1Departments of Pharmacology and Psychiatry, Faculty of Medicine, Université de Montréal, Montreal, Quebec H3T 1J4, Canada; and 2Laboratory of Cellular Signaling, Department of Zoology and Genetics, Iowa State University, Ames, Iowa 50010
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ABSTRACT |
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Trudeau, Louis-Eric, Vladimir Parpura, and Philip G. Haydon. Activation of neurotransmitter release in hippocampal nerve terminals during recovery from intracellular acidification. Intracellular pH may be an important variable regulating neurotransmitter release. A number of pathological conditions, such as anoxia and ischemia, are known to influence intracellular pH, causing acidification of brain cells and excitotoxicity. We examined the effect of acidification on quantal glutamate release. Although acidification caused only modest changes in release, recovery from acidification was associated with a very large (60-fold) increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons. This was accompanied by a block of evoked EPSCs and a rise in intracellular free Ca2+ ([Ca2+]i). The rise in mEPSC frequency required extracellular Ca2+, but influx did not occur through voltage-operated channels. Because acidic pH is known to activate the Na+/H+ antiporter, we hypothesized that a resulting Na+ load could drive Ca2+ influx through the Na+/Ca2+ exchanger during recovery from acidification. This hypothesis is supported by three observations. First, intracellular Na+ rises during acidification. Second, the elevation in [Ca2+]i and mEPSC frequency during recovery from acidification is prevented by the Na+/H+ antiporter blocker EIPA applied during the acidification step. Third, the rise in free Ca2+ and mEPSC frequency is blocked by the Na+/Ca2+ exchanger blocker dimethylbenzamil. We thus propose that during recovery from intracellular acidification a massive activation of neurotransmitter release occurs because the successive activation of the Na+/H+ and Na+/Ca2+ exchangers in nerve terminals leads to an elevation of intracellular calcium. Our results suggest that changes in intracellular pH and especially recovery from acidification have extensive consequences for the release process in nerve terminals. Excessive release of glutamate through the proposed mechanism could be implicated in excitotoxic insults after anoxic or ischemic episodes.
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INTRODUCTION |
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The final cascade of events leading to
neurotransmitter release from synaptic vesicles is known to be
initiated by a rise in intracellular calcium
([Ca2+]i) (Dodge and Rahamimoff
1967). Other intracellular ions have also been proposed to play
a critical role in the release process. Among others, a role has been
suggested for intracellular sodium (Adam-Vizi et al.
1993
; Nordmann and Stuenkel 1991) and hydrogen ions. Intracellular pH may be an important variable influencing neurotransmitter release in a number of ways such as 1)
influencing the uptake of some neurotransmitters in synaptic vesicles
(Fykse and Fonnum 1996
), 2) regulating
synaptic vesicle endocytosis (Lindgren et al. 1997
;
Wang et al. 1995
), and 3) possibly
influencing some of the multiple protein-protein interactions involved
in the process of synaptic vesicle exocytosis.
Neurotransmitter release could also be affected by intraterminal pH
through an indirect effect on [Ca2+]i. A
number of possible mechanisms may be proposed. First, intracellular pH
may affect the ability of endogenous buffers to bind calcium ions
(Zucker 1981). Second, calcium currents can be pH
sensitive (Mironov and Lux 1991
; Takahashi et al.
1993
), directly impacting neurotransmitter release
(Barnes et al. 1993
). Finally, the activity of ionic
pumps and exchangers may be pH sensitive. The sodium-hydrogen antiporter, for example, is activated by intracellular acidification, leading to sodium uptake from the extracellular medium and extrusion of
intracellular protons (Jean et al. 1985
;
Raley-Susman et al. 1991
). This is one mechanism through
which many cell types attempt to compensate for deviations from normal
intracellular pH levels. Because some synaptic terminals are known to
express particularly high levels of the sodium-calcium exchanger
(Fontana et al. 1995
; Luther et al. 1992
;
Michaelis et al. 1994
; Reuter and Porzig
1995
), one possible consequence of intracellular acidification
and such sodium loading may be to cause an increase in
[Ca2+]i through reverse activity of the
Na+/Ca2+ exchanger or diminished rate of
calcium efflux. In support of this type of mechanism, it has recently
been demonstrated that veratridine-mediated sodium loading may activate
neurotransmitter release by inducing calcium uptake independently of
the activation of voltage-gated calcium channels (Bouron and
Reuter 1996
). Evidence has also been presented to suggest that
under some conditions activation of Na+/Ca2+
exchange can trigger noradrenaline release from cultured sympathetic neurons (Wakade et al. 1993
) and dopamine release from
tuberoinfundibular hypothalamic neurons (Annunziato et al.
1992
). Functional coupling of Na+/H+
and Na+/Ca2+ exchangers has also been
demonstrated in non-neuronal systems (Urcelay et al.
1994
). One potential problem with the hypothesis that
acidification may initiate such a sequential activation of the
Na+/H+ and Na+/Ca2+
exchangers is that the activity of the latter is actually inhibited by
acidic pH (and activated by alkaline pH) (Doering and Lederer 1993
). However, because intracellular acidification is often
followed by rebound alkalinization on cessation of the acidic
challenge, this may provide ideal conditions for sodium loading and
reverse activation of the Na+/Ca2+ exchanger.
A better understanding of the influence of intracellular pH on
neurotransmitter release may provide data helpful in understanding neurotoxicity associated with conditions such as cerebral ischemia, anoxia, and hypoglycemia. Indeed these pathological conditions are
known to be associated with perturbations of intracellular pH in brain
cells. Ischemia and anoxia cause intracellular acidification and sodium
loading, which can be followed by rebound alkalinization during
recovery (Chen et al. 1992; Chopp et al.
1990
; Friedman and Haddad 1994
; Pirttila
and Kauppinen 1992
). In the heart, evidence has been presented
to suggest that acidification during ischemia may be associated with a
rise in intracellular sodium and a subsequent activation of
Na+/Ca2+ exchange (Murphy et al.
1991
). Reverse operation of the
Na+/Ca2+ exchanger has also been shown to occur
in response to sodium accumulation during anoxia in myelinated axons
(Lehning et al. 1996
; Stys et al. 1992
).
Hypoglycemia may also be associated with intracellular alkalinization
and is associated with excessive release of excitatory amino acids and
excitotoxic damage (Auer and Siesjo 1993
).
To determine more directly how intracellular acidification affects neurotransmitter release, we have recorded quantal glutamate release from cultured hippocampal neurons under such conditions. We find that recovery from acidification is associated with a substantial activation of the spontaneous release process under conditions where action potential-evoked release is mostly blocked.
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METHODS |
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Cell culture
Primary cultures of neonatal rat hippocampal neurons were
prepared as described previously (Trudeau et al. 1996).
Briefly, the hippocampus was dissected from newborn rats and
dissociated with papain. Neurons and astrocytes were plated onto
collagen/poly-L-lysine-coated glass coverslips (0.1 mg/ml). Culture medium contained 5% fetal calf serum (GIBCO BRL; Grand
Island, NY) and Mito + serum additives (Collaborative Biomedical
Products; Bedford, MA). A mitotic inhibitor was added after the 4th day
in culture to halt glial proliferation (5-fluoro-2-deoxyuridine and
uridine, 5 µM). One-third of the medium was replaced with fresh
medium twice a week. Neurons were used in physiological experiments
after 3-4 wk in culture. In preliminary experiments we noted that the
major observations reported in this paper appeared to be less robust in
2-wk-old cultures. In some experiments neuron-enriched cultures were
prepared, as previously described (Goslin and Banker
1991
), to permit better visualization of neuronal processes.
Electrophysiology
Whole cell recordings were performed on the stage of a Nikon Diaphot inverted microscope equipped with phase contrast optics. Signals were recorded through Axopatch-1D patch amplifiers (Axon Instruments; Foster City, CA), digitized at 5 kHz and acquired in pClamp software (version 6.0) (Axon Instruments). Miniature synaptic currents were further analyzed with ACSPLOUF software (Dr. P. Vincent, UCSD, CA). In most experiments it was possible to adequately resolve all unitary synaptic events. However, in a subset of experiments the frequency of miniature excitatory synaptic currents (mEPSCs) rose to very high levels, and it is possible that some proportion of elementary events failed to be detected. This may have caused a slight underestimation of maximal mEPSC frequencies but should not affect any of the conclusions reached in this paper. Normal extracellular saline contained (in mM) 140 NaCl, 2 MgCl2 , 2 CaCl2, 5 KCl, 10 HEPES, 6 sucrose, and 10 glucose (pH 7.35). High-divalent saline was used to decrease spontaneous synaptic activity and to facilitate recording of monosynaptic action potential-evoked EPSCs. It contained (in mM) 135.5 NaCl, 7 MgCl2, 3 CaCl2, 5 KCl, 10 HEPES, and 8 glucose (pH 7.35). Propionate saline used to acidify neurons was prepared by completely replacing NaCl with Na propionate in either normal or high divalent salines. Nominally calcium-free saline contained (in mM) 140 NaCl, 5 KCl, 4 MgCl2, 1 EGTA, 10 HEPES, 4 sucrose, and 10 glucose (pH 7.35). To record action potential-evoked EPSCs, 10 µM SR-95531 (RBI; Natick, MA) was added to the saline to block GABAA receptors. To record mEPSCs, normal saline was supplemented with 10 µM SR-95531 to block GABAA receptors and 0.5 µM TTX (Calbiochem) to block sodium channels. The internal patch pipette solution used to record action potential-evoked synaptic currents contained (in mM) 140 K gluconate, 10 EGTA, 4 Mg-ATP, 0.2 Tris-GTP, and 10 HEPES (pH 7.35). To record mEPSCs, the internal patch pipette solution consisted of (in mM) 117.5 Cs gluconate, 10 NaCl, 4 MgCl2, 5 EGTA, 2 Mg-ATP, 0.2 Tris-GTP, and 15 HEPES (pH 7.35). Statistical comparisons were performed with Student's t-test, unless otherwise indicated.
Imaging
[Ca2+]i, pH, and sodium levels
were monitored by fluorescence imaging with a silicon-intensified
target camera (Hamamatsu; Bridgewater, NJ), IC-300 intensified
charge-coupled device (CCD) camera (Photon Technology International;
Monmouth Junction, NJ), or CH-250 cooled CCD camera (Photometrics;
Tucson, AZ). Images were acquired with Metafluor (Universal Imaging;
West Chester, PA) or NeDLC software (Prairie Technologies;
Waunakee, WI). Signals were measured from regions representing cell
bodies and/or major neurites. Fluorescent dyes {fura-2AM,
fluo-3AM, 2',7'-bis-(2-carboxyethyl)-5-carboxyfluorescein-AM (BCECF-AM), and 1,3-benzenedicarboxylic acid,
4,4'-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]-AM (SBFI-AM)} were acquired from Molecular Probes (Eugene, OR). Calcium levels were estimated according to Basarsky et al. (1995). Calibration was performed in situ (Thomas and Delaville 1991
) with
the Ca2+ ionophore 4-BrA-23187 (Molecular Probes) at the
end of each experiment. Although it is known that the affinity for
calcium of fluorescent indicators such as fluo-3 may be sensitive to
acidic pH, this is not likely to have significantly affected the
results reported because intracellular pH never reached the critical
level of 5.5 (Lattanzio and Bartschat
1991
). Furthermore, the experiments performed in this
study investigated calcium changes during alkalinization of the
cytosol. Alkalinization by itself has not been reported to have a major
effect on fluo-3 fluorescence. This is experimentally supported by data
in Fig. 5, where, in the absence of external calcium, alkalinization
had no effect on fluo-3 fluorescence.
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RESULTS |
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To produce intracellular acidification, cultured hippocampal
neurons were exposed to saline in which NaCl was replaced with the weak
acid Na propionate. This approach has been previously shown to produce
reliable intracellular acidification in neurons and other cell types
(Chen et al. 1998; Lindgren et al. 1997
; Stuenkel and Nordmann 1993
). An advantage of this
approach is that intracellular alkalinization can be produced on
propionate saline washout, under conditions where the extracellular
solution is again normal, bypassing problems related to the
extracellular effects of alkalinizing agents such as ammonium chloride,
which is known, for example, to affect glutamate receptors (Fan
and Szerb 1993
; Fan et al. 1990
). Imaging
experiments were performed with the fluorescent pH indicator BCECF. We
found that, as expected, propionate produced a rapid intracellular
acidification followed by partial and gradual recovery (Fig.
1) (n = 10). Washout with normal saline caused a rapid rebound intracellular alkalinization within 1 min. This was followed by a gradual recovery to resting intracellular pH (Fig. 1).
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Effect on action potential-evoked and spontaneous neurotransmitter release
Dual whole cell recording experiments were performed to monitor
action potential-evoked neurotransmitter release. The presynaptic neuron was maintained at its resting potential, whereas the
postsynaptic neuron was voltage clamped at 60 mV. Action potentials
evoked rapid EPSCs (Fig. 2A),
which were blocked by the glutamate receptor antagonist
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (not shown). Intracellular
acidification produced by propionate-containing saline produced only
transient and variable decreases in the amplitude of EPSCs (Fig. 2,
A and B) (68 ± 22% of control, mean ± SE,
P > 0.05; n = 7). The amplitude of
EPSCs recovered completely within 3 min in the continued presence of
propionate (Fig. 2, A and B). Washout of
propionate rapidly produced an almost complete block of evoked EPSCs
(Fig. 2, A and B) (11 ± 5% of control,
P < 0.05, n = 7). This block of evoked
release occurred within 30 s after perfusion with normal saline
(Fig. 2C), in parallel to the timing of rebound
alkalinization (Fig. 1). Block of release was not caused by
irreversible damage to nerve terminals, as reintroduction of propionate-containing saline allowed an immediate and almost complete recovery (Fig. 2, A and B) (74 ± 5% of
control, n = 7). In the absence of such
re-acidification, EPSC amplitude was found to recover gradually but
very slowly (to 15% of basal level within 10 min of washout).
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Propionate-induced intracellular acidification was associated with a
modest but reliable increase in the frequency of spontaneous mEPSCs
(600 ± 136% of control, P < 0.05, n = 8) (Fig. 3,
A and B). This was short lasting, similar to the
observed decrease in evoked EPSC amplitude during acidification.
Considering the powerful block of action potential-evoked glutamate
release after removal of propionate saline, it was somewhat unexpected
to observe that the mEPSC frequency rose to very high levels 2-4 min
after washout (Fig. 3, A and B). This increase in
mEPSC frequency was not immediate, such as for the block of evoked
EPSCs, and was delayed in relation to the timing of the peak of
intracellular alkalinization, suggesting that alkalinization per se did
not stimulate exocytosis. After 4 min of propionate washout, the
frequency of mEPSCs was increased ~60-fold above baseline (5,940 ± 2,530% of control, P < 0.05, n = 8) (Fig. 3, A and B). Recovery was gradual and
reached levels close to baseline by 10 min (310 ± 131% of
baseline, P > 0.05, n = 8) (Fig. 3,
A and B). This massive increase in release was obtained without any significant depolarization of neuronal cell bodies. On average, resting potential during the control period was
55 ± 2 mV, whereas it was
52 ± 3 mV after 4 min of
propionate saline washout, which is at the peak of the increase in
mEPSC frequency.
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Role of [Ca2+]i
To determine whether the substantial rise in mEPSC frequency was
caused by a rise in [Ca2+]i in neurons,
imaging experiments were performed with the fluorescent calcium
indicators fluo-3 and fura-2. In experiments performed with fluo-3,
propionate-containing saline sometimes caused a small and unreliable
increase in [Ca2+]i (Fig.
4). However, washout of propionate was
associated with a delayed and significant rise in
[Ca2+]i (n = 8; ANOVA and
Tukey's post hoc test, P < 0.01) (Fig. 4). The time
course of this rise in [Ca2+]i was very
similar to the time course of the elevation in mEPSC frequency observed
on propionate saline washout (Fig. 3B). By using recorded
fluorescence changes in cells and standard calibration curves for
fluo-3 (Minta et al. 1989), we estimated a
[Ca2+]i accumulation of ~700 nM caused by
rebound alkalinization. In parallel experiments with fura-2, we
confirmed that similar [Ca2+]i accumulations
(~800 nM) occur in neuronal processes (n = 2).
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To determine whether extracellular calcium was required for the rebound rise in [Ca2+]i and mEPSC frequency, propionate saline was washed out with nominally calcium-free saline instead of normal saline. We found that, although rebound alkalinization was unimpaired under such conditions (Fig. 5B), both the rebound rise in [Ca2+]i (Fig. 5C) and mEPSC frequency (Fig. 5A) were prevented. Reintroduction of normal saline after calcium-free saline still caused a substantial rise in [Ca2+]i (Fig. 5C) and mEPSC frequency (2,606 ± 924% of control after 1 min, P < 0.05, n = 4) (Fig. 5A), thereby showing that the driving force behind the rise in mEPSC frequency was not dissipated by the absence of external calcium during rebound alkalinization. These results suggest that calcium influx is important for the rebound increase in mEPSC frequency. The observation that the rise in mEPSC frequency is blocked under conditions where the rebound intracellular alkalinization still occurs suggests that the latter phenomenon is not sufficient by itself to cause the rise in mEPSC frequency. Further support for this hypothesis comes from the observation that direct intracellular alkalinization with 15 mM ammonium chloride fails to cause a rise in mEPSC frequency (n = 2, mEPSC frequency at 60% of baseline after 3 min; results not shown).
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Because the delayed rise in [Ca2+]i and mEPSC
frequency requires extracellular calcium, it may be hypothesized that
this occurs through the activation of voltage-gated calcium channels.
Alternately, calcium influx could occur through a carrier such as the
Na+/Ca2+ exchanger. The role of calcium
channels was tested by determining whether washing out
propionate-containing saline with saline containing 100 µM cadmium
would prevent the rebound rise in mEPSC frequency. This concentration
of cadmium is sufficient to block most high-voltage-activated calcium
channels (Bouron and Reuter 1996) (results not shown) while sparing the activity of the Na+/Ca2+
exchanger (Bouron and Reuter 1996
). We found that the
rebound rise in mEPSC frequency that follows intracellular
acidification was still clearly present under such conditions (Fig.
6A). Additionally, the delayed
rise in [Ca2+]i that parallels rebound
alkalinization was unaffected by cadmium (Fig. 6B)
(n = 9). Both the rise in mEPSC frequency and the rise in [Ca2+]i were not significantly different
from those obtained under control conditions (P > 0.05). These results suggest that, although calcium influx may trigger
the rise in mEPSC frequency, this does not occur through voltage-gated
calcium channels.
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Role of ion exchangers
One hypothesis that may be proposed is that intracellular
acidification activates the Na+/H+ exchanger,
thereby driving Na+ accumulation in the cytoplasm. This
sodium load may then provide a driving force, allowing reverse
activation of the Na+/Ca2+ exchanger to cause
an accumulation of calcium in synaptic terminals. Such a hypothesis
finds support from the recent observation that synaptic terminals are
richly endowed with the Na+/Ca2+ exchanger
(Luther et al. 1992; Michaelis et al.
1994
; Reuter and Porzig 1995
). The contribution
of the Na+/H+ exchanger was tested in
preliminary experiments with the classical blocker amiloride. We found
that application of amiloride (1 mM) together with
propionate-containing saline prevented the rise in mEPSC frequency
during propionate washout (n = 3) (data not shown).
Because amiloride is known to have significant affinity not only for
the Na+/H+ exchanger but also for the
Na+/Ca2+ exchanger as well as other pumps
(Murata et al. 1995
), we performed a second series of
experiments with the more specific amiloride derivative
5-(N-ethyl-N-isopropyl)-amiloride (EIPA). This
compound can be used at much lower concentrations and is known to have close to one order of magnitude higher affinity for the
Na+/H+ exchanger than for the
Na+/Ca2+ exchanger (Murata et al.
1995
). We found that 10 µM EIPA prevented the ability of
propionate-containing saline to induce the rebound rise in mEPSC
frequency (Fig. 7A)
(n = 3). As expected for a block of
Na+/H+ exchange, this antagonist was also
effective at blocking the induction of rebound alkalinization (Fig.
7B) (n = 5). The delayed rise in
[Ca2+]i was also completely prevented by EIPA
(Fig. 7C) (n = 6). To provide additional
support for the hypothesis that a sodium load may be induced by
intracellular acidification, we used SBFI, a fluorescent ratiometric
sodium indicator to directly monitor intracellular sodium levels
(Minta and Tsien 1989
; Rose and Ransom
1997
). Propionate-containing saline was found to produce a
gradual and long-lasting rise in intracellular sodium in neurons (Fig.
8) (n = 6), compatible
with our results suggesting that acidification activates
Na+/H+ exchange that consequently leads to a
loading of neurons with Na+.
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If the delayed rise in [Ca2+]i and mEPSC
frequency is caused by activation of Na+/Ca2+
exchange during the rebound alkalinization phase, then blocking this
exchanger during the propionate saline washout period should block the
rise in calcium and mEPSC frequency. This was tested with
2',4'-dimethylbenzamil (DMB), a Na+/Ca2+
exchanger blocker that is not fully specific for the
Na+/Ca2+ exchanger at all concentrations but
that is known to have close to one order of magnitude more affinity for
this exchanger than for the Na+/H+ exchanger or
the sodium ATPase (Murata et al. 1995). At a
concentration of 10 µM, DMB was found to produce a complete block of
the delayed rise in mEPSC frequency (Fig.
9A) and
[Ca2+]i elevation (n = 7)
(Fig. 9B).
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DISCUSSION |
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Our results demonstrate that acidification and rebound
alkalinization may have considerable effects on quantal
neurotransmitter release from synaptic terminals. Although
acidification itself produces some modification of neurotransmitter
release (Figs. 2 and 3), removal of acidification has a more
significant impact on glutamate release. One major consequence of
recovery from temporary acidification is a pronounced but reversible
block of action potential-evoked neurotransmitter release (Fig. 2).
This effect is not caused by a block of postsynaptic CNQX-sensitive
glutamate receptors because mEPSCs are still easily detectable during
this period (Fig. 3). The block of release is also not caused by a
toxic reaction to rebound alkalinization because the effect is rapidly
reversible on re-acidification of neurons (Fig. 2). Although we have
not identified the specific mechanism responsible for the block of action potential-evoked EPSCs, the time course of this block follows that of rebound alkalinization (Fig. 2C). One possibility is
that intracellular alkalinization of synaptic terminals leads to a block or inactivation of voltage-dependent calcium channels. However, as mentioned previously, the opposite has been reported; alkalinization is known to facilitate calcium currents (Mironov and Lux
1991; Takahashi et al. 1993
). Although other
mechanisms may be proposed, two possibilities are that calcium channel
coupling to synaptic vesicle exocytosis is perturbed or that the
synaptic protein complex responsible for the release of
neurotransmitter is directly inactivated.
A second major consequence of recovery from acidification is a delayed
but massive increase in the frequency of mEPSCs (Fig. 3). In many
preparations, the stimulation of quantal secretion was large enough to
be reminiscent of the effects of powerful secretagogues such as
-latrotoxin (Capogna et al. 1996
; Krasnoperov et al. 1997
). This effect was notably delayed in all neurons
(Fig. 3), in contrast to the block of action potential-evoked EPSCs (Fig. 2C). This is compatible with the idea that the two
phenomena are mediated by distinct mechanisms. It also suggests that
the increase in mEPSC frequency is not induced solely by the
intracellular alkalinization that follows washout of propionate saline.
This increase in mEPSC frequency was reversible over time, suggesting that the effect was also not caused by an irreversible toxic reaction, although a partial depletion of readily releasable vesicles could have
occurred. Rather our calcium imaging experiments suggest that the
stimulation of spontaneous exocytosis may be caused by an intracellular
accumulation of free calcium (Fig. 4). Perfusion of
propionate-containing saline was initially associated with a brief and
relatively small rise in [Ca2+]i (Fig.
4B). This occurred in parallel to intracellular
acidification and could be caused by the previously described decrease
in the calcium-buffering power of cellular cytoplasm induced by
acidification (Zucker 1981
) or by release of calcium
from intracellular stores (Chen et al. 1998
), although
other mechanisms cannot be rejected. This modest and short-lasting rise
was followed by a more substantial and delayed increase in
[Ca2+]i on washout of propionate-containing
saline. The delay was usually between 2 and 4 min (Fig. 4B),
in close parallel to the delay observed for the rise in mEPSC frequency
(Fig. 3). Our observation that the delayed rise in mEPSC frequency does
not occur if propionate-containing saline is washed out with
calcium-free saline (Fig. 5) suggests that the rise in
[Ca2+]i is due to influx or transport of
calcium across the cellular membrane. The finding that re-introduction
of normal calcium-containing saline still causes a rise in mEPCS
frequency after exposure of cells to calcium-free saline (Fig.
5A) suggests that the driving force for the calcium entry,
whatever its nature, was still present after this delay. Washout of
propionate-containing saline with saline containing 100 µM cadmium
was also not effective at blocking the delayed rise in mEPSC frequency
(Fig. 6). Because this concentration of cadmium is sufficient to block
most high-voltage-activated calcium currents in neurons, this may be
taken as evidence that the intracellular accumulation of calcium is not
occurring through activation of voltage-dependent calcium channels.
This was important to determine because previous reports have suggested
that some classes of calcium channels may be facilitated by
alkalinization (Mironov and Lux 1991
; Takahashi
et al. 1993
).
If calcium channels do not cause the delayed rise in
[Ca2+]i and mEPSC frequency, then an
alternate hypothesis is that the rise in calcium is mediated by a
transporter-based mechanism. One hypothesis is that acidification
causes the activation of the Na+/H+ antiporter
and thus a rise in intracellular sodium. On washout of
propionate-containing saline, this rise in sodium may provide a driving
force sufficient to cause a reverse activation of the Na+/Ca2+ exchanger. Because this exchanger is
otherwise known to be highly concentrated in nerve terminals, it is
conceivable that such a mechanism may be responsible for the observed
delayed rise in mEPSC frequency. Alkalinization that follows washout of
propionate saline may play a permissive role in this process because it
has been shown that the Na+/Ca2+ exchanger can
be activated under alkaline conditions and inhibited under acidic
conditions (Doering and Lederer 1993). Nonetheless, the
current set of data does not allow us to conclude that alkalinization per se is required for the activation of
Na+/Ca2+ exchange.
Our data provide four independent pieces of evidence that support
the hypothesis that elevated exocytosis results from calcium influx
driven by Na+/Ca2+ exchange that is activated
by elevated intracellular Na that accumulates as a result of
Na+/H+ antiporter activity during acid
conditions. First, we show that two blockers of the
Na+/H+ exchanger, amiloride and EIPA, block
rebound alkalinization and the delayed rise in mEPSC frequency (Fig. 7)
when applied simultaneously with the acidifying saline. Second, EIPA
also blocks the delayed rise in calcium (Fig. 7C). Third,
propionate-containing saline causes the predicted rise in intracellular
sodium (Fig. 8). This rise is relatively long lasting, compatible with
the observation that the driving force for the increase in calcium and
mEPSC frequency is also relatively long lasting (Fig. 5). Fourth, a
blocker of the Na+/Ca2+ exchanger, DMB, is able
to block the rise in mEPSC frequency and
[Ca2+]i, even when applied after the
propionate-containing saline (Fig. 9), under conditions where the rise
in sodium has already occurred. Finally, we have found that direct
intracellular alkalinization with ammonium chloride does not cause an
increase in spontaneous release. This argues in favor of the idea that
an initial intracellular acidification is specifically required to
obtain the observed increase in secretion and that alkalinization is
not a sufficient condition. This is compatible with the hypothesis that
sodium loading of neurons during acidification provides a driving force for the subsequent rise in mEPSC frequency during the recovery period.
Although DMB has a ~10-fold higher affinity for the
Na+/Ca2+ exchanger than for other targets such
as the Na+/H+ antiporter or the sodium ATPase,
it is not fully selective for the Na+/Ca2+
exchanger (Murata et al. 1995). However, because the
block of Na+/H+ exchange by DMB displays an
IC50 above 60 µM (Murata et al. 1995
), it is likely
that at 10 µM DMB will not produce a substantial block of the
Na+/H+ exchanger. Nonetheless, taken by itself
the block of the rise in mEPSC frequency by DMB does not prove the
involvement of Na+/Ca2+ exchange. However,
taken with the rest of our results this observation provides support
for this hypothesis and can be considered the simplest explanation of
our findings.
It should be noted that because our experiments were performed in
nominally CO2 and HCO3-free medium we have not
evaluated the role of sodium-dependent Cl/HCO3 exchange in
rebound alkalinization. Such an exchange mechanism could possibly
contribute to rebound alkalinization in vivo (Schwiening and
Boron 1994).
Our results demonstrate a substantial influence of intracellular pH on
quantal glutamate release in hippocampal neurons. We find that
intracellular acidification has only modest effects on release, even
under conditions where pH decreases by more than one-half a unit (Fig.
2). However, recovery from acidification is associated with both a
powerful block of action potential-evoked EPSCs and a delayed but
substantial rise in mEPSC frequency. Although our experimental
conditions do not specifically mimic pathological conditions such as
ischemia and hypoxia, it is noteworthy that such conditions may be
associated with intracellular acidification and possibly with a
sequential acidification, alkalinization sequence (Chen et al.
1992; Pirttila and Kauppinen 1992
). It will be
important for future studies to examine the influence of in vitro
models of ischemic or hypoxic episodes on intracellular pH, calcium, and quantal glutamate release. A combined block and stimulation of
action potential-evoked and spontaneous glutamate release, respectively, could have dramatic influences of brain functions and
play some role in the observed deficits associated with conditions such
as ischemia or hypoxia. It is of interest to note that it has been
hypothesized that neuronal death in brain ischemia may be caused by two
phases of excessive glutamate release. The first may occur during the
ischemic episode and be caused by reverse operation of the glutamate
transporter. The second may occur after ischemia and be caused by
glutamate released from vesicular stores (Szatkowski and Attwell
1994
). One exciting possibility is that this second phase is
caused by reversed activation of the Na+/Ca2+
exchanger in synaptic terminals during recovery from the acidic challenge.
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ACKNOWLEDGMENTS |
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We thank Drs. Harald Reuter and Alexandre Bouron for helpful comments on this manuscript.
This work was supported in part by National Institute of Neurological Disorders and Stroke Grants NS-26650 and NS-24233 to P. G. Haydon and by postdoctoral fellowships from the Human Frontier Science Program and the Medical Research Council (MRC) of Canada to L.-E. Trudeau. L.-E. Trudeau is now a Michael Smith scholar of the MRC of Canada and is supported by grants from the MRC, the Fonds de la Recherche en Santé du Québec, and the EJLB Foundation. V. Parpura is supported by funds from the Whitehall Foundation.
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FOOTNOTES |
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Address for reprint requests: L.-E. Trudeau, Dept. of Pharmacology, Faculty of Medicine, Université de Montréal, 2900 Boul. Edouard-Montpetit, Montreal, Quebec H3T 1J4, Canada.
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.
Received 9 November 1998; accepted in final form 8 February 1999.
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REFERENCES |
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