1Department of Biomedical Engineering,
Bikson, Marom,
Rahul S. Ghai,
Scott C. Baraban, and
Dominique M. Durand.
Modulation of Burst Frequency, Duration, and Amplitude in the
Zero-Ca2+ Model of Epileptiform Activity.
J. Neurophysiol. 82: 2262-2270, 1999.
Incubation of hippocampal slices in zero-Ca2+ medium blocks
synaptic transmission and results in spontaneous burst discharges. This
seizure-like activity is characterized by negative shifts (bursts) in
the extracellular field potential and a K+ wave that
propagates across the hippocampus. To isolate factors related to
seizure initiation, propagation, and termination, a number of
pharmacological agents were tested. K+ influx and efflux
mechanisms where blocked with cesium, barium, tetraethylammonium (TEA),
and 4-aminopyridine (4-AP). The effect of the gap junction blockers,
heptanol and octanol, on zero-Ca2+ bursting was evaluated.
Neuronal excitability was modulated with tetrodotoxin (TTX), charge
screening, and applied electric fields. Glial cell function was
examined with a metabolism antagonist (fluroacetate). Neuronal
hyperpolarization by cation screening or applied fields decreased burst
frequency but did not affect burst amplitude or duration. Heptanol
attenuated burst amplitude and duration at low concentration (0.2 mM),
and blocked bursting at higher concentration (0.5 mM).
CsCl2 (1 mM) had no effect, whereas high concentrations (1 mM) of BaCl2 blocked bursting. TEA (25 mM) and low
concentration of BaCl2 (300 µM) resulted in a two- to
sixfold increase in burst duration. Fluroacetate also blocked burst
activity but only during prolonged application (>3 h). Our results
demonstrate that burst frequency, amplitude, and duration can be
independently modulated and suggest that neuronal excitability plays a
central role in burst initiation, whereas potassium dynamics establish
burst amplitude and duration.
The generation and spread of spontaneous
epileptiform discharges is generally attributed to synaptic excitatory
feedback. However, studies performed "in situ" using ion-selective
electrodes (Heinemann et al. 1977 Incubation of an acute hippocampal slice in calcium-free solution
results in neuronal hyperexcitability and enhanced synchronization (Taylor and Dudek 1982 Although much is known about the mechanisms of increased excitability
and synchronization that occur during zero-calcium bursting, the
mechanisms underlying burst initiation, termination, and the source of
the extracellular field shifts are largely unknown. We therefore tested
the effects of several pharmacological perturbations on burst
frequency, duration, and amplitude. To study the role of neuronal
excitability in the generation and propagation of zero-calcium burst
discharges, we examined the effects of cation screening, sodium channel
block, and application of electric fields. To investigate the role of
gap junction coupling, we applied heptanol and octanol. Several
potassium channel blockers were similarly tested. Our results
demonstrate that neuronal excitability plays a central role in
establishing burst frequency, whereas burst amplitude and duration are
modulated by gap junction coupling and potassium conductances.
Preparation of hippocampal slices
All experiments were performed in the CA1 pyramidal cell region
of hippocampal brain slices prepared from Sprague-Dawley rats (75-250
g). Rats were anesthetized using ethyl ether and decapitated. The brain
was then rapidly removed and one hemisphere glued to the stage of a
Vibroslicer (Vibroslice, Campden). Slicing was carried out in cold
(3-4°C), oxygenated sucrose-based artificial cerebrospinal fluid
(ACSF) consisting of (in mM) 220 sucrose, 3 KCl, 1.25 NaH2PO4, 2 MgSO4, 26 NaHCO3, 2 CaCl2, and 10 dextrose. Sucrose-based slicing
medium has been shown to increase cell viability in vitro
(Aghajanian and Rasmussen 1989 Solutions and drugs
All pharmacological studies were conducted in a submerged
recording chamber. Following transfer to the recording chamber, slices
were perfused in "zero-calcium" ACSF
(zero-Ca2+) bubbled with 95%
O2-5% CO2 (pH 7.4).
Zero-Ca2+ ACSF was of the following composition
(in mM): 123 NaCl, 4.75 KCl, 1.25 KH2PO4, 1.5 MgSO4, 26 NaHCO2, 10 dextrose, and 1 EGTA (osmolarity 300 ± 3 mosmol
l Generation and application of electric fields
For studies involving electric fields, slices were transferred
to a standard interface recording chamber (Durand 1986 Field recording
Extracellular recordings of field potentials were made using
glass micropipettes (2-5 M For each slice, control amplitude and duration was calculated by
sampling 10 bursts during stable control conditions (at least 25 min
after switching to low calcium medium). The amplitude and duration from
10 bursts 15-60 min after pharmacological perturbation were similarly
averaged. Results are presented as means ± SD. The effects of all
the drugs used in this study were completely reversible after 10-40
min of wash.
Whole cell recording
Tight seal (4-6 G Zero-Ca2+ epileptiform activity in a submerged
recording chamber
Ten to 20 min after perfusion with zero-Ca2+
ACSF, stable field "bursts" were recorded in the CA1 pyramidal cell
layer (Fig. 1A). Once fully
established, the activity remained stable for up to 8 h. The
average burst frequency for submerged chamber experiments was 6.2 ± 2.7 per 30 s (n = 30). Within a given slice,
interevent intervals varied by as much as 50%, however, the number of
bursts in a 30-s period was very stable over time. A saturating
concentration of cadmium chloride (100-300 µM), a nonselective
blocker of voltage-activated Ca2+ channels, had
no effect on bursting, as expected (n = 2).
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
; Pumain et al.
1985
) have shown that during an epileptic seizure extracellular
calcium concentration can decrease to levels where chemical synaptic
transmission is abolished. Interestingly, several laboratories have
demonstrated the development of synchronized epileptiform activity in
hippocampal slices when synaptic transmission is blocked with a
calcium-free artificial cerebrospinal fluid solution (Jefferys
and Haas 1982
; Konnerth et al. 1984
). This "nonsynaptic" epileptiform activity is characterized by negative shifts (burst discharges) in the extracellular field potential that
propagate slowly across the pyramidal cell layer and are always
accompanied by a transient increase in extracellular potassium (Konnerth et al. 1984
).
). This hyperexcitability is
generally attributed to reduced cation screening (Frankenhaeuser
and Hodgkin 1957
; Hahin and Campbell 1983
),
block of calcium-activated hyperpolarizing currents (Alger and
Nicoll 1980
; Hotson and Prince 1980
), and reduced synaptic GABAergic inhibition (Jones and Heinemann
1987
). Enhanced ephaptic and electrotonic interactions also
contribute to the observed synchrony (Perez-Velazquez et al.
1994
; Taylor and Dudek 1982
). These nonsynaptic
mechanisms, however, are considered to be too fast to account for the
slow propagation (0.5-100 mm/s) of burst discharges observed in the
zero-calcium model (Haas and Jefferys 1984
;
Konnerth et al. 1986
). It has been suggested that an
individual burst is initiated when intense neuronal firing results in a
local increase in extracellular potassium (Hounsgaard and
Nicholson 1983
). This slow increase in
[K+]o would in turn
depolarize neighboring cells that would be induced to fire. Thus it is
generally believed that neurons contribute in a feed-forward manner to
the waves of potassium observed in this model. The slow speed of
propagation is consistent with the diffusion of potassium passively and
through glial spatial buffering mechanisms (Yaari et al.
1986
).
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
). The resulting
350-µm-thick slices were immediately transferred to a holding chamber
filled with "normal" ACSF bubbled with 95%
O2-5% CO2 (pH 7.4). Normal ACSF consisting of (in mM) 124 NaCl, 3.75 KCl, 1.25 KH2PO4, 2 CaCl2, 2 MgSO4, 26 NaHCO2, and 10 dextrose (osmolarity 305 ± 4 mosmol l
1, mean ± SD). Slices
were held at room temperature for
60 min before being transferred to
the recording chamber (Warner Instrument), where they were perfused
(3-10 ml/min) with normal ACSF (temperature, 34 ± 2 °c)
bubbled with 95% O2-5% CO2. Fluid level in
the recording chamber was maintained under 1 mm. A total of 123 hippocampal slices were used in this study.
1). For all cation screening experiments
(including BaCl2) MgCl2 was
used in place of MgSO4. 1-Octanol and 1-heptanol
(Aldrich) were diluted in zero-Ca2+ ACSF and
sonicated immediately before use. Monofluroacetic acid (FAC),
4-aminopyridine (4-AP), tetraethylammonium ion (TEA), and 4,4'-diisothiocyanatostilbebe-s,s'-disulfonic acid (DIDS) were obtained
from Sigma. CdCl2, BaCl2,
and CeCl were obtained from Fisher. 5-Nitro-2-(3-phenylpropylamino)
benzoic acid (NPPB) was obtained from RBI.
4-4'-dinitrostilbene-2,2'disolfonic acid, disodium salt (DNDS) was
obtained from Molecular Probes. Tetrodotoxin (TTX) was obtained from
Calbiochem. Patch pipettes were filled with internal recording solution
consisting of (in mM) 140 KGluconate, 2 MgCl2, 2 CaCl2, 2 Na-ATP, 0.2 Na-GTP, 10 EGTA, and 10 HEPES (pH was adjusted to 7.25 with 10 M KOH and osmolarity adjusted to
285-290 mosmol l
1).
).
Electric fields were generated across individual slices by passing
current between two parallel AgCl-coated silver wires placed on the
surface of the ACSF in the interface chamber. Slices were always
aligned such that the dendritic-somatic axis was parallel to the
direction of the field. Pulses applied to the wires were generated by a voltage generator (Master-8 Programmable Pulse Generator, A.M.P.I.) and
converted to a current pulse by a stimulus isolation unit (Grass
Instrument). The electric field (mV/mm) in the chamber was calibrated
by measuring the voltage difference (mV) between two recording
electrodes of known distance (mm). During field application, two
recording electrodes were positioned to allow for differential
recording. One electrode was positioned in the CA1 pyramidal layer and
the other aligned along an isopotential line (Fig.
3D).
) filled with 150 mM NaCl. Recording electrodes were positioned in the somatic layer of the CA1 region. Signals were amplified and low-passed filtered (1 kHz) with an AxoClamp
2B amplifier (Axon Instruments) and an FLA-01 amplifier (Cygnus
Technology) and finally stored on a DAT (MicroData System). Monopolar
stimulating electrodes were placed on the surface of the alveus. KCl
was ejected by pressure (PV 800, WPI) in the CA1 pyramidal layer via a
microelectrode filled with 20 mM KCl. Pressure pulses of 500 ms ranged
between 5 and 20 psi. In control experiments, NaCl was similarly injected.
) whole cell voltage- and current-clamp
recordings were made with an Axopatch-1D amplifier (Axon Instruments). Patch pipettes were pulled from 1.5-mm borosilicate filament containing glass tubing (Warner Instrument) using a two-stage process,
firepolished, and coated with silicone elastomer (Sylgard; Dow
Corning). The pipette was positioned under visual control with
differential interference contrast optics and infrared light (IR-DIC).
During voltage clamp cells were held at a command potential of
60 mV. No holding current was used during current-clamp recordings. Data were
transferred directly to a computer (DELL XPS H266) using a DigiData
1200 board and pCLAMP software (Axon Instruments).
RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effects of incubation in zero-Ca2+ medium in a submerged
chamber. A: field recording in CA1 stratum pyramidale 25 min after switch to zero-Ca2+ medium. B:
frame grabber image of a CA1 pyramidal cell visualized with infrared
video microscopy. C: response of a patched pyramidal
cell to current step before and shortly after perfusion with
zero-Ca2+ media. Cells displayed increased spontaneous
firing, enhanced excitability, enhanced afterdepolarizations
(DAP), and reduced slow afterhyperpolarizations following a
spike train (AHP). D: simultaneous recordings of
current-clamped pyramidal cell (top) and extracellular
field (bottom) during zero-Ca2+ burst
discharges. E: simultaneous recordings of
voltage-clamped pyramidal cell (top) and extracellular
field (bottom) during zero-Ca2+ burst
discharges. F: frame grabber image of an interneuron
visualized with infrared video microscopy. G: response
of patched interneuron to current step in normal artificial
cerebrospinal fluid (ACSF). H: simultaneous recordings
of current-clamped interneuron cell (top) and
extracellular field (bottom) during
zero-Ca2+ burst discharges. I: simultaneous
recordings of voltage-clamped interneuron (top) and
extracellular field (bottom) during
zero-Ca2+ burst discharges.
CA1 pyramidal cells and interneurons were patched in normal ACSF before
perfusion with zero-Ca2+ media. Individual
pyramidal cells were identified in the pyramidal cell layer using
IR-DIC (Fig. 1B) and by their response to depolarizing current steps (Fig. 1C); pyramidal cells exhibit spike
frequency adaptation (Lacaille and Schwartzkroin 1988).
Immediately after perfusion with zero-Ca2+
solution, pyramidal cells displayed increased spontaneous firing, enhanced afterdepolarizations, and reduced afterhyperpolarization following a spike train (Fig. 1C). Whole cell current-clamp
recordings (n = 8) during bursting showed bursts of
action potential superimposed on a slow depolarizing shift (Fig.
1D). The depolarizing shift occurred simultaneously with
extracellular bursts as expected (Haas and Jefferys
1984
). Voltage-clamp recordings (n = 9)
revealed a slow persistent inward current that underlies this slow
depolarization shift (Fig. 1E).
Interneurons in stratum lacunosom-moleculare were identified by
infrared video microscopy (Fig. 1F), by their location in the slice (at least 200 µM from the pyramidal cell layer), and by
their response to depolarizing current pulses (Fig. 1G);
interneurons exhibit little spike frequency adaptation (Lacaille
and Schwartzkroin 1988). Immediately after perfusion with
zero-Ca2+ medium, interneurons, recorded in whole
cell current clamp (n = 3), began firing tonically and
continued to fire at a high rate during zero-Ca2+
bursting (Fig. 1H). However, no increase in firing rate was
observed coincident with field burst activity. Furthermore,
voltage-clamp recordings (n = 4) showed no change in
holding current during bursting (Fig. 1I). These results
suggest that stratum lacunosom-moleculare interneurons do not play a
significant role in the generation and modulation of bursting in the
zero-Ca2+ model.
Role of neuronal firing during zero-Ca2+ bursting
Addition of 1 µM TTX, a sodium channel antagonist at a
concentration that blocks action potential generation (Hille
1992), to the perfusate medium, completely blocked spontaneous
bursting (n = 3; Fig.
2A). Localized pressure
injection of 2 mM KCl in the stratum pyramidale has been shown to be
sufficient to initiate a zero-Ca2+ burst
(Yaari et al. 1986
). Following the block of bursting
with 1 µM TTX, subsequent injection of KCl resulted in a local
negative shift in the field potential (Fig. 2, B and
C). Multiple field electrodes positioned along the CA1
layer, however, showed that this shift decayed quickly over distance
(i.e., never measurable >0.5 mm away from injection site) and always
followed the injection with no delay (Fig. 2C). Varying the
amplitude of the KCl injection varied the size of the induced potential
shift. Pressure injection of 2 M NaCl did not produce a negative shift
in the field potential (n = 2). Thus, although neuronal
firing does not appear to be essential for the generation of a
burstlike shift in the field potential, neuronal firing is required for
burst propagation.
|
Effects of neuronal excitability on zero-Ca2+ burst frequency
Because neuronal firing is required for the propagation of
zero-Ca2+ bursts, we next studied how burst
activity is affected by changes in neuronal excitability. To modulate
neuronal excitability we used cation screening and electric fields to
directly polarize cells. Consistent with earlier findings (Haas
and Jefferys 1984), increasing extracellular magnesium
concentration ([Mg2+]o)
from 1.5 to 3.2 mM decreased burst frequency but did not affect burst
amplitude or duration (Fig. 3,
A and B) in 10 slices. In two other slices in
which high burst frequency was observed (defined as >1 per 3 s),
increasing ([Mg2+]o)
increased burst amplitude. This could be due to each burst no longer
arriving during the relative refractory period of the previous one
(Konnerth et al. 1986
). Increasing
[Mg2+]o above 3.7 mM
arrested bursting, reversibly, in all slices tested (n = 3). Figure 3C shows the changes in burst frequency
observed during incremental increases in
[Mg2+]o from a
representative slice. A similar linear relationship was seen with all
slices tested (average slope,
3.8 ± 2.0; n = 3).
|
Application of uniform electric fields has also been shown to modulate
neuronal excitability (Durand 1986). In the zero calcium model, exogenous electric fields caused changes in burst frequency but
not burst amplitude or duration (n = 10) except at high
burst frequencys (>1 per 3 s; Fig. 3, D and
E). Electric fields generating a depolarization of the
somatic membrane (anode near tip of apical dendrites) enhanced burst
frequency, whereas hyperpolarizing fields (anode near basal dendrites)
reduced burst frequency. Incremental steps in field strength resulted
in a linear modulation of burst frequency (Fig. 3F) with an
average slope of 1.5 ± 0.2 (n = 3).
Factors affecting zero-Ca2+ burst amplitude
Because amplitude was not effected by neuronal excitability, we tested the hypothesis that burst amplitude could be modulated by network properties. We therefore added heptanol and octanol to the perfusate media to study the role of intracellular gap junction coupling on zero calcium bursting. Heptanol (0.5-3 mM; n = 8) and octanol (0.3 mM; n = 3) reversibly blocked bursting in all slices tested. Lower concentrations of heptanol (0.2 mM) attenuated bursting in eight slices and blocked bursting in two (Fig. 4, A and B). Both burst amplitude and duration were significantly reduced at lower concentrations of heptanol (0.2 mM). Burst frequency, however, was not consistently altered at this concentration.
|
Because heptanol can antagonize sodium currents (Largo et al.
1997; Nelson and Makielski 1991
), we tested the
effect of heptanol perfusion on the intrinsic firing properties of
patched CA1 pyramidal neurons (Fig. 4C). We found that
resting membrane potential (97 ± 5.1% control), input resistance
(96 ± 10% control), action potential amplitude (97 ± 1.5%
control), threshold (106 ± 13% control), and width (94 ± 9.7% control) were not significantly effected by heptanol at a
concentration (0.2 mM) shown to modulate burst activity
(n = 3). Taken together, these results suggest that the movement of ions through either glial or neuronal gap junctions contributes to the generation of the extracellular field shift.
Factors affecting burst duration
Because potassium concentration is known to rise during a burst
(Yaari et al. 1986), we investigated the effect of
potassium channel blockers on zero-Ca2+ bursting.
Cesium and barium have been shown to reduce Kir
(Ransom and Sontheimer 1995
) and reduce potassium uptake
by astrocytes (Ballanyi et al. 1987
; Janigro et
al. 1997
; Walz and Hinks 1985
). To investigate
the role of the Kir channel and glial potassium uptake, we perfused slices with CeCl (0.1-1 mM) and
BaCl2 (0.3-1 mM).
Zero-Ca2+ bursting was not affected at any
concentration of cesium tested (n = 7). Similarly, 300 µM Ba2+ had no effect on bursting
(n = 2), whereas 700 µM Ba2+
increased burst duration two- to sixfold (n = 4; Fig.
5A). In experiments where
individual bursts became so wide that they followed one another with no
delay, a concomitant decrease in burst frequency was observed. Higher
concentrations of barium (1 mM) blocked bursting in all slices tested
(n = 3).
|
Interestingly, Agopyan and Avoli (1988) observed a
similar dramatic increase in burst duration after removal of
extracellular Cl
. Because Ba2+ has been shown
to inhibit KCl uptake by glia (Ballanyi et al. 1987
), we
tested the effect of DNDS, a glial Cl
channel blocker
(Muller and Schlue 1998
), on burst duration. We found
that low concentrations of DNDS (20-200 µM; n = 6) had no effect of bursting. A higher concentration of DNDS (2 mM;
n = 2) resulted in a 6- to 10-fold increase in
burst duration (Fig. 5C). Two other chloride channel
blockers (Muller and Schlue 1998
), NPPB (0.1-0.2 mM,
n = 2) and DIDS (10-200 µM,
n = 9), blocked bursting at all concentrations tested.
To determine the role of outward potassium currents, we tested the
effects of TEA, an antagonist of the voltage-activated delayed
rectifier channel (Ik) (Hille
1992). TEA (25 mM, n = 4) attenuated burst
amplitude and increased burst duration (Fig. 6A). 4-AP (5 mM,
n = 3), which blocks the fast transient
voltage-activated potassium current (IA)
(Storm 1990
) did not affect bursting
(n = 3; Fig. 6B). Preliminary data
indicate that the above effects were not due to changes in solution
osmolarity.
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DISCUSSION |
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The main finding of this study is that burst frequency, amplitude, and duration can be independently modulated in the zero-Ca2+ model of nonsynaptic bursting. The following discussion will address each aspect of this finding in greater detail.
Factors affecting burst frequency or initiation
Previous studies have shown that several drugs and transmitter
candidates, which presumably interfere with neuronal excitability, modified burst frequency but not burst amplitude or duration
(Jefferys and Haas 1982). Consistent with these studies,
using both cation screening and electric fields to polarize CA1 cells,
we were able to vary the frequency of zero-calcium field bursts without
affecting burst size or duration. The effect of
[Mg2+]o on action
potential threshold changes has been shown to be linear over the
concentration range used in this study (Hahin and Campbell
1983
). Similarly, the amplitude of the membrane polarization varies linearly with the strength of the applied electric field (Nakagawa and Durand 1991
). Interestingly, for both
electric fields and cation screening, the relationship between degree
of polarization and burst frequency was found to be linear. One
explanation for this finding is that cation screening and electric
fields polarize a pacemaker cell or group of cells, modulating their
intracellular burst rate in a linear fashion, and thus the rate of
field burst initiation.
Linear modulation of action potential frequency, by neuron
polarization, has been demonstrated for maximal dentate activation-like bursting (Pan and Stringer 1996) and for endogenously
bursting CA3 cells (Johnston and Brown 1984
).
Polarization of cells with electric fields has been shown to
specifically modulate the population burst frequency of
high-K+-induced epileptiform activity (Gluckman et
al. 1996
). Elevation of extracellular potassium, which has the
effect of depolarizing cell membranes, has been shown to increase burst
frequency linearly in the zero-Ca2+ (Yaari et al.
1986
), 4-AP, high-K+, and bicuculline
(Rutecki et al. 1985
, 1987
) models of
epilepsy. Taken together these results suggest that membrane
polarization plays a central role in establishing the frequency of
epileptiform bursting.
Factors effecting burst amplitude
Electric fields, cation screening, and drugs that effect neuronal
excitability (Haas and Jefferys 1984; Jefferys
and Haas 1982
) have been shown to alter the interburst interval
but not burst size or duration. Similarly, Watson and Andrew
(1995)
found no correlation between neuronal excitability and
burst amplitude. Thus once a nonsynaptic burst is generated, its
amplitude and duration appear to be independent of the level of
neuronal excitability. This suggests that neuronal action potentials
are not directly responsible for the generation of the slow field
shift. Treatments that enhanced neuronal excitability increased the
frequency of the population spikes that are sometimes observed
superimposed on the slow zero-Ca2+ bursts induced
in an interface chamber (Hass and Jefferys 1984
). There
is no doubt that these fast spikes are summations of synchronized neuronal action potentials. We propose, however, that the slow extracellular potential shift observed in the zero-calcium model is
due, at least in part, to an influx of potassium into glia (Dietzel et al. 1989
). Recently, it was shown that block
of a field burst is always coincident with block of the accompanying potassium wave (Ghai et al. 1998
). Moreover, local KCl
injection, in the presence of TTX, could still elicit a local negative
field shift. Several groups have correlated a slow shift in the
extracellular field potential with the influx of potassium into glia
(Gabriel et al. 1998
; Heinemann and Walz
1998
). These results suggest that the field shift observed
during zero-calcium bursting is due in part to glial spatial buffering
of extracellular potassium (Yaari et al. 1986
). Another
likely contributor to the extracellular field shift is a persistent
inward sodium current into neurons (Fig. 1E).
Consistent with earlier reports, we found that heptanol
(Perez-Velazquez et al. 1994) and octanol, two gap
junction blockers, annihilated bursting. Several lines of evidence
would suggest that gap junction blockers modulate nonsynaptic bursting
via a glial specific mechanism. First, CA1 pyramidal cells exhibit very restricted coupling (Haas and Jefferys 1984
) and exhibit
little connexin expression (a gap junction protein) (Dermietzel
and Spray 1993
; Shiosaka et al. 1989
). Second,
although it has been shown that perfusion with low calcium media can
enhance coupling between a small number of nearby CA1 pyramidal cells
(Perez-Velazquez et al. 1984
), CA1 astrocytes are
characterized by an extremely high degree of cell-to-cell coupling
among hundreds of cells (D'Ambrosio et al. 1998
) and
express high levels of connexins (Dermietzel and Spray
1993
; Yamamoto et al. 1990
). Third, each burst
is accompanied by a transient increase in extracellular
K+. Glial cell membranes exhibit a selective
permeability to K+ and have been postulated to
play a central role in the buffering of extracellular
K+ changes (Nilius and Reichenbach
1988
). Taken together, these results are consistent with our
hypothesis that direct block of glial gap junctions inhibits
zero-calcium bursting and suggests that movement of
K+ through glial gap junctions directly
contributes to the generation of extracellular field shifts.
Proliferating glial tissue is found in nearly all epileptogenic lesions
(Somjen 1980; Vital et al. 1994
).
Naus et al. (1991)
found that Connexin43, the principle
gap junction protein of astrocytes, is elevated in surgical samples
removed during epilepsy resection. Lee et al. (1995)
found that gap-junction coupling was more pronounced in cells isolated
from epileptic tissue than from normal tissue. Glial proliferation and
enhanced coupling have been proposed to be factors in the generation of
seizures, however, by what mechanisms remains unclear (Lee et
al. 1995
; Somjen 1980
). Our results suggests that electrotonic coupling between glia may be critical for the buffering of extracellular potassium waves associated with seizure activity.
Factors effecting burst duration or termination
In astrocyte cultures, inward-rectifier potassium
(Kir) currents were blocked by both cesium
(Kd = 189 µM) and barium
(Kd = 3.5 µM) (Ransom and
Sontheimer 1995). Extracellular application of 1 mM cesium or
300 µM barium, however, had no effect on bursting in our studies,
suggesting that the Kir current does not
contribute to zero-calcium bursting. Furthermore, it was recently shown
that potassium channels on most CA1 astrocytes are not affected by extracellular cesium (D'Ambrosio et al. 1998
),
suggesting that Kir channels are not expressed in
this region. Because barium had no effect on burst duration at
concentrations below 700 µM (a concentration that far exceeds the
Kd to block Kir
channels), the elongation and blockage of bursts at higher
concentrations is not likely to be mediated by an action at
Kir channels.
As shown above, application of TEA resulted in an elongation of
zero-Ca2+ burst duration similar to that observed
with 700 µM barium. The increase in burst integral observed during
these two treatments may reflect an increase in net potassium uptake by
glia. This paradoxical result is supported by work showing an increase
in stimulation-induced K+ efflux after addition
of barium or TEA (Ballanyi et al. 1987; Gabriel
et al. 1998
; Jones and Heinemann 1987
). The
failure of 25 mM TEA and 5 mM 4-AP to block bursting suggests that the
annihilation of bursting by 1 mM barium is not due to inhibition of
outward potassium channels.
Several researchers have found that in addition to glial spatial
buffering (i.e., Cl-independant
K+ uptake), KCl cotransport and accumulation by
glia accompanies neuronal discharges (Dietzel et al.
1989
; Walz and Hinks 1985
). During
zero-Ca2+ bursting, large decreases in
[Cl
]o are observed
(Heinemann et al. 1992
). Removal of extracellular Cl
(Agopyan and Avoli 1988
),
addition of the Cl
channel blocker DNDS (2 mM),
or addition of 700 µM Ba2+ results in a 6- to
10-fold increase in burst duration. As such, the increase in burst
duration by 700 µM Ba2+ and block by 1 mM
Ba2+ may be explained by the inhibition of KCl
uptake (Ballanyi et al. 1987
). The difference between
the actions of heptanol and Ba2+ could be due to
the former affecting spatial buffering (which requires coupling via gap
junctions) and the latter affecting KCl accumulation by glia. If this
were the case, however, it is unclear why TEA, which should not
interfere with glial KCl accumulation, would cause an increase burst
duration. The nonspecific effects of high concentrations of TEA on
glial potassium channels have not been investigated.
It has been suggested that monofluroacetic acid (FAC) interferes with
glial cell metabolism (Largo et al. 1997) and acts as a
"suicide" agent for glial cells (Clarke 1991
). To
directly test the role of glia on zero-Ca2+
bursting, slices were perfused in 100 µM FAC. Prolonged incubation in
FAC resulted in some attenuation of burst amplitude and block of
bursting in two slices (n = 5). However, this
attenuation was always followed by a decrease in the antidromic
response size, suggesting overall tissue damage. Therefore until glial
specific agents are developed that do not produce overall tissue
damage, we will be unable to determine the precise role of glia in the modulation of nonsynaptic bursting.
Summary: relevance to epileptogenisis in vivo
From our results we conclude the following. 1) Neuronal
firing plays a central role in zero-Ca2+ burst
initiation and is required for propagation, but the level of neuronal
excitability does not effect the generation of the slow shift in the
field potential. 2) Movement of ions through gap junctions
(presumably glial) contributes to the amplitude of the extracellular
field shift. 3) Zero-Ca2+ burst
termination is mediated by neuronal potassium efflux or glial KCl
accumulation. Most current treatments of epilepsy target seizure
activity by attempting to calm neurons. Our results suggest that these
agents interfere with seizure initiation. Furthermore, we have shown
that agents that interfere with burst termination are effective at
blocking epileptiform activity. Thus rational therapies for epilepsy
could be aimed at interfering with burst termination and propagation
mechanisms, or glial cell function (Armand et al. 1997;
Somjen 1980
).
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ACKNOWLEDGMENTS |
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This work was supported by The Whitaker Foundation and National Science Foundation Grant IBN93-19591.
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FOOTNOTES |
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Address for reprint requests: D. M. Durand, Dept. of Biomedical Engineering, 3510 Charles B. Bolton Building, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106.
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 18 March 1999; accepted in final form 12 July 1999.
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REFERENCES |
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