INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Most vagal afferent nerves can be activated by mechanical deformation of the receptive field. Mechanical activation is thought to occur via the opening of mechanically gated ion channels. Mechanically gated ion channels in vagal sensory neurons are nonselective cation channels that when opened result in the influx of ions including calcium from the extracellular space (Cunningham et al. 1997; Drummond et al. 1998; Sullivan et al. 1997). Calcium can also enter vagal sensory cell bodies during the action potential via both N- and L-type calcium channels (Danks et al. 1994). Calcium entering through these channels leads to the release of additional calcium from an endogenous calcium-induced calcium release pool (Cohen et al. 1997).

Increases in intracellular calcium may serve to inhibit airway vagal afferent nerve activity by entering the nerve and increasing the open times of certain types of potassium channels (Hay and Kunze 1994). The influx of extracellular calcium into vagal sensory neurons through voltage-gated N-type calcium channels during the action potential can lead to activation of a distinctive class of potassium channels that causes a slowly developing and long-lasting afterspike hyperpolarization (AHP-slow) of the membrane (Cordoba-Rodriguez et al. 1999; Undem and Weinreich 1993). This calcium-activated AHP-slow is effective in inhibiting the maximum frequency by which the nerve can elicit action potentials. Agonists of the large-conductance calcium-activated potassium current, such as NS1619, can also inhibit the excitability of vagal afferent nerve endings in guinea pig isolated airways by a mechanism that is blocked by the selective potassium channel blocker iberiotoxin (Fox et al. 1997).

On the other hand, extracellular calcium may serve to increase activity of airway afferent nerves. For example, if calcium is a significant charge carrier in the mechanically or chemically gated ion channels, extracellular calcium may have positive influence on excitability (Cunningham et al. 1997; Hunt et al. 1978). In some neurons, calcium can interact with an extracellular calcium-sensing receptor leading to increases in cation currents (Chattopadhyay et al. 1999). Calcium may also increase the excitability of vagal afferent nerves by activating an outward chloride current (Lancaster et al. 2001).

These types of electrophysiological considerations predict that a rise in intracellular calcium can have both inhibitory and excitatory effects on afferent nerve activity, depending on the composition of ion channels within the given nerve. Therefore the affect of decreasing extracellular calcium on the activity of a particular type of afferent nerve must be determined empirically. The affect of reducing extracellular calcium concentration in the activity of primary vagal afferent nerves innervating the airways has not been studied.

The vagal afferent innervation to the guinea pig trachea and bronchus can be conveniently categorized based on their mechanical sensitivity and location of the cell bodies (Undem and Carr 2001). Approximately half the nerve endings are low-threshold mechanosensors. These fibers adapt rapidly to a sustained stimulus, conduct action potentials in the A range and have cell bodies located nearly exclusively in the nodose ganglion. The remaining fibers are high-threshold mechanosensors that have cell bodies located in the jugular ganglion (Riccio et al. 1996a). The high-threshold mechanosensors comprise C and A fibers, both of which respond to bradykinin, capsaicin, and hypertonic solutions (Kajekar and Myers 2000; Pedersen et al. 1998; Riccio et al. 1996a). By analogy to the somatosensory system, the high-threshold mechanosensory are classified as nociceptive-like fibers.

In the present study, we evaluated the effect of reducing the concentration of extracellular calcium ions on the excitability of nociceptive and non-nociceptive afferent nerve endings in a guinea pig isolated innervated trachea/bronchus preparation and in jugular ganglion neuronal somata. The data support the hypothesis that modest reductions in extracellular calcium concentration significantly and selectively increases the excitability of nociceptive-like airway vagal afferent nerves.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Tissue preparation for extracellular recording

Male Hartley guinea pigs (200-400 g) were killed by asphyxiation with CO₂ and exsanguinated. The airways were prepared as previously described (Riccio et al. 1996a). Briefly, airways with intact right-side extrinsic innervation (including nodose and jugular ganglia) were removed and placed in a dissecting dish containing Krebs' bicarbonate buffer solution gassed with 95% O₂-5% CO₂ and composed of (in mM) 118 NaCl, 5.4 KCl, 1.0 NaH₂PO₄, 1.2 MgSO₄, 1.9 CaCl₂, 25.0 NaHCO₃, and 11.1 dextrose (pH 7.4). Connective tissue was trimmed away, leaving the trachea, larynx, and right bronchus with intact nerves (vagus, superior laryngeal, and recurrent), including nodose and jugular ganglia. A transverse cut was made along the ventral surface to open the larynx, trachea, and bronchus. Airways were then pinned to a silicone elastomer (Sylgard) lined Perspex chamber. The right nodose and jugular ganglia, along with the rostral most vagus and superior laryngeal nerves, were gently pulled through a small hole into an adjacent compartment of the same chamber for recording of single fibre activity. Both compartments were superfused separately with the Krebs' bicarbonate buffer solution. The temperature of the buffer was maintained at 37°C with a flow rate of 6-8 ml/min. Studies using blue dye revealed that the buffer solutions perfusing each compartment remained separate. The experiments were approved by the Johns Hopkins Animal Care and Use committee.

Extracellular recording of action potentials

Extracellular recordings were performed by manipulating a fine aluminosilicate glass electrode near cell bodies in either the jugular or nodose ganglion. The microelectrodes were pulled using a Flaming/Brown micropipette puller (Sutter Instrument, Novato, CA) and filled with 3 M sodium chloride. The recorded signal was amplified (A-M Systems) and filtered (low cut-off = 0.3 kHz; high cut-off = 1 kHz), and the resultant activity was displayed on an oscilloscope (TDS 340, Tektronix, Beaverton, OR) and a model TA240S chart recorder (Gould, Valley View, OH). The data were stored on digital tape (DT-120RA, Sony, Tokyo, Japan) for off line waveform analysis on a Macintosh computer using the software program The NerveOfIt (Phocis, Baltimore, MD).

Discrimination of single-fiber activity, location of receptive fields, and determination of conduction velocities

Single-fiber activity was discriminated by placing a concentric electrical stimulating electrode on the recurrent laryngeal nerve, through which the majority of fibers enter the trachea (Riccio et al. 1996a). The recording electrode was placed within the ganglion and manipulated until single-unit activity was detected. When electrically evoked action potentials were seen, the stimulator was switched off and the trachea and bronchi were gently probed with a von Frey filament. Mechanically sensitive receptive fields were revealed when a burst of action potentials was elicited in response to von Frey filament stimulation. Conduction velocity and amplitude of the action potential were then compared with responses elicited by electrical stimulation of the superior laryngeal, recurrent laryngeal, or vagus nerve trunks to determine the trunk that supplied the fiber.

Conduction velocities were calculated by electrically stimulating the receptive field and measuring the distance the action potential traveled along the nerve pathway divided by the time between the shock artifact and the recorded action potential. Fibers were classified as C fibers if their action potentials traveled <1.5 m/s. Fibers were classified as A fibers if their action potentials traveled at >2 m/s.

Mechanical stimulation

Mechanical thresholds were determined using calibrated von Frey filaments as described previously (Riccio et al. 1996b). The action potential pattern to a ramp-and-hold mechanical stimulus was determined as previously described (McAlexander et al. 1999). Briefly, a blunt cylindrical Plexiglas probe connected to a Grass model FT03C force transducer (Astra-Med, Warwick, RI) was attached to a motorized micro manipulator (MS 314, DC3001R, WPI, Sarasota, FL). The force transducer was connected to the second channel of the Gould chart recorder so that the degree of force applied to the tissue could be monitored on-line. The probe was lowered onto the receptive field until action potential discharge was noted. The threshold for stimulation with the blunt probe averaged ~1.0 g for nodose ganglion-derived fibers and 1.5 g for jugular ganglion-derived fibers. Because of the large diameter (~3 mm) of the probe, the threshold force was greater than that previously noted with von Frey filaments (Riccio et al. 1996a). After an individual fiber's threshold for action potential discharge was determined, the probe was lowered until a force of three times the threshold force was reached. This force was held for 10 s in a ramp-and-hold protocol.

Calcium reduction

After the mechanical receptive field and conduction velocity of a nodose or jugular nerve fiber has been established, von Frey fibers were used to obtain its mechanical threshold. The action potential pattern to a 10-s ramp and hold (3 times mechanical threshold) was determined and repeated after 5 min. The average of the two responses was taken as the baseline response. The buffer solution was then changed to one in which the calcium chloride was replaced with equi-molar magnesium chloride (in several experiments the calcium chloride was removed and not replaced with magnesium chloride and the results were noticeably different). The response of the nerve fibers to calcium-free buffer solution was recorded. If after 5 min of exposure to calcium-free solution the nerve fiber discharge was at baseline levels (less than ~0.2 Hz), the mechanical threshold was determined and the response to a ramp-and-hold mechanical stimulus was investigated two times at 5-min intervals. The average of the two responses was calculated. The buffer superfusing the tissue was then switched back to the normal buffer solution, and the mechanical responsiveness of the fibers was again analyzed. In some experiments, the response of the fiber to several concentrations of extracellular calcium was determined. After a baseline mechanical response was obtained, the tissue was superfused with calcium-free buffer solution for 5 min, and the mechanical response was obtained as described in the preceding text. The airway was then superfused with control buffer solution (Ca²⁺ 1.9 mM) for 15 min, and a new baseline response established to which the response in the presence of the next low-calcium solution was compared. This process was repeated with the calcium in the buffer solution reduced to 0.2, 0.4, and 1 mM. There was no significant difference in the control responses obtained between each low-calcium challenge.

Isolation of jugular ganglion neurons

Jugular ganglion neurons (JGNs) were dissociated enzymatically as described previously for nodose ganglion neurons (Lancaster et al. 2001). JGNs, adhered to 15-mm round glass polylysine-coated cover slips, were maintained in culture for 2-9 h at 37°C prior to recording.

Labeling airway projecting jugular ganglion neurons

We modified the airway labeling procedure of Christian (Christian et al. 1993). Guinea pigs were anesthetized with ketamine (50 mg/kg ip)/xylazine (10 mg/kg ip). To expose the trachea, a midline incision, ~1.5 cm, was made from the larynx caudally. Five hundred microliters of a 0.5 mg/ml DiC18 (diI; Molecular Probes, Eugene, OR) solution (in sterile saline) was injected into the tracheal lumen using a 27.5 gauge needle. The skin was sutured with 4.0 silk, and the animal was maintained in a supine position with its head tilted at ~30° until the animal awoke (~30 min). Neurons were studied 10-12 days after dye injection. DiI labeled vagal somata did not show any changes in their passive or active membrane properties when compared with non-labeled vagal neurons (unpublished observations).

Patch-clamp recording

Whole cell patch-clamp techniques were employed as described by Lancaster et al. (2001) using an Axopatch 200B amplifier and PCLAMP7 software (Axon Instruments, Union City, CA). Pipettes were filled with a solution composed of (in mM) 140 KCl, 2 MgCl₂, 10 HEPES, 11 EGTA, and 10 dextrose; titrated to pH 7.3 with KOH; 306 m/OsM. Pipette voltage offset was neutralized prior to the formation of a gigaseal. Membrane input resistance (R_in), series resistance (R_s), and capacitance (C_m) 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% R_s compensation were used. Criteria for cell inclusion in the study were: R_s < 10 M, R_in > 100 M, and stable recording with 80% R_s compensation during the entire experiment. Cover slips were superfused (2-4 ml/min) continuously during recording with Locke solution (32-35°C); composition (in mM): 10 dextrose; 136 NaCl; 5.6 KCl; 1.2 MgCl₂, 6H₂0; 2.2 CaCl₂, 2H₂0; 1.2 NaH₂PO₄; 14.3 NaHCO₃), equilibrated with 95% O₂-5% CO₂, pH ranged between 7.3 and 7.5). The recording chamber was grounded via a 3 M KCl agar bridge.

"Sharp" microelectrode recording

For "sharp" microelectrode recording, intact jugular ganglia were placed on the floor of the recording chamber, covered with gauze thread, and superfused with Locke solution (3-4 ml/min; 26-29°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 JGNs blindly. R_in was calculated from the peak amplitude of electronic voltage transients evoked by 100-pA hyperpolarizing currents at 60 mV. Resting membrane potential was determined as the membrane potential recorded with zero current injected, corrected for tip potential. Criteria for cell acceptance include: an action potential overshooting 0 mV and a R_in > 10 M (typically 20-100 M).

Statistics

Data are presented as means ± SE. The peak frequency in response to mechanical stimuli represents the largest number of action potentials in a 1-s bin. Electrophysiological data were compared using a one-way ANOVA followed by Student's non-paired t-test to locate any differences detected with the ANOVA.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Jugular (nociceptive-like) fibers

Jugular afferent fibers in the isolated airway preparation had no or very little (<1 Hz) background impulse activity. The occasional fibers that was spontaneously discharging action potentials at >1 Hz were not studied (<5%). When the calcium was removed from the Kreb's bicarbonate buffer solution superfusing the mechanical receptive field in isolated trachea/bronchus, 10 of 15 (67%) C fibers responded with a burst of action potentials. Typically, in the continuous absence of [Ca²⁺]_o, the response waned over ~5-10 min (e.g., Fig. 1), however, 3 of the 10 fibers responded with a persistent (>10 min) barrage of action potentials. In these persistent responding fibers, action potential discharge ceased within 1 min of superfusion with normal buffer solution. Jugular A fibers were less responsive to reduced [Ca²⁺]_o, as only 4 of 15 fibers (27%) responded with action potential discharge.

View larger version (46K): [in this window] [in a new window]   Fig. 1. A representative recording of a single afferent nerve responding to superfusion of the receptive field in the airway with zero-calcium buffer with barrage of action potentials. This fiber was a jugular fiber with a conduction velocity in the A range (3.6 m/s). Note that the response subsides despite the continued presence of calcium-free buffer. The horizontal bar represents 5 min.

We evaluated the response of jugular nerve endings in the airways to a mechanical force before and 5 min after switching the superfusion solution to calcium-free buffer solution. In those fibers that responded to zero calcium with action potential discharge, the mechanical response was studied after the frequency of action potential discharge was <1 Hz. If this did not occur by 15 min, the fiber was not no longer studied. In jugular C fibers, the number of action potential discharge evoked in response to a 10-s, three times threshold mechanical stimulus significantly increased from 88 ± 20 to 170 ± 45 when calcium was removed from the superfusing buffer solution (P < 0.05, n = 11). As with jugular C fibers, mechanically induced action potential discharge was also increased in jugular A fibers. The total number of action potentials evoked by a 10-s ramp-and-hold mechanical stimulus was increased from 100 ± 17 to 238 ± 46 by removing calcium from the superfusing solution (P < 0.05, n = 11). The pooled data from all jugular A and C fibers studied is illustrated in Fig. 2 and is presented in Table 1.

View larger version (32K): [in this window] [in a new window]   Fig. 2. A histogram showing the mean ± SE of the number of action potentials evoked by a ramp-and-hold mechanical stimulus applied to the receptive field of C and A fibers derived from jugular ganglion neurons and A fibers derived from nodose ganglion neurons. The mechanical stimulus was adjusted to 3 times threshold and held for 10 s then removed. The bars represent the number of evoked action potentials before () and after removing calcium from the superfusion buffer (). The n values were 11, 11, and 12 for jugular C fibers, jugular A fibers, and nodose A fibers, respectively. *, a significant difference between the responses obtained before and after calcium removal. Inset: a representative recording from a jugular A fiber showing the action potential discharge in response to a 10-s 3 times threshold ramp-and-hold mechanical stimulus () before (control), after 15 min of superfusion with nominally calcium-free buffer and again 5 min after switching to control Krebs' solution (2nd control). The number of action potentials evoked by the mechanical stimulus in the respective 3 consecutive responses were 58, 155, and 75. The respective peak frequencies in 1-s bins were 10, 27, and 11 Hz.

In 12 experiments (7 C fibers and 5 A fibers), we evaluated the reversibility of the effect of removing calcium on mechanical responsiveness of the nerve endings. In these experiments, removing extracellular calcium for 5 min increased the number of action potentials to the mechanical ramp-and-hold stimulus from a control value of 127 ± 27 to 235 ± 51 (P < 0.01). Within 5 min of returning calcium to the superfusion buffer, the number of mechanically provoked action potentials returned to control values (93 ± 18, P > 0.1; Fig. 2, inset).

We evaluated the concentration-response relationship for low-calcium-induced enhancement in mechanical excitability of jugular nerve endings. The receptive field was superfused with buffer containing a fixed concentration of calcium for 5 min prior to mechanical stimulation. The receptive field was then superfused with control buffer solution (containing 2 mM calcium) for 15 min to establish a new baseline for mechanically induced responses (see METHODS). Reducing the calcium concentration from 2 to 1 mM led to an increase in mechanically induced action potential discharge, and the maximum potentiating effect was observed at an extracellular calcium concentration of 0.2 mM. The half-maximal effect was estimated by extrapolation to occur at an extracellular calcium concentration of ~0.6 mM (Fig. 3).

View larger version (15K): [in this window] [in a new window]   Fig. 3. The concentration-response relationship between decreasing calcium concentration in the superfusion buffer and the increase in action potential discharge of jugular airway afferent fibers to a 3-times threshold ramp and 10-s hold mechanical stimulation. In 4 experiments (2 C fibers and 2 A fibers), the number of action potentials in control (2 mM calcium) buffer averaged 82 ± 20. This increased to 206 ± 33 in the presence of 0-calcium buffer. This increase was taken as 100% and the response to 0.2; 0.4 and 1 mM were calculated as a percentage of this increase. Each bar represents the mean ± SE (n = 4).

We tested whether the increase in mechanically-induced action potential discharge upon lowering [Ca²⁺]_o was explained by an increase in the peak frequency of discharge or a decrease in the degree of adaptation over the 10-s stimulation. In the jugular A fibers, the peak frequency of mechanically induced action potential discharge was statistically increased after treatment with calcium-free buffer solution (11 ± 2 vs. 23 ± 3 Hz, respectively P < 0.01, n = 11). This was not the case for jugular C fibers. The peak frequency of mechanically induced action potential discharge was not significantly different before and after treatment with calcium-free buffer solution (10.4 ± 2.1 vs. 13.6 ± 3.4 Hz, n = 11, P > 0.05).

The mechanical thresholds, as determined by von Frey analysis, of the jugular C fibers and jugular A fibers averaged 3.1 ± 0.1, and 3.3 ± 0.2 mg, respectively. The mechanical threshold of these fibers was unaffected by superfusion with calcium-free buffer solution tested after action potential activity has subsided (P > 0.1).

Neither the peak frequency of mechanically induced action potential discharge nor the total number of action potentials evoked was affected by the treating the tissues with the N-type calcium channel blocker (-conotoxin, GVIA, 1.0 µM). In seven jugular fibers (4 A and 3 C fibers), the number of action potentials to a 10-s mechanical ramp-and-hold stimulus averaged 55 ± 10 and 82 ± 12 before and after 30 min treatment with -conotoxin, respectively (P > 0.1). In these same seven fibers, subsequent removal of calcium from the buffer solution increased the number of action potentials evoked by the mechanical stimulus to 158 ± 38, P < 0.05. As mentioned in the preceding text, removing calcium from the superfusion solution was associated with an increase in the peak frequency of mechanically induced action potential discharge in jugular A fibers. By contrast, the peak frequency of action potential discharge was unaffected by -conotoxin. In the four A fibers the peak frequency of mechanically induced action potential discharge was 13 ± 3 and 15 ± 3 Hz before and after -conotoxin treatment, respectively. Subsequent removal of calcium from the buffer solution superfusing the tissue resulted in an increase in peak frequency in the four A fibers to 29 ± 3 Hz (P < 0.05). In an additional five experiments, combining the L-type calcium channel blocker (nifedipine 10 µM) with -conotoxin (1.0 µM) also failed to affect the number of action potentials evoked by the 10-s ramp-and-hold mechanical stimulus (Table 1).

The nonselective potassium channel blocker, TEA (10 mM), had no effect on mechanically evoked action potential discharge in two A and two C jugular afferent fibers (Table 1).

Nodose fibers (rapidly adapting low-threshold mechanosensors)

Consistent with our previous findings, the vast majority (10 of 12) of nodose fibers innervating the guinea pig trachea/bronchus conducted action potentials in the A range (conduction velocity = 5.2 ± 0.7 m/s). The other two fibers conducted action potentials at 1.1 and 1.2 m/s. The nodose fibers adapted rapidly to the ramp and hold stimulus, with >99% of the response occurring during the dynamic (ramp phase) of the stimulus. By contrast to the afferent fibers arising from cell bodies in the jugular ganglion, 0 of 12 nodose nerve fibers responded with action potential discharge when their receptive fields were superfused with calcium-free buffer solution. Superfusion with calcium-free buffer solution also had no effect on the mechanical threshold (averaging 2.1 ± 0.1 mg). Unlike the jugular fibers in the airways, superfusing the receptive fields of nodose nerve fibers with calcium-free buffer solution had no effect on the peak frequency or the total number of action potentials evoked by a three-times threshold ramp-and-hold mechanical stimulus (Fig. 2).

Dissociated neurons

The affects of reduced [Ca²⁺]_o on airway afferent nerve terminal excitability could be due to a direct action of Ca²⁺ on the nerves or an indirect affect on other cell types in the airway tissue. We thus examined the effects of reducing [Ca²⁺]_o on neurons acutely dissociated from the jugular ganglion. Dissociated JGNs are free of adherent satellite cells and are often used as a tractable model for investigation of voltage and ligand gated ion channels in sensory nerves. Accordingly, we applied sharp micropipette electrodes and whole cell patch-clamp techniques to study the effect of reducing [Ca²⁺]_o on electrophysiological membrane properties of JGNs.

Properties of 86 JGNs isolated from guinea pig jugular ganglia were studied using sharp microelectrodes. The neurons were superfused with a bicarbonate-based buffer solution similar in fashion to the experimental design used to study the nerve endings within the airways. Superfusion with calcium-free buffer solution depolarized the resting membrane potential in 41 of 86 neurons an average of 13 mV (Fig. 4). The membrane potential of these neurons averaged 62 ± 1 and 49 ± 1 mV before and after exposure to calcium-free buffer solution, respectively (P < 0.01). The membrane depolarization coincided with a significant decrease in R_in. The R_in was 58 ± 6 and 44 ± 4 M before and after exposure to calcium free buffer solution, respectively (P < 0.01). The decrease in R_in observed in current-clamp mode was unlikely to be secondary to opening of voltage-gated ion channels as the current-voltage relationship in these neurons revealed a linear I-V relation between resting potential and 40 mV (not shown). In two neurons, the sensitivity of the response to reduction in extracellular calcium solution was examined. Reducing the calcium from 2 to 1 mM was sufficient to cause membrane depolarization and a decrease in R_in. The transient membrane depolarization observed when the buffer solution was reduced from 2 to 1 mM averaged 70% of that observed with the calcium-free solution.

View larger version (52K): [in this window] [in a new window]   Fig. 4. A representative intracellular recording from an isolated guinea pig jugular ganglion neuron before and after switching the superfusion solution to 0-calcium. The vertical lines represent electrotonic voltage transients resulting from brief (100 ms) injections of hyperpolarizing current (100 pA). The decreasing amplitude of the voltage transient during the depolarization reflects a decrease in input resistance of the membrane. The resting membrane potential of this neuron was 65 mV, and the resting input resistance was 50 M. This response was representative of 41 of 86 neurons in which the average membrane depolarization was 13 mV after switching to zero [Ca2+]o solution (see text for details).

The whole cell patch-clamp technique was used to measure membrane currents and to estimate the reversal potential (E_rev) of the current evoked by exposing JGNs to calcium-free solution. In the first series of experiments, 25 (unlabeled) JGNs were studied. In ~50% of these neurons (12/25), voltage-clamped at 60 mV, application of external solution with nominally zero calcium produced an immediate inward current (Fig. 5A). The slight delay for onset time of the inward current reflects the time required for solution change. The peak amplitude of the current averaged 1.9 ± 0.5 nA. The E_rev for the low calcium-induced inward current was estimated by constructing I-V plots before and during the peak of the inward current using ramp-voltage commands. For the response shown in Fig. 5A, the E_rev was approximately 10 mV (Fig. 5C). In five experiments, E_rev ranged from 20 to +20 mV.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Effect of nominally 0 extracellular calcium on membrane currents recorded from voltage-clamped jugular neurons. A: effect of nominally 0 extracellular calcium recorded in an unlabeled jugular neuron voltage clamped to 60 mV. The inward current, recorded at (35°C), was unusually large in this neuron. Upward and downward deflections reflect current responses to a step ramp voltage command produced by 30-ms incremental steps (10 mV) from 40 to 100 mV. The peak current evoked by each voltage step was plotted against membrane potential to determine the reversal potential value (Erev), shown in C. Resting input resistance (Rin) and membrane capacitance (Cm) were 260 M and 52 pF, respectively. B: an inward current recorded in an identified jugular airway neuron (see METHODS) evoked by switching from control Locke solution to one containing nominally 0 extracellular calcium (horizontal bar). Cell was voltage-clamped at 60 mV. Rin and Cm were 960 M and 20 pF, respectively. Recording was made at 29°C. C: I-V relation recorded before (open circles) and after switching to 0 extracellular calcium (filled circles) for neuron shown in A. The Erev (upward arrow) was about 10 mV.

As with the airway nerve terminal studies, the effect of reducing calcium was noted even when the calcium concentration was reduced by only 50%. In four cells studied in current-clamp mode, the membrane potential depolarized an average of 8 ± 2 mV when the [Ca²⁺]_o was reduced from 2.2 to 1.1 mM. When the neurons were voltage clamped, this reduction in [Ca²⁺]_o was associated with 75 ± 20 pA inward current (n = 4). In an additional three responsive neurons, applying nifedipine (20 µM) plus -conotoxin GVIA (500 nM) did not evoke an inward current, whereas reducing [Ca²⁺]_o to zero caused an inward current (169 ± 54 pA) in each of the neurons (data not shown).

Using retrograde tracing techniques, eight airway-identified (dye-labeled) JGNs were studied. Four of eight showed an inward current upon switching to nominally zero extracellular calcium that averaged 0.9 ± 0.65 nA (range: 0.2-2.9 nA; Fig. 5B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study demonstrates that relatively modest reductions in extracellular calcium concentration substantially increases the excitability of nociceptive-like vagal afferent nerve fibers in the airways. The increase in excitability was manifest by a consistent increase in mechanically induced impulse generation and, occasionally, in the overt induction of action potential discharge. This was a selective effect on afferent nerve fibers in the airways derived from jugular ganglia, as reducing extracellular calcium concentration did not affect the excitability of airway afferent nerve fibers derived from nodose ganglion neurons.

The phenotype of vagal afferent fibers in the guinea pig trachea/bronchus have been categorized based on the ganglionic location of the cell body, fiber conduction velocity, and physiological properties (Kajekar et al. 1999; Kummer et al. 1992; Riccio et al. 1996a). The vast majority of fibers in the guinea pig extrathoracic airways conduct action potentials in the C range (<1 m/s) or A range (3-12 m/s). Neurons in the jugular ganglion project equal numbers of C and A fibers to the airways. These jugular fibers have relatively high threshold for mechanical activation and adapt slowly to ramp-and-hold suprathreshold mechanical stimulation. Nearly all jugular airway C fibers and the majority of jugular A fibers respond to capsaicin and bradykinin. Neurons located in the nodose ganglia project primarily A fibers to the airways. Unlike C and A fibers arising from the jugular ganglia, nodose A fibers are low-threshold mechanosensors that rapidly adapt to a ramp-and-hold mechanical stimulus (McAlexander et al. 1999; Riccio et al. 1996a). Guinea pig airway nodose A fibers are insensitive to capsaicin and bradykinin and relatively insensitive to hypertonic saline. These observations support the conclusion that neurons in the jugular ganglia project fibers to the airways that have characteristics of nociceptors, whereas the afferent nerves in the airways arising from nodose ganglia are rapidly adapting low-threshold mechanosensitive receptors (often referred to as RARs). That reducing extracellular calcium affected the excitability of afferent fibers in the airway arising from neurons in the jugular ganglia but not those arising from nodose ganglion neurons supports the conclusion that the effect is selective for nociceptive-like fibers.

Mechanical stimulation of vagal afferent neurons activates a nonselective cation channel through which extracellular calcium ions can flow (Cunningham et al. 1997; Raybould et al. 1999; Sharma et al. 1995). Elegant studies on mechanical transduction in muscle spindle fibers suggest that calcium ions may contribute to the mechanically induced generator potential (Hunt et al. 1978), although others have noted an inverse relationship between extracellular calcium and the afferent responsivity to mechanical activation of cat muscle spindles (Fischer and Schafer 2000). Reducing extracellular calcium concentration had no effect on mechanically induced afferent discharge in low-threshold RAR-type mechanosensors in the airway, and an inverse relationship was observed between extracellular calcium concentration and mechanical responsivity in high-threshold mechanosensors. This argues against the hypothesis that extracellular calcium contributes in a positive fashion to the mechanically induced generator potential in airway afferent nerves. This is consistent with findings in vagal afferent fibers innervating rat atrial and arterial baroreceptors, where decreasing extracellular calcium concentration increased the action potential discharge in response to increases in pressure (Andresen and Kunze 1987; Andresen et al. 1979).

We considered several mechanisms by which reducing extracellular calcium selectively excites nociceptive fibers in the airways. The increase in excitability could be explained by a nonspecific reduction in surface charge (i.e. the so-called surface potential surface potential theory). Second, reducing extracellular calcium could decrease calcium-mediated inhibition of sodium channels. Third, a decrease in calcium-activated potassium currents may lead to an increase in afferent nerve excitability in the airways. Finally, extracellular calcium could be responsible for inhibition of a specific excitatory cation current in nociceptive-like airway vagal afferent fibers afferent fibers.

The basis of so-called electric field theory is that local adsorption of calcium to the nerve membrane increases the gradient of local transmembrane electric fields resulting in changes in ion channel function (Armstrong 1999; Frankenhaeuser 1957; McLaughlin 1989; Zhou and Jones 1995). The electric field theory has been forwarded as an explanation by which reducing extracellular calcium increases baroreceptor activity (Andresen and Kunze 1987). Three considerations indicate that the increase nociceptor excitability observed when extracellular calcium was reduced is not due to nonselective effects on channel activity through changes in the electric field. First, in our experiments the extracellular cation concentration was kept constant by replacing the calcium ions with magnesium. Second, the affect on mechanical excitability was observed even when the calcium concentration in the buffer bathing the airways was only modestly reduced from 2 to 1 mM. Third, the selectivity of the effect indicated by the fact that reducing extracellular calcium had no influence on the low-threshold RAR type fibers is inconsistent with a nonspecific effect on ion channel function.

Calcium has long been known to enter sodium channels and affect their gating characteristics (Armstrong and Cota 1999). Again, the observation that decreasing extracellular calcium only increased excitability of a subset of airway afferent nerve endings argues against an effect on voltage-gated sodium channels. Mechanically evoked action potential discharge in both the jugular-ganglion-derived nociceptive fibers and the nodose-ganglion-derived low-threshold mechanosensors in our preparation is abolished by tetrodotoxin. It remains possible, however, that extracellular calcium has an inhibitory effect on a particular type of sodium channel only present in the nociceptive population. For example, in hippocampal neurons, reducing extracellular calcium concentration increases a persistent sodium current leading to increase in an after spike depolarization and increases in bursting activity (Su et al. 2001). We have observed afterdepolarizations in isolated nodose ganglion neurons, but unlike the hippocampal neurons, these potentials appear to be due to calcium-activated chloride and are abolished by removing extracellular calcium (unpublished observations).

The third hypothesis considered to explain our results was that under normal conditions extracellular calcium controls afferent excitability by activating potassium currents. By decreasing the concentration of extracellular calcium ions, less calcium enters the neuron and less calcium-activated potassium current may be realized. Consistent with this hypothesis is the finding that reduction in extracellular calcium concentration was selective for nociceptive-type fibers. Thus the calcium-activated potassium current responsible for the AHP-slow in guinea pig vagal sensory neurons appears to be selectively expressed in nociceptive C-fiber neurons and rarely observed in nodose A-fiber neurons (Undem and Weinreich 1993). Also consistent with a potassium channel hypothesis is the observation that pharmacologically activating the calcium-gated potassium current with NS1619 inhibits afferent nerve activity in guinea pig airways (Fox et al. 1997). On balance, however, our results fail to support a role of potassium channels in the effect of reducing extracellular calcium concentration on jugular fiber excitability. The majority of calcium ions entering guinea pig vagal sensory neurons during the action potential go through L- and N-type voltage gated calcium channels, and the AHP-slow observed in vagal afferent C fibers is abolished by the N-type calcium channel blocker -conotoxin (Cordoba-Rodriguez et al. 1999). We found that blocking both L and N channels with -conotoxin and nifedipine, respectively, had no affect on the excitability of jugular afferent fibers. A lack of effect of voltage-gated calcium channel blockers has also been reported in studies on low-calcium-induced increased excitability of aortic baroreceptors. The observation that the nonselective potassium channel blocker TEA, at a concentration that inhibits the iberiotoxin-sensitive potassium channel (Hay and Kunze 1994), did not mimic the effect of low extracellular calcium concentration on jugular fiber excitability also argues against a role for inhibition of calcium-activated potassium currents in this response. Finally, if reducing extracellular calcium blocks a tonically active potassium current, there should be a conductance decrease associated with removal of extracellular calcium. We observed a consistent increase in membrane conductance accompanying membrane depolarization or inward current brought about by reducing extracellular calcium.

The hypothesis that best explains our collective results is that extracellular calcium inhibits an excitatory current in nociceptive-like airway afferent fibers and that by reducing extracellular calcium, this inhibition is removed leading to an increase in nerve excitability. This hypothesis is consistent with the observation that the affect of low calcium was selective for nociceptive fibers, as there are other ion channels known to be selectively expressed on nociceptive-like afferent nerves (e.g., the TRPV1 channel, some TTX-resistant sodium channels). Moreover, this hypothesis is directly supported by the electrophysiological studies on neurons isolated from jugular ganglia that reveal pronounced calcium-inhibited inward currents in these neurons.

Reducing extracellular calcium has many effects on electrophysiological membrane properties of neurons (Xiong and MacDonald 1999). Characterizing the specific ion channel(s) responsible for the calcium-inhibited inward current in vagal sensory neurons is beyond the scope of this study. Nevertheless, the reversal potential observed in the whole cell patch configuration is consistent with calcium-inhibiting channels that are relatively nonselective for cations. It is also noteworthy that our data indicate that relatively large concentrations of calcium are required to inhibit the cation current. Thus modestly reducing the calcium concentration in the superfusing buffer solution to 1 mM was sufficient to depolarize the membrane potential of the cell body. This observation was in accord with the results obtained on mechanical excitability of the airway afferent nerve endings.

It should be noted that an inward cation current associated with reduction in extracellular calcium concentration is not a novel observation. It is known, for example, that in some voltage-gated calcium channels there is an anomalous mole fraction effect such that the channel becomes nonselectively permeant to sodium and other monovalent cations when the extracellular calcium concentration is reduced (Almers 1984). In chick dorsal root ganglion (DRG) neurons and mouse hippocampal neurons, modest reductions in extracellular calcium concentration evoked an inward cation current (Hablitz et al. 1986; Xiong et al. 1997). It was argued that calcium channels were involved in the inward current in the chick DRG neurons. Reducing extracellular calcium concentrations causes an inward cation current in cardiac myocytes that is not affected by nifedipine (Mubagwa 1997). A critical evaluation of this current in the cardiac myocytes lead the authors to hypothesize that some novel ion channel normally permeant to monovalent ions exist in the cardiac cell membranes. Calcium (or other divalent cations) may bind to this channel at a site required for the translocation of monovalent cations. Upon reducing extracellular calcium concentration, the block is removed and an inward current caused by sodium (and other monovalent cations) is uncovered. Our observations in vagal sensory nociceptive nerves would be consistent with this type of hypothesis.

The physiological relevance of calcium inhibition of vagal airway afferent fiber excitability is not known. Baseline extracellular calcium concentration is not a constant. Many of the jugular derived afferent nerve endings are found in the airway epithelium where water and ions are in constant flux (Hunter and Undem 1999). Increased nerve activity itself has been found to lead to local reduction in extracellular calcium concentrations. In the somatosensory cortex of cats, for example, neuronal discharge has been shown to lead to a transient decrease in extracellular calcium concentration from ~1.5 to 0.8 mM (Nicholson et al. 1978). If this was to occur in airway nociceptive nerve fibers, a positive feedback loop could lead to inordinate increases in action potential discharge. Although the present study focussed on airway sensory nerves, it is unlikely that the inhibitory effect of extracellular calcium on nociceptor excitability is specific for airway nerves. In studies on isolated vagal sensory cell bodies, we noted that the majority of sensory nerves randomly selected showed inward depolarizing currents in response to reduction in extracellular calcium. This suggests the effects observed regarding calcium inhibition of afferent nociceptor excitability may be extrapolated to visceral nociception in general.

Regardless of physiological consequences, the observation that the effect of calcium on afferent nerve excitability was selective for the nociceptive phenotype nerve endings may be useful to those interested in mechanisms whereby visceral nociceptors can be pharmacologically modulated independently of low-threshold mechanosensory fibers. The observations that modest reductions in extracellular calcium can activate certain airway afferent nerve terminals may also have practical relevance to those studying airway pharmacology. Thus increased afferent nerve excitability may explain some of the reflex physiology induced by the aerosol delivery of ethylenediamine, citric acid, and other calcium ion chelators.