INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Carotid body chemoreceptors respond to a decrease in arterial oxygen tension by increasing afferent spiking activity on the sinus nerve, which is a branch of the glossopharyngeal nerve. Somata of the chemoreceptor afferents are located in the petrosal ganglion. The increase in nerve activity is developmentally dependent-small in the newborn period and increasing to adult levels by 2 wk of age (Blanco et al. 1984; Kholwadwala and Donnelly 1992). At present it is unclear how the nerve afferent action potential is initiated; it is perhaps due to episodic depolarization events at the nerve terminal (Hayashida et al. 1980; Zhang et al. 2000) or perhaps due to a process that is intrinsic to the nerve terminals (Donnelly et al. 1998). In either case, excitability of the nerve terminals would be expected to play a critical role in determining afferent spike initiation, and the major cation that determines nerve excitability is sodium. The dependence of excitability on Na⁺ currents is supported by observations showing a high sensitivity of chemoreceptor afferent activity to isosmotic reductions in extracellular sodium concentration and to the application of low doses of TTX (Donnelly et al. 1998). In contrast, a reduction or removal of calcium causes little change in nerve activity or even an increase in afferent activity (Fidone et al. 1982; Lahiri et al. 1996).

At present, little is known about Na⁺ channel expression in the petrosal ganglia. Electrophysiologically, both TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) currents are readily recorded in dissociated petrosal neurons, but age, modality, and cell size were not previously considered (Stea and Nurse 1992). Anatomical studies and measurement of conduction velocity have shown that rat chemoreceptor afferents are primarily C-fibers with conduction velocities around 0.5 m/s (Donnelly 1999; Finley et al. 1992). Since direct data on petrosal ganglia are currently lacking, it might be expected that petrosal ganglia neurons express Na⁺ currents with a similar profile to dorsal root ganglia (DRG), which have been more extensively studied. Small DRG neurons express Na⁺ channel isoforms Na_v1.6, Na_v1.7, Na_v1.8, and Na_v1.9 at moderate to high levels (Black et al. 1996), and expression of Na_v1.3 decreases in the postnatal period (Waxman et al. 1994). Thus the working hypothesis for the present study was that the petrosal ganglia expresses multiple subtypes of TTX-S and TTX-R currents, that both are present in chemoreceptor afferent neurons, and that developmental changes in Na⁺ channel subtypes may help to explain the developmental increase in chemoreceptor responsiveness with age.

Besides sensitivity to TTX, TTX-R and TTX-S type currents differ in their activation and inactivation characteristics. Compared with TTX-S currents, TTX-R currents typically activate at more depolarized potentials and development of inactivation is slower than for TTX-S currents. For instance, the TTX-S-type current Na_v1.7 activates near 50 mV (Cummins et al. 1998) while the TTX-R-type current Na_v1.8 activates above 40 mV (Dib-Hajj et al. 1998). Similarly, differences in activation for TTX-S and TTX-R current have been reported for the whole petrosal ganglia (Stea and Nurse 1992). Therefore, in discussing currents in our cells, we will refer to them as TTX-S or TTX-R, although pharmacologic identification was not undertaken in every instance. A reference to TTX-R currents should be taken as referring to currents that activate at a more depolarized potential, have a slower development of inactivation, and have a lower sensitivity to TTX.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Labeling and isolation of petrosal ganglion neurons

Chemoreceptor complexes (carotid body, sinus nerve, glossopharyngeal nerve, and petrosal ganglion) were isolated from rats as previously described under a protocol approved by the Yale Animal Care and Use Committee (Donnelly et al. 1998). In brief, Sprague-Dawley rats (Harlan, Chicago, IL) were anesthetized by placement in a chamber in which the atmosphere was saturated with methoxyfluorane vapor. The anesthetized rats were removed from the chamber and decapitated. The glossopharyngeal nerve was identified at its junction with the vagus nerve near the petrosal/nodose complex and vagus/glossopharyngeal nerve was cut central to the ganglia and reflected over the carotid bifurcation. The glossopharyngeal nerve and carotid bifurcation were dissected free and placed in oxygenated (95% O₂-5% CO₂) saline. The carotid body, glossopharyngeal nerve, and petrosal ganglia were cleaned of surrounding tissue, including removal of the vagus nerve and nodose ganglia.

For labeling of petrosal neurons with projections to the carotid body, several crystals of hydroxystilbamine methanesulfonate (Molecular Probes, Eugene, OR), the active component of Fluorogold, a retrogradely transported fluorescent label, were pushed into the carotid body and the area was enclosed in paraffin film or rapid-curing sylgard. In four control experiments, the crystals were applied to the cut glossopharyngeal nerve distal to the sinus nerve to label axons that did not project toward the carotid body. The chemoreceptor complex was placed in culture tubes containing oxygenated DMEM and cultured at 35°C with gentle agitation for about 24 h. On the following day, the petrosal ganglia was cut from the glossopharyngeal nerve and placed in enzyme solution to aid in cell dissociation. Cells were dissociated by trituration following exposure to collagenase A, collagenase D, and papain as described by Rizzo et al. (1994). Cells were suspended in culture solution containing DMEM and F-12 (1:1) and 10% fetal calf serum, 1.5 mg/ml trypsin inhibitor, 1.5 mg/ml bovine serum albumin, 100 U/ml penicillin, and 0.1 mg/ml streptomycin and plated on polyornithine-laminin-coated coverslips. Cells were maintained for 1-6 h at 37°C in a humidified 95% air and 5% CO₂ incubator. Cells with presumed projection to the carotid body were identified by the appearance of fluorescence marker in the soma.

RT-PCR

After harvesting, the petrosal ganglia were cut free from the glossopharyngeal nerve and total cellular RNA was extracted from five adult rats using RNeasy columns (Qiagen). First-strand cDNA was reverse transcribed in a 25-µl final volume using 1 mM random hexamer (Boehringer Mannheim), 500 units SuperScript II reverse transcriptase (Life Technologies), and 100 units of RNase Inhibitor (Boehringer Mannheim). The buffer consisted of (in mM) 50 Tris-HCl (pH 8.3), 75 KCl, 3 MgCl₂, 10 DTT, and 5 dNTP. The reaction was allowed to proceed at 37°C for 90 min., 42°C for 30 min, and then was terminated by heating to 95°C for 5 min. Control templates (RT) were prepared in an identical fashion except that the RT enzyme was omitted from the reaction.

For PCR, we used primers designed against highly conserved sequences in domain 1 (D1) to amplify products from multiple  subunits that might have been present in the cDNA pool (Dib-Hajj et al. 1998; Fjell et al. 1997). The amplified products contain the terminal part of the conserved transmembrane segment D1-S3 and extend into the first half of D1-SS1. The core of this region shows significant sequence and length polymorphism (Dib-Hajj et al. 1998; Fjell et al. 1997). Due to codon degeneracy, four forward primers are used to ensure efficient priming from all templates that may be present in the cDNA pool; however, any of these primers may bind to multiple templates depending on the stringency of the reaction.

Amplification was performed in 60 µl volume using 1 µl of the first-strand cDNA, 0.8 µM of each primer, and 1.75 units of Expand Long Template DNA polymerase enzyme mixture (Boehringer Mannheim). PCR reactions in which the template was substituted by water or a -RT control template produced no amplification products (data not shown). As described previously (Dib-Hajj et al. 1996), amplification was carried out in two stages using a programmable thermal cycler (PTC-200, MJ Research, Cambridge, MA.): first, a denaturation step at 94°C for 4 min, an annealing step at 58°C for 2 min, and an elongation step at 72°C for 1 min. Second, a denaturation step at 94°C for 30 s, an annealing step at 58°C for 30 s, and an elongation step at 72°C for 1 min. The second stage was repeated 33 times for a total of 35 cycles, with the elongation step in the last cycle extended to 10 min.

Biolistic transfection of Na_v1.8-null DRG neurons

The Helios Gene Gun System (Bio-Rad Laboratories) was used for biolistic transfection of DRG neurons as previously described (Cummins et al. 2001). Briefly, the L4 and L5 DRG ganglia were harvested from adult Na_v1.8-null mice (Akopian et al. 1999), dissociated using collagenase and papain, and plated on glass coverslips. Na_v1.8-null neurons were kept under standard tissue culture conditions for 5-6 days before biolistic transfections. Gold particles (1.6 µm) were coated with 4.5 µg of Na_v1.8 DNA mixed with 2.5 µg green fluorescent protein (GFP) DNA. Just before biolistic transfection, the culture medium was removed from the petri dish. The gene gun was held 1 cm above the cells and a pressure of approximately 100 psi was used to deliver the gold particles to the cells. Within 24 h the cells usually showed expression of GFP, indicating a successful biolistic transfection. The majority of cells transfected with Na_v1.8 exhibited slow-inactivating TTX-R currents. Electrophysiologic studies of these Na_v1.8 currents were conducted 40-72 h after transfection in the presence of 250 nM TTX. Slow-inactivating TTX-R sodium currents were not observed in untransfected Na_v1.8-null neurons or Na_v1.8-null neurons transfected with only GFP-coated gold particles.

Whole-cell patch-clamp recordings

Whole-cell patch-clamp recordings were conducted at room temperature (22°C) using a HEKA EPC-9 amplifier (HEKA Electronics, Germany) controlled by a PC running the HEKA Pulse program. Electrodes were fabricated from 1.5 mm Drummond capillary glass using a Sutter P-97 puller (Sutter Instruments, Novato, CA), fire polished and used without any coatings. Pipettes were filled with saline containing (in mM) 140 CsF, 1 EGTA, 10 NaCl, and 10 HEPES at pH 7.3. With this solution, electrode resistance was 0.8-1.5 M and access resistance was 1.6 ± 0.1 M (n = 106). Voltage errors were minimized by using 80-85% series resistance compensation with the feedback compensation speed set at 10 µs. Electrode current signal was hardware filtered at 5 kHz and sampled at 20 kHz. Linear leak subtraction was used for all recordings. The extracellular solution for all recordings was (in mM) 140 NaCl, 3 KCl, 1 MgCl₂, 1 CaCl₂, and 10 HEPES at pH 7.3. Cadmium (50 µM) was included to block calcium currents and osmolarity was adjusted to 310 mOsm, which matched the pipette solution. Current densities were estimated by dividing the peak current amplitude by the cell capacitance.

Statistical analysis

Data are expressed as mean ± SE. One-way ANOVA was used to test for significant differences between the experimental groups. Multiple comparison test was used to determine the effect of age on cellular properties, and Fisher's least significant difference (LSD) at  = 0.05 values were determined.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Restriction enzyme analysis

The identity of candidate Na⁺ channel isoforms in the petrosal ganglia was based on examination of the  subunits as determined by restriction enzyme analysis of their PCR products. For restriction enzyme analysis, typically 1/12 of the PCR reaction is digested for 1 h at the recommended temperature and the products are resolved by electrophoresis in a 1.7% agarose gel. Fragment sizes were determined by comparison to a 100-bp ladder molecular weight marker (Pharmacia). DNA was visualized by ethidium bromide fluorescence. The gel images were digitized using a GelBase 7500 system (UVP).

Restriction enzyme profile (REP) analysis of amplification products from D1 from petrosal ganglia was used to determine which voltage-gated Na channel  subunits are present in this cDNA pool. Multiple amplification products (Fig. 1; lane 1) are consistent with the presence of Na_v1.1 (558 bp), Na_v1.2 and Na_v1.3 (561 bp), Na_v1.6, Na_v1.7, Na_v1.5 and Na_x (507, 501, 518, and 501 bp, respectively), Na_v1.8 and Na_v1.9 (479 and 468 bp, respectively). Lanes 2-10 (Fig. 1) show the result of cutting this DNA with EcoRV, EcoNI, AvaI, SphI, BamHI, AccI, AflII, EcoRI and XbaI, respectively. As expected, Na_v1.1 product appears to constitute most of the band migrating faster than the 600-bp marker (lane 2). Na_v1.2 (lane 3) and Na_v1.3 (lane 4) are not evident by this analysis. Na_v1.6 and Na_v1.7 products (lanes 5 and 6) are in agreement with the predicted results. The doublet migrating faster than the 400-bp marker in lane 6 is due to the presence of a restriction site for BamHI in both Na_v1.7 and Na_v1.8. Both Na_v1.9 and Na_v1.5 contain the recognition site of the restriction enzyme AccI; however, the restriction enzyme products in lane 7 are consistent with the presence of Na_v1.9 (expected products: 185 and 283 bp) in this cDNA pool but not with the presence of Na_v1.5 (expected products: 173 and 345 bp). This is consistent with the absence of Na_v1.5 transcripts from other adult sensory peripheral ganglia (Akopian et al. 1999; Black et al. 1998; Donahue 1995). Na_v1.8, Na_v1.9, and Na_x are cleaved by AflII (lane 8), EcoRI (lane 9), and XbaI (lane 10), respectively.

View larger version (80K): [in this window] [in a new window]   Fig. 1. REP analysis of Na channel domain 1 RT-PCR products from petrosal ganglia. M lanes: 100-bp ladder marker (Pharmacia). Lane 1: amplification product from petrosal ganglia cDNA. Lanes 2-10: result of cutting this DNA with EcoRV, EcoNI, AvaI, SphI, BamHI, AccI, AflII, EcoRI, and XbaI, which are specific to subunits Nav1.1, Nav1.2, Nav1.3, Nav1.6, Nav1.7/Nav1.8, Nav1.5/Nav1.9, Nav1.8, Nav1.9, and Nax, respectively. The image was digitized using GelBase 7500 system (UVP).

Thus REP analysis indicates that petrosal ganglia from old (18 days) rats express the Na_v1.1, Na_v1.6, and Na_v1.7 TTX-S sodium channel isoforms and the Na_v1.8 and Na_v1.9 TTX-R sodium channel isoforms. Message for the atypical channel Na_x is also found in petrosal ganglia from 18-day rats. However, message for the TTX-S neuronal sodium channels Na_v1.2 and Na_v1.3 was not detected by REP analysis.

Activation/inactivation characteristics

Whole-cell patch-clamp recordings were obtained on petrosal neurons that had been prelabeled with hydroxystilbamidine crystals placed in the carotid body or on the cut distal glossopharyngeal nerve approximately 24 h before dissociation. Following dissociation and plating, fluorescent cells could be readily discerned from nonfluorescent cells. In general the fluorescent cells were small diameter cells that had a whole-cell capacitance of 14, 13, and 18 pF for cells harvested from 3-, 9-, and 18-day-old ganglia (Table 1). The change in cell capacitance with age was significant (P < 0.05, ANOVA analysis with age as a grouping variable). Pairwise comparisons (Fisher's LSD multiple comparison test) revealed that the capacitances of neurons from 18-day rats were significantly different from the capacitances in neurons from both 3- and 9-day rats (P < 0.05).

Depolarization from a holding potential of 120 mV activated a rapidly activating and inactivating current with a threshold voltage near 70 mV in all cases (Fig. 2). The peak inward current was obtained with depolarizations to near 20 mV (Fig. 2) with values for the peak Na⁺ current of 18, 15, and 30 nA for 3-, 9-, and 18-day age groups (Fig. 2). The change in peak current amplitude with age was significant (P < 0.001, ANOVA analysis with age as a grouping variable). Pairwise comparisons (Fisher's LSD multiple comparison test) revealed that the peak currents in the 18-day neurons were significantly different from those in neurons from either 3- or 9-day neurons (P < 0.05). However, when normalized to cell size, peak Na⁺ current density was 1.3, 1.2, and 1.6 nA/pF with no significant effect of age (Table 1). The half-activation voltage was near 23 mV for all ages with no significant effect of age. However, half-activation was more negative at all ages than that measured for Na_v1.8 (previously termed SNS or PN3) TTX-R currents expressed in DRG neurons (Table 1).

View larger version (29K): [in this window] [in a new window]   Fig. 2. Activation characteristics of sodium currents in petrosal ganglion neurons that innervate the carotid body. A: family of sodium current traces from representative labeled petrosal ganglion neurons at day 3 (left), day 9 (middle), and day 18 (right). Currents were elicited by 40-ms test pulses to various potentials from 80 to 40 mV. Cells were held at 120 mV. B: normalized peak current-voltage relationship for sodium currents in labeled petrosal ganglion neurons at day 3 (left; n = 11), day 9 (middle; n = 10), and day 18 (right; n = 9). Threshold and reversal potential of sodium currents in labeled petrosal ganglion neurons were similar at all ages.

The current waveforms produced by depolarizations from 120 to 10 mV and 30 mV were fit to a Hodgkin-Huxley m³h model of channel activation and inactivation and the activation (m) and inactivation (h) time constants were recorded for each cell. The average activation time constant (_m) and inactivation time constants (_h) were not significantly influenced by age (ANOVA analysis with age as a grouping variable). However, these time constants were significantly different (Fisher's LSD multiple comparison test, P < 0.05) from those of Na_v1.8 TTX-R currents recorded from DRG neurons (Table 1).

Steady-state inactivation

Previous work in DRG neurons has shown that the trajectory of the steady-state inactivation curve can be used as an indication of whether multiple sodium channel transcripts are present in the recorded cell (Cummins and Waxman 1997). For instance, the slowly inactivating TTX-R sodium current, which is generated by the Na_v1.8 transcript in DRG sensory neurons (Akopian et al. 1999), has a half-inactivation voltage near 30 mV (Table 1). Coexpression of this current with TTX-S currents results in a biphasic or inflected steady-state inactivation curve (Roy and Narahashi 1992). Accordingly, the steady-state inactivation curves were developed for petrosal neurons and examined for evidence of inflection points, which would implicate the presence of both TTX-S and TTX-R isoforms. The protocol employed a 500-ms prepulse at potentials between 130 and 10 mV from a holding potential of 120 mV, followed by a 20-ms test pulse to 10 mV to determine the fraction of current that remains available for activation.

The steady-state inactivation curves for most labeled petrosal neurons showed no evidence of inflection and a half-inactivation voltage near 70 mV (Table 1). This was true at 3 days (Fig. 3A), 9 days (Fig. 3B), and 18 days (Fig. 3C). The steady-state inactivation curves for these neurons were well-fit with a single Boltzman equation and the midpoint of steady-state inactivation was 73 ± 1, 70 ± 1, and 72 ± 2 mV for labeled neurons at 3 days (n = 18), 9 days (n = 19), and 18 days (n = 18). In a small subpopulation of labeled neurons there was evidence of a small inflection in the steady-state inactivation curve. This was true in 4 of 20 labeled 3-day neurons, 5 of 19 labeled 9- day neurons, and 3 of 20 labeled 18-day neurons.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Comparison of steady-state inactivation of sodium currents in petrosal ganglion neurons. Representative steady-state inactivation data are shown for sodium currents from labeled petrosal ganglion neurons at 3 days (A), 9 days (B), and 18 days (C). D: fraction of current available at prepulse potentials ranging from 130 to 10 mV is plotted for labeled petrosal ganglion neurons at 3 days (filled squares; n = 11), 9 days (open triangles; n = 11), and 18 days (open circles; n = 11). The steady-state inactivation curves for labeled neurons were well fit with a single Boltzman function and did not show any developmental changes. E: representative steady-state inactivation data are shown for sodium currents from an unlabeled petrosal ganglion neuron. F: steady-state inactivation curves for unlabeled neurons typically were inflected (arrow) and could not be well fit with a single Boltzman function. Steady-state inactivation was estimated by measuring the peak current amplitude elicited by 20-ms test pulses to 10 mV after 500-ms prepulses to potentials over the range of 130 to 10 mV. Current is plotted as a fraction of the maximum peak current.

In contrast to the labeled cells, the sodium currents in petrosal ganglion neurons that were not labeled following injection of Fluorogold into the carotid body exhibited complex inactivation kinetics (Fig. 3E) and an inflected steady-state inactivation curve (Fig. 3F). Nine of 12 unlabeled neurons had both fast and slowly inactivating sodium currents, and the current profiles were not well fit to a single Boltzman equation (Fig. 3D). To determine whether the lack of TTX-R currents in labeled neurons was the direct effect of Fluorogold label, recordings were undertaken on 12 nonchemoreceptor neurons, which were labeled by placement of the crystals on the glossopharyngeal nerve distal to the sinus nerve. Eight of 12 labeled neurons evidenced TTX-R current based on their steady-state inactivation profiles (data not shown). This demonstrates that the relative lack of TTX-R currents in neurons labeled following injection of Fluorogold into the carotid body was not a result of the Fluorogold label.

Sensitivity to tetrodotoxin

To determine whether the fast-inactivating sodium current in carotid body labeled petrosal ganglion cells was TTX-sensitive, we exposed eight neurons to 250 nM TTX. In six of the labeled neurons only fast-inactivating currents were observed before application of TTX, and TTX reduced the current amplitude by 97.8 ± 0.4% (Fig. 4A). Assuming a single binding/blocking site for TTX and using a Langmuir inhibition isoform equation, this corresponds to a half-maximal inhibition of 5.5 nM for TTX, which is similar to that reported for recombinant rat Na_v1.1 (9.6 nM) (Smith and Goldin 1998), Na_v1.6 (6.4 nM) (Smith et al. 1998), and Na_v1.7 (4 nM) (Safo et al. 2000) channels expressed in Xenopus oocytes. Two of the labeled petrosal ganglion cells exposed to TTX exhibited both fast-inactivating current, which was blocked by TTX, and slowly inactivating current, which was not (data not shown). In unlabeled petrosal ganglion cells that expressed both fast and slowly inactivating sodium currents, the fast-inactivating current was also blocked by TTX and the slowly inactivating current was not (Fig. 4B). As has been shown for dorsal root ganglion neurons, TTX-R currents could be subdivided into slowly inactivating and persistent TTX-R sodium currents (Cummins et al. 1999). While persistent sodium currents could be identified in 40% of unlabeled neurons, persistent currents were observed in only about 10% of labeled neurons. Figure 4C shows a persistent TTX-R sodium current isolated in an unlabeled petrosal ganglion neuron using the prepulse inactivation and digital subtraction technique, as previously described (Cummins et al. 1999, 2000). In control neurons that were labeled from the glossopharyngeal nerve distal to the sinus nerve, addition of TTX to the bath eliminated a portion of the Na⁺ current (n = 8). Two of these neurons exhibited only fast-inactivating current, and 250 nM TTX eliminated 94% of the sodium current in these cells. However, the other six cells exhibited both fast and slowly inactivating currents. In these cells 42 ± 8% of the original current amplitude remained in the presence of TTX, further demonstrating that Fluorogold did not inhibit expression of TTX-R current in petrosal ganglion neurons. In every instance in which TTX was used, the fast-inactivating currents were sensitive to TTX and the slowly inactivating currents were resistant to TTX. Fast-inactivating TTX-R sodium currents were not observed in labeled or unlabeled petrosal ganglion neurons.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Most of the sodium current in labeled petrosal neurons, but not in unlabeled neurons, is TTX-S. A: sodium current traces from representative labeled petrosal ganglion neuron are shown before (left) and after (right) exposure to 250 nM TTX in the bath solution. TTX blocked 98% of the inward current in this neuron. B: sodium current traces from representative unlabeled petrosal ganglion neuron are shown before (left) and after (right) exposure to 250 nM TTX in the bath solution. TTX only blocked about 50% of the current in this neuron. Remaining current displays slow macroscopic inactivation. Currents in A and B were elicited by 40-ms test pulses to various potentials from 80 to 40 mV. Cells were held at 120 mV. C: persistent TTX-R sodium currents recorded from an unlabeled petrosal ganglion neuron. Persistent currents were obtained by subtraction of currents recorded when the neuron was held at 50 mV and a 500-ms step to 120 mV preceded the test pulses (which attenuates the persistent current) from total currents recorded from a holding potential of 120 mV. This prepulse inactivation and digital subtraction reveals the TTX-R persistent current (Cummins et al. 1999, 2000; Sleeper et al. 2000). For comparison, the time scale is the same in A, B, and C.

We compared the amplitudes of the slowly inactivating (Fig. 5, left) and persistent (Fig. 5, right) TTX-R sodium currents in unlabeled neurons and labeled chemoreceptor neurons at 3, 9, and 18 days. The amplitude of the slowly inactivating TTX-R sodium current was estimated by measuring the sodium current elicited with a test pulse to 10 mV after the cells had been held at 50 mV for 500 ms to inactivate the fast TTX-S and persistent TTX-R sodium currents. While the amplitude of the slowly inactivating TTX-R current was <2 nA in most of labeled neurons (Fig. 5, A-C), the majority (75%) of the unlabeled petrosal ganglion neurons had a slowly inactivating TTX-R current amplitude that was >2 nA (Fig. 5D, left). Persistent sodium current amplitudes were also examined in labeled and unlabeled petrosal neurons using the prepulse inactivation and digital subtraction technique (Cummins et al. 1999, 2000). While the amplitude of the persistent current was <1 nA in the majority of labeled neurons at all three ages (Fig. 5, A-C), many (40%) of the unlabeled petrosal ganglion neurons had a persistent current amplitude that was >1 nA (Fig. 5D, right).

View larger version (19K): [in this window] [in a new window]   Fig. 5. Amplitude distribution of slowly inactivating and persistent TTX-R currents in petrosal ganglion neurons. Amplitude of the slowly inactivating (left column) and persistent (right column) TTX-R sodium currents were estimated in labeled petrosal chemoreceptor neurons at 3 days (A; n = 20), 9 days (B; n = 19), and 18 days (C; n = 16) and in unlabeled neurons (D; n = 12). While the majority of labeled neurons expressed low levels of slowly inactivating and persistent TTX-R currents, 75% of unlabeled neurons displayed slowly inactivating current amplitudes > 2 nA and 42% of unlabeled neurons displayed persistent current amplitudes > 1 nA.

Recovery from inactivation

In small DRG sensory neurons the TTX-S fast-inactivating sodium current has been characterized as having slow ( ~ 145 ms at 80 mV) recovery from inactivation (Cummins and Waxman 1997; Elliott and Elliott 1993; Everill et al. 2001). By contrast the TTX-S fast-inactivating current in large cutaneous afferent DRG neurons exhibits fast ( ~ 25 ms at 80 mV) recovery from inactivation (Everill et al. 2001). Because repriming kinetics can have important implications for repetitive firing properties, we studied repriming in labeled petrosal neurons at 3, 9, and 18 days. The repriming time course at 80 mV for sodium currents in a typical 18-day neuron is shown in Fig. 6A. The time course was fit with a single exponential function (solid curve,  = 33 ms) and a dual exponential function (dotted curve, ₁ = 7 ms, ₂ = 103 ms). The fit with two exponentials gave a better match to the repriming time course in this neuron and in the majority of labeled neurons at 3, 9, and 18 days. Figure 6B shows the averaged repriming time course at 80 mV for labeled petrosal neurons at 3 days (n = 10), 9 days (n = 10), and 18 days (n = 9). The time course was slightly faster for the 18-day neurons, although this difference did not reach significance. We measured the time course for repriming at voltages ranging from 140 to 60 mV and fit the time course with two exponentials. The averaged fast (Fig. 6C) and slow (Fig. 6D) recovery time constants for labeled cells at 3, 9, and 18 days indicate that recovery from inactivation is slightly faster at 18 days at all potentials. However, the only significant effect of age was an increase in the speed of recovery from inactivation at 140 and 120 mV but not at other potentials. At 80 mV the time constant was approximately 6-9 ms for the fast component and approximately 80-110 ms for the slow component. The two components had roughly equal amplitudes at recovery potentials ranging from 120 to 70 mV at all three ages (data not shown). These results are consistent with the possibility that chemoreceptor petrosal neurons express two types of TTX-S sodium channels.

View larger version (31K): [in this window] [in a new window]   Fig. 6. Recovery from inactivation in labeled petrosal ganglion neurons shows complex kinetics. A: time course of recovery from inactivation at 80 mV for sodium currents recorded from a representative labeled petrosal ganglion neuron is shown (open circles). Data were fit with a single exponential function (solid curve;  = 33 ms) and a dual exponential function (dotted curve; 1 = 7 ms, 2 = 103 ms). The dual exponential function gave a better fit in the majority of labeled neurons. This neuron, like most labeled neurons, expressed only fast-inactivating sodium currents. Cells were prepulsed to 20 mV for 20 ms to inactivate all of the current and then brought back to the recovery potential for increasing recovery durations prior to the test pulse to 20 mV. Maximum pulse rate was 0.5 Hz. Inset illustrates superimposed records of Na+ currents evoked at 20 mV after each recovery interval. B: comparison of the time course of recovery from inactivation at 80 mV for sodium currents recorded from 3-, 9-, and 18-day labeled petrosal ganglion neurons is shown. C: fast time constants for recovery from inactivation (1) are shown plotted as a function of recovery voltage for sodium currents recorded from 3-, 9-, and 18-day labeled petrosal ganglion neurons. Time constants were estimated from dual exponential fits to time courses measured with the protocols shown in A. D: slow time constants for recovery from inactivation (2) are shown plotted as a function of recovery voltage for sodium currents recorded from 3-, 9-, and 18-day labeled petrosal ganglion neurons.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The principal observation from this study is that, while petrosal ganglia neurons express multiple Na⁺ current isoforms, similar to DRG, expression in chemoreceptor petrosal neurons is nearly exclusively TTX-S, suggesting a close link between sensory modality (chemoreception) and Na⁺ channel characteristics, at least in the rat. However, contrary to our original hypothesis, developmental changes in Na⁺ channel characteristics do not appear to take place. The apparent specificity of TTX-S to chemoreceptor neurons is not due to a lack of TTX-R expression in the petrosal ganglia or due to an inhibition of TTX-R expression by Fluorogold. TTX-R currents could be readily recorded in unlabeled neurons or neurons labeled following placement of Fluorogold crystals on nonchemoreceptor afferent fibers.

Na⁺ channel subtypes in petrosal ganglia

This is the first study to examine the Na⁺ channel characteristics of rat chemoreceptor afferent neurons and demonstrates a modal specificity in Na⁺ channel characteristics in chemoreceptor cells. In the vast majority of neurons studied, >95% of the total Na⁺ current was carried by fast-inactivating, TTX-S Na⁺ current with little or no evidence of slow or TTX-R current. In contrast, previous electrophysiologic recordings from rat petrosal neurons, as a whole, showed that approximately 50% of cells had predominantly TTX-R current to the extent that addition of 2 µM TTX to the bath had no effect on magnitude of total Na⁺ current (Stea and Nurse 1992). However, in the previous study, neither cell size nor sensory modality was considered and the entire ganglion was considered as a single sample. In agreement, our RT-PCR analysis examined channel expression in the entire ganglia and demonstrated the expression of both TTX-S and TTX-R Na⁺ channel transcripts at high levels.

In partial contrast to our results, previous results in cat demonstrated TTX-R currents in some neurons projecting to the sinus nerve (Gallego 1983). However, this study did not examine the response to natural stimuli and thus did not discriminate between baroreceptor and chemoreceptor afferent fibers. It also limited its scope to myelinated afferent fibers and did not examine C-type afferent fibers. Our previous results indicate that all rat chemoreceptor afferent fibers are nonmyelinated (Donnelly 1999). Thus differences in methodology, fiber type, or species may account for the apparent presence or lack of TTX-R currents in the two studies.

Overall, the distribution of Na⁺ current isoforms in the petrosal ganglia is similar to that previously obtained in DRG (Black et al. 1996; Felts et al. 1997). DRG neurons express TTX-S transcripts Na_v1.1, Na_v1.6, and Na_v1.7 at high levels (Felts et al. 1997) and all of these transcripts were present in the petrosal ganglia. DRG also express TTX-R transcripts for Na_v1.8 and Na_v1.9 in predominantly small DRG neurons (Amaya et al. 2000) and both of these transcripts are found in petrosal ganglia. Transcript for the atypical sodium channel Na_x (previously termed NaG or Na_v2) is also found in both DRG (Felts et al. 1997) and petrosal ganglia; however, the function of this putative voltage-gated sodium channel is not clear. There was no transcript that was not previously shown to be present in DRG found in petrosal ganglia.

Electrophysiologic characterization of Na⁺ currents in chemoreceptor neurons

The results of the RT-PCR analysis showed that both TTX-R and TTX-S transcripts are expressed at high levels. However, in the majority of petrosal ganglia neurons that were identified as projecting to the carotid body by retrograde labeling, slowly inactivating and persistent TTX-R currents were not evident. If these currents were expressed in the soma of chemoreceptor neurons, then they should have been readily identified. For instance Na_v1.8, which produces the slowly inactivating TTX-R current of DRG ganglion neurons (Akopian et al. 1999; Cummins et al. 1999), has a V_1/2 for steady-state inactivation of 30 to 40 mV, which is much different from the ca. 70 mV values observed in chemoreceptor neurons in this study. If Na_v1.8 had been present at significant levels, then a discernable inflection near 50 mV would have been expected in the steady-state inactivation curves, but this was not usually observed in the chemoreceptor neurons. A second type of TTX-R current, Na_v1.9, is also expressed at high levels in small DRG neurons (Cummins et al. 1999, 2000; Sleeper et al. 2000), but the current is noninactivating or very slowly inactivating, which is much different from the fast-inactivating current observed in chemoreceptor cells. Thus it is unlikely that Na_v1.9 contributes significantly to the Na⁺ current of chemoreceptor neurons.

Although the proportion was considerably smaller than in unlabeled cells, there was clear evidence of the presence of a TTX-R current in approximately 20% of the neurons labeled from the carotid body. This may indicate the presence of TTX-R currents in a subpopulation of chemoafferent neurons but may also indicate some label contamination. Baroreceptor fibers, which share the sinus nerve, pass over the surface of the carotid body on their way to the carotid sinus. During cleaning of the preparation, these fibers would have been cut near the carotid body and thus may have picked up label from crystals of Fluorogold placed in the carotid body. At present, we cannot differentiate between these possibilities.

Several studies indicate that the majority of small DRG neurons express TTX-R sodium currents (Akopian et al. 1999; Cummins and Waxman 1997; Gold et al. 1996). However, while 95% of 18- to 25-µm diam DRG neurons express TTX-R current (Cummins and Waxman 1997), Rush et al. (1998) reported that 50% of DRG neurons with diameters < 20 µm expressed exclusively TTX-S currents. This suggests that there is also a subpopulation of small DRG neurons that express predominantly TTX-S currents. It is not known if these small DRG neurons with predominantly TTX-S current are associated with a specific modality. Gold et al. (1996) reported that, although the majority of DRG neurons with TTX-R sodium currents responded to capsaicin, which is associated with nociception in vivo, the majority of neurons without TTX-R sodium currents did not respond to capsaicin. These data, and our data on petrosal neurons, are consistent with the idea that Na_v1.8 and Na_v1.9 TTX-R sodium currents are expressed predominantly in nociceptive neurons.

The TTX sensitivity and voltage dependence of activation and steady-state inactivation for the large TTX-S currents recorded from labeled petrosal neurons were similar to those described for TTX-S currents in small DRG neurons (Cummins and Waxman 1997; Elliott and Elliott 1993) and neocortical neurons (Cummins et al. 1994; Huguenard et al. 1988). These currents were clearly distinct from the slow-inactivating currents generated by Na_v1.8 channels (Table 1). The TTX-S currents in the labeled petrosal neurons exhibited complex recovery from inactivation kinetics. Roughly equal amounts of rapidly ( ~ 8 ms at 80 mV) and slowly ( ~ 90 ms at 80 mV) repriming TTX-S currents were recorded in the majority of labeled neurons at all ages. By contrast, the TTX-S sodium currents in neocortical neurons exhibit predominantly rapid repriming ( ~ 8 ms at 80 mV) (Huguenard et al. 1988) and the TTX-S sodium currents in small DRG neurons exhibit predominantly slow repriming ( ~ 70-160 ms at 80 mV) (Cummins et al. 1998). Small DRG neurons express high levels of Na_v1.7, a TTX-S sodium channel isoform that is almost exclusively found in peripheral neurons (Toledo-Aral et al. 1997). Since Na_v1.7 channels display slow recovery from inactivation kinetics when expressed in HEK293 cells ( ~ 144 ms at 80 mV) (Cummins et al. 1998), Na_v1.7 is a likely candidate to underlie the slowly repriming TTX-S current in labeled petrosal ganglion neurons.

If Na_v1.7 is likely to underlie the slowly repriming TTX-S sodium current in labeled petrosal neurons, which isoform(s) might underlie the rapidly repriming sodium current? The best candidates are Na_v1.1 and Na_v1.6, which are TTX-S sodium channel isoforms and which are expressed at high levels in the petrosal ganglion as well as in other peripheral and cortical neurons (Felts et al. 1997). However, the repriming characteristics of these channels have not been fully investigated in a mammalian expression system. In the CNS, immunoreactivity of Na_v1.1 is observed in both the soma and proximal dendrites of neocortical neurons (Gong et al. 1999). Na_v1.6 appears to be the predominant sodium channel isoform found at the nodes of myelinated axons (Caldwell et al. 2000; Tzoumaka et al. 2000) and also found in the soma and apical dendrites of neocortical neurons (Krzemien et al. 2000). Therefore, at present, both Na_v1.1 and Na_v1.6 are viable candidates, but a more complete resolution may depend on the future availability of isoform-specific transgenic animals and characterization of the repriming kinetics of these channels.

Interpretative assumptions and possible importance of Na⁺ channel isoforms in chemotransduction

One limitation of the present study is that our data were obtained at the soma of chemoreceptor neurons and not at the nerve terminals, which are the critical site of spike generation. Since the nerve terminals are small (ca. 0.1 uM) and fairly inaccessible, direct recordings are difficult. Thus an interpretative assumption is how well the soma does or does not reflect the Na⁺ channel complement of the nerve terminal. Differential targeting of Na⁺ channel isoforms has been reported for central neurons where type Na_v1.1 is preferentially located in the soma while Na_v1.2 is preferentially located in the axons (Gong et al. 1999; Westenbroek et al. 1989). In cultured DRG neurons, Na_v1.7 immunoreactivity was mainly observed in the soma and neurite terminals but not along the neurites (Toledo-Aral et al. 1997). The pertinent question here is whether TTX-R channels might be present in the terminals of the axons but absent from the soma. There is, at present, no evidence for such separation, and, in fact, the opposite may be true. In C-fibers innervating the cornea, TTX-R currents do not support axonal conduction but are present at both the nerve terminals and soma (Brock et al. 1998), suggesting that the soma might best reflect channels at the nerve terminal compared with the conducting portion of the axon. Thus the absence of TTX-R at the soma suggests that this current is not critical in initiating the afferent action potential. In support of this argument, we have previously demonstrated a large decrease in spontaneous spiking activity from the nerve terminal on exposure to nanomolar concentrations of TTX, indicating that TTX-S Na⁺ channels are critical to nerve terminal function (Donnelly et al. 1998).

The predominant expression of TTX-S currents over TTX-R currents is typically found in cells that evidence spontaneous spiking activity. TTX-S channels are conducive to repetitive discharge and spontaneous nerve activity because of the more negative position of their activation curve and more rapid inactivation compared with TTX-R channels. Indeed, in nodose ganglion neurons, a study by Schild and Kunze (1997) indicated that spontaneous activity only occurs with maximal expression of TTX-S current and minimal expression of TTX-R currents. Thus a prevalence of TTX-S over TTX-R at the chemoreceptor nerve terminal may confer the ability to generate afferent spikes in the absence of synaptic input. However, if somal sodium currents are representative of sodium currents at the nerve terminal, then changes in Na⁺ current voltage-dependence and kinetic properties do not appear to account for the developmental increase in chemoreceptor sensitivity. This is consistent with the finding that there are no developmental changes in rheobase or repetitive firing characteristics during sharp electrode, intracellular recording of petrosal chemoreceptor neurons (Donnelly 1999). Our results cannot rule out the possibility that there might be developmental changes in the modulation of sodium channels at the nerve terminal that impact chemoreceptor sensitivity. However, while our data suggest a close association between TTX-S currents and the chemoreceptor modality, there are other changes within the carotid body that could account for the developmental increase in chemoreceptor sensitivity, such as an increase in axonal branching with age (Kondo 1976) or an increase in the magnitude of neurotransmitter release from cells that are presynaptic to the nerve terminals (Donnelly and Doyle 1994).

In conclusion, the present results demonstrate that petrosal ganglion neurons express Na⁺ channel isoforms similar to that found in DRG. However, chemoreceptor afferent neurons predominantly express TTX-S isoforms in contrast to baroreceptor neurons, which express a mixture of TTX-R and TTX-S isoforms with extensive variability among neurons in the proportion of each current (Schild and Kunze 1997). Taken together, the low variability in the type and magnitude of chemoreceptor Na⁺ currents suggest they play an essential role in determining the sensory modality of the receptor.