Transient outwardly rectifying A currents are involved in the firing rate response to altered CO₂ in chemosensitive locus coeruleus neurons from neonatal rats

Abstract

The effect of hypercapnia on outwardly rectifying currents was examined in locus coeruleus (LC) neurons in slices from neonatal rats [postnatal day 3 (P3)–P15]. Two outwardly rectifying currents [4-aminopyridine (4-AP)-sensitive transient current and tetraethyl ammonium (TEA)-sensitive sustained current] were found in LC neurons. 4-AP induced a membrane depolarization of 3.6 ± 0.6 mV (n = 4), while TEA induced a smaller membrane depolarization of 1.2 ± 0.3 mV (n = 4). Hypercapnic acidosis (HA) inhibited both currents. The maximal amplitude of the TEA-sensitive current was reduced by 52.1 ± 4.5% (n = 5) in 15% CO₂ [extracellular pH (pH_o) 7.00, intracellular pH (pH_i) 6.96]. The maximal amplitude of the 4-AP-sensitive current was reduced by 34.5 ± 3.0% (n = 6) in 15% CO₂ (pH_o 7.00, pH_i 6.96), by 29.4 ± 6.8% (n = 6) in 10% CO₂ (pH_o 7.15, pH_i 7.14), and increased by 29.0 ± 6.4% (n = 6) in 2.5% CO₂ (pH_o 7.75, pH_i 7.35). 4-AP completely blocked hypercapnia-induced increased firing rate, but TEA did not affect it. When LC neurons were exposed to HA with either pH_o or pH_i constant, the 4-AP-sensitive current was inhibited. The data show that the 4-AP-sensitive current (likely an A current) is inhibited by decreases in either pH_o or pH_i. The change of the A current by various levels of CO₂ is correlated with the change in firing rate induced by CO₂, implicating the 4-AP-sensitive current in chemosensitive signaling in LC neurons.

there are a number of neurons within various brain regions, called chemosensitive neurons, that respond to altered CO₂ and/or pH. One such region that contains a large percentage of chemosensitive neurons is the locus coeruleus (LC). The LC is an important noradrenergic region in the pons and has been implicated to play a role in many pathological conditions, including panic disorder, Rett syndrome, and posttraumatic stress disorder (1, 55, 65, 66). In addition, chemosensitive LC neurons are believed to be a part of a distributed network of neurons that are involved in the control of breathing (18, 35). In addition to the LC (13, 15, 16, 20, 44), this network includes neurons from the ventral medullary surface (32, 54), the medullary raphé (3, 49, 50), the pre-Bötzinger complex (56), the retrotrapezoid nucleus (14, 19, 52), and the nucleus tractus solitarius (NTS) (8, 11). There has, thus, been great interest in the properties of chemosensitive neurons within the brain.

The cellular and molecular mechanisms underlying the response of these chemosensitive neurons are not well elucidated. Increased CO₂ (hypercapnia) results in decreased outside pH (pH_o) and intracellular pH (pH_i). It is believed that decreased pH alters ion channel activity, resulting in decreased neuronal membrane potential (Vm) and increased neuronal firing rate (47). However, it is also clear that the pH response of a neuron does not define it as a chemosensitive neuron (4, 46). It is likely that the properties and types of channels on the membrane of a neuron determine whether it is chemosensitive or not (46).

A variety of channels can potentially serve as chemosensitive channels, including K⁺ channels (44), acid-sensing ion channels (22), transient receptor potential (TRP) channels (10), a Ca²⁺-activated nonselective ion channel (9), Cl⁻ currents (44), and L-type Ca²⁺ channels (16, 23). We focus here on the K⁺ channel targets of pH changes, since these channels, if active at or near resting Vm, can regulate Vm and determine neuronal excitability (41). Several such K⁺ channel targets have been identified. Decreased pH has been shown to inhibit inwardly rectifying K⁺ (Kir) channels, Ca-activated K⁺ (KCa) channels, and TASK channels (2, 43, 63, 64). In LC neurons, Kir channels have been shown to respond to hypercapnic acidosis (HA) (45, 65). Filosa and Putnam (16) reported that HA inhibited a tetraethyl ammonium (TEA)-sensitive K⁺ channel and probably a TASK channel in LC neurons. No report has been made of a potential role for a 4-aminopyridine (4-AP)-sensitive transient A current in the chemosensitive response of LC neurons, even though these channels have been shown to be present in LC neurons (17).

In the current study, whole cell patch-clamp was used to study the role of outwardly rectifying K⁺ channels in the chemosensitive response to HA in LC neurons in brain stem slices from neonatal rats. Our main goals in this study were 1) to determine which outward currents are present in LC neurons from neonatal rat, to demonstrate which, if any, of these outward currents are involved in the firing rate response of LC neurons to hypercapnia; and 2) to see whether these currents are inhibited by decreases in either pH_i or pH_o. We found that a transient A current is involved in the firing rate response to hypercapnia of LC neurons from neonatal rats.

Preliminary reports of this work have previously been published (31, 48).

MATERIALS AND METHODS

Slice Preparation

All procedures involving animals were reviewed and approved by the Wright State University Institutional Animal Care and Use Committee and are in agreement with standards set out in the National Institutes of Health Guide for Care and Use of Laboratory Animals. Wright State University is accredited by Association for the Assessment and Accreditation of Laboratory Animal Care and is covered by National Institutes of Health assurance (no. A3632–01).

Pontine LC slices were prepared from neonatal Sprague-Dawley rats (postnatal; P, aged P3–P15), as previously described (15, 28, 51). Briefly, rats were anesthetized either by hypothermia (<P10) or by 100% CO₂ (≥P10) and rapidly decapitated. The brain stem was subsequently removed and placed onto a vibratome (Pelco Vibratome 1000). Coronal brain stem slices (300 μm) were cut in ice-cold artificial cerebral spinal fluid (aCSF) and then incubated at room temperature in aCSF until used.

Solutions

aCSF contained (in mM): 3 KCl, 124 NaCl, 1.3 MgSO₄, 26 NaHCO₃, 1.25 NaH₂PO₄, 10 glucose, and 2.4 CaCl₂ and was equilibrated with 5% CO₂/95% O₂ (pH_o ∼ 7.45). Various hypercapnic acidotic (HA) solutions were identical in composition but were equilibrated with either 10% CO₂/90% O₂ (pH_o ∼ 7.15) or 15% CO₂/85% O₂ (pH_o ∼ 7.0). We also employed a solution that was hypercapnic but with a normal pH_o, isohydric hypercapnia (IH). This solution was made by modifying aCSF by elevating external NaHCO₃ to 77 mM (isosmotically substituting for an equal amount of NaCl) and equilibrating with 15% CO₂/85% O₂ so that pH_o remained constant at 7.45 (15). A solution that was hypocapnic and alkalotic was also used and consisted of aCSF equilibrated with 2.5% CO₂/97.5% O₂ (pH_o ∼ 7.75). Synaptic blockade medium (SNB) contained (in mM): 3 KCl, 121 NaCl, 11.4 MgSO₄, 26 NaHCO₃, 1.25 NaH₂PO₄, 10 glucose, and 0.2 CaCl₂, plus carbenoxolone (CAR; 100 μM) to block gap junctions (8, 37). In many experiments, tetrodotoxin (TTX; 1 μM) and CoCl₂ (2 mM) were added to aCSF to block sodium channels and voltage-gated calcium channels, respectively (16). Whole cell patch pipettes were filled with an internal solution designed to reduce washout of the chemosensitive response (16) that contained (in mM): 130 K-gluconate, 0.4 EGTA, 1 MgCl₂, 0.3 Na₂GTP, 2 Na₂ATP, 10 HEPES; pH_o 7.45. When pH_i was to be measured, 200 μM pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid) was added to the pipette filling solution (51). K⁺ channel inhibitors tetraethyl ammonium chloride (TEA-Cl; 20 mM) or 4-AP (5 mM) were added to aCSF isosmotically replacing NaCl. All chemicals were from Fisher Scientific except for K-gluconate, carbenoxolone, Na₂ATP, Na₂GTP, EGTA, HEPES, TEA-Cl, and 4-AP (which were from Sigma-Aldrich), glucose (VWR Scientific), pyranine (Molecular Probes), TTX (Tocris Bioscience), and Na-acetate (EMD Scientific).

Recording Chamber

Slices were placed on the floor of a perfusion chamber and held with a nylon grid (16, 51). Brain slices were continuously superfused through stainless-steel tubes via a gravity-fed system at a rate of 3–5 ml/min. Cells were visualized with an upright microscope (Nikon Optiphot-2) using near-infrared illumination and a 40× water-immersion objective (Hoffmann modulated contrast, N.A. 0.55, 3.0-mm working distance). All experiments were done at a temperature of about 35°C (16), achieved by maintaining solution reservoirs in a water bath at around 40°C and adjusted with an in-line thermoelectric Peltier assembly (27).

Electrophysiological Recordings

Patch pipettes (3–6 MΩ) were pulled from thin-walled (1.5-mm outer diameter, 1.12-mm inner diameter) borosilicate glass capillaries (TW150–3; World Precision Instruments) on a vertical electrode puller (Narishige PP-830). Electrical signals from individual neuronal soma were obtained in whole cell patch clamp configuration with an Axopatch 200B amplifier, a Digidata 1440A A/D converter and pCLAMP 10.2 software (all from Molecular Devices). An Ag-AgCl electrode connected to the bath solution via a KCl-agar bridge served as the reference electrode. Data were filtered at 2 kHz, sampled at 10 kHz, and saved on a computer to be analyzed offline. Data were discarded if the series resistance (10.1 ± 0.4 MΩ) changed >20% over the duration of the recording.

Current-clamp recordings.

Membrane potential (Vm) and firing frequency were measured from whole cell patched LC neuronal soma in current clamp mode. A recording was used if the neuron exhibited a stable Vm and a steady firing frequency in the presence of SNB and CAR. Occasionally, a small depolarizing DC current was injected into the cell to maintain an appropriate firing frequency, but this was not necessary in most neurons. We employed episode mode to inject 30-pA hyperpolarizing current for 300 ms every 50 s to calculate the input resistance (Rin) of the neuron. Rin was calculated from Ohm's law by dividing the hyperpolarizing change in Vm by the injected hyperpolarizing current (30 pA). To record Vm in the absence of action potentials, we added TTX (1 μM) and CoCl₂ (2 mM) to the aCSF to block fast sodium channels and voltage-gated calcium channels, respectively (16). Membrane potential was corrected for the liquid junction potential of 16-mV (liquid junction potentials were calculated from the Henderson equation using Clampex).

Voltage-clamp recordings.

To determine the presence of specific K⁺ channels, we studied individual LC neurons within the slice using voltage clamp. In the presence of TTX (1 μM) and CoCl₂ (2 mM), the whole cell outwardly rectifying current was elicited by command pulses from −116 to +34 mV with 10-mV increments (300 ms) from an initial holding potential (Vh) of −96 mV. The records were not compensated for series resistance because even attempting modest compensation often resulted in electrode ringing and the loss of the patch. Membrane potential was corrected for the liquid junction potential of 16–18 mV. The membrane steady-state leak current was subtracted with Clampfit software.

We recognize the inherent issues of space clamping in intact neurons within slices. As others have done, we chose to employ slices to obtain at least a qualitative description of the ionic currents (5, 25, 60). We opted not to use isolated or cultured neurons to avoid the potential that they could have altered properties compared with in vivo neurons and to maintain as much of the neuropil intact as possible.

Various techniques were used to divide out individual currents from the whole cell outwardly rectifying current. A TEA-sensitive portion of this whole cell current was isolated by subtracting current in the presence of TEA (20 mM) from that in the absence of TEA. The plateau amplitude at the end of the TEA-sensitive current was measured and was plotted as a function of the conditioning step potentials (I-V plot). Similarly, a 4-AP-sensitive portion of the current was isolated by subtracting current in the presence of 4-AP (5 mM) from that in the absence of 4-AP. Maximum 4-AP-sensitive current was measured as the peak current. A second protocol was used to study the 4-AP-sensitive current, based on the protocol of Sonner et al. (57). Initially, a neuron, in the presence of 1 mM TTX, 2 mM CoCl₂, and 20 mM TEA, was held at a holding Vm of −98 mV (to eliminate any K⁺ channel inactivation), and then currents were measured in response to depolarizing command pulses (from −98 to +42 mV, 10-mV increments, 300 ms). A second set of currents was collected, which included an initial depolarized (−40 mV) conditioning pulse (200 ms), whose purpose was to inactivate any transient K⁺ currents, before the depolarizing command pulses (from −98 to +42 mV, 10-mV increments, 300 ms). These two sets of currents were digitally subtracted offline using Clampfit 10.2 (Axon Instruments), and the maximum current was plotted as a function of the conditioning step pulse.

Measurement of Intracellular pH

Intracellular pH (pH_i) was measured as described previously (20, 51). All pH_i measurements were recorded from the cell soma. Briefly, the pH-sensitive dye pyranine (100 μM) was loaded into individual LC neurons with whole cell pipettes. Pyranine-loaded LC neurons were excited (with light from a xenon arc lamp) alternately at 450 ± 10 nm (pH-sensitive excitation wavelength) and 415 ± 10 nm (pH-insensitive wavelength) using a Sutter Lambda 10–2 filter wheel. Image acquisition was achieved within 2 s and was repeated at 60-s intervals. There was no excitation light between acquisitions to minimize photobleaching. Emitted fluorescence at 515 ± 10 nm (all filters were obtained from Omega Optical) was directed to the Nikon multi-image port module and was then directed to a GenIISys image intensifier and a CCD 100 camera (both obtained from Dage-MTI). The subsequent fluorescence images were acquired using a Gateway 2000 E-3100 computer and were collected and processed using Metafluor 4.6r5 software (Universal Imaging), and the 450/415 fluorescence ratios (Rfl) were determined. To normalize Rfl values, we calibrated pyranine using the high-K⁺/nigericin technique (59) to obtain Rfl values at pH 7.4. Rfl values were normalized to the Rfl value at pH 7.4, yielding normalized Rfl (Nfl). Nfl values were converted to pH_i values using the following equation (51): pH_i = 7.5561 + log[(Nfl − 0.1459)/(2.0798 − Nfl)].

Intracellular pH was clamped using the technique of Hartzler et al. (20). Briefly, a patched neuron was loaded with a weak acid (acetic acid) by adding K-acetate (50 mM, isosmotically replacing K-gluconate) to the pipette-filling solution. A stable pH_i of typical value was obtained by superfusing the neuron with aCSF containing 45 mM Na-acetate (isosmotically replacing NaCl). When the neuron was exposed to hypercapnic acidotic solution (CO₂ 15%, pH_o 7.0), pH_i acidified normally when the solution contained 45 mM Na-acetate, but pH_i did not change when the aCSF contained only 15 mM Na-acetate due to a counterbalancing efflux of H⁺ in the form of acetic acid (20).

Data Analysis and Statistical Treatment

The spontaneous firing rates over a 1-min period before and during exposure to acidic or alkalotic stimuli were calculated to get the mean value for every neuron. Three successive input resistances in control and during exposure to acidic or alkalotic stimuli were averaged for every neuron. All values are expressed as means ± SE. Changes in Vm, Rin, or firing rate were tested for being different from zero using a t-test. All comparisons of two means were done using either a two-tailed unpaired or paired Student's t-test, and comparisons of three or more means were done using a one-way ANOVA with multiple paired comparisons done using Tukey's method. The effects of TEA, 4-AP, and the two together on Vm were tested for differences using a one-way repeated-measures ANOVA with Tukey's studentized range for paired comparisons. Differences were assumed to be statistically significant at a level of P ≤ 0.05.

RESULTS

Firing Rate Response of LC Neurons to Hypercapnia or Hypocapnia

The vast majority (>80%) of LC neurons that we patched responded to hypercapnia with an increased firing rate, as previously observed (15, 40, 45). We studied only LC neurons that showed an intrinsic, reversible increase in firing rate in response to HA. All neurons were tested for their intrinsic firing rate response to various levels of CO₂ in aCSF that had elevated Mg²⁺ and reduced Ca²⁺ for blockade of chemical synapses and contained 100 μM CAR to block gap junctions (e.g., Ref. 8). As shown in Fig. 1, the firing rate response of LC neurons to CO₂ was dependent on the level of CO₂. Compared with firing in normocapnic solutions (5% CO₂), hypocapnic solutions (2.5% CO₂) resulted in decreased firing (from 1.0 ± 0.3 to 0.6 ± 0.2 Hz; n = 6, P = 0.04) (Fig. 1A), while firing rate was increased in 10% CO₂ (from 1.1 ± 0.4 to 2.6 ± 0.6 Hz; n = 9, P = 0.002) (Fig. 1B) and in 15% CO₂ (from 0.5 ± 0.1 to 1.2 ± 0.2 Hz; n = 33, P < 0.001) (Fig. 1C). Note that the % firing rate increase had nearly reached a plateau by 10% CO₂, not significantly increasing further at 15% CO₂ (Fig. 1D). Changes of CO₂ not only affected firing rate but they significantly altered the Rin of LC neurons as well. The resting Rin of LC neurons under normocapnic (5% CO₂) conditions was 446.6 ± 33.2 MΩ (n = 40). Hypocapnia (2.5% CO₂) significantly (n = 5; P = 0.01) reduced Rin by 8.1 ± 1.8% (Fig. 1E), while hypercapnia significantly increased Rin by 10.2 ± 4.2% (n = 8; P = 0.04) and by 10.7 ± 1.2% (n = 27; P = 0.001) at 10 and 15% CO₂, respectively (Fig. 1E).

Fig. 1.

The effects of hypocapnia and hypercapnia on the spontaneous firing rate and input resistance in locus coeruleus (LC) neurons. A: traces of membrane potential (Vm) vs. time showing action potential frequency. Compared with control (5% CO₂), firing rate went down in 2.5% CO₂. B: compared with control, firing rate increased in 10% CO₂. C: compared with control, firing rate increased in 15% CO₂. D: relationship between the CO₂ and the change in firing rate induced by different levels of CO₂ in LC neurons. The response is presented as the percentage of change of firing rate in altered CO₂ vs. control (5% CO₂). Symbols represent the mean changes in firing rate ± SE. Note the large effect of CO₂ on firing rate between 2.5 and 10% but the plateau of firing rate above 10% CO₂. The % change in firing rates at various levels of CO₂ were significant at *P < 0.05, **P < 0.01, or ***P < 0.001. E: effect of CO₂ on input resistance (Rin). The response is plotted as paired data for Rin at 5% CO₂ vs. 2.5%, 10%, or 15% CO₂. Each pair shows the response of Rin for a given neuron in response to the indicated change of CO₂. Rin is significantly different from the values at 5% CO₂ at either *P < 0.05 or ***P < 0.001 based on paired t-tests.

Effects of TEA and 4-AP on Membrane Potential, Input Resistance, and Firing Rate

To determine whether voltage-gated K⁺ channels are active at resting membrane potentials in LC neurons from neonates, we studied the effects of the Kv channel blockers TEA (20 mM) and 4-AP (5 mM) on resting Vm (control Vm was −53.5 ± 0.7 mV; n = 58). In the presence of SNB and CAR, we first showed that an LC neuron was chemosensitive (increased firing rate in response to 15% CO₂) (data not shown). TTX and Co²⁺ were then added to eliminate fast Na⁺ channels and voltage-gated Ca²⁺ channels, respectively, which yielded a Vm trace free of action potentials (Fig. 2A). Under these conditions, exposure to 4-AP induced a large and significant (P = 0.0001) depolarization (∼3.6 mV), TEA induced a smaller but significant (P = 0.026) depolarization (∼1.2 mV), and exposure to both 4-AP and TEA induced a depolarization (P < 0.0001) that was equal to the sum of the effects of the two drugs (∼4.8 mV) (Fig. 2, A and B). 4-AP and the combination of 4-AP+TEA caused a significantly larger depolarization than TEA alone (Fig. 2B). The depolarization induced by 4-AP was not significantly different from that induced by 4-AP+TEA (P = 0.17, based on one-way repeated-measures ANOVA with Tukey's studentized range for paired comparisons), but these values differed on the basis of a two-tailed paired Student's t-test (P < 0.05). These results suggest that both 4-AP-sensitive and TEA-sensitive K⁺ channels are active at resting Vm in chemosensitive LC neurons.

Fig. 2.

The effects of TEA, 4-AP, and CO₂ on the membrane potential, input resistance, and firing rate in LC neurons. A: LC neuron in the presence of synaptic blockade medium (SNB), carbenoxolone (CAR), tetrodotoxin (TTX), and Co²⁺, which shows no action potentials. TEA induced a small, and 4-AP induced a larger, reversible depolarization. The combination of TEA and 4-AP resulted in the largest reversible depolarization, which was the sum of the effects of each drug alone. B: summary of the effects of TEA and 4-AP on Vm. The depolarization induced by 4-AP alone and in combination with TEA was significantly larger than the depolarization induced by TEA alone. The average effects of TEA, 4-AP, and the two together on Vm are shown for 4 LC neurons (all in the presence of 5% CO₂). C: effect of 15% CO₂, 4-AP, and the combination of the two on the change of Rin. Rin was significantly increased by increasing CO₂ from 5% to 15% and was also significantly increased by the addition of 4-AP (in the presence of 5% CO₂). However, in the maintained presence of 4-AP, raising CO₂ from 5% to 15% resulted in no additional increase of Rin. Thus, the increase in Rin induced by increasing CO₂ from 5% to 15% in the presence of 4-AP was significantly smaller than when CO₂ was increased in the absence of 4-AP. The average effects for 8 LC neurons is shown. D: effect of 15% CO₂, 4-AP, and the combination of the two on the change of firing rate. The firing rate of LC neurons was significantly increased by changing CO₂ from 5% to 15% and was also significantly increased by the addition of 4-AP (in the presence of 5% CO₂). The addition of 4-AP alone caused a significantly greater increase in firing rate than increasing CO₂ to 15%. However, in the maintained presence of 4-AP, increasing CO₂ from 5% to 15% resulted in no additional increase in firing rate. Thus, the increase in firing rate induced by increasing CO₂ from 5% to 15% in the presence of 4-AP was significantly smaller than when CO₂ was increased in the absence of 4-AP. The average effects for 8 LC neurons is shown. E: effect of 15% CO₂, TEA, and the combination of the two on the change of Rin. Rin was significantly increased by increasing CO₂ from 5% to 15% and was also significantly increased by the addition of TEA (in the presence of 5% CO₂). In the maintained presence of TEA, increasing CO₂ from 5% to 15% resulted in a significant increase of Rin. The increase in Rin induced by increasing CO₂ from 5% to 15% in the presence of TEA was significantly smaller than when CO₂ was increased in the absence of TEA. The average effects for five LC neurons are shown. F: effect of 15% CO₂, TEA, and the combination of the two on the change of firing rate. The firing rate was significantly increased by increasing CO₂ from 5% to 15% and was also significantly increased by the addition of TEA (in the presence of 5% CO₂). In the maintained presence of TEA, with the firing rate adjusted back to its initial value by injecting negative current, increasing CO₂ from 5% to 15% resulted in a significant increase of firing rate. Both increasing CO₂ from 5% to 15% and the addition of TEA (in 5% CO₂) resulted in a significant and nearly identical increase in firing rate. Further, in the maintained presence of TEA, increasing CO₂ from 5% to 15% caused a nearly identical increase in firing rate to that caused by raising CO₂ in the absence of TEA. The average effects for five LC neurons is shown. In all figures, bars show means ± SE. Differences were significant at either *P < 0.05 or at **P < 0.01.

Because hypercapnia can induce an increase in Rin and firing rate in LC neurons (Fig. 1), we next examined the effects of 4-AP and TEA on these parameters. The initial Rin for all neurons in 5% CO₂ was 393 ± 44.8 MΩ. Exposure of LC neurons that were in 5% CO₂ to 15% CO₂ caused a significant (8.8%) increase in Rin that was similar to the significant increase of Rin of 12.2% caused by exposing LC neurons to 4-AP (in 5% CO₂) (Fig. 2C). However, in the maintained presence of 4-AP, exposure of LC neurons that were in 5% CO₂ to 15% CO₂ caused no additional increase in Rin (Fig. 2C). Thus, increasing CO₂ from 5% to 15% caused a significantly larger increase in Rin in the absence of 4-AP than in the presence of 4-AP. In parallel with these findings, LC neuron firing rate was increased significantly by increasing CO₂ from 5% to 15% or by the addition of 4-AP in 5% CO₂, with the increase induced by 4-AP being significantly larger than the increase induced by hypercapnia (Fig. 2D). In the maintained presence of 4-AP at 5% CO₂, we injected negative current to return firing rate to its initial value. When these neurons, in the presence of 4-AP and 5% CO₂, were exposed to 15% CO₂, firing rate showed no increase (Fig. 2D). Thus, the firing rate increase normally seen upon raising CO₂ from 5% to 15% CO₂ was completely inhibited in the presence of 4-AP (Fig. 2D). These findings show that 4-AP can recapitulate the effects of 15% CO₂ on Rin and firing rate and that the effects of 4-AP and 15% CO₂ on firing rate appear to be mediated by a similar process since the firing rate response to CO₂ is eliminated in the presence of 4-AP.

In a different set of LC neurons, exposure of neurons in 5% CO₂ to either TEA (also in 5% CO₂) or to a hypercapnic solution (15% CO₂) resulted in similar increases in Rin (10.9 and 16.9%, respectively) (Fig. 2E). In the maintained presence of TEA (in 5% CO₂), exposure to 15% CO₂ resulted in a significant change in Rin, but this change was significantly less than in the absence of TEA (Fig. 2E). Exposure of LC neurons in 5% CO₂ to either TEA (still in 5% CO₂) or to 15% CO₂ resulted in a similar increase in firing rate (Fig. 2F). In the maintained presence of TEA (in 5% CO₂), we injected negative current to bring firing rate back down to a value similar to firing rate before exposure to TEA. When we then exposed these neurons to solutions of 15% CO₂ (still containing TEA), we observed a similar increase in firing rate to that caused by 15% CO₂ in the absence of TEA (Fig. 2F). Thus, the firing rate increase seen upon raising CO₂ from 5% to 15% CO₂ was the same in the presence and in the absence of TEA (Fig. 2F). These results show that TEA can increase firing rate to the same extent as 15% CO₂ but that TEA-inhibited channels do not appear to be significantly involved in the firing rate response of LC neurons to hypercapnia.

Effect of Ba²⁺ on Firing Rate and Input Resistance

It has been suggested that Kir channels play a role in the CO₂ response of LC neurons (45, 65). To test whether Kir channels are also involved in the response of our LC neurons, we studied the response of firing rate and Rin to hypercapnia in the presence of 200 μm Ba²⁺, which has been shown to inhibit Kir channels (45, 65). Firing rate increased in response to 15% CO₂ by 0.54 ± 0.12 Hz (from 0.35 ± 0.07 to 0.89 ± 0.11 Hz; n = 7). Exposure to Ba²⁺ also increased firing rate in 5% CO₂ by 0.65 ± 0.10 Hz (from 0.36 ± 0.09 Hz to 1.01 ± 0.12 Hz; n = 7), indicating that Kir channels are present at rest in LC neurons. We injected negative current to bring control firing rate down to 0.50 ± 0.08 Hz. In the presence of Ba²⁺, 15% CO₂ once again increased firing rate by 0.67 ± 0.17 Hz (to 1.17 ± 0.18 Hz; n = 7). The hypercapnia-induced increased firing rate was not significantly different (P = 0.52) in the presence vs. the absence of Ba²⁺.

Similarly, Ba²⁺ had no effect on the hypercapnia-induced increase in Rin. Hypercapnia (15% CO₂) increased Rin by 43.5 ± 14.7 MΩ (from 345.8 ± 70.8 to 389.2 ± 85.2 MΩ; n = 7) in the absence of Ba²⁺ and by 43.4 ± 10.7 MΩ (from 399.4 ± 84.4 to 442.7 ± 90.1 MΩ; n = 7) in the presence of Ba²⁺. These data show that while LC neurons from neonatal rats contain Kir channels, these channels do not appear to play a role in the response of our LC neurons to hypercapnia.

Characterization of and the Effect of Hypercapnia on the TEA-Sensitive K⁺ Current Using Voltage Clamp

We used whole cell patch clamp of individual LC neurons within a slice to characterize TEA-sensitive K⁺ current. In the presence of TTX (1 μM) and Co²⁺ (2 mM), currents were elicited by command pulses in 10-mV increments (300 ms) from a holding potential of −96 mV. The resulting currents had a transient peak component and a sustained component (Fig. 3A). TEA (20 mM) partially inhibited the sustained current but did not affect the transient peak current (Fig. 3B). The difference current, obtained by subtracting the currents in the presence of TEA from control currents in the absence of TEA, is the TEA-sensitive current (Fig. 3C). This current is slowly activating without apparent inactivation, similar to a delayed-rectifying K⁺ current. The I-V plot for this current is consistent with an outwardly rectifying K⁺ current (Fig. 3C, inset).

Fig. 3.

TEA-sensitive outwardly rectifying K⁺ current in LC neurons. A: representative family of outwardly rectifying K⁺ currents elicited by command pulses (300-ms duration) from −116 to +34 mV with 10-mV increments; Vh = −96 mV. B: representative family of outwardly rectifying K⁺ currents elicited by the same command pulses in the presence of TEA (20 mM). C: representative family of TEA-sensitive outwardly rectifying K⁺ currents was obtained by subtracting the currents in B from those in A. Arrow shows time at which the current amplitude was quantified. Inset: activation current-voltage (I-V) plot for TEA-sensitive outwardly rectifying K⁺ current (n = 4). Symbols represent means ± SE. D: representative family of TEA-sensitive outwardly rectifying K⁺ currents. E: family of TEA-sensitive outwardly rectifying K⁺ currents from the same neuron as in D but in the presence of 15% CO₂. Note the markedly smaller currents in the hypercapnic solution. F: LC neurons showing paired currents for each neuron in control (5% CO₂) and in 15% CO₂. Currents were quantified at the points indicated by the vertical arrows in D and F. Note that 15% CO₂ significantly (**P < 0.01) reduced the TEA-sensitive outwardly rectifying K⁺ current.

We studied the effect of HA (15% CO₂, pH_o 7.0) on the TEA-sensitive K⁺ current. This TEA-sensitive current was significantly (P = 0.010) inhibited by 15% CO₂ (Fig. 3, D and E), decreasing the maximal current by 52.1 ± 4.5% (Fig. 3F). These results show that the TEA-sensitive outwardly rectifying current can be inhibited by hypercapnia, but because our previous results suggest that inhibition of this current does not play a role in the chemosensitive response of LC neurons (Fig. 2F), we did not study this current further at more physiological values of CO₂.

Characterization of and the Effect of Hypercapnia on the 4-AP-Sensitive K⁺ Current Using Voltage Clamp

For the rest of our study, we focused on the transient K⁺ current due to the large effect of 4-AP on Vm (Fig. 2B) and the ability of 4-AP to block the hypercapnia-induced increased firing rate (Fig. 2D). We did experiments similar to the TEA experiments but using 4-AP (5 mM). The control current had a transient and sustained component (Fig. 4A). 4-AP resulted in the loss of the transient component but not the sustained current (Fig. 4B). The difference current (current in the absence minus current in the presence of 4-AP) shows the 4-AP-sensitive K⁺ current (Fig. 4C), which has a rapid activation and a rapid inactivation, like an A current (IA).

Fig. 4.

4-AP-sensitive outwardly rectifying K⁺ current in LC neurons. A: representative family of outwardly rectifying K⁺ currents elicited by command pulses (300-ms duration) from −116 to +34 mV with 10-mV increments; Vh = −96 mV. B: representative family of outwardly rectifying K⁺ currents elicited by the same command pulses in the presence of 4-AP (5 mM). C: representative family of 4-AP-sensitive outwardly rectifying K⁺ currents were obtained by subtracting the currents in B from those in A. Arrow shows time at which the current amplitude was quantified. D: representative family of A currents elicited by a different voltage-clamp protocol (inset) designed to identify the activation characteristics of the A current. TTX, Co²⁺, and TEA were added to the perfusate to eliminate Na⁺, Ca²⁺, and TEA-sensitive K⁺ currents. E: A currents in the presence of 4-AP (5 mM). Arrows show the times at which the current amplitudes were quantified. Inset: paired plot of A current amplitude in the absence (control) and the presence (4-AP) of 4-AP for 4 LC neurons. Note that 4-AP significantly inhibited I_max of the transient A current (*P < 0.05). F: activation I-V relationship for 4-AP-sensitive outwardly rectifying K⁺ current (n = 4) in the absence (●) and the presence (○) of 4-AP. The I–V plots were fit by Boltzmann curves. Symbols represent means ± SE.

We used a different electrophysiological protocol combined with pharmacological methods (TTX, Co²⁺, and TEA) to study the transient K⁺ current (Fig. 4D). In this case, activation was measured as the difference in the currents using the standard protocol and the currents using a conditioning pulse of −40 mV for 200 ms before repeating the activation protocol (Fig. 4D, inset) (54). The difference current showed a rapidly activating and inactivating current (Fig. 4D), similar to the 4-AP-sensitive current shown previously (Fig. 4C). The maximal amplitude of the difference current was significantly (P = 0.02) reduced (Fig. 4E) by 74.7 ± 1.9% by 4-AP (Fig. 4E, inset). The I-V plot for this curve is consistent with an outward current and was fit by a Boltzmann curve (Fig. 4F). The I-V plot showed that the A current is activated at around −50 mV, has a maximal current of 6.7 ± 1.3 nA with a voltage for half-activation of −10.4 ± 0.9 mV (n = 7).

We studied the effect of HA (15% CO₂, pH_o 7.0) on the 4-AP-sensitive K⁺ current. The maximum transient K⁺ current was significantly (P = 0.01) inhibited by hypercapnia (Fig. 5, A and B) by 34.5 ± 3.0% (n = 5) (Fig. 5C). However, a stimulus of 15% CO₂ is a very large hypercapnic stimulus. We repeated the measurements of inhibition of transient K⁺ currents using a milder HA (10% CO₂, pH_o 7.15). This milder HA also significantly (P = 0.01) decreased the maximal amplitude of the transient K⁺ current (Fig. 5, D and E) by 29.4 ± 6.8% (n = 6) (Fig. 5F). We also determined the effect of hypocapnic alkalotic solutions (2.5% CO₂, pH_o 7.75) on the transient K⁺ current. Hypocapnia significantly (P = 0.002) increased the maximal amplitude of this current (Fig. 5, G and H) by 29.0 ± 6.4% (Fig. 5I). Taken together, these results suggest that the transient K⁺ current can be modulated by different levels of CO₂.

Fig. 5.

The effect of 15%, 10%, and 2.5% CO₂ on the transient A current in LC neurons. A and B: representative family of transient A currents in control (5% CO₂) (A) and hypercapnia (15% CO₂) (B) aCSF. 15% CO₂ decreased the transient A current. Arrows show times at which the current amplitudes were determined. C: paired plot of A current amplitude in control (5% CO₂) and in 15% CO₂ for 6 LC neurons. Note that 15% CO₂ significantly inhibited I_max of the transient A current (**P < 0.001). D and E: representative family of transient A currents in control (5% CO₂) (D) and hypercapnia (10% CO₂) (E) aCSF. 10% CO₂ decreased the transient A current. Arrows show times at which the current amplitudes were determined. F: paired plot of A current amplitude in control (5% CO₂) and in 10% CO₂ for 6 LC neurons. Note that 10% CO₂ significantly inhibited I_max of the transient A current (*P < 0.05). G and H: representative family of transient A currents in control (5% CO₂) (G) and hypocapnia (2.5% CO₂) (H) aCSF. 2.5% CO₂ increased the transient A current. Arrows show times at which the current amplitudes were determined. I: paired plot of A current amplitude in control (5% CO₂) and in 2.5% CO₂ for 6 LC neurons. Note that 2.5% CO₂ significantly increased I_max of the transient A current (**P < 0.001).

Effect of Changes of CO₂ on pH_i and Maximum Transient K⁺ Current

We next measured pH_i in LC neurons in response to the various hypercapnic and hypocapnic challenges that we used. pH_i varied from 6.96 in the presence of 15% CO₂ (HA) to 7.35 in the presence of 2.5% CO₂ (Fig. 6). The normalized maximum transient K⁺ current varied sharply from 2.5% to 10% CO₂ but appeared to reach a minimum plateau above 10% CO₂ (HA) (Fig. 6). This pattern of the effects of CO₂ on transient K⁺ current is similar to the effects of CO₂ on firing rate (Fig. 1). This relationship suggests that the inhibition of the transient K⁺ current by hypercapnia plays a role in the increased firing rate induced by hypercapnia in LC neurons, which supports the findings of the effect of 4-AP on the hypercapnia-induced increased firing rate (Fig. 2D).

Fig. 6.

Summary of the effects of hypercapnia and hypocapnia on the transient A current in LC neurons. The transient A currents in various levels of CO₂ were expressed as a function of the maximum current at 5% CO₂. Each bar represents the mean ± SE for the maximum transient A current in 2.5% (black bar), 5% (white bar), 10% (gray bar), 15% HA (upward hatched bar) CO₂ and 15% isohydric hypercapnia (IH) solution (downward hatched bar). The number of neurons is given in each bar. For each level of CO₂, the maximum transient A current was significantly different than in 5% CO₂ (***P < 0.001) and 15% CO₂-IH was also significantly different than in 5% CO₂ (*P < 0.05). Further, the maximum transient A current was significantly smaller in 15% CO₂-HA vs. 15% CO₂-IH (#P < 0.05). Below each bar is the value of pH_o measured for aCSF equilibrated with the corresponding value of CO₂ and of the mean ± SE of the pH_i measured using pyranine fluorescence in LC neurons exposed to aCSF equilibrated with the corresponding level of CO₂.

Inhibition of Transient K⁺ Currents by pH_i or pH_o?

The decrease in the maximal transient K⁺ current in response to HA is accompanied by decreases in both pH_o and pH_i (Fig. 6). To initially determine whether the transient K⁺ current is inhibited by changes of pH_o or by changes of pH_i, we employed IH solutions, whereby a neuron is exposed to hypercapnia (15% CO₂) but with no change of pH_o. We directly measured the effect of IH solutions on LC neuron pH_i and found that pH_i acidified from a value of about 7.23 in 5% CO₂ to 7.12 in IH (Fig. 6). This acidification is smaller than the intracellular acidification seen in HA solutions equilibrated with 15% CO₂ (Fig. 6), as expected (15), and in fact is nearly identical to the fall in pH_i seen with HA solutions equilibrated with only 10% CO₂ (Fig. 6). IH solutions significantly (P = 0.01) reduced the transient K⁺ current by 17.4 ± 3.9% (Fig. 7, A–C). Interestingly, the reduction of the transient K⁺ current caused by IH solutions was significantly less than the reduction caused by HA solutions, even though both solutions were equilibrated with 15% CO₂ (Fig. 6). This smaller inhibition of transient K⁺ current by IH vs. HA solutions is correlated with the smaller pH_i change induced by IH vs. HA solutions (Fig. 6). However, both the degree of inhibition of the transient K⁺ current and the fall of pH_i are similar in LC neurons exposed to IH solutions (equilibrated with 15% CO₂) vs. those exposed to HA solutions (equilibrated with 10% CO₂) (Fig. 6). These data clearly indicate that a fall of pH_i is sufficient to inhibit the transient K⁺ current in LC neurons even without a change of pH_o.

Fig. 7.

The effects of 15% CO₂ on the transient A current with either pH_o or pH_i held constant. A and B: representative family of transient A currents in control (5% CO₂) (A) and isohydric hypercapnia (IH) (15% CO₂) (B) aCSF. IH solution decreased the transient A current. Arrows show times at which the current amplitudes were determined. C: paired plot of A current amplitude in control (5% CO₂) and in IH (15% CO₂) solutions for six LC neurons. Note that 15% CO₂ IH solutions significantly inhibited I_max of the transient A current (*P < 0.05) despite the fact that pH_o did not change. D: trace of the effect of HA (15% CO₂) on the pH_i of an LC neuron. pHi was measured by fluorescence imaging microscopy of the pH-sensitive dye pyranine, injected into the neuron from the patch pipette. Note the reversible hypercapnia-induced acidification of the neuron when pH_i was not clamped. The color images show a pyranine-loaded LC neuron soma. A shift from green to blue fluorescence indicates acidification. Bottom: associated transient A current traces in control (5% CO₂; left traces) and in 15% CO₂ (right traces). Note that when hypercapnia was allowed to acidify the neuron, the transient A current was decreased. E: trace of the effect of HA (15% CO₂) on the pH_i of an LC neuron when pH_i was clamped. Note the lack of an effect of hypercapnia on pH_i. The color images show a pyranine-loaded LC neuron soma whose pH_i was clamped during hypercapnic exposure. Bottom: associated transient A current traces in control (5% CO₂; left traces) and in 15% CO₂ (right traces). Note that when the LC neuron was exposed to hypercapnia while its pH_i was maintained constant (i.e., clamped), the transient A current was still decreased by hypercapnia. F: inhibition of the maximum transient A current upon exposure to hypercapnic aCSF (15% CO₂) with pH_i clamped (gray bar) or not clamped (black bar). The bars represent the mean % inhibition of the maximum transient A current ± SE; n = 3. Note that hypercapnia inhibited the maximum transient A current to the same extent regardless of whether pH_i was allowed to acidify or not.

We tested the opposite possibility, that a change of pH_o, in the absence of any change of pH_i, could also inhibit the transient K⁺ current in LC neurons. We used a technique that we previously developed (20) to clamp pH_i at a constant value in the face of changes of CO₂ and pH_o. When pH_i was not clamped, HA resulted in a reversible fall of pH_i (from 7.24 ± 0.008 to 7.02 ± 0.014; n = 3; P = 0.007) (Fig. 7D, top). Under these conditions, both pH_i and pH_o were acidified during hypercapnia and the magnitude of the transient K⁺ current was decreased (Fig. 7D, bottom). When pH_i was clamped, hypercapnia did not result in any change of pH_i (from 7.21 ± 0.003 to 7.21 ± 0.009; n = 3, P = 0.784) (Fig. 7E, top). Under these conditions, hypercapnia resulted in a fall of pH_o, but no change of pH_i, and yet the magnitude of the transient K⁺ current still was decreased by hypercapnia (Fig. 7E, bottom). The degree of inhibition of the maximum value of the transient K⁺ current was about 30% whether pH_i was clamped or not (Fig. 7F). These data clearly indicate that the transient K⁺ current can also be inhibited by a decrease of pH_o, even in the absence of a decrease in pH_i. These findings, combined with the IH data above, show that the transient K⁺ current in LC neurons can be inhibited by either intracellular or extracellular acidosis.

DISCUSSION

The main conclusions of this study are that 1) the firing rate response of chemosensitive LC neurons to altered CO₂ from neonatal rats is very similar to the response seen in LC neurons from adult rats; 2) there are at least two outwardly rectifying currents in LC neurons from neonatal rats: a 4-AP-sensitive transient current (the A current) and a sustained TEA-sensitive delayed-rectifying K⁺ current; 3) both the 4-AP and the TEA-sensitive currents are active at resting Vm and both outwardly rectifying currents are inhibited by hypercapnic acidosis in LC neurons; 4) altered activity of the A current in response to altered levels of CO₂ is largely responsible for the CO₂-induced changes of firing rate in LC neurons; and 5) hypercapnic acidosis inhibits the A current by either decreased pH_o or decreased pH_i in LC neurons (Fig. 7).

LC Firing Rate Response to Changes of CO₂ in LC Neurons

The pattern of our firing rate response to altered levels of CO₂ (Fig. 1D) is remarkably similar in our LC neurons from neonatal rats compared with the pattern previously shown in LC neurons from adult rats (Fig. 1C in Ref. 45). The data are reported as % change in firing rate induced by various levels of CO₂ vs. the initial firing rate in 5% CO₂, which was the same in both studies (0.7 Hz in 45 vs. 0.5–1.1 Hz in the present study). However, the maximal firing rate in response to hypercapnia was smaller (∼150%) in LC neurons from adults vs. the response in LC neurons from neonates (∼300%). This finding is consistent with previous reports of a smaller chemosensitive response in LC neurons from older vs. younger rats (18, 36). The similarity in the pattern of response in neurons from neonates vs. adults suggests that the general pattern of the chemosensitive response of LC neurons is established early in development and maintained into adulthood.

Ion Channels in LC Neurons

The chemosensitive response of a neuron is most likely determined by its complement of ion channels that are responsive to changes of CO₂ and/or pH (14). A number of different ion channels have been described in LC neurons. These include a 4-AP-inhibitable A current (17, 63), a TTX-sensitive Na⁺ channel (16), an inwardly rectifying K⁺ channel (45, 65), a Ca²⁺-activated K⁺ (KCa) channel (29, 34, 53), an L-type Ca²⁺ channel (16, 21, 23), TASK channels (2, 61), a TRP channel (10), and a delayed-rectifying K⁺ channel (17). Any or all of these channels could be affected by hypercapnia and ultimately contribute to the chemosensitive firing response of LC neurons. It is not clear that all of these channels coexist in single LC neurons. For instance, TRPC5 channels are expressed in only 35% of LC neurons (10). Further, some of the K⁺ channels have only been shown to be expressed in adult rodents, such as the A current (63), Kir (45, 65), and TASK 1 channels (61), although A currents have been demonstrated from neonatal LC neurons in culture for up to 6 mo. Thus, we did not assume that the LC neurons from our preparation expressed a given channel without evidence for it.

In the current study, we found clear evidence for a 4-AP-sensitive transient A current (Figs. 2, A and B, and 4) and a TEA-sensitive sustained K current (Figs. 2, A and B and 3) in LC neurons from neonatal rats. Further, these channels were expressed in the same neurons (Fig. 2A). Given space clamp issues with studying neurons in slices, we were concerned about our characterization of these channels using voltage clamp. Thus, we compared our values with those of Forsythe et al. (17), who studied these currents in cultured LC neurons, a preparation that has better space clamping than our slice preparation. They found a sustained current that we estimate had a magnitude of 3.5 nA at 0 mV and a slope conductance of 140 nS (Fig. 1 from 17), which compares with our values of 1.5 nA and 40 nS (Fig. 3C, inset). Our measured properties for the A current from LC neurons in neonatal slices also compare favorably to those measured in cultured LC neurons (17). Our maximum whole cell A current was 6.7 nA (Fig. 4F) compared with 5–10 nA in cultured neurons, and our voltage for half maximum activation was −10 mV (Fig. 4F) compared with about −20 mV for cultured neurons. Further, Forsythe et al. (17) found that with 2.5 mM 4-AP the A current was inhibited by 36%, whereas we noted a 75% inhibition with 5 mM 4-AP (Fig. 4E, inset). The similarity in the properties of these two currents in these two very different preparations suggest that the channel properties are robust and imply that we may have adequate space clamping to study them. One way that this could occur in intact neurons within a brain slice is if the channels that constitute these currents are largely localized to the soma. Somal localization would be consistent with the findings, using localized acidification, that the chemosensitive response of LC neurons is largely restricted to the soma (51), which would require that the chemosensitive channels be largely restricted to the soma.

Our I-V plots suggest that the TEA-sensitive sustained current is not active at resting Vm (Fig. 3C, inset), but the 4-AP-sensitive current should have some activity (Fig. 4F). In fact, on the basis of our inhibitor studies, both TEA-sensitive and 4-AP-sensitive currents have activity at resting Vm, since both drugs resulted in membrane depolarization (Fig. 2B), an increase in Rin (Fig. 2, C and E), and an increase in firing rate (Fig. 2, D and F). 4-AP resulted in a three-fold higher membrane depolarization and increased firing rate compared with TEA (Fig. 2, B, D, and F). These findings suggest that the 4-AP-sensitive current is considerably larger under resting conditions than the TEA-sensitive current in LC neurons from neonates.

CO₂ and pH Sensitivity of Outwardly Rectifying Channels in LC Neurons

In the current study, both outwardly rectified currents observed were inhibited by a large HA challenge (15% CO₂, pH_o 7.0) (Fig. 5), the TEA-sensitive sustained current by ∼50% (Fig. 3, D and E) and the 4-AP-sensitive A current by about ∼35%. Despite both channels being inhibited by 15% CO₂, our data with inhibitors also clearly show that inhibition of the TEA-sensitive current does not appear to play a role in hypercapnia-induced increased firing rate (Fig. 2F), while the 4-AP-sensitive A current appears to account for all of the increased firing rate induced by 15% CO₂ (Fig. 2D). This lack of a contribution from the TEA-sensitive current is consistent with the much smaller membrane depolarization induced by TEA vs. 4-AP. Thus, while a high dose of TEA can inhibit the sustained channels sufficiently to increase firing rate, we believe that the effect of hypercapnia on normal TEA-sensitive channel activity is sufficiently small as to contribute very little, if at all, to the increased firing rate induced by high CO₂. For this reason, we focused on the effect of altered CO₂ on the A current.

The maximum A current was inhibited by 35%, 30%, and increased by 25–30% by HA solutions equilibrated with 15% CO₂ (Fig. 5, A and B), 10% CO₂ (Fig. 5, D and E) and 2.5% CO2 (Fig. 5, G and H), respectively. Thus, the A current is affected by both increases and decreases in CO₂ over the physiological range (Fig. 6). This range of sensitivity of the maximum A current matches quite well the sensitivity of LC neuron firing rate at CO₂ values between 2.5 and 15% (Fig. 1D). As expected, the relationship is inverse (Fig. 6). At CO₂ values where A current is high, firing rate is low and firing rate increases as A current decreases. The relationship between the two is further strengthened by the fact that between 10 and 15% CO₂, the firing rate saturates, increasing only slightly, and, in parallel, inhibition of the A current flattens out (Fig. 1D and 6). These data indicate that inhibition of A current by HA seems to play an important role in determining the firing rate of LC neurons, which is consistent with our findings that the effect of hypercapnia on LC neuron firing rate is eliminated in the presence of 4-AP (Fig. 2D). These are the first data to indicate an important role for the A current in chemosensitive signaling in mammalian neurons, although the A current has been shown to play a major role in the response of chemosensitive neurons from the snail, Helix aspersa (12).

It may seem strange that a common K⁺ current like the A current, which is present in nearly all neurons, should be involved in the chemosensitive response of a neuron. However, common channels, like Kir channels (45), TASK channels (2), and KCa channels (62) have been implicated in central chemosensitive signaling in mammalian neurons. Further, the involvement of an A current and a delayed-rectifying K⁺ current in chemosensitivity in snail neurons has been shown (12). The fact that these channels are widely distributed but contribute to central chemosensitivity in select neurons suggests that these channels come in a variety of forms involving different subunits or splice variants with differing sensitivities to elevated CO₂/H⁺. For instance, the Kir current from chemosensitive LC neurons is believed to derive from a highly pH-sensitive hybrid channel formed from the Kir4.1-Kir5.1 subunits (64, 65). Thus, in many chemosensitive neurons, their pH responsiveness may indeed derive from altered forms of channels commonly found in many neurons. It may also seem odd that we find no evidence here for a role of Kir channels in the chemosensitive response of our LC neurons given that they appear to be present (based on the Ba²⁺ experiments) and given previous reports of a role for this channel in LC neuron chemosensitivity (45, 65). These previous studies were done in adult rats and mice, while we are working with neonates, so it is possible that the basis of the chemosensitive response in LC neurons shifts from one based largely on the A current in neurons from neonates to one involving Kir currents in neurons from adults. This would also suggest a shift in the molecular basis for Kir channels in LC neurons from non-pH-sensitive subunits to more highly pH-sensitive subunits during development.

The degree of inhibition of the A current by HA seems rather small, with at most about a 35% inhibition of the maximum current. This small inhibition of the A current may account for the small increase of Rin seen in response to hypercapnia in LC neurons (Figs. 1E and 2, C and E). However, a mathematical model of chemosensitive neurons from the snail showed that firing rate could be increased nearly 4-fold with about a 10% inhibition of K⁺ conductance (see Fig. 3 in Ref. 6). In LC neurons from neonatal rats, the Rin is rather high (400–500 MΩ) under normocapnic conditions, and thus, small changes in Rin induced by hypercapnia may result in substantial changes in firing rate. This high value for Rin is about twice previously measured values of around 200–250 MΩ, but all of these values were measured in neurons from adult rats using sharp-tipped electrodes (7, 26, 33, 38), so it may be that LC neurons from neonates have higher Rin values. Thus, it appears that hypercapnia does not need to inhibit K⁺ conductance greatly to have a substantial impact on neuronal firing rate.

Finally, we addressed whether it is the change of pH_i or pH_o associated with HA that is responsible for the inhibition of the A current (15, 46, 47). We have previously suggested that a decrease in either pH_i or pH_o is sufficient to increase LC firing rate with hypercapnia (20). A currents have commonly been observed with channels made from the voltage-gated K⁺ channel subunit Kv1.4 (24, 39, 42, 58). Ishii et al. (24) have shown that the maximum A current from channels made from Kv1.4 can be inhibited by decreases in pH_o. In contrast, Padanilam et al. (42) showed an inhibition of the maximum A current from channels made from Kv1.4 by decreases in pH_i, with a pK for inhibition in the physiological range (pK= 7.5). We used IH solutions and a technique that we had previously developed to clamp pH_i at a constant value in neurons exposed to HA solutions (20) to determine whether the A current in LC neurons is inhibited by decreased pH_i or pH_o. Our results show that the A current in LC neurons can be inhibited by a fall of either pH_o or pH_i (Fig. 7). This is consistent with our previous suggestion that either decreased pH_o or pH_i can induce increased firing in LC neurons (18). We do not know which Kv subunit(s) are responsible for the A current in LC neurons nor do we know whether the A current derives from a single channel type that is sensitive to changes of both pH_o and pH_i or whether multiple channel types lead to the A current in LC neurons, some of which are inhibited by decreased pH_o and others by decreased pH_i. A similar pattern of sensitivity to both decreased intracellular and extracellular pH has been shown recently for TRP channels in LC neurons (10).

Perspectives and Significance

Our findings are the first direct evidence that the A current is involved in chemosensitive signaling in mammalian neurons. In LC neurons, we find not only that the A current is affected by HA, but that the sensitivity of the current to various levels of CO₂ is inversely correlated to the firing rate of LC neurons at those same levels of CO₂ (Figs. 1D and 6), suggesting an important role for the A current in the chemosensitive response of LC neurons. Thus, in mammalian central chemosensitive neurons, the A current plays a role in chemosensitive signaling in LC neurons and likely in NTS neurons as well (31). Combined with previous work that showed a role for an A current in the response of chemosensitive neurons from the invertebrate Helix aspersa (12), our data suggest that the A current may play a widespread role in chemosensitive neurons from many different animals from various taxa.

The presence of multiple hypercapnia-sensitive channels, as well as multiple signals (e.g., changes of pH_o and pH_i) is consistent with a polyphyletic origin for pH-sensing in neurons, arising many different times and based on different channels. It is curious that numerous CO₂ and acid-sensitive neurons are present in LC neurons. Although our work suggests that A currents play a predominant role in LC neurons from neonates, other work suggests that Kir channels may be important in LC neurons from adults (45, 65). There is precedence for a developmental change in the channel involved in the chemosensitive response of neurons. In medullary raphé neurons, it has been shown that TASK 1 and TASK 3 channels mediate the small firing rate response to hypercapnia in young neonates (<P10) but that a Ca-activated nonspecific cation current mediates the much larger firing rate response to hypercapnia in raphé neurons from older rats (9). The ion-channel basis for the development of chemosensitivity in LC neurons merits further study since there appears to be a change in the channel basis with age, but the overall firing rate pattern in response to different levels of CO₂ is very similar in LC neurons from neonates vs. adults.

Our understanding of the cellular basis of chemosensitive signaling has presented a large number of potential targets for drug therapy aimed at altering central chemosensitive gain and anxiety/panic disorders. Further, the fact that the cellular basis for the chemosensitive response seems to differ among neurons from different chemosensitive regions offers an approach to help answer the question of why there are so many different chemosensitive regions and what are the roles of these various regions.

In summary, the ability to sense acid involves different channels in different neurons, and our work suggests that the A current plays a major role in LC neurons from neonates. Since LC neurons have been implicated both in the control of breathing and in mediating anxiety, modulation of acid-sensitive A currents could potentially alter either or both of these important neural functions.

GRANTS

This work was supported by National Heart, Lung and Blood Institute Grant R01 HL-56683 and a Research Challenge Augmentation Grant from Wright State University to R. W. Putnam.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: K.-Y.L. and R.W.P. conception and design of research; K.-Y.L. performed experiments; K.-Y.L. analyzed data; K.-Y.L. and R.W.P. interpreted results of experiments; K.-Y.L. prepared figures; K.-Y.L. drafted manuscript; K.-Y.L. and R.W.P. edited and revised manuscript; K.-Y.L. and R.W.P. approved final version of manuscript.