1Department of Neurological Surgery, Northwestern University Medical School, Chicago, Illinois 60611; and 2Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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Schweitzer, Jeffrey S., Haiwei Wang, Zhi-Qi Xiong, and Janet L. Stringer. pH Sensitivity of Non-Synaptic Field Bursts in the Dentate Gyrus. J. Neurophysiol. 84: 927-933, 2000. Under conditions of low [Ca2+]o and high [K+]o, the rat dentate granule cell layer in vitro develops recurrent spontaneous prolonged field bursts that resemble an in vivo phenomenon called maximal dentate activation. To understand how pH changes in vivo might affect this phenomenon, the slices were exposed to different extracellular pH environments in vitro. The field bursts were highly sensitive to extracellular pH over the range 7.0-7.6 and were suppressed at low pH and enhanced at high pH. Granule cell resting membrane potential, action potentials, and postsynaptic potentials were not significantly altered by pH changes within the range that suppressed the bursts. The pH sensitivity of the bursts was not altered by pharmacologic blockade of N-methyl-D-aspartate (NMDA), non-NMDA, and GABAA receptors at concentrations of these agents sufficient to eliminate both spontaneous and evoked synaptic potentials. Gap junction patency is known to be sensitive to pH, and agents that block gap junctions, including octanol, oleamide, and carbenoxolone, blocked the prolonged field bursts in a manner similar to low pH. Perfusion with gap junction blockers or acidic pH suppressed field bursts but did not block spontaneous firing of single and multiple units, including burst firing. These data suggest that the pH sensitivity of seizures and epileptiform phenomena in vivo may be mediated in large part through mechanisms other than suppression of NMDA-mediated or other excitatory synaptic transmission. Alterations in electrotonic coupling via gap junctions, affecting field synchronization, may be one such process.
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INTRODUCTION |
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The occurrence of large changes
in [Ca2+]o and
[K+]o during seizures and
seizure-like phenomena both in vitro and in vivo has been well
documented (Hablitz and Lundervold 1981;
Heinemann et al. 1977
; Krnjevic et al.
1982
; Pumain and Heinemann 1981
; Somjen and Giacchino 1985
). The significance of these changes, and
particularly their role as either cause or effect of the associated
electrophysiological excitatory activity, has been a matter of
considerable debate. Changes in neuronal excitability resulting from
such ionic changes must be considered in developing a complete model of
seizure propagation (Jensen and Yaari 1997
;
Schweitzer and Williamson 1995
; Schweitzer et al.
1992
). Another extracellular ion, H+, is
an important regulator of myriad physiological processes, and its
relationship to epilepsy and epileptiform events has also been well
recognized (Balestrino and Somjen 1988
; Caspers
and Speckmann 1972
; Velisek et al. 1994
), but
the effects of [H+]o on
mechanisms of propagation and synchronization other than classical
synaptic transmission have not been fully characterized.
We have previously developed an in vitro model that in many respects
resembles "maximal dentate activation," a phenomenon described in
the intact rat that is closely related to limbic seizures
(Patrylo et al. 1994; Schweitzer et al.
1992
; Stringer and Lothman 1989
; Stringer
et al. 1989
). The in vitro model is based on alteration of the
slice extracellular environment to approximate levels of
[Ca2+]o and
[K+]o measured during
seizures in vivo. This manipulation by itself is capable of
producing full-blown seizure-like events, and both extracellular and
intracellular techniques have been applied to demonstrate that these
events are independent of fast amino acid-mediated synaptic
transmission (Pan and Stringer 1996
; Schweitzer
et al. 1992
). Moreover, we have previously demonstrated that
intense synaptic activity in the perforant path of this slice system is capable of modifying the extracellular ionic environment sufficiently to create the conditions that support such nonsynaptic bursts in the
dentate granule cell layer (Schweitzer and Williamson
1995
).
Effects of [H+]o on
seizure-like phenomena have sometimes been ascribed to modulation of
N-methyl-D-aspartate (NMDA) channel activity,
although this has not been directly demonstrated (Gottfried and
Chesler 1994; Velisek et al. 1994
; Yoneda
et al. 1994
). Excitatory synaptic transmission via the NMDA
ligand-gated Ca2+ channel is sensitive to
[H+]o (Taira et
al. 1993
; Tang et al. 1990
), but to what extent
NMDA-dependent synaptic transmission is involved in synchronization of
the ictal seizure event is not known. Other pH-sensitive mechanisms
might also participate in seizure synchronization. Among such
mechanisms is electrotonic coupling via gap junctions (Connors
et al. 1984
; Deitmer and Rose 1996
;
Dermietzel and Spray 1993
; Giaume and McCarthy 1996
; Lee et al. 1995
; Pappas et al.
1996
; Venance et al. 1998
), the role of which in
burst synchronization has been examined using qualitative measures to
alter intracellular pH in CA1 (Perez-Velazquez et al.
1994
).
In this report we show, in quantitative fashion, the pH sensitivity of an in vitro model of maximal dentate activation. The results suggest that the pH sensitivity of these events is independent of synaptic transmission. These findings may provide clues to how pH alters seizure activity in vivo and may provide information about the mechanisms of intercellular communication involved in these nonsynaptic seizures.
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METHODS |
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Preparation of rat hippocampal slices
Rat hippocampal slices were prepared in standard fashion as
previously described (Schweitzer et al. 1992). Briefly,
adult male Sprague-Dawley rats (weights 100-350 g) were anesthetized with 150 mg/kg phenobarbital sodium and decapitated. The brain was
rapidly removed and placed in artificial cerebrospinal fluid (ACSF, see
following text) at 0-4°C. A block containing the hippocampus was cut
and mounted on a vibratome slicer (Campden Instruments, Sileby,
UK) where 500-µm slices were made. The hippocampus was removed from each slice by trimming with a sliver of razor blade. Slices were placed in an interface chamber at 34°C with a 95% O2-5% CO2 atmosphere in
ACSF containing (in mM) 124 NaCl, 1.4 NaHPO4, 26 NaHCO3, 3.0 KCl, 1.3 MgSO4,
1.3 CaCl2, and 11 glucose. Chamber flow rate was
~1.5-2.0 ml/min.
pH of test solutions was set by altering the concentration of NaHCO3, and adjustments in NaCl concentration were made to maintain constant Na+ concentration of the ACSF. For pH 7.3, NaHCO3 was 26 mM and NaCl 124 mM; for pH 7.0, NaHCO3 was 13 mM and NaCl 137 mM; and for pH 7.6, NaHCO3 was 39 mM and NaCl 111 mM. Osmolality of the resulting solutions was tested using an osmometer (Wescor) and remained unchanged with these pH adjustments. Test solutions were substituted for the control during continuous recording from the slices. Recordings were made 1-2 h after application of the test solutions. Maintenance of the adjusted pH level was confirmed by measuring the pH of the effluent from the chamber.
Oleamide (Sigma) was nearly insoluble in water and was added to ACSF containing 10% fetal calf serum, as in the original description of its assay. Appropriate controls using fetal calf serum alone were performed for this set of experiments.
Recording and stimulation
Stimulating electrodes were made from insulated platinum-iridium
wire twisted into a pair (75 µm). A Winston Electronics SC-100 isolation box was used; stimulus intensities ranged from 100 to 300 µA and duration of the stimulus pulse was typically 0.3 ms. Extracellular recording was performed with glass pipettes filled with 1 M NaCl and broken to a tip resistance of 5-10 M. Intracellular recording was performed with sharp electrodes (resistance 50-80 M
)
filled with 4 M potassium acetate. Unit and multiunit recordings were
made with ~2 M
platinum-iridium electrodes (model PTM23B20; WPI).
Field and intracellular signals were amplified with an Axoclamp 2B
amplifier (Axon Instruments). Unit and multiunit recordings were
obtained with a DAM-80 AC differential amplifier (WPI). All recordings
were made near the apex of the dentate granule cell body layer and data
were collected via a Digidata 1200 A-D converter board (Axon
Instruments) and analyzed using pClamp 7.0 software on a PC computer. A
low-pass filter at 3 kHz was used for extracellular recordings, and
low-pass of 3 kHz, highpass of 30 Hz for unit and multiunit recordings.
No filters were applied with intracellular recording. Records were
stored on an Iomega Jaz drive for off-line analysis.
Measurements of DC field potential shift and population spike amplitude were made for each field burst. The negative field potential shift amplitude was measured from the preburst baseline to the point of greatest baseline negativity during the burst, and the population action potential height (which is a function of cellular action potential amplitude, number of cells firing, and synchronization) was measured from the baseline field potential between population spikes to the peak of the spikes. The largest amplitude sustained spikes (i.e., 10 successive equally large spikes) that occurred during the burst formed the basis of this measurement.
Extracellular pH measurements
DC-coupled recording of field potentials and pH were measured with double-barreled ion-sensitive electrodes. One barrel was silanized with 15% tri-N-butylchlorosilane (Alfrebro; Monroe, OH) in chloroform, and the tip was filled with the hydrogen ion-selective resin (Fluka hydrogen ionophore II-Cocktail A). The electrode was then backfilled with (in mM) 100NaCl , 10 HEPES, and 10vNaOH, pH 7.5. The reference barrel was filled with 2 M NaCl. The reference and pH signals were amplified (Axoprobe 1A, Axon Instruments) and displayed on a chart recorder (Astro-Med). The electrode was calibrated before each experiment in standard solutions of artificial cerebrospinal fluid with pH 6.6, 7.0, 7.3, and 7.6. The pH-sensitive electrodes had a response of 50-55 mV per unit change in pH (6.6-7.6).
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RESULTS |
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Prolonged field bursts are sensitive to pHo
Extracellular pH was altered by changing the concentration of NaHCO3 added to the ACSF and adjusting the sodium appropriately. Increasing the pH from 7.3 to 7.6 increased the amplitude of the population spikes and the DC potential shift, while decreasing the pH from 7.3 to 7.0 decreased the population spike amplitude and DC shift (n = 18, Fig. 1, Table 1). In most instances, field bursts were nearly or totally suppressed at nominal pH 7.0 compared with pH 7.3. Changing slice perfusion bath using a similar protocol without altering the pH ("sham" procedure) had no significant effect on burst amplitude. Bursts evoked by antidromic stimulation at the hilus were similarly suppressed (not shown). The suppression of bursts by low pH was completely reversible after return to nominal pH 7.3 (Fig. 1A).
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Measurement of pH near the center of the slices during perfusion with
different test solutions revealed a small acid shift in pH at the
center of the slice relative to the bath (Table 1) with a corresponding
gradient from the outside to the center of the slice. There was also
some buffering of the nominal pH solutions by the slice, so that the
actual range of pH was smaller both at the surface and within the slice
compared with the nominal solutions. The effects on field bursts
observed here in the nominal solution pH range 7.0-7.6 therefore
occurred in the range 7.07-7.53 at the surface and 6.88-7.26 at the
center of the slice. Thus these differences in physiological response
occur over 0.3-0.5 pH units, which corresponds to a
[H+]o change from 8.5 to
3.0 × 108 M at the
surface of the slice or from 13.1 to 5.5 × 10
8 M at the center.
Burst pH sensitivity is not a synaptic effect
Zero-added-Ca2+ ACSF eliminated all
detectable evoked synaptic activity recorded both extracellularly and
intracellularly in the dentate granule cell layer (Fig.
2). In normal ACSF, addition of 30 µM
each of 6,7-dinitroquinoxaline-2,3-dione (DNQX),
D,L2-amino-5-phosphonopentanoate (AP-5), and bicuculline
methiodide (BMI) also completely blocked evoked and spontaneous
activity (Fig. 2). We did not observe spontaneous synaptic potentials
in the dentate granule cell layer in our slices, but these did occur in
CA1. This spontaneous activity was also eliminated either by deletion
of calcium or by addition of the blockers.
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In zero-added Ca2+, 9 mM K+ ACSF, spontaneous prolonged field bursts continued to occur after NMDA, non-NMDA, and GABAA blockade at the doses that eliminated evoked and spontaneous activity (30 µM). Bursts in these conditions showed no difference in pH sensitivity compared with bursts occurring without the agonists (n = 5, Fig. 3) and the effects were similarly reversible. Therefore ligand-dependent NMDA, AMPA, and GABAA receptor activation did not appear to be necessary for the appearance of the prolonged field bursts and their pH sensitivity did not depend on synaptic transmission via these transmitter systems.
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Other synaptic properties not affected by the pharmacologic blockade
might be sensitive to pH, including other putative transmitters or
other receptor types. However, pHo adjustment
from 7.3 to 7.0 did not suppress evoked or spontaneous postsynaptic
potentials recorded in normal
[Ca2+]o and
[K+]o ACSF either
extracellularly or intracellularly (Fig.
4, n = 8). Thus even if a
small amount of synaptic transmission were still occurring during the
field bursts, it would be unlikely to account for the large changes in
burst amplitude that occurred in this pH range. Granule cell resting
membrane potential and action potential amplitude or frequency are
known to be altered in the high
[K+]o, low
[Ca2+]o environment
(Pan and Stringer 1996), but the resting membrane potentials in granule cells of the dentate gyrus and pyramidal cells in
CA1 were not significantly affected by pHo in the
range 7.0-7.3. In granule cells of the dentate gyrus, the resting
membrane potential was 76.4 ± 1.5 (SD) mV in nominal pH 7.3 and
75.9 ± 2.5 mV in nominal pH 7.0 (n = 8). In
pyramidal cells of CA1, the resting membrane potential was 66.7 ± 1.5 mM in nominal pH 7.3 and 67.5 ± 1.5 mM in pH 7.0 (n = 12).
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Role of gap junctions in the effect of pH on field bursts
Since NMDA channel blockade by hydrogen ions does not appear to
explain the pH sensitivity of the prolonged field bursts, the possible
role of gap junctions, the patency of which is modulated by
pHi, was tested. To determine if dentate granule
cell field bursts are dependent on gap junction patency, slices in low
[Ca2+]o, high
[K+]o and pH 7.3 were
treated with 100 µM concentrations of octanol (n = 5), carbenoxolone (n = 5), or oleamide [an endogenous
amidated lipid derived from the cerebrospinal fluid of sleep-deprived
cats, that has been shown to block glial gap junctions with greater specificity than octanol (Guan et al. 1997);
n = 8]. These gap junction blockers reversibly
suppressed the field bursts in a manner similar to exposure to pH 7.0. In addition, the gap junction blockers and pH 7.0 exposure all acted
primarily on synchronization rather than cellular excitability
that
is, they blocked the field bursts without suppressing the individual
cellular epileptiform activity seen in single or multiple unit
recordings (Fig. 5). These findings are
consistent with the hypothesis that gap junctions are important in the
synchronization of the field bursts and that their modulation by pH
underlies the pH sensitivity of the bursts.
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DISCUSSION |
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A complete understanding of the mechanisms of seizure
synchronization and propagation must take into account
neurophysiological events not only at "normal," resting
extracellular ionic conditions but under conditions of altered
[Ca2+]o and
[K+]o that are known to
occur during seizure activity in vivo (Hablitz and Heinemann
1987; Heinemann et al. 1977
; Krnjevic and
Morris 1974
; Pumain and Heinemann 1981
;
Somjen and Giacchino 1985
). These changes in
[Ca2+]o and
[K+]o occur only
transiently in vivo as part of a dynamic mechanism containing both
synaptic and nonsynaptic components (Jensen and Yaari
1997
; Schweitzer and Williamson 1995
;
Schweitzer et al. 1992
). In this study, we have used
nominally zero-calcium solutions to eliminate synaptic contributions to
the bursts so as to dissect out and isolate the nonsynaptic mechanisms
involved in seizure synchronization. In particular, we have studied the
role of pH in the regulation of the synchronized field bursts in this model.
The effects of pH on epileptiform phenomena have sometimes been
attributed to modulation of NMDA channel activity. Since NMDA-mediated and other fast amino acid-dependent synaptic transmission are not
active in this model of epileptiform activity, modulation of NMDA
channel activity by pH cannot explain the pH sensitivity of the field
bursts. It could be argued that an undetected but still significant
amount of transmitter release is occurring, perhaps by a
calcium-independent mechanism, and that NMDA receptors might still be
involved in synchronizing the field bursts. However, addition of NMDA,
non-NMDA and GABAA antagonists, which did block synaptic transmission, did not alter the pH sensitivity of the field
bursts. Altogether, this suggests that the pH sensitivity of
epileptiform events in vivo may be due in large part to effects other
than NMDA receptor antagonism by hydrogen ions. Hydrogen ion
concentration changes per se may also not be the sole agent of the
effects shown here. In a C02-bicarbonate buffer
system, pH alterations are accompanied by significant alterations in
bicarbonate concentration such as those used to prepare the
experimental solutions used here. Bicarbonate ion itself may have
significant effects on both the passive and the active properties of
hippocampal neurons (Grover et al. 1993; Perkins
and Wong 1996
) although the observation that the pH-dependent
suppression of bursts occurred in the presence of
GABAA blockade makes it unlikely that this
particular ligand-gated ion channel is involved in the effect. Because
we could not test the effects of pH on synaptic potentials in
zero-Ca2+ conditions (where none were observed),
it is possible that pH somehow alters the efficacy of the receptor
antagonists or that sensitivity of synaptic potentials to pH is altered
in low Ca2+. However, both these possibilities
would have to be true simultaneously to negate our conclusion that low
pH burst suppression is a nonsynaptic phenomenon. We believe this
scenario to be highly unlikely.
pH affects many physiological processes that could be involved in
nonsynaptic synchronization, potentially including cell volume
regulation (Hansson and Ronnback 1992; Plesnila
et al. 1998
), nonligand-dependent ion channel patency
(Church et al. 1998
; Negulyaev and Vedernikova
1985
; Tombaugh and Somjen 1996
), or nonsynaptic
intercellular communication pathways such as gap junctions
(Connors et al. 1984
; Perez-Velazquez et al.
1994
; Rorig et al. 1996
; Spray et al.
1981
; Valiante et al. 1995
) or calcium signaling
(Trapp et al. 1996
). Osmolality changes seem an unlikely explanation, though we have not ruled out the possibility that pHo alters water transport across the cell
membrane, affecting the size of the extracellular space. The
second possibility, a pH effect on ion channel patency, awaits testing
by examining pH effects on specific ion channels. Our results favor the
third hypothesis, that the pH changes affect the efficacy of
intercellular communication via nonsynaptic signaling mechanisms such
as calcium waves or gap junctions. Octanol has been used to block
gap junctions in many systems (Bernardini et al. 1984
;
Charles et al. 1996
; Fujita et al. 1998
;
Pappas et al. 1996
; Venance et al. 1998
)
and is efficacious here, but its actions may be relatively nonspecific and in fact it may act at least partially via intracellular
acidification also (Pappas et al. 1996
). Oleamide is a
recently described endogenous gap junction modulator, derived from the
cerebrospinal fluid of sleep deprived cats (Boger et al.
1998
). This substance blocks gap junctions in glial systems at
low concentrations but does not affect glial calcium waves (Guan
et al. 1997
). Oleamide and carbenoxolone (a more traditional
gap junction modulator) were effective in blocking the field bursts
in our system. The continued appearance of unit activity, including
burst behavior, during field blockade with low pH or gap junction
blockers demonstrates that burst activity at the cellular and field
levels are separable processes and suggests both of these manipulations
act on burst synchronization.
pH measurements in the slice demonstrated a small pH gradient
from the outside to the center, confirming the work of other authors
(Walz 1989). This gradient may affect the pattern of
burst propagation in the slice and similar gradients could affect
seizure propagation properties in vivo. The pH measurements also
demonstrated a small acid shift in the slice as a whole compared with
the perfusing medium. We have not addressed directly the issue of
whether pHo or pHi is the
critical determining factor in affecting burst amplitude or which
cellular element (neuronal, glial, or both) is involved. However, the
relationship between pHo and
pHi in similar systems at normal
[Ca2+]o has been
established and shows that relatively large changes in
pHo are associated with relatively small ones in
pHi (Mellergard et al. 1994a
,b
;
Pappas et al. 1996
; Siesjo et al. 1985
).
The pH ranges involved are within biologically meaningful limits.
Recent work (Perez-Velazquez et al. 1994
; Xiong
et al. 2000
) has suggested that pHi is
the significant parameter. Since gap junctions are modulated by
pHi, this would be consistent with the proposed
role of gap junctions in burst synchronization.
The data in this study suggest that seizure propagation and
synchronization "co-opt" existing pathways of intercellular
communication under specific ionic and pH conditions. Such a hypothesis
is attractive because it does not require novel machinery at the
cellular or tissue levels to support seizure synchronization. This is
consistent with the observations that seizures can occur not only in
the context of epilepsy but also in normal cortex under the appropriate conditions as well as in acutely injured cortex. It fits
well with other recent data on the role of pH, gap junctions, and
epilepsy (de Curtis et al. 1998; Dermietzel and
Spray 1993
; Elisevich et al. 1997
; Laxer
et al. 1992
; Lee et al. 1995
; Valiante et
al. 1995
). The alteration of seizure-like activity by pH
suggests a novel set of physiological changes that could explain
lowered or raised seizure susceptibility in a number of systems
independent of NMDA or other synaptic transmission. Changes in
Na+-H+ transporters
and electrogenic Na+-HCO3
pumps (Deitmer and Rose 1996
; O'Connor et
al. 1994
; Pizzonia et al. 1996
; Shrode
and Putnam 1994
), or altered composition, numbers, or
function of gap junction channels could be involved (Elisevich
et al. 1997
; Giaume and McCarthy 1996
). We hope
that this train of investigation may stimulate interest in new targets for therapeutic intervention.
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ACKNOWLEDGMENTS |
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This work was supported in part by National Institute of Neurological Disorders and Stroke Grant NS-39941 to J. L. Stringer.
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FOOTNOTES |
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Present address and address for reprint requests: J. S. Schweitzer, Kaiser Los Angeles Medical Center, 1505 N. Edgemont St., 4th Floor, Los Angeles, CA 90027 (E-mail: Jeffrey.S.Schweitzer{at}kp.org).
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 15 December 1999; accepted in final form 3 May 2000.
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REFERENCES |
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