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Intracellular pH Buffering Shapes Activity-Dependent
Ca 2+ Dynamics in Dendrites of CA1 Interneurons
Geoffrey C. Tombaugh
J Neurophysiol 80:1702-1712, 1998.
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Intracellular pH Buffering Shapes Activity-Dependent Ca 2/ Dynamics
in Dendrites of CA1 Interneurons
GEOFFREY C. TOMBAUGH
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
INTRODUCTION
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
Ca 2/ through voltage-gated channels (Ahmed and Conner
1980; Ballanyi et al. 1994; Rose and Deitmer 1995; Trapp et
al. 1996a,b). Beyond this Ca 2/ 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.
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1702
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 Ca 2/ influx and mobilization supports the idea that cellular acidosis serves a neuroprotective role by limiting the accumulation of cytosolic
Ca 2/ 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. Highthreshold Ca channels are the principal route of neuronal
Ca 2/ entry during strong depolarization, and cells must restrict this Ca 2/ 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 Ca 2/
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
[Ca 2/ ]i while allowing Ca 2/ 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
Ca 2/ 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 Ca 2/ 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 Ca 2/ .
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.
0022-3077/98 $5.00 Copyright q 1998 The American Physiological Society
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Tombaugh, Geoffrey C. Intracellular pH buffering shapes activity-dependent Ca 2/ dynamics in dendrites of CA1 interneurons. J.
Neurophysiol. 80: 1702–1712, 1998. Voltage-gated calcium (Ca)
channels are highly sensitive to cytosolic H / , and Ca 2/ 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 Ca 2/ influx. To examine
this possibility, whole cell current-clamp recordings were taken
from rat hippocampal interneurons, and dendritic Ca 2/ transients
were monitored fluorometrically during spike trains evoked by
brief depolarizing pulses. In cells dialyzed with elevated internal
pH buffering (high b ), trains of ú15 action potentials (Aps) provoked a significantly larger Ca 2/ 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 Ç20%. APs always broadened during a spike train, yet this
broadening was significantly greater in high b cells during rapid
but not slow firing rates. This effect of internal b on spike repolarization could be blocked by cadmium. High b 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 activitydependent pHi shifts can blunt voltage-gated Ca 2/ influx and retard
submembrane Ca 2/ clearance, suggesting a novel feedback mechanism by which Ca 2/ signals are shaped and coupled to the level
of cell activity.
PH
SENSITIVITY OF DENDRITIC CA 2/ SIGNALS
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-(oaminophenoxy)-N,N,N *,N *,-tetraacetic acid (BAPTA), 2 Na2ATP, and 0.01 Na-GTP. The ‘‘low b’’ solution contained (in mM)
135 K-gluconate, 5 K-HEPES, 2 MgCl2 , 0.5 K4-BAPTA, 2 Na2ATP, and 0.1 Na-GTP. Solution pH was adjusted to 7.2 with gluconic acid. In some cases, the pH of the low b solution was adjusted
to 6.9; in other experiments, K-methylsulfate (ICN Biomedicals)
was used in place of K-gluconate. For Ca current recording, Csgluconate, Cs4-BAPTA, and the free-acid of HEPES were substituted for their respective potassium salts, and 5 mM TEA-Cl was
added (high b: 95 mM Cs-glu; low b: 130 Cs-glu); pH was adjusted to 7.2 with CsOH. Junction potentials for both K- and Csbased solutions were determined to be Ç10 mV (Neher 1992).
The concentration of BAPTA in the pipette solutions was estimated
to yield a free [Ca 2/ ] of Ç15–20 nM (Tsien and Pozzan 1989).
The normal internal pH buffering capacity ( b ) of hippocampal
interneurons is not known. However, b 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,
METHODS
Slice preparation
Transverse hippocampal slices ( 300 mm) were prepared from
male Sprague-Dawley rats ( 16 – 24 days old, 20 – 40 g ) with a
vibrating tissue slicer ( 47C ) 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 307C. 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 mM, Calbiochem) , tetraethylammonium chloride ( 5 mM ) , and 4-aminopyridine ( 2 mM )
were added to block Na and K currents. In cases where 200 mM
CdCl2 was added to block high-threshold Ca channels, NaH2PO4
was omitted. Unless otherwise indicated all drugs were obtained
from Sigma ( St. Louis, MO ) .
Intracellular recordings
Interneurons located in the s. radiatum region of CA1 were
viewed through a Zeiss 401 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). Highresistance seals ( ¢2 GV ) and subsequent whole cell current-clamp
recordings were made with patch pipettes (3–4 mV ) filled with
K-gluconate-based solutions containing either a nominally ‘‘high’’
or ‘‘low’’ concentration of pH buffers ( b ). The ‘‘high b’’ solution
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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 b solution). B: graphs illustrate the relationships between the size
of the current pulse and action potential number [ top panel; n Å 14 (high
b ), n Å 20 (low b )] or firing frequency (bottom panel; n Å 11 (high b ),
n Å 6 (low b )]. Firing frequency was determined for both the 1st and last
interspike interval (ISI).
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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 b 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 Ca 2/ . Solution pH
was adjusted with concentrated HCl or NaOH before the addition of 10
mM OGB-1. Data are presented as mean { SE (n Å 4). The Boltzmann
fit and pK were derived with PClamp 6.0 software.
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G. C. TOMBAUGH
intracellular HCO 03 is Ç18–20 mM and corresponds to a b value
of Ç45 mM (Chesler et al. 1994). Intrinsic pH buffering, provided
primarily by large dialysis-resistant proteins and organelles, is Ç10
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 b solutions, respectively. Thus both solutions yield
a higher nominal internal b (high, 78; low, 58 mM) than thought
to exist in vivo ( Ç40 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 Ca 2/ buffers (e.g., BAPTA, Fura-2) on cytosolic Ca 2/
gradients. The relative importance of HEPES/MOPS buffering becomes more striking when one considers that the speed of
HCO 03 buffering will be reduced as soluble carbonic anhydrase is
washed out by internal dialysis.
All cells were initially held in voltage clamp (Vh Å 060 mV)
to measure input resistance (Rin ), series resistance (Rs ), and cell
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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 060 mV. Actual
Vm was estimated to be near 070 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 Å 060
mV); Ca currents were then evoked by a series of depolarizing
voltage steps (50 ms) between 050 mV and /20 mV in 10-mV
increments at 20-s intervals. For determination of Ca current rundown, repeated voltage steps (50 ms, 010 mV) were applied every
20 s for 20 min. Capacitive artifacts were canceled electronically
and series resistance (6–15 mV ) was compensated 60–85%. Current and voltage records were sampled at 5–10 kHz, filtered at 5
kHz, and stored on a PC.
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FIG . 3. Internal pHi buffering does not directly affect whole cell Ca
conductance or Ca 2/ current rundown. A: examples
of whole cell Ca currents evoked 10 min after break-in (Vh Å 070 mV) in the presence of tetrodotoxin and TEA-Cl.
Depolarizing test pulses (50 ms) were preceded by a 50-ms hyperpolarizing pulse to 080 mV. Scale bars: 400 pA, 20 ms.
B: summary current-voltage (I-V) curve for Ca currents evoked from both high b (n Å 5) and low b cells (n Å 6) or from
cells in which the high b pipette solution was deliberately acidified to pH 6.9 ( n Å 7). C: currents were evoked by 50-ms
voltage steps to 010 mV every 20 s for 20 min (n Å 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.
PH
SENSITIVITY OF DENDRITIC CA 2/ SIGNALS
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FIG . 4. Dendritic Ca
transients are larger
and decay more rapidly than somatic transients.
A: fluorescence image of a hippocampal interneuron (s. radiatum) dialyzed with 100 mM
OGB-1 (high b 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 400ms 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.
2/
For optical recording of Ca transients, patch pipette tips were
filled with internal solution and then backfilled with the same solution containing 100 mM of the Ca 2/ indicator Oregon Green 488
BAPTA-1 (OGB-1; Molecular Probes, Eugene, OR). Reducing
the dye concentration to 50 mM 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 b internal solutions containing
200 nM free Ca 2/ plus 10 mM of the Ca 2/ 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 mm 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
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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 mV 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 [%DF Å (F 0 Fo )/For100]. For between-group comparisons, DF values were normalized by grouping them according to the
number of associated APs. The decay of Ca 2/ transients was fit with
a single exponential and used as an index of Ca 2/ 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
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 Ca 2/ chelator BAPTA. Fluorescence data
obtained in vitro in the presence of 200 nM free Ca 2/ 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
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Fluorescence imaging
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G. C. TOMBAUGH
low b: 15 { 1 pF, n Å 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 b 87 { 1 mV, low
b 87 { 6 mV; ú25 APs: high b 76 { 3 mV, low b 78 {
1 mV, n Å 10).
To assess whether differences in b of the internal solutions
directly affected Ca channel function, whole cell Ca currents
were monitored 10 min after membrane rupture with Csbased pipette solutions containing the identical type and concentration of pH buffers used in the current-clamp experiments (see METHODS ). In both high and low b groups, Ca
currents activated near 040 mV, peaked near 010 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 b ) from pH 7.2 to 6.9 depressed Ca currents by Ç20%
(Fig. 3B).
Effects of phi buffering on Ca 2/ transients
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 b and low b-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 b: 260 { 9
mV; low b: 267 { 15 mV ), Rs (high b: 11 { 1 mV; low
b: 10 { 1 mV ), or cell capacitance (high b: 14 { 1 pF;
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2/
FIG . 5. Dendritic Ca
transients are amplified in high b cells. A: examples of Ca transients evoked by 400-ms current pulses (0–250 pA) in a
high b and low b cell. B: peak amplitude of these transients were pooled
and normalized for the number of action potentials (AP) evoked during
the current pulse; * P õ 0.05, ** P õ 0.01 (n Å 6, high b; n Å 9, low
b ). C: Ca transient decay (tau) was plotted against the number of APs
evoked by the current pulse (derived from data in B).
Depolarizing current pulses large enough to trigger a train
of APs also evoked Ca 2/ 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). Ca 2/ transients in the soma
decayed with a time constant of 2.8 { 0.2 s (n Å 5, high
b, 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 Ca 2/ changes
between high and low b cells were not made.
When dendritic Ca transients were normalized for the
number of APs, high b cells exhibited larger transients than
those recorded in low b 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 Å 7, low b )
did not significantly alter the amplitude of Ca transients (6–
15Aps: Kgluc 42 { 4%, KMeSO4 42 { 4%; ú25 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 b and low b cells. The initial AP repolarized with a
half-time (RT) of 1.5 { 0.1 ms in both high b and low
b groups. In high b 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
mM) spike broadening still occurred in both groups, but the
effect of elevated pHi buffering on AP repolarization was
abolished.
PH
SENSITIVITY OF DENDRITIC CA 2/ SIGNALS
1707
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 b cells ( Ç15%, Fig.
7) but was unaffected by substitution of KMeSO4 for Kgluconate (250 pA: Kgluc 06.7 { 1 mV; KMeSO4 06.8 {
1 mV, n Å 12). No significant difference in sAHP amplitude
between low and high b 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 b 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
/ 9k2d$$oc25 J260-8
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 Å 5).
DISCUSSION
The key findings are that dendritic Ca 2/ 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 Ca 2/
dynamics very close to dendritic membranes.
During cell activity H / may rise gradually in response to
continued Ca 2/ influx (Ahmed and Conner 1980; Ballanyi
et al. 1994; Rose and Deitmer 1995; Trapp et al. 1996b),
and this rise in H / may limit Ca 2/ accumulation by blunting
Ca channel activity. According to this model, H / inhibition
of Ca 2/ 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
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FIG . 6. Spike repolarization occurs more slowly in high b cells. A: examples of individual APs from spike trains evoked
by small (50 pA) or large (250 pA) current injection (high b ). 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 b and
low b cells at 2 levels of spike frequency. Note that cadmium (200 mM) abolished the difference in RT between groups by
reducing DRT in high b cells. * P õ 0.05; n Å 10 (control), n Å 5 (cadmium).
1708
G. C. TOMBAUGH
spike train in high b 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 b groups clearly indicates that this
FIG . 8. Early component of the sAHP decays more rapidly in high b
cells. The sAHP evoked in both high and low b cells could be fit with 2
exponentials. Note that, like the amplitudes of both Ca 2/ transients and the
sAHP, a statistically significant difference for tau1 emerged with increasing
current amplitude. *P õ 0.05 (n Å 5–9).
/ 9k2d$$oc25 J260-8
phenomenon is not dependent on Ca influx. However, the
increase in RT caused by elevated internal b 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
b 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 b 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 Ca 2/ dynamics
Ca 2/ transients in the proximal dendrites were consistently larger and decayed more rapidly than those evoked in
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FIG . 7. Slow afterhyperpolarization (sAHP) is enhanced in high b cells. A: peak sAHP values were measured °100 ms
from the end of the current pulse. *P õ 0.05, **P õ 0.01 (n Å 15). B: examples of sAHP wave forms were evoked with
50-, 100-, and 250-pA current pulses. Scale bars: 2 mV, 0.5 s.
PH
SENSITIVITY OF DENDRITIC CA 2/ SIGNALS
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
Ca 2/ -buffer gradients between the soma and neighboring
dendrite could also explain differences in both peak Ca 2/
fluorescence and its rate of decay.
In hippocampal interneurons Ca entry through voltagegated 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 Ca 2/ (Lancaster and
Zucker 1994; Sah 1992), the sAHP can be exploited as a
submembrane Ca 2/ sensor (Tucker and Fettiplace 1996).
That sAHP amplitude was larger in high b cells is consistent
with enhanced Ca 2/ influx and the larger Ca 2/ transients
/ 9k2d$$oc25 J260-8
that were detected fluorometrically. One early component of
the sAHP also decayed more rapidly in high b cells, suggesting that, in addition to limiting Ca influx, H / accumulation interfered with Ca 2/ sequestration or buffering near the
cell membrane. As possible mechanisms, proton accumulation may have impaired H / –Ca 2/ exchange (Trapp et al.
1996a) or competed with Ca 2/ 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 highaffinity mobile Ca 2/ buffers (e.g., OGB-1, BAPTA). These
compounds enhance Ca 2/ diffusion away from the membrane (Zhou and Neher 1993), where Ca 2/ ions activate
KCa channels, and into the bulk cytosol, where the fluorescent
Ca 2/ signal was actually measured. Such an increase in Ca 2/
mobility may have obscured fluorescent detection of a more
subtle effect of H / on Ca 2/ clearance near the membrane.
Thus fluorescent Ca 2/ measurements averaged across even
relatively confined regions of a cell may be inadequate for
drawing conclusions about the behavior of Ca 2/ signals
close to their source.
The presence of mobile Ca 2/ buffers presumably contributes to the generally slow decay of Ca 2/ transients reported
here and in other studies (Jaffe et al. 1994). Prolonged Ca 2/
transients may also reflect the delayed contribution of Ca 2/ induced Ca 2/ 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 Ca 2/ 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 Ca 2/ influx
(Church 1992), but they need not preclude other mechanisms. For example, the depression of sAHP in low b 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
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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 b internal solution
(current pulse Å 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 b ) 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.
1709
1710
G. C. TOMBAUGH
Possible physiological significance
Active dendritic Ca 2/ 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
/ 9k2d$$oc25 J260-8
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 Ca 2/ 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 Ca 2/ transients during synaptic activity (Miyakawa et al. 1992). If synaptically evoked H / transients
arise in these spatially restricted regions, the resulting reduction of Ca 2/ 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 Ca 2/
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 b cells dendritic
pH may have fallen transiently by °0.5 pH units, an estimate
based on the degree to which Ca 2/ transients were influenced
by internal b, 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 Ca 2/
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 Ca 2/ permeable AMPA receptors and
Ca 2/ -binding proteins implies an especially strong need for
tight Ca 2/ 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 Ca 2/
signals in excitable cells.
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 NS18670.
Address for reprint requests: UMDNJ, RWJ Medical School, Dept. of
Neuroscience, CABM bld., 3rd Fl., 675 Hoes Lane, Piscataway, NJ 088545635.
Received 6 April 1998; accepted in final form 22 June 1998.
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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 Ca 2/ 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 ( õ1.0 pH unit) to affect OGB-1 fluorescence directly.
The Ca 2/ affinity of BAPTA-based indicators such as OGB1 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 b cells had a
lower resting pHi than high b cells, which could have in
turn reduced 1) baseline Ca channel activity or 2) the transmembrane driving force by elevating resting [Ca 2/ ]i (Ouyang et al. 1994). Neither of these possibilities appear likely
given that Ca currents evoked in low and high b 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 Ca 2/ 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 b
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
b cells.
PH
SENSITIVITY OF DENDRITIC CA 2/ SIGNALS
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