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INTRODUCTION |
Chemosensitive neurons respond to changes in PCO2 and/or pH with a change in membrane potential and/or in spike frequency (Kawai et al. 1996
; Neubauer et al. 1991
; Richerson 1995
). Whereas the general neuronal response to acidic stimuli is depression of activity (Balestrino and Somjen 1988
; Jodkowski and Lipski 1986
), specialized neurons have been identified that respond to increases in PCO2 with a membrane depolarization and/or increases in spike frequency.
CO2 sensitivity appears to be a widespread property of neurons within areas involved in control or modulation of respiration. CO2-sensitive neurons, which are proposed to mediate the central chemosensitivity of breathing, have been identified below the ventral surface of the medulla oblongata (Issa and Remmers 1992
; Kawai et al. 1996
). In addition, CO2-stimulated neurons have been recorded in the medullary raphe (Richerson 1995
) and in the locus coeruleus (Ballantyne et al. 1997
), suggesting that CO2 sensitivity also is present in nuclei that are not primarily involved in the control of respiration. Beside the modulation of respiration, CO2 is shown to affect other CNS functions such as cardiovascular regulation (Millhorn and Eldridge 1986
), cerebral blood flow (Madden 1993
), pain sensitivity, and arousal (Gronroos and Pertovaara 1994
).
Several studies have demonstrated an intrinsic chemosensitivity of neurons, i.e., a response to changes in CO2/H+ even after all synaptic inputs has been blocked. Such neurons have been found in the ventrolateral medulla (Kawai et al. 1996
; Onimaru et al. 1989
), the nucleus tractus solitarii (Dean et al. 1990
), and rostral medullary raphe (Richerson 1995
). These data give rise to the hypothesis that CO2/H+ may modulate ion channels expressed in chemosensitive cells. However, the underlying ionic mechanisms of these neuronal responses have not yet been identified. Voltage-clamp recordings of Richerson and Pizzonia (1995)
in medullary raphe neurons demonstrated a transient outward current (IA), calcium currents, Ca2+-activated K+ currents, and an inward rectifying K+ current. Any of these currents could be the site of action of CO2 and H+, but a detailed analysis of CO2-induced modulation of these ion currents has not been performed. Dean et al. (1989)
reported a hypercapnia-induced decrease in membrane conductance in dorsal medullary neurons attributed to a decreased outward K+ conductance which was, however, not further identified. Experiments on H+-sensitive type I cells of the rat carotid body (Peers 1990
) indicated that H+ effects on Ca2+-activated K+ channels result in a reduction of K+ efflux and membrane depolarization. In their study of chemosensitive neurons of the solitary complex and the ventrolateral medulla, Southard et al. (1995)
determined the reversal potential of the CO2-sensitive current at
90 mV and found that the application of K+ channel blocker 4-aminopyridine abolished the CO2-induced membrane depolarization in these cells. These data suggest an inhibitory effect of CO2 on IA.
However, a comprehensive study investigating the contribution of different ion currents to the neuronal chemosensitivity is not yet available. We performed experiments on cultivated CO2-stimulated neurons of the fetal rat medulla to study which ion currents are modulated by CO2 and/or H+ and thus might be responsible for the chemosensitivity of these neurons.
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METHODS |
Slice preparation
Organotypic cultures of the fetal rat medulla were prepared as described earlier (Wellner-Kienitz and Shams 1998
). Briefly, pregnant Sprague-Dawley rats were anesthetized with halothane and killed by cervical dislocation on the 16th day of gestation. After removing the fetuses, further preparation steps were performed under sterile conditions. The fetuses were transferred to sterile Hank's balanced salt solution (HBSS without NaHCO3 and phenol red) and decapitated. The medulla was prepared from its rostral part (adjacent to the pons, near the outlet of the hypoglossus) to the caudal region, where both vertebral arteries merge into the basilar artery (near the outlet of N. abducens) and cut into 225-µm-thick slices using a conventional tissue chopper. The slices were transferred to the Hank solution and kept there for 1 h at 4°C. After this incubation time, the slices were attached on sterile glass slides by using 20 µl thrombin and 40 µl chicken plasma (Sigma). The attached slices were placed into 15 ml centrifuge tubes containing medium A [59% Dulbecco's modified Eagle's medium (DMEM, GIBCO), 29% HBSS (GIBCO), 9% fetal calf serum (FCS, GIBCO), 2% glucose (20 g/l), 1% antibiotic/antimycotic solution (GIBCO) and 20 µg/l nerve growth factor (Biermann, Germany) and cultivated at 37°C and 5.3% CO2 under continuous rotation. Five days after preparation, medium A was substituted by medium B (89% DMEM, 10% FCS, 1% antibiotic/antimycotic solution, 5 µg/ml nerve growth factor) supplemented with a solution containing 10 µM 5-fluoro-2-desoxyuridine, 10 µM uridine, and 10 µM cytosine-
-D-arabino-furanoside hydrochloride (referred to as FUA; all substances from Sigma) to reduce growth of glial cells. Medullary slices were incubated with FUA for 24 h, then stored in medium B for further cultivation. During cultivation, medium B was refreshed all 3 days.
As described previously (Wellner-Kienitz and Shams 1998
), outgrowing cells that originated from slices, cultivated for
5 days, were identified as glial cells and immature neurons that exhibited a large sodium inward current but only small potassium outward currents. The membrane potential of these cells varied between
10 and
30 mV (hyperpolarizing with increasing cultivation time). During further cultivation time (days 11-17), outgrowing cells formed a neuronal network at the dorsal and to a smaller extent at the ventral side of the medullary slice. The membrane potential of these outgrowing cells was significantly more negative (around
50 mV) and some of these cells exhibited a spontaneous, regular firing pattern.
Electrophysiological experiments presented in this study were predominantly performed on neurons that originated from rostral medullary slices, cultivated for
11 days.
Electrophysiological measurements
Electrophysiological recordings were performed in the current- and voltage-clamp configuration of the patch-clamp technique. Electrodes were made from borosilicate glass (Clark Electromedical Instruments, Reading, UK; 5-6.5 M
tip resistance) and filled with a solution containing (in mM) 120 K-gluconate, 10 KCl, 10 ethylene glycol-bis(
-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), N-(2-hydroxyethyl) piperazine-N'-(2-ethane-sulfonic acid) (HEPES), 1 CaCl2, and 1 MgCl2 or with a solution containing 50 KCl, 80 K-gluconate, 10 EGTA, 10 HEPES, 1 CaCl2, and 1 MgCl2, adjusted to pH 7.2 with KOH. Signals were amplified (Axopatch 200A, Axon Instruments), filtered (1 kHz), and stored on disk for further analysis. Spikes and integrated firing rate also were recorded with a multichannel pen-recorder (model 2800S, Gould). Membrane potential, action potential parameters, and membrane currents were analyzed with the software ISO2 (MFK, Germany). All experiments were performed at 37°C.
For recordings, the attached medullary slices were transferred into the experimental chamber (volume 300-500 µl) and superfused with an artificial cerebrospinal fluid (CSF I) at a flow rate of 3 ml/min. CSF I contained (in mM) 125 NaCl, 0.5 NaH2PO4, 4 KCl, 10 MgSO4, 26 NaHCO3, 30 glucose, and 2 CaCl2 and was equilibrated with gas mixtures of low (70% N2, 28% O2, 2% CO2, superfusate pH 7.8) or high PCO2 (70% N2, 21% O2, 9% CO2, superfusate pH 7.2). Although the Ca2+ concentration was not reduced to preserve Ca2+-dependent cell functions, increasing the bath Mg2+ concentration to 10 mM was sufficient to block synaptic transmission. As described previously (Wellner-Kienitz and Shams 1998
), the increase in the bath Mg2+ concentration from 1 to 10 mM completely blocked both the electrical activity and the response to CO2 in one type of chemosensitive medullary neuron. Although in these cells, both the spike generation and the chemosensitivity were entirely dependent on synaptic inputs, other chemosensitive neurons retained their spontaneous electrical activity and sensitivity to changes in the bath CO2 in the presence of 10 mM Mg2+. In this type of neuron, we frequently observed that the regular firing pattern was superimposed by synaptically transmitted spikes and excitatory postsynaptic potentials (EPSPs) in the presence of 1 mM Mg2+. Increasing the bath Mg2+ concentration from 1 to 10 mM abolished the superimposing spikes and EPSPs without affecting the spike generation or chemosensitivity. In the present study, electrophysiological experiments were performed on neurons perfused with a bath solution containing 10 mM Mg2+ for
20 min. Under these experimental conditions, superimposed spikes were not observed.
Electromagnetic valves controlled the solution flow from each reservoir, allowing rapid changes of CO2 by switching from one perfusion solution to the other. In another series of experiments, Ca2+-activated K+ channels were blocked by superfusion of the modified CSF (CSF II) containing (in mM) 10 tetraethylammonium chloride (TEA), 120 NaCl, 0.5 NaH2PO4, 4 KCl, 10 MgSO4, 30 glucose, 2 CaCl2, and 10 piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) (Na2-salt), adjusted to pH 6.8 or 7.8 with NaOH and equilibrated with a gas mixture of 60% N2 and 40% O2. Control experiments for this series were performed with CSF II in the absence of TEA.
Space clamp problems that cause distortions of membrane currents might result in misinterpretation of the I-V relationships. To ensure that the neurons investigated in this study were appropriately clamped, we determined both the spike rise time and spike amplitude. Only neurons that exhibited spike amplitudes of >60 mV and a dV/dt of >50 V/s (at 2% CO2) were selected for analysis. In the voltage-clamp configuration, the constancy of the INa threshold indicated that the cells were appropriately clamped. However, despite these precautions to select cells that were adequately clamped, in a few experiments, small current deflections superimposed the membrane currents that occurred in response to depolarizing voltage steps. Because we cannot exclude the possibility that outgrowing neurons are electrically coupled, these current deflections might be due to the activation of sodium channels in neighboring neurons.
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RESULTS |
Electrophysiological experiments were performed on neurons that were cultivated for 11-17 days. Most of the cells studied were localized within groups of 10-15 cells. Within these cell groups, several types of neuron were distinguished in respect of their firing pattern and CO2/H+ sensitivity. In addition to chemosensitive cells in which both the electrical activity and the response to CO2 were dependent on synaptic inputs, CO2-inhibited and -stimulated neurons with a regular firing pattern were identified that retained their electrical activity and CO2/H+ sensitivity in the absence of synaptic transmission (see Wellner-Kienitz and Shams 1998
). For this study, only CO2-stimulated cells that generated action potentials after blockade of synaptic transmission and thus revealed an intrinsic chemosensitivity were investigated(n = 42). A high percentage of these regularly firing neurons (60%, n = 25) was found in the outgrowing margins of the rostroventral medulla (in contrast, 80% of the CO2-inhibited cells were located in the dorsal part of the tissue explant).
CO2-stimulated neurons
The spontaneously active neuron in Fig. 1A displayed a regular firing pattern at 2% CO2 with a spike frequency averaging 141/min. As shown on an expanded time scale in Fig. 1B, each spike was preceded by a gradual interspike ramp that depolarized the neuron up to the threshold for action potential generation, and was followed by an afterhyperpolarization. This neuron responded to a reduction in the superfusate pH (by increasing the bath CO2-level to 9%) with an increase in spike frequency, due to increases in the interspike ramp slope, and membrane depolarization (from
55 to
49 mV, see Fig. 1B, right). The comparison of two typical spikes in Fig. 1C shows that the spike amplitude decreased with high CO2 and that the spike duration was prolonged. In addition, the afterhyperpolarization level was reduced at 9% CO2.

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| FIG. 1.
Responses of a medullary pacemaker neuron to different levels of CO2 in the absence and presence of Ca2+ channel blockers. A: pen-recording of the electrical activity of a CO2-stimulated cell at 2 and 9% CO2. B: computer playbacks of action potentials in an expanded time scale. Spikes were shown at 2% CO2 (left) and 9% CO2 (right). C: comparison of single spikes at different CO2 levels. D: pen-recording of the same neuron in the presence of 50 µM Cd2+ and Ni2+. Membrane potential was set to the same level as in A by constant injection of 10 pA. E: single spikes in the absence and presence of Cd2+ and Ni2+ were superimposed (CO2 level: 2%). F: membrane currents recorded during a depolarizing test pulse from a holding potential of 80 mV to a test potential of 0 mV (duration, 100 ms) at 2 and 9% CO2 (different cell as in A). Note the superimposition of INa with K+ outward currents. CO2 increase from 2 to 9% has no effect on INa amplitude but decreases the amplitude of outward currents.
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The reduction in spike amplitude is suggestive of a hypercapnia-induced decrease of a sodium inward current, but as shown in voltage-clamp experiments (Fig. 1F), the amplitude of the Na+ current was not affected by hypercapnia. Therefore, the decrease in spike amplitude is most likely due to partial inactivation of sodium channels as a result of depolarized membrane potential at the high level of CO2. Both the prolongation of repolarization and the reduction of afterhyperpolarization may be caused by the inhibition of K+ outward currents. Because it is known that the activation of Ca2+-activated K+ channels contribute to the repolarization and afterhyperpolarization in neurons (e.g., in rat CA1 hippocampal interneurons) (Zhang and McBain 1995
), we investigated whether hypercapnia inhibits Ca2+-activated K+ channels and thus blocks the observed CO2 effects on spike frequency and membrane depolarization.
Indirect block of Ca2+-activated K+ channels
APPLICATION OF CA2+ CHANNEL BLOCKERS CD2+ AND NI2+.
To inhibit Ca2+-activated K+ channels indirectly by reducing the Ca2+ influx, we blocked voltage-dependent Ca2+ channels by Cd2+ and Ni2+ and recorded the neuronal activity in the presence of both blockers. Application of both CdCl2 (50 µM) and NiCl2 (50 µM) resulted in a membrane depolarization accompanied by an increase in spike frequency and a reduction in afterhyperpolarization (not shown here). For an appropriate comparison of action potential parameters recorded during control with those recorded in the presence of Cd2+ and Ni2+, we corrected the Cd2+- and Ni2+-mediated change in membrane potential by injection of a constant negative current and then tested the neuronal response to CO2 (Fig. 1D). In comparison with control, the spike frequency was significantly reduced at both levels of CO2 (compare A with D), and the CO2 effects on spike frequency and membrane potential were abolished completely after application of Cd2+ and Ni2+ (Fig. 1D, n = 5). In addition, the level of afterhyperpolarization was diminished and the repolarization of action potentials was prolonged by Cd2+ and Ni2+ (Fig. 1E).
To determine whether these effects were produced alone by one of these ions, Ni2+ or Cd2+, or by both, we applied these agents separately. The chemosensitive neuron of Fig. 2A responded to decreases in bath CO2 with hyperpolarization (from
50 to
54 mV) and a decrease in spike frequency. Application of Ni2+ alone (Fig. 2B) resulted in a decrease in spike frequency due to a slope reduction of interspike depolarization, but neither the action potential duration nor the level of afterhyperpolarization were significantly affected (Fig. 2C). The effects of CO2 on spike frequency as well as the CO2-mediated membrane depolarization were retained in the presence of Ni2+ (Fig. 2B). In all four cells tested, application of Ni2+ (50-100 µM) was ineffective in blocking neuronal chemosensitivity.

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| FIG. 2.
CO2-induced effects on membrane potential and spike frequency maintained in the presence of Ni2+. A: electrical activity of a CO2-stimulated neuron under control conditions. B: pen-recording of the same neuron in the presence of 50 µM Ni2+. C: superimposition of spikes in the absence and presence of Ni2+. Note the reduction in slope of the interspike depolarization in the presence of Ni2+ (CO2 level: 9%).
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The effect of Cd2+ alone is shown in Fig. 3. Under control conditions, this neuron responded to hypercapnia with a slight membrane depolarization (from
64 to
60 mV) and an increase in spike frequency. These effects were reversible when switching back to 2% CO2. Superfusion with Cd2+ resulted in a membrane depolarization that was corrected by injection of a negative DC current. Cd2+ induced a reduction in the afterhyperpolarization level (compare Fig. 3, A with B) and completely blocked the CO2 sensitivity of the neuron (Fig. 3B). The CO2-dependent modulation of spike frequency and membrane potential as well as a significant afterhyperpolarization reappeared when Cd2+ was removed by washout (Fig. 3C). The Cd2+-mediated block of the CO2 effects was repeatable and independent of the level of neuronal activity at different membrane potentials (compare Fig. 3, B and D). These effects of Cd2+ were observed in all four spontaneously active neurons tested.

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| FIG. 3.
Block of chemosensitivity by Cd2+. A: pen-recording of neuronal activity under control conditions. B: electrical activity in the presence of 50 µM Cd2+ during constant current injection of 15 pA. C: washout. D: electrical activity (at a different membrane potential as in B during 2nd application of Cd2+.
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APPLICATION OF BA2+.
Ba2+ is known to permeate Ca2+ channels instead of Ca2+, but in contrast to Ca2+, it does not activate the Ca2+-dependent K+ channels (Yellen 1987
). The neuron in Fig. 4A responded to an increase in the bath CO2 level with an increase in spike frequency without a significant membrane depolarization. Substituting external Ca2+ with equimolar concentration of Ba2+ depolarized the cell and increased its spike frequency, but the CO2 sensitivity of the cell was abolished (Fig. 4B). Even the correction of membrane potential back to its control level (induced by current injection of
25 pA) did not restore the CO2 sensitivity of the neuron (Fig. 4C). Again, as Ba2+ was replaced by Ca2+ (washout, Fig. 4D) the CO2 effects were restored. The comparison of single spikes in the absence and presence of Ba2+ (Fig. 4E) demonstrates that the repolarization of action potentials was significantly prolonged, and the afterhyperpolarization was diminished markedly by Ba2+, whereas the Ba2+-induced changes in spike amplitude were small and the spike rise time was not affected. Qualitatively identical results were obtained in all cells studied (n = 5).

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| FIG. 4.
CO2 sensitivity is reversibly blocked by Ba2+. A: control conditions. B: pen-recording of neuronal activity in the presence of 2 mM Ba2+. C: same neuron recorded during constant current injection of 25 pA. D: washout. E: comparison of single spikes in the absence (control) and presence of Ba2+ at a CO2 level of 9%.
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Direct block of Ca2+-activated K+ channels by TEA
In these experiments, the spontaneous activity and H+ sensitivity of the neurons were recorded in a CO2-free bath solution (CSF II, n = 28) while the H+ concentration was modified by fixed acids. Jarolimek et al. (1990)
proposed that hypercapnia potentiated the pH-induced responses of medullary neurons and demonstrated that the same pH decrease was much more effective when raising PCO2 than when decreasing [HCO
3]. Because we performed our experiments in a CO2-free bath solution, we decided to enhance the pH reduction (by using bath solutions adjusted to pH 7.8 and pH 6.8) to maintain adequate H+-induced effects on neuronal activity in comparison with those experiments in which the bath PCO2 was increased.
In the experiment shown in Fig. 5A, increasing bath pH from 6.8 to 7.8 resulted in membrane hyperpolarization and an almost complete cessation of firing. Subsequent reduction of bath pH to 6.8 depolarized the membrane from
54 to
50 mV and increased the spike frequency of the neuron back to its control level (35 spikes/min).

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| FIG. 5.
Tetraethylammonium (TEA)-induced block of H+ effects on membrane potential and spike frequency. A: neuronal responses to pH reduction under control conditions and during application of 10 mM TEA. Note the strong membrane depolarization and increase in spike frequency after application of TEA. B: pen-recording of electrical activity during current injection of 5 pA in the presence of TEA. C: washout of TEA. D: superimposition of spikes in the absence and presence of TEA at pH 6.8.
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Application of TEA (10 mM) further depolarized the cell membrane to
44 mV accompanied with increases in the action potential frequency and a reduction in the afterhyperpolarization level (see Fig. 5A). In addition, the repolarization of the action potentials was significantly prolonged (Fig. 5D). TEA induced also a slight increase in the spike amplitude (Fig. 5D), which could be due to the delayed spike repolarization, which in turn allows the full appearance of Na+ current amplitude.
In the presence of TEA, changes in bath pH had no effect on either the membrane potential or spike frequency of the neuron (Fig. 5A). Even at more negative membrane potentials (induced by current injection, B), at which pH-induced effects on neuronal activity have been shown to be augmented (Wellner-Kienitz and Shams 1998
), changes in pH did not modulate the regular firing pattern or membrane potential of the neuron (Fig. 5B). The H+ effects on neuronal activity were restored after TEA was removed during washout (Fig. 5C). TEA either completely blocked the cellular response to pH (9 cells) or attenuated the H+-induced effects (1 cell).
Voltage-clamp experiments
Voltage-clamp experiments were performed to investigate whether acidification is accompanied with a decrease in K+ currents. H+-stimulated neurons were depolarized by voltage ramps from
120 to + 80 mV within 3 s (Fig. 6IA) or from
120 to 0 mV within 5 s (Fig. 6IC). At 2% CO2, the resulting I-V curve reversed at
73 mV [see Fig. 6IA,bottom; mean value of 0 current potential:
70 ± 10(SD) mV, n = 5] and exhibited a strong outward rectification (Fig. 6IA, top). In the same cell, increasing CO2 induced a prominent decrease in outward currents and a shift in the zero current potential to more positive membrane potentials (
63 mV, see Fig. 6IA, bottom; mean value:
63 ± 9 mV). The I-V curves recorded at 2 and 9% CO2 crossed each other at approximately
75 mV (mean
72 ± 9 mV, n = 5). Although the reversal potential of the H+-sensitive current (see difference trace in Fig. 6IB) is more positive than the calculated K+ equilibrium potential, EK =
88 mV, a H+-induced modulation of ion conductances other than K+ (e.g., a chloride channel) is extremely unlikely because increasing the chloride concentration in the pipette from 14 to 54 mM (thereby shifting ECl from
56 to
22 mV) did not significantly shift the reversal potential of the H+-sensitive current (n = 2).

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| FIG. 6.
Voltage-clamp recordings of CO2/H+-stimulated cells. IA, top: I-V curves of a CO2-stimulated neuron at 2 and 9% CO2. Voltage ramps polarized the cell from 120 to +80 mV within 3 s (holding potential 80 mV). I-V curves revealed a crossover at 75 mV (see arrow). IA, bottom: I-V curves at increased vertical resolution to show the 0 current potential at different levels of CO2. IB: difference trace of the I-V curves in IA (2 9% CO2). H+-sensitive current reversed at 75 mV. IC: I-V curves obtained during a voltage ramp from 120 to 0 mV (duration 5 s, holding potential 80 mV) at pH 7.8 and 6.8. IC, bottom: I-V curves at increased vertical resolution. II: same neuron as in IC. IIA: I-V curves recorded during application of 10 mM TEA at pH 7.8 and 6.8. IIB: difference trace.
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In another series of experiments, the bath pH was reduced from 7.8 to 6.8 (Fig. 6IC). The pH reduction shifted the zero current potential from
54 to
49 mV (Fig. 6IC, bottom) and reduced the outward currents. The reversal potential of the H+-sensitive current was
60 mV (Fig. 6ID). Applying TEA in the same cell results in a strong decrease in outward membrane currents (compare Fig. 6, IC and IIA). In the presence of TEA, membrane currents were identical at both levels of pH (Fig. 6, IIA and IIB), e.g., H+-sensitive currents were blocked completely by TEA. In addition, TEA induced a positive shift of the zero current potential, consistent with the membrane depolarization recorded under current-clamp conditions. While the zero current potential was
54 mV at pH 7.8 during control (Fig. 6IC), it was shifted to
46 mV in the presence of TEA.
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DISCUSSION |
We investigated the effects of CO2/H+ on spontaneously active neurons with intrinsic chemosensitivity in cultures of the fetal rat medulla. A stimulatory effect on the neuronal activity was observed irrespective of whether changes in bath pH were induced by fixed acids or CO2. Increasing H+ induced increases in the slope of the interspike depolarization and prolonged the spike repolarization phase together with a reduction in the afterhyperpolarization. Furthermore, hypercapnia induced a significant reduction in outward current (voltage-clamp experiments) with a reversal potential of the H+-sensitive current of
72 mV (Fig. 6). Similar results were described in a study of Buckler and Vaughan-Jones (1994)
in type I cells of rat carotid body in which a reversal potential for the H+-sensitive current of
75 mV was found. Our data suggest a CO2/H+-induced inhibition of K+ currents that contribute to the resting potential, which, in turn, results in a positive shift in the zero current potential (see Fig. 6), membrane depolarization and increasing firing rate.
The modulation of a chloride influx as a cause of the cell response to H+ is unlikely because shifting the ECl from
56 to
22 mV did not affect the reversal potential of the H+-sensitive current.
In several studies, the CO2/H+-mediated inhibition of K+ channels is proposed to account for H+ chemosensitivity of these cells. The H+-inhibitory effect on a K+ channel is suggested to be the primary step in transduction of hypercapnic and/or acidic stimuli in type I cells of the carotid body (for review, see Peers and Buckler 1995
) as well as in neurons of the nucleus tractus solitarii (Dean et al. 1989
) and of the ventrolateral medulla (Southard et al. 1995
). Whereas the H+-induced inhibition of the large Ca2+-activated K+ channel appears to be responsible for the H+ chemoreception in rat type I cells (Peers 1990
), the H+-induced inhibition of the A current contributes to the H+ sensitivity in neurons of the solitary complex and the ventrolateral medulla (Southard et al. 1995
).
In our study, however, Ca2+-sensitive K+ channels were involved in the process of neuronal H+ sensitivity in as much as the neuronal response to pH was abolished by the application of Cd2+. In the neurons of our study, the Cd2+ block of Ca2+ channels induced prolongation of spike duration without affecting the spike rise time and amplitude. The lack of Cd2+ effects on spike rise time and amplitude suggests that Ca2+ influx did not contribute to the generation of action potentials in CO2/H+-stimulated neurons. The application of Cd2+ was accompanied by a membrane depolarization and a reduction of the afterhyperpolarization, indicating that K+ channels were blocked in this condition. Because it is known that Cd2+ has no direct blocking effect on K+ channels, the effect of Cd2+ is proposed to be mediated indirectly by blocking the Ca2+ influx, which would result in an inhibition of one or more types of Ca2+-activated K+ channel.
In contrast to Cd2+, application of Ni2+ (50-100 µM) alone affected neither the spike duration nor the amplitude of afterhyperpolarization and failed to block neuronal chemosensitivity. Therefore the contribution of Ni2+-sensitive Ca2+ channels to the chemosensitivity of CO2/H+-stimulated neurons can be ruled out, but we hypothesize that Ni2+-sensitive Ca2+ currents contribute to the spontaneous interspike depolarization because the application of Ni2+ reduced the spike frequency by a reduction in the slope of the interspike depolarization (Fig. 2).
It is known that Cd2+ in a concentration of 50 µM preferentially blocks the Ca2+ current through L-type channels, whereas Ni2+ in a concentration of 100 µM effectively blocks T-type Ca2+ channels without affecting L-type Ca2+ channels (Fox et al. 1987
, experiments on chick sensory neurons). However, both Cd2+ and Ni2+ are not absolute selective blockers for the different types of Ca2+ channel. Therefore, additional voltage-clamp experiments and the application of more specific blockers (e.g., dihydropyridines,
-conotoxin) are required to identify the types of Ca2+ channel that contribute to the K+ conductance in our study. Although our data suggest that one type of these channels may be a L-type Ca2+ channel, we have to assume that at least one more type of Ca2+ channel contributes to the activation of Ca2+-dependent K+ channels, one that is active near the resting potential. A persistent Ca2+ current that can account for the slow ramp-like depolarization has been described in dopaminergic neurons of the substantia nigra (Kang and Kitai 1993
). It remains to be investigated whether a similar Cd2+-sensitive Ca2+ current is also present in our preparation.
We have, however, experimental evidence that Ca2+-activated K+ currents contribute to the membrane currents at negative membrane potentials because the application of TEA induced a decrease of membrane currents at potentials negative to
30 mV [Fig. 6: reduction from +42 pA (without) to +24 pA (with TEA) at
40 mV, pH 7.8]. Although we propose that Ca2+-activated K+ channels are sufficiently active near the resting potential such that its H+-induced inhibition can account for a membrane depolarization, the activity of Ca2+-activated K+ channels at negative membrane potentials in vivo may be greater than assumed here. Because our recording solution strongly buffered the intracellular free Ca2+ concentration, Ca2+-dependent K+ channels only may be activated partially. Although the slow Ca2+ buffer EGTA does not modify the IK(Ca) in other preparations (Protti and Uchtel 1997, experiments on mouse motor nerve terminals), we cannot exclude that, in our preparation, the neuronal response to increases in the bath CO2 and pH reduction may be enhanced in vivo or under experimental conditions that reduce Ca2+ buffering.
The idea that CO2/H+-induced inhibition of Ca2+-activated K+ channels primarily accounts for central chemosensitivity is further supported by our observation that Ba2+ and TEA completely block the neuronal response to CO2/H+. Both TEA and Ba2+ are known to block Ca2+-activated K+ channels in various tissues either directly (TEA) or indirectly (Ba2+) (Yellen 1987
). From our data, we cannot determine whether the neuronal chemosensitivity is attributed to an indirect inhibition of Ca2+-activated K+ channels after H+-induced block of voltage-dependent Ca2+ channels or if H+ ions are acting directly on the Ca2+-activated K+ channel. Although an H+-induced modulations of L-type Ca2+-currents has been described in rat CA1 neurons, in which extracellular acidosis (pH 6.9-6.0) reversibly depressed the Ca2+ current amplitude and caused a positive shift in the voltage dependence of current activation (Tombaugh and Somjen 1996
), further voltage-clamp experiments have to investigate whether a similar CO2/H+-induced inhibition of one or more types of Ca2+ channel occurs in our preparation.
In the case of a direct H+ inhibition of Ca2+-activated K+ channels, it remains to be elucidated whether the site of H+ action is extracellular or intracellular. In rat type I cells of carotid body, the K+ inhibition appears to be mediated by a fall in intracellular pH (see Peers and Buckler 1995
for review). Because Erlichman et al. (1995)
observed a hypercapnia-induced decrease in pHi in medullary neurons, a similar mechanism might be proposed for the inhibition of Ca2+-activated K+ channels in our preparation, but this hypothesis remains to be tested.