 |
INTRODUCTION |
In medullary respiratory neurons of cats in vivo,
-aminobutyric acid- (GABA) and/or glycine-induced periodic membrane hyperpolarization (Ballantyne and Richter 1986
; Richter 1996
; Richter et al. 1992
) is accompanied by a prominent decrease of intracellular pH (pHi) (Ballanyi et al. 1994a
). It was hypothesized (Ballanyi et al. 1994a
) that this intracellular acidosis is secondary because of HCO
3 efflux via the receptor-coupled anion pore (Bormann et al. 1987
), as was originally shown for crayfish muscle fibers and neurons (Kaila 1994
; Kaila and Voipio 1987
; Voipio et al. 1991
).
A Cl
-mediated inspiration-related hyperpolarization is also a characteristic feature of several types of respiratory neurons in the isolated brain stem-spinal cord preparation of neonatal rats (Ballanyi et al. 1994b
; Onimaru and Homma 1992
; Onimaru et al. 1990
, 1996
; Smith et al. 1992
). This in vitro respiratory network remains functionally active in a transverse slice, containing ventral respiratory group (VRG) neurons of the pre-Bötzinger complex, which is the core of the respiratory network (Smith et al. 1991
). We have further reduced this preparation to a nonrhythmic, 150-µm thin brain stem slice to measure fluorometrically pHi in voltage-clamped neurons of the region of the VRG, which were dialyzed via the patch electrode with the fluorescent indicator 2
,7
-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF) (Trapp et al. 1996a
).
The results show that GABA and glycine cause a prominent and sustained fall of pHi, which depends on the presence of extracellular CO2/HCO
3 and is reversed into an intracellular alkalosis at positive membrane potential (Em). This substantiates the above assumption that rhythmic fluctuations of pHi during physiological activity of respiratory neurons in vivo are due to HCO
3 efflux through GABAA- or glycine receptor-associated Cl
channels (Ballanyi et al. 1994a
). Parts of the results have been published in abstract form (Lückermann et al. 1995
; Trapp et al. 1995
).
 |
METHODS |
Preparation and solutions
Wistar rats (1- to 4-day-old) of either sex were anesthetized with ether and decapitated. The brain was removed and the brain stem with the cerebellum was isolated. After removal of the cerebellum, the brain stem was glued to the stage of a vibratome (FTB Vibracut, Weinheim, Germany) and three transverse slices (150 µm thick) were cut at levels of 200 µm caudal to the obex (see Smith et al. 1991
). After transfer and immobilization of individual slices with a net, the recording chamber (volume 3 ml) was superfused with oxygenated standard saline (temperature 30°C, flow rate 5 ml/min) of the following composition (in mM): 118 NaCl, 3 KCl, 1 MgCl2, 1.5 CaCl2, 25 NaHCO3, 1 NaH2PO4, and 10 glucose, pH adjusted to 7.4 by gassing with 95% O2-5% CO2. In some experiments, a CO2/HCO
3-free superfusate of the following composition was used (in mM): 118 NaCl, 3 KCl, 1 MgCl2, 1.5 CaCl2, 25 N-2-hydroxy-ethylpiperazine-N
-2-ethane sulphonic acid (HEPES), and 10 glucose, pH adjusted to 7.4 with 1 N NaOH. This solution was gassed with 100% O2. Drugs, purchased from Sigma (Deisenhofen, Germany), were added from stock solutions to the superfusion fluid.
Intracellular recordings
Patch pipettes were made from borosilicate glass capillaries (GC 150TF, Clark Electromedical Instruments, Pangbourne, UK) with the use of a horizontal electrode puller (Zeitz, Munich, Germany). The standard ("high-Cl
") patch electrode solution contained (in mM) 130 KCl, 1 NaCl, 1 MgCl2, 0.5 CaCl2, 1 K-1,2 bis(2-aminophenoxy)ethane-N,N,N
,N
-tetraacetic acid, 10 HEPES, and 1 Na-ATP, pH adjusted to 7.4 with 1 N KOH. In some experiments, KCl was replaced by 130 mM potassium gluconate ("low-Cl
solution") or a solution containing the K+ channel blockers Cs+ and tetraethylammonium (TEA), with KCl replaced by 100 CsCl and 30 TEA-Cl. These solutions had an osmolarity of between 270 and 290 mosmol and were adjusted to a pH of 7.4 with 1 N KOH. DC resistance of patch electrodes ranged from 3-8 M
, depending on the composition of the filling solution. The pH-sensitive dye BCECF (50 µM) was added to the pipette solution. In some experiments, 15 or 30 µM carbonic anhydrase (CA) was also added.
Whole cell recordings were performed on superficial neurons in the region of the VRG, located in the ventrolateral reticular formation near the nucleus ambiguus (Arata et al. 1990
; Onimaru and Homma 1992
; Richter 1996
; Smith et al. 1991
). The cells were visualized through a ×40 water immersion objective under an upright microscope (Standard-16, Zeiss, Oberkochen, Germany), with the use of an EPC-9 patch-clamp amplifier (HEKA, Lambrecht, Germany). Seal resistances were between 1.5 and 3 G
. Series resistance (8-20 M
) and cell capacitance (21.83 ± 9.9 pF) were compensated by >70%. Membrane conductance (gm) was measured by regular injection of hyperpolarizing direct current or voltage pulses (duration 500 ms, amplitude
20 mV or
10 to
100 pA in current-clamp mode). During voltage-clamp recordings, holding potential (Vh) was typically
60 mV. Steady-state current-voltage relations were determined by application of depolarizing or hyperpolarizing voltage steps (duration 300 ms) and analysis of membrane current (Im) responses. Averages of responses to three consecutive pulses were analyzed.
pHi measurements
The microscope was equipped with an epifluorescence optics, a xenon lamp, and a photomultiplier system (Luigs & Neumann, Ratingen, Germany). A pinhole diaphragm limited the region from which emitted light was collected to a circular spot of 40 µm diam. The ratio of the fluorescence (F) signals was measured at 540 nm (band-pass filtering 515-565 nm) in response to alternating excitation at 440 and 490 nm (F440, F490). Once the whole cell configuration was established, steady-state loading of BCECF was achieved after a maximum of 10 min as indicated by the stable signals of the excitation fluorescence signals and a smooth ratio trace, representing pHi. Signals were calibrated according to a modified (Trapp et al. 1996a
) "nigericin" method (Thomas et al. 1979
). For this purpose, cells were exposed to 4 µM of the K+/H+ ionophore nigericin in a high-K+ solution consisting of (in mM): 118 KCl, 3 NaCl, 1 MgCl2, 1.5 CaCl2, 25 HEPES, and 10 glucose, pH varied between 6 and 8 with 1 N KOH. For further details, see Trapp et al. (1996a)
.
Fluorescence signals were sampled at 2 Hz by a Mega STE-4 computer (Atari Corporation, Sunnyvale, CA) with the use of the Fura-2 Data Acquisition System (Luigs & Neumann). Electrophysiological data were sampled via an additional ITC-16 interface (Instrutech, Elmont, NY) and into a second Mega STE-4 computer with the use of the EPC-9 software (HEKA). Analysis of data was performed with Review (HEKA) and the Fura-2 Data Acquisition System. For production of figures, pHi traces were low-pass filtered at 0.5 Hz. Values are means ± SD.
 |
RESULTS |
Hyperpolarizing inhibitory postsynaptic potentials (IPSPs)are a characteristic feature of VRG neurons in the isolated brain stem-spinal cord preparation of neonatal rats (Ballanyi et al. 1994b
; Onimaru et al. 1996
; Smith et al. 1992
; see also Feldman and Smith 1989
). The contribution of GABAA and glycine receptors to these responses and their pharmacology has, however, not been analyzed in detail yet. Therefore in an initial series of experiments we tested the electrical response of voltage-clamped neurons in the region of the VRG to bath-applied GABA and glycine and to the specific antagonists bicuculline and strychnine.
Effects of GABA and glycine on Im and gm
AGONIST-EVOKED CURRENTS.
For these measurements, either the standard high-Cl
electrode solution or a high-Cl
solution containing Cs+ and TEA+ for improving voltage-clamp conditions by reduction of K+ currents (Onimaru et al. 1996
) was used. Bath application of 0.1-1 mM GABA produced an inward current 0.1-1.3 nA in amplitude, accompanied by an increase in gm of several hundred percent in 12 cells tested. These currents with a reversal potential (Erev) of between
8 and +9 mV (n = 4) were blocked by 50-100 µM bicuculline (Fig. 1, A and B). In nine of these neurons, glycine evoked an inward current 0.05-0.4 nA in amplitude and a concomitant gm increase, which was suppressed by strychnine (Fig. 1, C and E). Similar to the GABA response, Erev of the glycine-evoked inward currents was close to 0 mV (Fig. 1D). Two cells, in which GABA evoked an inward current and gm increase, did not respond to glycine. As tested in a total of eight cells, the GABA responses were not markedly affected by 25 µM strychnine and the glycine responses persisted in the presence of 50 µM bicuculline (Fig. 1E).

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| FIG. 1.
-Aminobutyric acid (GABA)- and glycine-evoked membrane currents. A: bicuculline block of a GABA-induced inward current (membrane current, Im) and membrane conductance (gm) increase, measured by regular application of 20-mV voltage steps. B: current-voltage relation of 4 cells revealing a reversal potential (Erev) of the GABA-evoked current of ~0 mV. C: strychnine block of a glycine-induced inward current and gm increase. D: current-voltage relation of 3 cells shows a mean Erev of the glycine response of ~0 mV. E: effects of bicuculline and strychnine on spontaneous inhibitory postsynaptic currents and Im response to bath-applied glycine. All measurements were performed with Cs-tetraethylammonium (TEA)-Cl intracellular solution, except in C, where a KCl solution was used.
|
|
SPONTANEOUS INHIBITORY POSTSYNAPTIC CURRENTS.
In ~50% of recordings in which the standard high-Cl
electrode solution was used (n = 25), and, in particular, during recordings with the Cs/TEA-filled electrodes (n = 13), spontaneous synaptic inward currents were observed at a Vh of
60 mV. These currents, which had an amplitude of up to 0.5 nA and reversed polarity at ~0 mV, were substantially attenuated by either 10-50 µM bicuculline or 10-25 µM strychnine (Fig. 1E). Whereas the bicuculline block was reversible within 5 min, recovery of such spontaneous inhibitory postsynaptic currents (IPSCs) from strychnine was still incomplete after >10 min of washout of the drug. These results demonstrate that the majority of neonatal neurons in the region of the VRG has functional GABAA and glycine receptors.
pHi measurements
EFFECTS OF GABA AND GLYCINE.
Subsequent to this pharmacological characterization it was examined whether exposure to GABA or glycine for 1 min affects pHi. In 10 neurons, recorded at a Vh of
60 mV with the standard high-Cl
intracellular solution containing the fluorescent pH indicator BCECF, a mean pHi baseline of 7.33 ± 0.13 (SD) was revealed. The calculated equilibrium potential for H+ and, thus, bicarbonate ranged between
10 and +9 mV. In nine of these cells, the GABA-evoked inward current and gm decrease were accompanied by a delayed fall of pHi (Fig. 2A) by up to 0.4 pH units in individual cells. The average values for the GABA-induced pHi decrease were 0.25 ± 0.11 pH units (n = 5) for 1 mM GABA and 0.17 ± 0.07 pH units (n = 4) for 0.1 mM GABA. On washout of GABA, pHi recovered from the intracellular acidosis within ~2 min. A smaller mean fall of pHi by 0.18 ± 0.1 pH units (1 mM, n = 4) and 0.12 ± 0.06 pH units (0.1 mM, n = 3) was observed on administration of glycine. The GABA-induced inward current and gm increase as well as the accompanying fall of pHi were reversibly blocked by 100 µM bicuculline, whereas 10 µM strychnine effectively suppressed the pHi responses to glycine (Fig. 3). In 11 different cells tested for glycine (n = 10) and GABA (n = 1), pHi remained unaffected despite a profound effect on Im and gm (not illustrated).

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| FIG. 2.
Effects of GABA, NH+4, and membrane depolarization on intracellular pH (pHi). A: GABA evoked an inward current and a concomitant delayed fall of pHi, whereas an inward current in response to NH+4 was accompanied by an initial intracellular alkalosis, turning into a fall of pHi after washout of the drug. Recording was performed with a high-Cl intracellular solution. B and C: whereas GABA (B) or glycine (C) did not lead to a fall of pHi in this cell, depolarization to 0 mV led to a progressive profound intracellular acidosis. Recording was performed with a low-Cl intracellular solution.
|
|

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| FIG. 3.
Pharmacological blockade of GABA- and glycine-induced decreases of pHi. A: bicuculline suppressed the current response and the intracellular acidosis evoked by bath application of GABA. B: strychnine led to a diminution of the inward current and to a full blockade of the accompanying pHi fall on exposure to glycine, whereas the corresponding responses to GABA were not affected. Recordings were made with a KCl intracellular solution (which also contained 30 µM carbonic anhydrase in B).
|
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EFFECTS OF NH+4 AND DEPOLARIZATION.
To test whether this lack of a GABA- or glycine-evoked acidification was due to impaired pH sensitivity of BCECF (see DISCUSSION), alternative procedures to displace pHi were used. For this purpose, the cells were either exposed to NH+4 or depolarized via current injection. Bath application of 10 mM NH+4, which is an established tool to perturb pHi for an analysis of pH regulatory mechanisms (Thomas 1984
), led to an inward current up to 0.3 nA in amplitude. The inward current was accompanied by an initial intracellular alkalinization by maximally 0.3 pH units, followed by a delayed acidification of 0.15-0.4 pH units in three cells tested. These effects of NH+4 are illustrated for a cell in which GABA led to a prominent pHi decrease (Fig. 2A). In a total of five neurons, depolarization to 0 or +30 mV for 1 min evoked an intracellular acidosis by 0.12-0.5 pH units (Fig. 2B, see also Fig. 5).

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| FIG. 5.
Voltage dependence of the GABA-induced pHi transient. At a holding potential (Vh) of 60 mV, GABA led to the typical inward current, gm increase, and pHi fall in a cell recorded with a KCl electrode. After depolarization to 0 mV and, subsequently, to +30 mV, the GABA current was reduced and changed polarity, and also the pHi fall was reversed into an intracellular alkalosis. Note the prominent and sustained decrease of pHi baseline on membrane depolarization.
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EFFECTS OF CARBONIC ACID.
A second possible explanation for the lack of the GABA- or glycine-related intracellular acidosis in a subpopulation of these medullary neurons would be washout of carbonic acid (CA), which is essential for the generation of GABA-evoked decreases of pHi (Pasternack et al. 1993
); 15-30 µM of this enzyme was added to the patch pipette solution. In this series of experiments, administration of 1 mM GABA for only 30 s produced a fall of pHi by 0.18 ± 0.05 pH units in 15 neurons loaded with 15 µM CA, whereas in 6 control cells, the mean GABA-related pHi fall was 0.09 ± 0.03 pH units. In addition to this significant (P < 0.05) potentiation of the amplitude of the pHi response inthe cells dialyzed with CA, the initial rate (0.39 ± 0.14 pH units/min) of the GABA-induced fall of pHi, which was linear during the first 20 s of occurrence, was also significantly (P < 0.05) faster than in the control neurons (0.14 ± 0.05 pH units/min). In this set of experiments, pHi remained unaffected by GABA in two of the CA-dialyzed neurons and in four cells of the control.
EFFECTS OF Cl
.
In a different series of experiments, it was investigated whether the intracellular acidosis on activation of the receptor-associated Cl
channels was affected by the Cl
gradient, determining chloride equilibrium potential (ECl) and, thus, the direction of Cl
fluxes through the receptor-coupled Cl
channel. In eight cells, dialyzed with a low-Cl
electrode solution, resting pHi (7.37 ± 0.15) did not significantly differ from pHi baseline in cells recorded with the standard high-Cl
solution (see above). Figure 4A illustrates that GABA evoked a substantial gm increase, but no major inward current, at a Vh of
60 mV in these cells. After switch to current clamp, GABA induced a gm increase, which was accompanied by a small depolarization. However, neither the amplitude nor the kinetics of the GABA-associated intracellular acidosis was changed with respect to the response under voltage clamp. The mean Erev of the Em response to GABA was
64 ± 5.1 mV (n = 4). The average values of the pHi decreases accompanying the GABA-induced currents in voltage clamp (which had an amplitude of ±30 pA) in low-Cl
cells were 0.25 ± 0.07 pH units (1 mM, 1 min, n = 10) and 0.18 ± 0.09 pH units (0.1 mM, n = 5), and thus were almost identical to those observed with high-Cl
electrodes. However, pHi remained unaffected during the action of GABA on Im and gm in seven different neurons (Fig. 2B). As tested in three of these cells, depolarization to 0 or +30 mV for 1 min evoked an intracellular acidosis by 0.1-0.4 pH units (Fig. 2, B and C), similar to that revealed in cells recorded with the high-Cl
electrodes (see above). A comparable depolarization-induced acidosis was also revealed in cells in which GABA or glycine led to the typical fall of pHi.

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| FIG. 4.
Effects of CO2/HCO 3-free solution on GABA-induced pHi changes. A: in a neuron recorded with a potassium gluconate patch electrode, GABA evoked a fall of pHi and a major gm increase but no major change in Im. An almost identical GABA-induced intracellular acidosis was observed after switch to current clamp. B: continuation of the recording in A shows that introduction of a CO2/HCO 3-free, N-2-hydroxy-ethylpiperazine-N -2-ethane sulphonic acid (HEPES) pH-buffered saline caused a sustained intracellular alkalosis. In this situation, the GABA-evoked pHi fall, but not the gm increase, was suppressed.
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EFFECTS OF CO2/HCO
3-FREE SUPERFUSATE.
Furthermore, the effects on GABA-induced pHi decreases of CO2/HCO
3-free HEPES pH-buffered solution were tested. As exemplified in Fig. 4B for a recording with a low-Cl
patch electrode, superfusion of the HEPES solution produced a stable rise of pHi baseline by 0.24 ± 0.16 pH units (n = 5). In three cells, recorded with high-Cl
electrodes, HEPES increased steady-state pHi by 0.15 ± 0.04 pH units. The absence of CO2/HCO
3 did not substantially affect resting Em, Im, or gm, or the membrane response to GABA. However, the pHi decreases accompanying the GABA-induced Im (n = 3) or Em (n = 2) responses were completely blocked (Fig. 4B). The HEPES-induced alkalinization and the supression of the GABA-induced pHi decrease were fully reversible after reintroduction of the standard CO2/HCO
3-containing solution (not shown).
REVERSAL OF THE GABA RESPONSE.
In a final set of experiments, the voltage-dependence of the GABA-induced intracellular acidosis was analyzed. In five cells, recorded with high-Cl
electrodes, the GABA-induced inward current and gm increase were accompanied by a pHi fall by 0.1-0.22 pH units. Subsequent change of Vh to either 0 or +30 mV led to a steady outward current and a sustained fall of pHi by up to 0.5 pH units. Under these conditions, the GABA-induced current was decreased in amplitude (0 mV) or reversed polarity (+30 mV). At +30 mV, GABA now led to an intracellular alkalinization by between 0.09 and 0.15 pH units in three neurons (Fig. 5), whereas pHi did not change in two cells. In the example of Fig. 5, a minor alkalinization was also observed during the action of GABA at 0 mV.
 |
DISCUSSION |
GABAA and glycine receptors in VRG neurons
RHYTHMICALLY ACTIVE PREPARATIONS.
Respiratory activity in mammals in vivo critically depends on mutual Cl
-mediated inhibition within the neuronal network of the VRG (Richter 1996
). In isolated preparations from rodents, however, GABAAergic or glycinergic inhibition does not appear to be essential for generation of the primary rhythm, because respiratory activity is not suppressed by bicuculline or strychnine (Feldman and Smith 1989
; Onimaru et al. 1990
; Richter et al. 1992
, 1997
). It is assumed that conditional burster neurons of the VRG mediate rhythmic Em fluctuations in such reduced respiratory networks, which are devoid of afferent (peripheral) synaptic inputs (Feldman and Smith 1989
; Onimaru et al. 1990
; Richter et al. 1997
; Smith et al. 1991
).
Nevertheless, GABAA and glycine receptors are functional in these in vitro preparations. During the inspiratory phase, different types of rhythmically active VRG neurons are hyperpolarized by synchronized Cl
-mediated IPSPs (Ballanyi et al. 1994b
; Onimaru and Homma 1992
; Onimaru et al. 1996
; Ramirez et al. 1996
; Smith et al. 1992
). Furthermore, bicuculline and strychnine selectively block spontaneous IPSPs of VRG neurons in rhythmically active, tilted sagittal (Paton and Richter 1995
; Paton et al. 1994
) or transverse (Ramirez et al. 1996
) slices. In functionally identified VRG cells of the brain stem-spinal cord preparation of neonatal rats, bath application of GABA and glycine evoked prominent bicuculline- and strychnine-sensitive membrane hyperpolarizations (Brockhaus and Ballanyi 1995
). In individual respiratory neurons of the latter study, the responses to both GABA and glycine were not affected by addition of tetrodotoxin. This almost excludes the possibility that these responses are due to indirect effects of the agonists, caused by modulation of spontaneous activity of presynaptic cells within the network (e.g., Khazipov et al. 1993
).
THIN SLICES.
For fluorometric measurements of GABA- and glycine-mediated changes of pHi, it was necessary to use brain stem slices with a rostrocaudal thickness of not more than 200 µm. These slices contained only subregions of the pre-Bötzinger complex and, thus, not the entire core (~350 µm diam) of the respiratory network, which is necessary to produce respiration-like activity in transverse slices (Richter 1996
; Richter et al. 1992
; Smith et al. 1991
). Accordingly, functional identification of neurons in the region of the VRG was not possible in the present study. However, in the vast majority of cells in the nonrhythmic thin slices the responses to GABA and/or glycine were very similar to those of the rhythmically active VRG neurons described above. In future studies, a more precise determination of the localization and properties of pre-, post-, or extrasynaptic (Brown 1979
) components of Cl
-mediated inibitory responses in these cells deserves immunohistochemical analysis in combination with synaptic stimulation of identified afferents rather than agonist application.
pHi measurements
METHODOLOGICAL CONSIDERATIONS.
The pHi measurements showed that bath application of GABA or glycine as well as membrane depolarization or administration of NH+4 produces a major intracellular acidosis of the VRG neurons. For monitoring of pHi, we have used the rather novel technique of intracellular application of the fluorescent dye BCECF via the patch electrode (Trapp et al. 1996a
). Thus some methodological aspects, which are discussed in detail in the latter study, should be considered before discussion of the mechanisms of the observed pHi changes. The steady increase of both the F440 and F490 fluorescence signals within ~10 min after establisment of the whole cell configuration indicates ongoing loading with BCECF. However, the ratio trace, representing pHi, was typically stable after <2 min of intracellular recording (compare Fig. 2 of Trapp et al. 1996a
). Therefore HEPES, which should enter the cells similarly to BCECF during this phase, does not appear to affect pHi baseline. Nevertheless, 10 mM intracellular HEPES should, at physiological levels of pHi, contribute by ~5 mM/pH unit to intrinsic buffering power, which was 18 mM/pH unit in dorsal vagal neurons studied under comparable conditions (Trapp et al. 1996a
). As in these cells, removal of extracellular CO2/HCO
3 by superfusion of HEPES-buffered solution led to a stable alkalinization of the VRG neurons by maximally 0.5 pH units (Fig. 4). This suggests that diffusion of "fresh" HEPES buffer from the pipette does not clamp pHi and should only have a minor influence on the kinetics or amplitude of experimentally induced displacements of pHi.
GABA- AND GLYCINE-INDUCED INTRACELLULAR ACIDOSIS.
Physiological relevance. The observed GABA- and glycine-induced pHi decreases are not caused by a nonspecific effect of these inhibitory amino acids, because bicuculline or strychnine completely abolished the agonist-evoked current, the gm increase, and also the accompanying fall of pHi. These findings are consistent with previous measurements in mammalian brain slices, which showed that GABA-induced increases of extracellular pH are blocked by the GABAA receptor antagonist picrotoxin (Chen and Chesler 1992
; Kaila et al. 1992a
; Taira et al. 1995
; see also Chen and Chesler 1991
). The blocking effects of bicuculline in the present study almost exclude a contribution of activation of GABAB receptors to the observed intracellular acidosis.
The magnitude of the intracellular acidosis on activation of receptor-coupled Cl
channels could exceed 0.3 pH units in individual cells. Even larger pHi decreases will possibly be detected in future studies with the use of imaging techniques, allowing for a higher spatial resolution and thus analysis of pHi changes in the vicinity of the GABAergic and glycinergic synapses (see also Trapp et al. 1996a
). It might be argued that receptor activation for periods of ~1 min by bath application of the agonists does not refer to the physiological situation. It should, however, be considered that maximum currents, conductance changes, and thus pHi decreases might not be observed at all because of partial desensitization of the GABA and glycine receptors during such a slow rate of agonist exposure (Kaila 1994
). In expiratory neurons of the in vivo cat, trains of synchronized IPSPs are responsible for inspiratory inhibition, which persists for periods of up to several seconds. Such hyperpolarizations can reach amplitudes of >20 mV within several hundred millseconds (Ballantyne and Richter 1986
; Richter 1996
). As measured with pH-sensitive microelectrodes in these cells, these hyperpolarizations are accompanied by rapid periodic decreases of pHi by >0.1 pH units (Ballanyi et al. 1994a
). However, the pHi of mammalian neurons is not only perturbed during such massive, albeit physiological activity. It was recently demonstrated that tonic spike activity with a frequency of ~1-6 Hz produces ongoing acidification of medullary dorsal vagal neurons in vitro (Trapp et al. 1996a
) by a mechanism that is discussed below. It remains to be determined with fluorometrical pHi measurements of higher spatial and/or time resolution whether spontaneous IPSCs, as revealed in the VRG neurons on recording with (Cs/TEA-containing) high-Cl
electrodes, also affect pHi.
HCO
3 permeability. The observation that the GABA-induced pHi decrease, but not the current response and gm increase, was blocked in CO2/HCO
3-free solution suggests efflux of the base equivalent HCO
3 through the anion pore as the ionic mechanism of this acidosis. This assumption is supported by the observation that the pHi fall turned into an intracellular alkalosis when the cells were depolarized. Because the equilibrium potentials for H+ and bicarbonate were close to 0 mV in the present study (see also Chesler 1990
; Chesler and Kaila 1992
; Kaila 1994
), an influx of bicarbonate was expected at positive Em. It is, indeed, established that GABAA receptor-coupled anion channels have a substantial permeability to HCO
3 (Bormann et al. 1987
; Kaila et al. 1993
). In a series of elegant experiments, it was originally demonstrated in crayfish muscle fibers and neurons (Kaila 1994
; Kaila and Voipio 1987
; Voipio et al. 1991
) that HCO
3 efflux through the GABAA receptor-coupled anion pore can produce a fall of pHi by several tenths of a pH unit in the presence of extracellular CO2 and bicarbonate. Similar GABA-induced intracellular acidifications have currently also been shown for mammalian neurons (Pasternack et al. 1993
; Trapp et al. 1996a
) or glial cells (Kaila et al. 1991
).
A major contribution of a HCO
3 permeability could theoretically also explain that the Erev of the GABA or glycine response was ~25 mV more positive than the ECl, which is typically close to
90 mV during recording with low-Cl
electrodes (Kaila 1994
; Kaila et al. 1993
). However, as suggested by preliminary findings, the average Erev of the GABA response is not considerably more negative in HEPES-buffered solutions, in which the HCO
3-mediated acidifications were fully blocked. It could well be that the depolarizing action of a putative GABA uptake counteracts the receptor-mediated hyperpolarization (for references, see Kaila et al. 1992b
). The presence of an electrogenic GABA uptake is, indeed, suggested by the observation that GABA still produced an inward current, but no conductance increase, after blockade of GABAA receptors with bicuculline (Fig. 3A). A further explanation for the discrepancy of Erev and calculated ECl could be incomplete equilibration of cellular Cl
with that of the patch electrode, possibly combined with ongoing activity of an inwardly directed Cl
pump leading to a depolarizing shift of ECl (Ballanyi and Grafe 1985
).
Role of CA. A proportion of cells in our study did not show a major acidification on exposure to GABA or glycine despite a considerable current and/or conductance response. The observation that administration of NH+4 or membrane depolarization led to a substantial change of pHi in these cells excludes the possibility of loss of pH sensitivity of the dye, e.g., by interference of intracellular constituents in these recordings (see also Trapp et al. 1996a
). In isolated hippocampal neurons, it was found that acetazolamide, a blocker of CA, strongly attenuated the GABA-induced decreases of pHi in hippocampal neurons (Pasternack et al. 1993
). The importance of CA for the generation of these HCO
3-related neuronal acidifications is also suggested by our observation of significantly faster and also larger GABA- and glycine-induced decreases of pHi in cells dialyzed with the enzyme. Although no information is available at present on the content of soluble CA in neurons, an intracellular concentration of 15-30 µM as provided by the patch pipette might be considerably higher than in the intact cell (Chesler 1990
; Kaila 1994
). It remains to be determined whether those neurons in which GABA or glycine did not change pHi even after dialysis of CA express receptors of a specific subunit composition (McKernan and Whitting 1996
) that might not be permeable to HCO
3.
Role of Cl
. With the use of high-Cl
electrodes, the average Erev of the GABA- and glycine-induced currents was ~0 mV. In this situation, a prominent efflux of Cl
is likely to accompany the HCO
3 efflux at a Vh of
60 mV. Such Cl
efflux, which was shown to decrease intracellular Cl
by >10 mM in rat sympathetic neurons (Ballanyi and Grafe 1985
), does not seem to hamper the efflux of HCO
3, because GABA- and glycine-evoked acidifications of very similar magnitude were revealed with low-Cl
patch electrodes. Because the apparent ECl was close to
60 mV under these conditions (see previous paragraphs), only a minor transmembrane flux of Cl
is expected despite a prominent increase of gm. The latter consideration implicates that the agonist-induced acidosis is not due to redistribution of Cl
, which could potentially involve pHi regulatory mechanisms like Cl
/HCO
3 exchange (Ballanyi and Grafe 1985
; Kaila 1994
).
Depolarization-induced acidosis. Whereas the mechanism of the biphasic change of pHi on application of NH+4 is well established (Chesler 1990
; Thomas 1984
), the origin of the depolarization-induced intracellular acidosis needs to be discussed briefly. It was recently demonstrated in hippocampal pyramidal neurons that depolarization-induced Ca2+ influx via voltage-gated Ca2+ channels leads to a delayed intracellular acidosis (Trapp et al. 1996b
). This depolarization-evoked fall of pHi appears to be due to activation of a vanadate- and eosin-sensitive plasma membrane pump, which extrudes intracellular Ca2+ in exchange for extracellular H+, as was originally described for snail neurons (Schwiening et al. 1993
; see also Paalasmaa and Kaila 1996
; Trapp et al. 1996b
). Because almost identical results were recently reported for dorsal vagal neurons of medullary slices from rats (Trapp et al. 1996a
), we hypothesize that activation of the plasmalemmal Ca2+/H+ pump is responsible for the depolarization-induced neuronal acidosis in the present study.
Conclusions
Our findings support recent assumptions that decreases in pHi during periodic trains of IPSPs, as occurring in respiratory neurons in vivo (Ballanyi et al. 1994a
), are due to GABAA and/or glycine receptor-mediated HCO
3 efflux. Because a variety of cellular processes, including membrane channel function, critically depends on pH (Chesler and Kaila 1992
; Kaila 1994
; Takahashi and Copenhagen 1996
), it is a challenge of future studies to illuminate to what extent these intracellular acidifications might contribute to modulation of neuronal function.