
al. 1997
). Modified pain responses are consistent with the
observation that vagal stimulation can either enhance or inhibit
nociception, depending on the circumstances (Randich and Gebhart
1992
; Ren et al. 1988
). If vagal afferents
became hyperexcitable following vagotomy, this would suggest continual
pro-nociceptive input from the nodose ganglia is enhancing pain
responses. However, our data suggest that hyperalgesia in vagotomized
rats is more likely a consequence of decreased vagal anti-nociceptive
input resulting from a profound decrease in NGN excitability, combined
with the removal of normal stimuli.
Profound changes in voltage-gated sodium, potassium, and calcium
currents have been reported in axotomized DRG neurons (Baccei and Kocsis 2000; Cummins and Waxman 1997
;
Everill and Kocsis 1999
) and are thought to underlie
changes in excitability in these neurons. While changes in passive
membrane properties in vagotomized NGNs (3 mV more negative
Em, 35% increased
Cm, and 23% decreased
Rin) may contribute slightly to
increased rheobase, the 200% increase in rheobase and substantial
decreases in AP discharge are very likely the results of changes in
voltage-dependent ionic currents. The ionic current(s) altered to
support decreased excitability in vagotomized NGNs are presently
unknown. Based on the data presented here and the roles of sodium and
potassium currents in shaping NGN AP discharge (Schild and Kunze
1997
; Schild et al. 1994
), a reduction in sodium
current and/or an augmentation of potassium current is likely. Either
change could produce increased AP threshold, decreased AP overshoot,
and decreased AP discharge. Additionally, the most likely explanation
for more negative resting membrane potentials in vagotomized NGNs is an
increased potassium conductance at rest. The relatively homogenous
response of NGNs, a population composed mostly (~90%) of C-fiber
afferents (Gallego and Adrover 1990
; Gallego and
Eyzaguirre 1978
; Mei 1983
), to nerve injury provides a tractable model system for studying the role of ionic currents in shaping AP discharge after injury.
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ACKNOWLEDGMENTS |
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The authors thank Drs. M. S. Gold and D. R. Matteson for reviewing an earlier version of the manuscript.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-22069.
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FOOTNOTES |
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Address for reprint requests: D. Weinreich, Dept. of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine, Rm. 4-002, Bressler Research Bldg., 655 West Baltimore St., Baltimore, MD 21201-1559 (E-mail: dweinrei{at}umaryland.edu).
Received 26 June 2000; accepted in final form 25 September 2000.
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