Intracellular pH Buffering Shapes Activity-Dependent Ca2+ Dynamics in Dendrites of CA1 Interneurons

Geoffrey C. Tombaugh

Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Tombaugh, Geoffrey C. Intracellular pH buffering shapes activity-dependent Ca2+ dynamics in dendrites of CA1 interneurons. J. Neurophysiol. 80: 1702-1712, 1998. Voltage-gated calcium (Ca) channels are highly sensitive to cytosolic H+, and Ca2+ influx through these channels triggers an activity-dependent fall in intracellular pH (pHi). In principle, this acidosis could act as a negative feedback signal that restricts excessive Ca2+ influx. To examine this possibility, whole cell current-clamp recordings were taken from rat hippocampal interneurons, and dendritic Ca2+ transients were monitored fluorometrically during spike trains evoked by brief depolarizing pulses. In cells dialyzed with elevated internal pH buffering (high beta ), trains of >15 action potentials (Aps) provoked a significantly larger Ca2+ transient. Voltage-clamp analysis of whole cell Ca currents revealed that differences in cytosolic pH buffering per se did not alter baseline Ca channel function, although deliberate internal acidification by 0.3 pH units blunted Ca currents by ~20%. APs always broadened during a spike train, yet this broadening was significantly greater in high beta  cells during rapid but not slow firing rates. This effect of internal beta  on spike repolarization could be blocked by cadmium. High beta  also 1) enhanced the slow afterhyperpolarization (sAHP) seen after a spike train and 2) accelerated the decay of an early component of the sAHP that closely matched a sAHP conductance that could be blocked by apamin. Both of these effects on the sAHP could be detected at high but not low firing rates. These data suggest that activity-dependent pHi shifts can blunt voltage-gated Ca2+ influx and retard submembrane Ca2+ clearance, suggesting a novel feedback mechanism by which Ca2+ signals are shaped and coupled to the level of cell activity.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Neuronal function in the mammalian brain depends critically on the tight regulation of intracellular pH (pHI) (Caspers and Speckmann 1972; Chesler 1990; Somjen and Tombaugh 1998), a fact hardly surprising given the narrow pH range in which most enzymes, ion channels, and other macromolecules operate. More provocative are reports that cytosolic pH fluctuates in response to cell activity. Intracellular acid transients as large as 0.2 pH units were recorded during neuronal firing and appear to be triggered by the influx of Ca2+ through voltage-gated channels (Ahmed and Conner 1980; Ballanyi et al. 1994; Rose and Deitmer 1995; Trapp et al. 1996a,b). Beyond this Ca2+ dependence, activity-driven acidosis remains poorly understood and is still often viewed as a simple metabolic consequence of cell activity that plays no active role in a functional sense.

Intracellular H+, like other short-lived messengers, acts as an important chemical signal in a variety of cell types, including neurons (Barnes and Bui 1991; Brown and Meech 1979; Busa and Nuccitelli 1984; Deitmer and Rose 1996; Dixon et al. 1993). Although many molecular substrates of H+ modulation presumably exist in neurons, membrane ion channels are cited frequently as H+ targets based on the high pH sensitivity of intrinsic neuronal excitability (Somjen and Tombaugh 1998). Among the multitude of ion channels in neuronal membranes, high-threshold voltage-gated Ca channels (e.g., L, N types) are particularly pH sensitive. Ca channel conductance is most sensitive to H+ near resting internal pH (7.1-7.3) and can be markedly depressed by small (0.1-0.2 pH) increases in cytosolic H+ (cf. Tombaugh and Somjen 1998).

The general H+-sensitivity of Ca2+ influx and mobilization supports the idea that cellular acidosis serves a neuroprotective role by limiting the accumulation of cytosolic Ca2+ during ischemic insults, during which brain pH can fall by as much as an order of magnitude (Siesjo 1988; Tombaugh and Sapolsky 1993). However, this autoprotective strategy may not be limited to pathological states. High-threshold Ca channels are the principal route of neuronal Ca2+ entry during strong depolarization, and cells must restrict this Ca2+ signal both temporally and spatially to ensure its specificity. Because Ca currents are most sensitive to H+-inhibition near resting pHi (Dixon et al. 1993), Ca channels may be part of a negative feedback circuit in which Ca2+ influx during intense periods of activity triggers a local acid shift that then restricts Ca channel conductance. Such negative feedback could prevent a potentially dangerous rise in [Ca2+]i while allowing Ca2+ to fulfill its varied role as an intracellular messenger. In this scenario, intracellular acidosis would effectively act as a low-pass filter for a coincident Ca2+ signal.

The goal of this study was to test the possibility of such a feedback circuit by examining the impact of internal pH buffering on dendritic Ca2+ transients triggered by brief spike trains. The working hypothesis predicts that submembrane acidosis during cell firing should be less pronounced in strongly pH buffered cells, resulting in reduced Ca channel blockade and facilitating a greater rise in cytosolic Ca2+. Because the size of activity-driven H+ transients has been previously correlated with firing rate, hippocampal interneurons were chosen for study as they exhibit high firing rates with little spike frequency adaptation. Overall, the results support a physiological role for pH shifts in mammalian neurons and sharpen the growing view that H+ ions act as important regulatory signals in excitable cells.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Slice preparation

Transverse hippocampal slices (300 µm) were prepared from male Sprague-Dawley rats (16-24 days old, 20-40 g) with a vibrating tissue slicer (4°C) and were maintained in a submerged holding chamber at room temperature in the following solution (in mM): 130 NaCl, 3.5 KCl, 1.2 CaCl2, 1.2 MgCl2, 3.5 NaH2PO4, 24 NaHCO3, 10 dextrose, 0.05 picrotoxin, pH 7.4, equilibrated with 95%O2-5%CO2. Slices were allowed to recover for 30-45 min before any recording. Slices were transferred as needed to a temperature-controlled perfusion chamber (Medical Systems) in which they were perfused with the same solution at 2 ml/min and kept at 30°C. The perfusion chamber was mounted on the stage of a compound microscope (Zeiss Axioskop) coupled to a BioRad MRC600 confocal system. In some cases, 50 nM apamin (Research Biochemicals) was added to block small conductance (SK) potassium channels. For Ca current recordings, tetrodotoxin (0.5 µM, Calbiochem), tetraethylammonium chloride (5 mM), and 4-aminopyridine (2 mM) were added to block Na and K currents. In cases where 200 µM CdCl2 was added to block high-threshold Ca channels, NaH2PO4 was omitted. Unless otherwise indicated all drugs were obtained from Sigma (St. Louis, MO).


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FIG. 1. OGB-1 fluorescence is not affected by H+ ions in a physiological pH range. The Hill plot depicts background-corrected OGB-1 fluorescence at various pH values in free solution normalized to that recorded at pH 7.2 (F/F7.2). In vitro measurements were performed in a slide-mounted cuvette filled with the same high beta  internal solution (K-gluconate) used for intracellular recording. This solution also contained 0.5 mM bis-(o-aminophenoxy)-N,N,N',N',-tetraacetic acid and 200 nM free Ca2+. Solution pH was adjusted with concentrated HCl or NaOH before the addition of 10 µM OGB-1. Data are presented as mean ± SE (n = 4). The Boltzmann fit and pK were derived with PClamp 6.0 software.

Intracellular recordings

Interneurons located in the s. radiatum region of CA1 were viewed through a Zeiss 40× WI objective (0.75 NA) with transmitted light. No rigorous cell selection criteria were applied, except that large pyramidal shaped cells recently described as "radiatum giant cells" were avoided (Maccaferri and McBain 1996). High-resistance seals (>= 2 GOmega ) and subsequent whole cell current-clamp recordings were made with patch pipettes (3-4 mOmega ) filled with K-gluconate-based solutions containing either a nominally "high" or "low" concentration of pH buffers (beta ). The "high beta " solution contained (in mM) 100 K-gluconate, 40 K-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 5 3-(N-morpholino)propanesulfonic acid (MOPS), 2 MgCl2, 0.5 K4-bis-(o-aminophenoxy)-N,N,N',N',-tetraacetic acid (BAPTA), 2 Na2-ATP, and 0.01 Na-GTP. The "low beta " solution contained (in mM) 135 K-gluconate, 5 K-HEPES, 2 MgCl2, 0.5 K4-BAPTA, 2 Na2-ATP, and 0.1 Na-GTP. Solution pH was adjusted to 7.2 with gluconic acid. In some cases, the pH of the low beta  solution was adjusted to 6.9; in other experiments, K-methylsulfate (ICN Biomedicals) was used in place of K-gluconate. For Ca current recording, Cs-gluconate, Cs4-BAPTA, and the free-acid of HEPES were substituted for their respective potassium salts, and 5 mM TEA-Cl was added (high beta : 95 mM Cs-glu; low beta : 130 Cs-glu); pH was adjusted to 7.2 with CsOH. Junction potentials for both K- and Cs-based solutions were determined to be ~10 mV (Neher 1992). The concentration of BAPTA in the pipette solutions was estimated to yield a free [Ca2+] of~15-20 nM (Tsien and Pozzan 1989).


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FIG. 2. Internal intracellular pH (pHi) buffering differences do not affect cell firing behavior. A: representative examples of spike trains evoked with 400-ms depolarizing current pulse of varying amplitude (cell dialyzed with low beta  solution). B: graphs illustrate the relationships between the size of the current pulse and action potential number [top panel; n = 14 (high beta ), n = 20 (low beta )] or firing frequency (bottom panel; n = 11 (high beta ), n = 6 (low beta )]. Firing frequency was determined for both the 1st and last interspike interval (ISI).

The normal internal pH buffering capacity (beta ) of hippocampal interneurons is not known. However, beta  can be estimated during whole cell recording if one makes the following two assumptions. First, the rate of buffer exchange from the pipette is slow relative to the kinetics of internal pH transients; second, rapid pH buffering depends primarily on intrinsic buffers rather than membrane transporters, which operate on a second-to-minute timescale. Under these assumptions, the cell can be viewed as a closed system from a pH buffering standpoint. In the presence of 5% CO2, which is about twice the PCO2 in vivo, and assuming a normal pHi of 7.2, intracellular HCO-3 is ~18-20 mM and corresponds to a beta  value of ~45 mM (Chesler et al. 1994). Intrinsic pH buffering, provided primarily by large dialysis-resistant proteins and organelles, is~10 mM (Chesler 1990). Additional buffering provided by the monobasic pH buffers in the pipette solution are 23 and 3 mM for the high and low beta solutions, respectively. Thus both solutions yield a higher nominal internal beta  (high, 78; low, 58 mM) than thought to exist in vivo (~40 mM), owing largely to the higher concentrations of CO2 present. HEPES and MOPS are rapid diffusable buffers that act to collapse H+ gradients, an effect analogous to that of mobile Ca2+ buffers (e.g., BAPTA, Fura-2) on cytosolic Ca2+ gradients. The relative importance of HEPES/MOPS buffering becomes more striking when one considers that the speed of HCO-3 buffering will be reduced as soluble carbonic anhydrase is washed out by internal dialysis.


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FIG. 3. Internal pHi buffering does not directly affect whole cell Ca2+ conductance or Ca2+ current rundown. A: examples of whole cell Ca currents evoked 10 min after break-in (Vh = -70 mV) in the presence of tetrodotoxin and TEA-Cl. Depolarizing test pulses (50 ms) were preceded by a 50-ms hyperpolarizing pulse to -80 mV. Scale bars: 400 pA, 20 ms. B: summary current-voltage (I-V) curve for Ca currents evoked from both high beta  (n = 5) and low beta  cells (n = 6) or from cells in which the high beta  pipette solution was deliberately acidified to pH 6.9 (n = 7). C: currents were evoked by 50-ms voltage steps to -10 mV every 20 s for 20 min (n = 6). Current amplitude is expressed relative to that of the 1st test pulse (I/Io). Inset depicts currents evoked at the beginning and end of the 20-min period. Scale bars: 400 pA, 20 ms. All voltages are corrected for junction potential.


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FIG. 4. Dendritic Ca2+ transients are larger and decay more rapidly than somatic transients. A: fluorescence image of a hippocampal interneuron (s. radiatum) dialyzed with 100 µM OGB-1 (high beta  solution). Rectangular regions of interest are positioned over the cell soma (S) and proximal dendrite (D). B: transient changes in the normalized fluorescence signal (background corrected) from these 2 regions evoked by a 400-ms current pulse (250 pA). Peak OGB-1 fluorescence in the soma was scaled to that in the dendrite; the fluorescence decay in each region was fit with a single exponential. Tau values represent the time constant of decay.


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FIG. 5. Dendritic Ca2+ transients are amplified in high beta  cells. A: examples of Ca transients evoked by 400-ms current pulses (0-250 pA) in a high beta  and low beta  cell. B: peak amplitude of these transients were pooled and normalized for the number of action potentials (AP) evoked during the current pulse; * P < 0.05, ** P < 0.01 (n = 6, high beta ; n = 9, low beta ). C: Ca transient decay (tau) was plotted against the number of APs evoked by the current pulse (derived from data in B).


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FIG. 6. Spike repolarization occurs more slowly in high beta  cells. A: examples of individual APs from spike trains evoked by small (50 pA) or large (250 pA) current injection (high beta ). The 1st and last APs are superimposed to illustrate the general feature of spike broadening during a current pulse. B: change in spike repolarization time (RT) of the last spike is expressed as a percent of the RT of the 1st spike. This index of spike repolarization was determined for both high beta  and low beta  cells at 2 levels of spike frequency. Note that cadmium (200 µM) abolished the difference in RT between groups by reducing Delta RT in high beta  cells. * P < 0.05; n = 10 (control), n = 5 (cadmium).


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FIG. 7. Slow afterhyperpolarization (sAHP) is enhanced in high beta  cells. A: peak sAHP values were measured <= 100 ms from the end of the current pulse. *P < 0.05, **P < 0.01 (n = 15). B: examples of sAHP wave forms were evoked with 50-, 100-, and 250-pA current pulses. Scale bars: 2 mV, 0.5 s.


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FIG. 8. Early component of the sAHP decays more rapidly in high beta  cells. The sAHP evoked in both high and low beta  cells could be fit with 2 exponentials. Note that, like the amplitudes of both Ca2+ transients and the sAHP, a statistically significant difference for tau1 emerged with increasing current amplitude. *P < 0.05 (n = 5-9).


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FIG. 9. Early component of the sAHP can be blocked by apamin. A: dual-component sAHP evoked in a cell dialyzed with low beta  internal solution (current pulse = 200 pA). Exponential fits (dashed lines) approximate the fast and slow components of the AHP decay and were derived with the Chebyshev curve-fitting routine of PClamp 6.0. B: sAHP in a different cell (high beta ) before and after bath application of 50 nM apamin. Apamin caused a large leftward shift in the I-f relationship (not shown); the AHPs illustrated were evoked by different current pulses but were preceded by a comparable number of APs [control: 26 APs (250 pA); + apamin: 27 APs (30 pA)]. The difference trace (diff.), obtained by subtraction, is larger than the control sAHP because the 2 parent traces diverge before Vm recovers to its prepulse potential. The fast component of the sAHP before apamin treatment decayed with a time constant (166 ms) similar to that of the difference trace (154 ms). Scale bars: 1 mV (A), 4 mV (B), 0.5 s.

All cells were initially held in voltage clamp (Vh = -60 mV) to measure input resistance (Rin), series resistance (Rs), and cell capacitance. After these measurements, the amplifier (Axopatch 200A) was switched to current-clamp mode, and injected current was adjusted to keep the pipette potential near -60 mV. Actual Vm was estimated to be near -70 mV caused by the junction potential offset, although no posthoc correction for this offset was performed. Depolarizing current pulses (400 ms) of varying amplitude (20-250 pA) were applied at 30-s intervals to evoke trains of action potentials (APs). In experiments designed to record Ca currents, cells were held continuously in voltage clamp (Vh = -60 mV); Ca currents were then evoked by a series of depolarizing voltage steps (50 ms) between -50 mV and +20 mV in 10-mV increments at 20-s intervals. For determination of Ca current rundown, repeated voltage steps (50 ms, -10 mV) were applied every 20 s for 20 min. Capacitive artifacts were canceled electronically and series resistance (6-15 mOmega ) was compensated 60-85%. Current and voltage records were sampled at 5-10 kHz, filtered at 5 kHz, and stored on a PC.

Fluorescence imaging

For optical recording of Ca2+ transients, patch pipette tips were filled with internal solution and then backfilled with the same solution containing 100 µM of the Ca2+ indicator Oregon Green 488 BAPTA-1 (OGB-1; Molecular Probes, Eugene, OR). Reducing the dye concentration to 50 µM or less noticeably reduced signal/noise, whereas omitting BAPTA resulted in elevated baseline fluorescence signals that limited detection of small activity-driven transients. The pH sensitivity of OGB-1 was determined in vitro by recording its fluorescence in high beta  internal solutions containing 200 nM free Ca2+ plus 10 µM of the Ca2+ indicator. The pH of these solutions was adjusted between 4 and 8 by the addition of concentrated HCl/NaOH.

Cells were allowed to "fill" with the indicator for 5-10 min before laser scanning. Cells were illuminated with 488 nm light from a krypton-argon laser; the emitted light was reflected by a dichroic mirror (560LP) and collected by a photomultiplier tube through a 520/16 nm band-pass filter. Rectangular regions of interest (ROIs) were positioned along a proximal dendritic segment 10-50 µm from the cell soma and over an adjacent area (for recording background fluorescence). ROI selection in this region was arbitrary, except that dendritic branch points or varicosities were deliberately avoided. In many cases, the dendritic ROI was limited by dendrite orientation or the extent of dye diffusion. Dendrites generally lacked spines, but scattered spines could be clearly resolved along both primary and secondary dendrites in several well-filled cells. Occasionally, ROIs were also placed over sections of the cell soma well separated from the nucleus. Average fluorescence intensities were collected by "time-course" software (TCSM, BioRad) at 3 Hz for short periods (3-10 s) to minimize bleaching and phototoxicity.

Data analysis

AP height, firing frequency, and 50% repolarization time (RT) were measured as described elsewhere (Zhang and McBain 1995). For a given spike train, changes in the RT of the last spike were expressed as a percent of the RT of the first spike. Peak sAHP amplitude (occurring <= 100 ms after the current pulse) was measured relative to membrane voltage before the current pulse.

Input resistance (Rin) was determined by applying small negative voltage steps (20 mV) or hyperpolarizing current pulses (10-50 pA). Series resistance (Rs) and cell capacitance were read directly from the amplifier controls. Cells in which Rs exceeded 20 mOmega or which displayed abrupt shifts in Rin, Vm, or holding current during the course of an experiment were rejected. For voltage-clamp records of Ca currents, steady-state leak currents were subtracted off-line, and any references in the text made to a given command voltage have been corrected for junction potential (10 mV). Poorly clamped cells, identified by uncontrolled current activation and prolonged tail currents, were rejected. In spite of this selection it is almost certain that membrane clamping was imperfect in these extensively arborized cells. For the purposes of this study, however, a quantitative comparison of Ca current amplitude proved a useful way to assess whether gross differences existed between experimental groups.

Raw fluorescence values were corrected for background fluorescence and expressed as a percent change in fluorescence relative to baseline [%Delta F = (F - Fo)/Fo·100]. For between-group comparisons, Delta F values were normalized by grouping them according to the number of associated APs. The decay of Ca2+ transients was fit with a single exponential and used as an index of Ca2+ clearance (Markram et al. 1995). All analyses of voltage and current records, including curve fitting, were performed with PClamp software (v. 6.0). Unpaired statistical comparisons were made with a two-tailed Student's t-test (Statview 4.0). All data are presented as means ± SE.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Effects of pHi buffering on membrane and dye properties

Because the emission spectra of fluorescein-based dyes tend to be highly pH sensitive, it was important to determine the pH sensitivity of OGB-1, a fluorescent indicator composed of F2-fluorescein (Oregon Green; Molecular Probes) conjugated to the Ca2+ chelator BAPTA. Fluorescence data obtained in vitro in the presence of 200 nM free Ca2+ were used to construct a Hill plot in which average fluorescence at a given pH was normalized to that measured at pH 7.2. pH adjustments within the range expected to occur in living cells (pH 6.5-7.5) caused no detectable change in dye fluorescence (Fig. 1). Analysis of the Hill plot yielded a pK of 4.9, very close to the pK value of 4.7 reported by the supplier (Molecular Probes) for the unconjugated fluorophore (Oregon Green).

To validate comparisons between high beta  and low beta -dialyzed cells, intrinsic membrane properties were measured in the two groups. Differences in the nominal pHi buffering capacity had no detectable effect on Rin (high beta : 260 ± 9 mOmega ; low beta : 267 ± 15 mOmega ), Rs (high beta : 11 ± 1 mOmega ; low beta : 10 ± 1 mOmega ), or cell capacitance (high beta : 14 ± 1 pF; low beta : 15 ± 1 pF, n = 34). Similarly, AP number or frequency did not differ significantly between the two experimental groups (Fig. 2). AP height was nearly identical regardless of firing rate (6-10 APs: high beta  87 ± 1 mV, low beta  87 ± 6 mV; >25 APs: high beta  76 ± 3 mV, low beta  78 ± 1 mV, n = 10).

To assess whether differences in beta  of the internal solutions directly affected Ca channel function, whole cell Ca currents were monitored 10 min after membrane rupture with Cs-based pipette solutions containing the identical type and concentration of pH buffers used in the current-clamp experiments (see METHODS). In both high and low beta  groups, Ca currents activated near -40 mV, peaked near -10 mV, and exhibited a small degree of rundown as reported previously (Lambert and Wilson 1996). No differences in either peak Ca current amplitude, voltage dependence, or rundown rate (0.7%/min) were detected between the two groups (Fig. 3). In contrast, deliberate acidification of the internal solution (high beta ) from pH 7.2 to 6.9 depressed Ca currents by~20% (Fig. 3B).

Effects of phi buffering on Ca2+ transients

Depolarizing current pulses large enough to trigger a train of APs also evoked Ca2+ transients in both the cell body and proximal dendrites. The size of these transients were well correlated to the number of spikes, although dendritic transients were consistently larger and decayed more rapidly than those in the soma (Fig. 4). Ca2+ transients in the soma decayed with a time constant of 2.8 ± 0.2 s (n = 5, high beta , 250-pA pulse), which did not vary significantly with the current pulse or spike number. Unstable fluorescence signals in the cell soma sometimes precluded reliable optical recordings, a problem rarely encountered in dendrites. Therefore rigorous unpaired comparisons of somatic Ca2+ changes between high and low beta  cells were not made.

When dendritic Ca transients were normalized for the number of APs, high beta  cells exhibited larger transients than those recorded in low beta  cells (Fig. 5), a difference that became more pronounced at higher firing rates (i.e., during larger current pulses). In contrast, Ca transients decayed with a time constant that was not significantly affected by the difference in pHi buffering and that did not vary significantly with spike number (Fig. 5). Whole cell dialysis with gluconate-based solutions was reported to depress ICa in CA1 pyramidal neurons (Zhang et al. 1994); however, substitution of K-gluconate with K-methlysulfate (n = 7, low beta ) did not significantly alter the amplitude of Ca transients (6-15Aps: Kgluc 42 ± 4%, KMeSO4 42 ± 4%; >25 APs: Kgluc 71 ± 6%, KMeSO4 69 ± 5%).

APs in a train tended to increase in duration and decrease slightly in amplitude (Fig. 6A), a feature observed in both high beta  and low beta  cells. The initial AP repolarized with a half-time (RT) of 1.5 ± 0.1 ms in both high beta  and low beta  groups. In high beta  cells spike broadening became more pronounced at higher firing rates (26-35 Aps, Fig. 6B). This difference in RT between groups did not occur at lower firing rates (6-10mAPs). In the presence of cadmium (200 µM) spike broadening still occurred in both groups, but the effect of elevated pHi buffering on AP repolarization was abolished.

Effects of phi buffering on the sAHP

Trains of APs were usually followed by a slow afterhyperpolarization (sAHP) that lasted for several seconds and could be blocked by cadmium (not shown). The size of this component was determined by measuring its peak amplitude within 100 ms after the current pulse. After "strong" cell depolarization (current pulse >= 150 pA), peak sAHP amplitude was moderately enhanced in high beta  cells (~15%, Fig. 7) but was unaffected by substitution of KMeSO4 for K-gluconate (250 pA: Kgluc -6.7 ± 1 mV; KMeSO4 -6.8 ± 1 mV, n = 12). No significant difference in sAHP amplitude between low and high beta  cells was seen with smaller depolarizing pulses. sAHP decay could usually be described by two exponentials. The fast AHP component (tau1) decayed more rapidly in high beta  cells with increasing cell depolarization, and the decay of the slower AHP component (tau2) was not affected (Fig. 8).

In a few cells, two distinct components of the sAHP could be seen, a rapidly decaying potential followed by one that activated and decayed more slowly (Fig. 9A). In the majority of cells, however, this visual separation was less clear. In such cases, the SK-channel antagonist apamin (50 nM) selectively blocked the early component of the sAHP, which peaked within 50-100 ms after the current pulse. This apamin-sensitive component, after isolation by subtraction (Fig. 9B), could be fit with a single exponential and decayed with a time constant of 150-200 ms (174 ± 12 ms, n = 5).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The key findings are that dendritic Ca2+ transients, sAHP amplitude, and sAHP decay in hippocampal interneurons are sensitive to pHi buffering. Each of these effects emerged with increased cell activity and could not be ascribed to baseline differences in Ca channel function. These data suggest that during rapid cell firing H+ transients occur with a magnitude and in a time domain capable of modulating intracellular Ca2+ dynamics very close to dendritic membranes.

During cell activity H+ may rise gradually in response to continued Ca2+ influx (Ahmed and Conner 1980; Ballanyi et al. 1994; Rose and Deitmer 1995; Trapp et al. 1996b), and this rise in H+ may limit Ca2+ accumulation by blunting Ca channel activity. According to this model, H+ inhibition of Ca2+ influx would be expected to be most pronounced near the end of the spike train. Although Ca currents could not be measured directly during current-clamp recordings, a modest increase in AP RT was detected at the end of a spike train in high beta  cells during rapid firing rates. This is consistent with an enhanced high-threshold Ca current that can broaden the repolarization of the AP (Llinas and Yarom 1981). That progressive spike broadening occurred in the presence of Cd in both beta  groups clearly indicates that this phenomenon is not dependent on Ca influx. However, the increase in RT caused by elevated internal beta  was abolished in the presence of cadmium (which blocks Ca influx during each spike) and was not observed at slow firing rates, when H+ shifts were presumably small. This suggests that internal beta  influenced Ca channels via a mechanism that was sensitive to the level of cell activity.

The most likely explanation for these data is a H+-mediated depression of Ca channel conductance, as changes in pHi do not to alter the kinetics of macroscopic Ca currents (Mironov and Lux 1991). However, one cannot rule out inhibitory H+ effects on other channel types. In general, H+ modulation of Na and K channels in mammalian neurons is small within a physiological pH range (pH 6.5-8.0) (Tombaugh and Somjen 1998). As an exception, rapid H+ inhibition of large-conductance BK channels can occur at physiological pH and could in principle increase spike width (Habartova et al. 1994; Zhang and McBain 1995). If such inhibition occurred during cell firing, one would expect that it would be more pronounced in low beta  cells, promoting a greater increase in spike width. However, in this study the opposite was observed, arguing against K channel blockade as a dominant mechanism. In the presence of any inhibitory effect of H+ on K channels, measurements of AP repolarization may have actually underestimated the degree of H+-mediated Ca channel blockade.

Submembrane H+ and Ca2+ dynamics

Ca2+ transients in the proximal dendrites were consistently larger and decayed more rapidly than those evoked in the soma. Similar observations were made in CA1 pyramidal and neocortical neurons and may reflect regional variation in Na or Ca channel density (Jaffe et al. 1992, 1994; Magee and Johnston 1995; Regehr et al. 1989; Schiller et al. 1995; Svoboda et al. 1997; Westenbroek et al. 1990, 1992). However, differences in either the surface area:volume ratio or Ca2+-buffer gradients between the soma and neighboring dendrite could also explain differences in both peak Ca2+ fluorescence and its rate of decay.

In hippocampal interneurons Ca entry through voltage-gated channels activates a number of K conductances, some of which underlie the sAHP (Zhang and McBain 1995). Because these K conductances are governed by the local submembrane concentration of free Ca2+ (Lancaster and Zucker 1994; Sah 1992), the sAHP can be exploited as a submembrane Ca2+ sensor (Tucker and Fettiplace 1996). That sAHP amplitude was larger in high beta  cells is consistent with enhanced Ca2+ influx and the larger Ca2+ transients that were detected fluorometrically. One early component of the sAHP also decayed more rapidly in high beta  cells, suggesting that, in addition to limiting Ca influx, H+ accumulation interfered with Ca2+ sequestration or buffering near the cell membrane. As possible mechanisms, proton accumulation may have impaired H+-Ca2+ exchange (Trapp et al. 1996a) or competed with Ca2+ ions for nonselective binding sites.

In contrast to its effect on sAHP decay, pHi buffering had no apparent effect on the decay of OGB-1 fluorescence after a spike train. This discrepancy may reflect the action of high-affinity mobile Ca2+ buffers (e.g., OGB-1, BAPTA). These compounds enhance Ca2+ diffusion away from the membrane (Zhou and Neher 1993), where Ca2+ ions activate KCa channels, and into the bulk cytosol, where the fluorescent Ca2+ signal was actually measured. Such an increase in Ca2+ mobility may have obscured fluorescent detection of a more subtle effect of H+ on Ca2+ clearance near the membrane. Thus fluorescent Ca2+ measurements averaged across even relatively confined regions of a cell may be inadequate for drawing conclusions about the behavior of Ca2+ signals close to their source.

The presence of mobile Ca2+ buffers presumably contributes to the generally slow decay of Ca2+ transients reported here and in other studies (Jaffe et al. 1994). Prolonged Ca2+ transients may also reflect the delayed contribution of Ca2+-induced Ca2+ release (CICR) from internal stores. CICR was proposed to explain the slowly decaying sAHP component seen in rat CA1 pyramidal cells and vagal motoneurons (Lancaster and Zucker 1994; Lasser-Ross et al. 1997; Sah 1992; Sah and McLachlan 1991). Whether CICR occurs in hippocampal interneurons is not known.

The KCa channels that were presumably affected by a H+-mediated reduction in Ca2+ influx are likely to include SK channels. This conclusion is based on the kinetic identity between an early, rapidly decaying component of the sAHP and that component that was blocked by apamin. This component is similar if not identical to GKCa,1 previously described in sympathetic ganglion neurons (Cassel and McLachlan 1987). In addition, SK channels appear to be functionally coupled to N-type Ca channels (Davies et al. 1996), which are more sensitive to internal H+ than the structurally related L-type Ca channel (Tombaugh and Somjen 1997). These findings strengthen the idea that H+ can depress the sAHP indirectly by reducing Ca2+ influx (Church 1992), but they need not preclude other mechanisms. For example, the depression of sAHP in low beta  cells could reflect a direct inhibitory action of H+ on SK channels themselves.

Possible sources of error

In this study, Ca fluorimetry yielded some important insights into the physiological effects of internal pH buffering, but the conclusions regarding H+ dynamics per se remain indirect. Attempts to measure dendritic pH shifts directly with the fluorescent dye SNARF-1 were empirically difficult. In retrospect this was not surprising given the results of some pilot experiments with cultured hippocampal neurons in which SNARF's pK exhibited an apparent alkaline shift and its isosbestic point shifted to longer wavelengths when introduced into cells (G. Tombaugh, unpublished observations). Such changes have been described elsewhere (Owen 1992) and not only limit one's ability to detect small acid transients but, with a fixed wavelength detection system, also prevent one from making reliable ratiometric measurements.

In considering the results of this study, three additional points should be made. First, fluorescein-based Ca2+ indicators can be highly pH sensitive, which raises a valid question about the ion specificity of OGB-1 fluorescence. However, in this study intracellular H+ shifts were presumably too small (<1.0 pH unit) to affect OGB-1 fluorescence directly. The Ca2+ affinity of BAPTA-based indicators such as OGB-1 are essentially unaffected by protons between pH 6 and 8 (Tsien 1980), and OGB-1 fluorescence is insensitive to H+ ions within a wide physiological pH range (Fig. 1). Nevertheless, it should be stated that the pK of OGB-1 within cells may differ from that recorded in vitro.

Second, intracellular dialysis with HEPES-buffered solutions may have impaired normal pH regulation via washout of carbonic anhydrase. Moreover, one could argue that, in the face of ongoing metabolic activity, low beta  cells had a lower resting pHi than high beta  cells, which could have in turn reduced 1) baseline Ca channel activity or 2) the transmembrane driving force by elevating resting [Ca2+]i (Ou-yang et al. 1994). Neither of these possibilities appear likely given that Ca currents evoked in low and high beta  cells exhibited nearly identical peak amplitudes, I-V profiles, and rundown rates.

Third, pHi buffering may have somehow affected the ability of Na+ APs to invade the proximal dendrites, a process that dictates the size and range of Ca2+ signals in the dendrites of CA1 pyramidal neurons (Jaffe at al. 1992, 1994). A rise in internal H+ capable of blocking Ca channels would, however, have little direct effect on Na+ channels, whose unitary conductance, activation, and inactivation properties are relatively insensitive to internal H+ in a physiological acid pH range (Carbone et al. 1981; Daumas and Anderson 1993). This is important in light of recent reports that backpropagating APs exhibit an activity-dependent decrease in amplitude arising from prolonged Na channel inactivation (Colbert et al. 1997; Jung et al. 1997).

The above argument applies if dendritic APs propagated actively but not in the extreme case in which dendritic Na channels were absent and APs propagated passively. Passive AP propagation into dendrites can be limited by large (2- to 3-fold) differences in internal resistivity (Jaffe et al. 1994), although it is difficult to imagine that such a large difference existed between cells dialyzed with the low and high beta  internal solutions used in this study. The time course of an AP also influences the extent of its attenuation, with sharper spikes traveling shorter distances (Spruston et al. 1994). However, no significant difference in spike amplitude or rise time (data not shown) was detected between high and low beta  cells.

Possible physiological significance

Active dendritic Ca2+ conductances, specifically those in the proximal dendrites, have been implicated in the integration and amplification of coincident synaptic potentials (Markram et al. 1995; Seamans et al. 1997). By limiting GCa, a rise in dendritic H+ may reduce excitatory postsynaptic potential amplification and thus the efficiency with which dendrites conduct electric signals to the cell soma. In addition, when APs invade dendritic membranes, they generate sizable submembrane Ca2+ signals that appear necessary for at least one form of associative synaptic plasticity (Magee and Johnston 1995). Such signal processing may not be limited to parent dendrites but might also occur in dendritic spines and presynaptic terminals, which experience large voltage-gated Ca2+ transients during synaptic activity (Miyakawa et al. 1992). If synaptically evoked H+ transients arise in these spatially restricted regions, the resulting reduction of Ca2+ influx may limit transmitter release or postsynaptic receptor modification in an activity-dependent manner.

Are the size of activity-driven pHi transients measured previously consistent with the depression of GCa in the present study? Internal acid shifts are generally correlated with cell activity but are relatively small (<= 0.1 pH), probably reflecting the slow firing rates (<= 10 Hz) of the cell types studied (Ahmed and Conner 1980; Ballanyi et al. 1994; Rose and Deitmer 1995; Trapp et al. 1996b). However, as these authors point out, even the most reliable measurements of rapid H+ transients may represent spatially and temporally filtered reflections of true local changes in pHi, especially in the narrow confines of the submembrane space (Chesler 1990). Thus localized H+ transients, much like brief Ca2+ transients that can occur in restricted dendritic regions (Eilers et al. 1995), may greatly exceed estimates based on lumped tissue averages or even fluorescent pHi measurements recorded from cell bodies. In low beta  cells dendritic pH may have fallen transiently by <= 0.5 pH units, an estimate based on the degree to which Ca2+ transients were influenced by internal beta , the observed 20% depression of ICa by internal acidification of 0.3pH units, and earlier findings in isolated neurons (Dixon et al. 1993; Tombaugh and Somjen 1997).

In sum, activity-dependent decreases in pHi appear to modulate intracellular Ca dynamics in part by limiting Ca2+ influx through voltage-gated Ca channels. Given the correlation between firing rate and acidification, this feedback mechanism may represent an important strategy for inhibitory and perhaps other fast-spiking neurons in which the expression of both Ca2+ permeable AMPA receptors and Ca2+-binding proteins implies an especially strong need for tight Ca2+ regulation (Gulyas and Freund 1996; Isa et al. 1996). To what extent the present findings apply to other cell types and to adult neurons in situ remains to be tested. On a broader level, however, these results expand the concept of pH regulation in the nervous system and present H+ ions in a new light, specifically as a conditioner of Ca2+ signals in excitable cells.

    ACKNOWLEDGEMENTS

  I am grateful to G. Somjen and L. McMahon for technical advice and critical readings of the manuscript.

  Support for this work was provided to G. C. Tombaugh by the American Heart Association and the Carl J. Herzog Foundation, and to G. Somjen by National Institute of Neurological Disorders and Stroke Grant 5R01 NS-18670.

    FOOTNOTES

  Address for reprint requests: UMDNJ, RWJ Medical School, Dept. of Neuroscience, CABM bld., 3rd Fl., 675 Hoes Lane, Piscataway, NJ 08854-5635.

  Received 6 April 1998; accepted in final form 22 June 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society