Download Physiological and Pharmacological Alterations in Postsynaptic

Physiological and Pharmacological Alterations in Postsynaptic GABAA
Receptor Function in a Hippocampal Culture Model of Chronic
Spontaneous Seizures
JOHN W. GIBBS III, 4 SOMPONG SOMBATI, 1,5 ROBERT J. DELORENZO, 1,2,3,5
AND DOUGLAS A. COULTER 1,3,4,5
1
Department of Neurology, 2 Department of Biochemistry and Molecular Biophysics, 3 Department of Pharmacology and
Toxicology, and 4 Department of Anatomy, Medical College of Virginia; and 5 Medical College of Virginia
Comprehensive Epilepsy Institute of Virginia Commonwealth University, Richmond, Virginia 23298-0599
to induce the spontaneous epileptiform activity, which was also
a consequence of this treatment.
INTRODUCTION
Human limbic epilepsy affects ú1 million people in the
United States alone, and thus represents a major health problem (Hauser and Hersdorf 1980). Limbic epilepsy is one of
the most devastating forms of epilepsy in the adult population. About 60% of patients with intractable epilepsy (i.e.,
seizures that are not adequately controlled by medication)
have partial seizures, and a significant proportion of these
patients have seizures that originate in the limbic system
(Mikati and Holmes 1993). Limbic epilepsy frequently is
characterized electroencephalographically by complex partial seizures originating in the temporal lobe, with or without
secondary generalization to adjacent and distant cortical
areas (reviewed in Lothman et al. 1991). The limbic system,
particularly the hippocampus, is uniquely vulnerable to develop the pathological synchronized electrical activity within
populations of neurons that constitute epilepsy (Dichter and
Ayala 1987; Lothman et al. 1991; McNamara 1994). One
of the major long-term goals of research into the pathophysiology of limbic epilepsy is to understand the cellular and
molecular mechanisms underlying this limbic vulnerability
to develop seizure activity. Elucidating the mechanisms that
cause limbic epilepsy may provide important insight into the
treatment and prevention of this debilitating condition.
One research direction that has provided valuable information concerning mechanisms underlying limbic epilepsy
has been the development of in vitro limbic model systems
that can sustain epileptiform activity. For example, exposure
of hippocampal/entorhinal cortical brain slices, thalamocortical slices, or hippocampal neurons in culture to an extracellular media containing no added Mg 2/ induces sustained
spontaneous epileptiform activity in vitro during the treatment (Coulter and Lee 1993; Sombati and DeLorenzo 1995;
Walther et al. 1986; Wilson et al. 1988; Zhang and Coulter
1996; Zhang et al. 1996a,b). Despite the ability to induce
‘‘acute’’ epileptiform activity through pharmacological and
electrical manipulations, efforts directed toward induction
of an epileptic state in normal medium have been largely
unsuccessful in vitro. One such preparation has been de-
0022-3077/97 $5.00 Copyright q 1997 The American Physiological Society
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Gibbs, John W., III, Sompong Sombati, Robert J. DeLorenzo, and Douglas A. Coulter. Physiological and pharmacological alterations in postsynaptic GABAA receptor function in
a hippocampal culture model of chronic spontaneous seizures. J.
Neurophysiol. 77: 2139 – 2152, 1997. Cultured rat hippocampal
neurons previously exposed to a media containing no added
Mg 2/ for 3 h begin to spontaneously trigger recurrent epileptiform discharges following return to normal medium, and this
altered population epileptiform activity persisted for the life of
the neurons in culture ( ú2 wk ) . Neurons in ‘‘epileptic’’ cultures
appeared similar in somatic and dendritic morphology and cellular density to control, untreated cultures. In patch-clamp recordings from hippocampal pyramidal cells from ‘‘epileptic,’’
low Mg 2/ pretreated hippocampal cultures, a rapid ( within 2 h
of treatment ) , permanent ( lasting ¢8 days ) and statistically
significant 50 – 65% reduction in the current density of functional g-aminobutyric acid-A ( GABAA ) receptors was evident
when the GABA responses of these cells were compared with
control neurons. Functional GABA receptor current density was
calculated by determining the maximal response of a cell to
GABA 1 mM application and normalizing this response to cellular capacitance. Despite the marked GABA efficacy differences
noted above, the potency of GABA in activating chloride currents was not significantly different when the responses to control and ‘‘epileptic’’ pyramidal cells to multiple concentrations
of GABA were compared. The EC50 for GABA was 4.5 { 0.2
( mean { SE ) for control neurons and 3.5 { 0.4 mM, 5.2 { 0.5
mM, 3.7 { 0.3 mM, and 4.6 { 0.3 mM for epileptic neurons
2 h, 2 days, 3 days, and 8 days after low Mg 2/ pretreatment,
respectively. Modulation of GABA responses by the benzodiazepine, clonazepam, was significantly reduced in epileptic neurons compared with controls. The kinetically determined clonazepam 100 nM GABA augmentation efficacy decreased from
44.1% in control neurons to 9.3% augmentation in neurons recorded from cultures 10 days posttreatment. The kinetics of
GABA current block by the noncompetitive antagonist picrotoxin were determined in hippocampal cultured neurons, and an
IC50 of 14 mM determined. Bath application of picrotoxin at half
of the IC50 concentration ( 7 mM ) induced epileptiform activity
in control cultures and this activity appeared very similar to the
epileptiform activity induced by prior low Mg 2/ treatment. This
concentration of picrotoxin was determined experimentally to
block 30% of the GABAA-mediated receptor responses in these
cultures, and this level of block was sufficient to trigger spontaneous epileptiform activity. The 50% reduction of GABA responses induced as a permanent consequence of low Mg 2/ treatment therefore was determined to be sufficient in and of itself
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METHODS
Hippocampal culture
Hippocampal neuronal cultures were prepared from hippocampal tissue isolated from 2-day-old Sprague-Dawley rats. Neurons
were isolated, plated at a density of 2 1 10 5 /cm2 onto a confluent
astroglial support layer, and maintained in a culture incubator for
°3 wk at 377C as has been described previously (Coulter et al.
1992; Sombati and DeLorenzo 1995; Sombati et al. 1991). Although cultures were of mixed cellular morphologies, only neurons
with medium to large pyramidally shaped somas were recorded in
the present study. Cultures were exposed to culture media containing no added Mg 2/ for 3 h and then returned to a regular
culture media containing normal Mg 2/ levels, in which the cultures
were maintained. Concentrations of Mg 2/ of ú0.5 mM during the
3-h pretreatment were sufficient to block the subsequent development of epileptogenesis. Shorter exposures to low Mg 2/ ( õ1 h)
did not consistently produce a permanent alteration in neuronal
excitability. A preexposure to low Mg 2/ for 3 h and subsequent
return to a regular culture media produced optimal epileptiform
discharge activity. At the time of electrophysiological recording,
the extracellular culture medium was replaced with a N-2-hydroxyethylpiperazine-N*-2-ethane sulfonic acid (HEPES) solution composed of (in mM) 155 NaCl, 3 KCl, 1 MgCl2 , 3 CaCl2 , 0.0005
tetrodotoxin, and 10 HEPES-Na / , with pH adjusted to 7.4 with
NaOH.
Voltage-clamp recordings
Whole cell voltage-clamp recordings were conducted as previously described (Coulter et al. 1990; Gibbs et al. 1996a,c; Oh et
al. 1995). The intracellular (pipette) solution contained (in mM)
100 Trizma phosphate (dibasic), 28 trizma base, 11 ethylene glycol
bis-( b-aminoethylether)-N,N,N *,N *-tetraacetic acid, 2 MgCl2 , and
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0.5 CaCl2 , with pH adjusted to 7.35 with NaOH. Whole cell patchclamp recording techniques were conducted on a Nikon inverted
microscope equipped with Hoffman modulation contrast optics.
Electrodes (4–8 MV ) were pulled on a Narishige PP-83 microelectrode two-stage puller with thin-walled borosilicate capillary glass
(WPI, Sarasota, FL). The pipette solution also contained an intracellular ATP reconstitution system, consisting of 50 U/ml creatinine phosphokinase, 22 mM phosphocreatinine, and 4 mM MgATP. The intracellular ATP maintenance solution was used to fill
the shank of the electrode but was omitted from the solution that
was used to back-fill the tip of the electrode. Recordings were
amplified using an Axopatch 200A amplifier (Axon Instruments,
Burlingame, CA) and filtered at 5 kHz with a 4-pole Bessel filter
before digitization. All data were displayed on a chart recorder online (Gould, Model 2107, Cleveland, OH; frequency response DC50 Hz) and stored on a VCR after digitization (at 44 kHz) with a
PCM interface (Neurodata Instrument, New York). For subsequent
off-line analysis, data were played back on a chart recorder with
a frequency response of DC-25 kHz (Astro-Med DASH IV, Warwick, RI).
Drug concentrations and method of application
g-Aminobutyric acid (GABA) was prepared as a 10 mM stock
solution dissolved in the HEPES extracellular solution. Clonazepam first was dissolved in dimethyl sulfoxide (DMSO) at 100
mM and then diluted in HEPES to the final concentration. The
maximum concentration of DMSO used in cellular perfusion was
õ0.001%, with application of DMSO alone (0.1%) not altering
GABA responses. The applied drug concentrations were as follows:
1–1,000 mM GABA, 0.1–100 nM clonazepam, and 1–300 mM
picrotoxin (all from Sigma, St. Louis, MO). Solution changes were
accomplished using a modified 13 barrel ‘‘sewer pipe’’ perfusion
technique (Gibbs et al. 1996a,c), in which several solutions flowed
out of parallel Teflon tubes (0.2 mm ID) in a laminar pattern.
Rapid (50–200 ms) and complete solution changes at a constant
flow rate then were effected by moving the tube assembly laterally
in relation to the neuron under study with no cross contamination
evident between tubes. After breaking the seal and allowing Ç2–
5 min to pass to establish stable leak currents (0 to 0200 pA),
GABA was applied for 5–6 s and washed out with control external
solution for 30–40 s, all at a holding potential of 024 mV. The
cell was pretreated with test drugs without GABA solutions for
50–60 s and then test solutions were applied together with GABA.
Clonazepam drug effects were expressed as percentage augmentation of control 10 mM GABA-evoked outward currents, recorded
at a VHOLD of 020 mV. Experiments were performed at room temperature (22–247C).
Current density
Current density was calculated based on the maximal response
of a neuron to GABA 1 mM, which then was divided by the
membrane capacitance of the cell, read directly off the capacitance
compensation potentiometer on the patch amplifier, which, assuming a capacitance/membrane area relationship of 1 mF/cm2 , should
be proportional to membrane area.
Statistics
All data were analyzed by calculating the current amplitude of
test solutions relative to currents evoked by GABA application
alone, and only reversible effects were analyzed with all data expressed as means { 1 SE. Both the GABA concentration/response
and the clonazepam concentration/GABA augmentation curves
were fitted by the Marquardt-Levenberg nonlinear least squares
method. The significance of differences in fit parameter values
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scribed recently, in which repetitive ‘‘kindling’’ stimuli trigger spontaneous recurrent electrographic seizure discharges
in hippocampal-entorhinal cortical slices (Rafiq et al. 1993,
1995). Recently, two neuronal hippocampal culture models
capable of supporting long-term, spontaneous self-sustained
epileptiform activity in normal extracellular medium have
been developed (Segal and Furshpan 1990; Sombati and
DeLorenzo 1995). In the Sombati and DeLorenzo model,
these spontaneous seizurelike events persisted for the life of
the cultures ( ú2 wk) and were controlled effectively by
clinically useful partial and generalized tonic-clonic anticonvulsant drugs, but not those effective in control of absence
seizures (Sombati and DeLorenzo 1995). In this latter
model, the epileptic state is created by kindling the cultures
with a 3-h pretreatment in medium containing no added
Mg 2/ . This treatment elicits sustained seizure activity in the
cultures and this activity then resolves after return to normal
medium into recurrent electrographic seizure discharges activating large populations of neurons within the culture. This
type of neuronal culture model capable of sustaining
‘‘chronic,’’ prolonged in vitro epileptiform activity may provide a powerful tool for the study of the cellular and molecular mechanisms potentially involved in limbic epileptogenesis.
In the present study, the potential role of diminished inhibitory efficacy within the ‘‘epileptic’’ hippocampal cultures
in the generation of the epileptic state is examined using
whole cell patch-clamp recording techniques.
ALTERED GABA RECEPTORS IN EPILEPTIC LIMBIC CULTURES
between curves was assessed using constrained simultaneous curve
fitting testing the equality of parameters, and ALLFIT, as described
in De Lean et al. (1987). This method involves testing for equality
of parameters by examining the statistical consequences (via an F
test) of forcing them to be equal.
RESULTS
Characteristics of the low Mg 2/ hippocampal culture
model of epileptogenesis
action potential firing (Sombati and DeLorenzo 1995). After
the readdition of extracellular Mg 2/ after 3 h of low Mg 2/
treatment, the tonic, high-frequency discharge activity
ceased. However, a permanent ‘‘epileptic’’ alteration in electrophysiological behavior remained, consisting of spontaneous bursting reminiscent of recurrent paroxysmal depolarizing shifts recorded in epileptic preparations both in vivo and
in vitro (e.g., Coulter and Lee 1993; Schwartzkroin and
Prince 1978; Traub and Wong 1988), intermixed with prolonged (20-s to 2-min duration) seizurelike depolarizations
with overriding multispike epileptic discharges, which
ranged in frequency from 3 to 20 Hz during the seizurelike
event, as described previously (Sombati and DeLorenzo
1995) (Fig. 1B). The majority of epileptic seizures were
20–40 s in duration, but some were °2 min in duration.
This epileptic neuronal activity has been shown previously
to be a population phenomenon using paired recordings and
intracellular calcium fluorescence measurements, which con-
FIG . 1. Induction of epileptiform discharge
activity in cultured hippocampal neurons. A:
a representative intracellular recording from a
control neuron showing spontaneous excitatory and inhibitory postsynaptic potentials
(EPSPs and IPSPs) and occasional spontaneous action potentials. Expansion trace (right)
illustrates a faster speed expansion of activity
indicated (r ). Resting potentials of individual
neurons ranged from 050 to 065 mV. B: a
representative intracellular recording from an
‘‘epileptic’’ neuron obtained after a 3-h Mg 2/ free treatment, showing 4 electrographic seizure discharges arising and ceasing spontaneously. Seizure is shown at faster sweep speeds
to demonstrate progressive depolarization at
beginning of each episode, which initiated
burst discharges of increasing amplitude and
accelerating frequency. Further expansion of
seizure discharge illustrates numerous action
potentials associated with each depolarization.
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Intracellular current-clamp recordings from cultures
grown and recorded in media containing normal levels of
Mg 2/ exhibited modest levels of spontaneous activity, with
frequent spontaneous excitatory and inhibitory synaptic potentials that sometimes elicited individual action potentials
(Fig. 1A). During the 3-h low Mg 2/ extracellular exposure,
neuronal firing behavior changed from occasional individual
action potentials to spontaneous, continuous, high-frequency
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firmed simultaneous cyclical elevations of intracellular calcium concentrations in large populations of neurons in a
time scale exactly overlapping that is seen in intracellular
recordings of seizurelike epileptiform activity (Sombati and
DeLorenzo 1995).
Hippocampal neuronal cultures appeared similar in morphology and cellular density when preexposed to the low
Mg 2/ media or to normal levels of extracellular Mg 2/ (Fig.
2). Extensive dendritic arborizations with no noticeable differences in dendritic morphology or length or alterations in
gross soma morphology were observed in both control and
epileptic cultures. The photomicrographs in Fig. 2 show the
same field at different time points in normal Mg 2/ media
before epileptogenic treatment, 1 day post low Mg 2/ treatment, and 3 days posttreatment. There were no noticeable
differences in cell density or morphology of individual neurons in this culture accompanying the transition of the culture
to a chronic epileptic condition characterized by recurrent
spontaneous epileptiform activity. Hippocampal neurons
were selected for electrophysiological recording in an unbiased manner in both control and low Mg 2/ cultures. Only
medium to large pyramidally shaped cultured hippocampal
neurons were recorded, using identical criteria in both control and treatment conditions. Bipolar and multipolar neurons
or neurons that were not overtly pyramidal in shape were
not recorded. There were no grossly noticeable differences
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2/
FIG . 2. Photomicrographs of normal and low Mg
pretreated hippocampal neurons in same field at different
time points. A: neurons treated with normal Mg 2/ media.
Note extensive dendritic arborizations present in neurons
in culture. B: neurons 1 day after low Mg 2/ treatment.
C: neurons 3 days after low Mg 2/ treatment. Note that
there were no noticeable gross anatomic differences in
somatic size, dendritic length, or neuronal density between neurons in a normal Mg 2/ media and neurons preexposed to a media containing no added Mg 2/ , despite
the fact that this treatment resulted in generation of spontaneous recurrent epileptiform activity, which persisted
for life of neurons in culture. Calibration bar represents
100 mm.
ALTERED GABA RECEPTORS IN EPILEPTIC LIMBIC CULTURES
Reduced postsynaptic GABAA receptor current density
associated with epileptogenesis
Conversion of the culture to the epileptic state by low
Mg 2/ pretreatment induced a significant decrement in the
postsynaptic response to increasing concentrations of exogenously applied GABA (1–1,000 mM) (Figs. 3, 4, and 6).
Hippocampal culture neurons were voltage-clamped at a
VHOLD of 024 mV, providing a driving force of 046 mV
from the theoretical EGABA of 070 mV, as calculated by the
Goldman-Hodgkin-Katz equation for a chloride conductance
(Goldman 1943; Hodgkin and Katz 1949), assuming a phosphate to chloride permeability ratio of 0.025 and an activity
coefficient of 0.75 for the 166 mM external chloride solution
(Bormann et al. 1987). The response to exogenous GABA
application was entirely due to activation of GABAA receptors because the intracellular electrode solution contained
no potassium, which would preclude outward movement of
potassium ions; the reversal of GABA-activated conductance
was identical to that predicted for a chloride conductance
using the Goldman-Hodgkin-Katz equation (data not
shown) (see Gibbs et al. 1996a; Oh et al. 1995) and the
response was blocked completely by picrotoxin at higher
concentrations (see below) (Coulter et al. 1990). In hippocampal pyramidal neurons in all cultures, the GABA response always was found to increase as increasing concentrations of GABA were applied to cultured hippocampal neurons. The effect of increasing concentrations of applied
GABA was found to peak at 1 mM as has been seen in
acutely isolated rat (Gibbs et al. 1996a; Oh et al. 1995)
and human cortical neurons (Gibbs et al. 1996b,c). GABA
concentration response curves were fitted using a nonlinear
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least squares method assuming a monophasic sigmoidal
GABA concentration/response relationship with the equation
I Å ImaxC n /(C n / EC n50 )
where C is the GABA concentration, I is the current elicited
by a given GABA concentration normalized to the GABAA
current elicited by application of GABA 1 mM in the same
neuron, Imax is the maximal GABAA current expressed as a
percentage of the GABA 1 mM response, EC50 is the GABA
concentration eliciting half-maximal current, and n is the Hill
coefficient. A significant reduction in postsynaptic GABAA
receptor current density was observed as rapidly as 2 h after
low Mg 2/ treatment (Fig. 3), but not at a 10-min time point
during low Mg 2/ treatment (before the exchange to normal
medium, Fig. 5). The amplitude of GABAA responses to
GABA 1 mM were reduced Ç50% in low Mg 2/ treated
neurons compared with control cultures. Similar changes in
the efficacy of GABA were measured in epileptic cultures
1–4 and 8 days after low Mg 2/ pretreatment, associated
with similar (50–65%) decrements in postsynaptic functional GABAA receptor current density (Figs. 4 and 5). At
each time interval after low Mg 2/ pretreatment, GABAA
receptor current density was decreased significantly in comparison with controls and stayed depressed for the life of
the culture (Fig. 5). The current density decreased from
2.15 { 0.27 pA/ mm2 (n Å 8) in normal Mg 2/ media to
1.03 { 0.22 pA/ mm2 (n Å 6) 2 h after low Mg 2/ treatment,
1.16 { 0.27 pA/ mm2 (n Å 5) 1 day after treatment, and to
a final GABAA postsynaptic current density of 0.75 { 0.19
pA/ mm2 (n Å 5) 8 days after low Mg 2/ treatment. Additionally, current density was determined after only 10 min of
low Mg 2/ treatment to examine the short-term effects of
low-Mg 2/ treatment (n Å 4). No difference was observed
between the 10-min low Mg 2/ treatment and the control
cultures. This short 10-min low Mg 2/ treatment was not
associated with the development of spontaneous epileptiform
activity.
Postsynaptic GABAA responses
To assess whether the epileptogenesis-associated reductions in functional GABAA receptor current density were
accompanied by alterations in the properties of the residual
receptors compared with controls, GABA concentration/response curves were plotted for data obtained in response to
GABA application in concentrations ranging from 1 to 1,000
mM to cultured hippocampal neurons at various time points
after the low Mg 2/ treatment. To examine the potency of
GABA in activating GABAA receptors as it potentially varied across the various treatments, all data were normalized
to the GABA response to GABA 1 mM (so as to directly
compare potencies in differing efficacy responses). Curves
were fitted kinetically using the equation presented above.
The potency or EC50 of GABA did not change after low
Mg 2/ treatment and the induction of epileptogenesis (Fig.
6). Control GABA-evoked responses showed an EC50 of
4.5 { 0.2 mM (n Å 8; mean { SE), whereas low Mg 2/
cultures had EC50s of 3.5 { 0.1 (n Å 6), 5.2 { 0.5
(n Å 6), 3.7 { 0.3 (n Å 6), and 4.6 { 0.3 mM (n Å 5)
for 2 h, 2 days, 3 days, and 8 days after treatment, respec-
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or alterations in soma or dendritic morphology or cellular
density in control and low Mg 2/ treated neurons, and no
evidence of cell swelling, even in cells within 10 min of the
low Mg 2/ treatment, where acute effects of the treatment
would be expected to be maximal. Membrane capacitance
in control neurons was 35.1 { 1.1 pF (mean { SE, n Å 7),
compared with 30.2 { 2.0 pF in low Mg 2/ exposed neurons
10 min after treatment (n Å 5) and 45.6 { 4.0 pF at 2 h
after treatment (n Å 6), 48 { 6 pF 1 day posttreatment
(n Å 5), 33.1 { 4.3 pF 2 days posttreatment (n Å 5),
45.8 { 3.6 pF 3 days posttreatment, and 50.1 { 4.2 pF
8 days posttreatment (n Å 5). There was no statistically
significant difference in the capacitance of these populations
of neurons, even at the 10-min time point, when acute swelling effects of the low Mg 2/ treatment would be expected to
be maximal if present (P ú 0.05; 1-way analysis of variance). In contrast to the low Mg 2/ treatment, glutamate
exposure-induced excitotoxicity causes acute cell swelling
and delayed massive neuronal death (Choi 1987; Rothman
et al. 1987). In the present study, to examine the issue of
potential excitotoxicity engendered by the experimental
treatment, neuronal counts showed that the low Mg 2/ treatment induced a 6.8 { 0.6% (n Å 3 cultures) loss 48 h after
exposure compared with control cultures. This modest level
of low Mg 2/ -induced neuronal loss contrasts significantly
with glutamate exposure studies where 90–100% loss of
neurons is observed within 48–72 h after exposure (Choi
1987).
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tively. These EC50s did not significantly differ (P ú 0.05;
F test, ALLFIT).
Effects of clonazepam on GABA-induced Cl 0 currents
Cultured hippocampal neurons were voltage-clamped at
024 mV, and GABA 10 mM was applied alone and concurrently with varying concentrations of clonazepam (CNZ)
to examine CNZ augmentation of GABAA-evoked chloride
currents. This concentration of GABA was on the rising
phase of the concentration/response curve and produced
minimal desensitization (Gibbs et al. 1996a; Oh et al. 1995).
Application of CNZ in concentrations from 0.1 to 100 nM
resulted in a concentration dependent, sigmoidally increasing potentiation of GABAA current amplitude in control cultures. The equation used to fit the CNZ concentration/GABA
augmentation curve was
H
% Augmentation Å M1 C H /(C H / EC 50
)
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where M1 is the maximal CNZ effect, C is the concentration
of CNZ, H is the Hill coefficient, and EC50 is the CNZ
concentration at which half-maximal effect is seen. Clonazapam augmentation of GABA responses virtually was abolished in hippocampal cultures 10 days after low Mg 2/ pretreatment (Fig. 7). The calculated values of M1 for CNZ
augmentation of GABA responses varied between normal
and low Mg 2/ treated neurons (Fig. 7). The efficacy (M1 )
of CNZ decreased from 44.1 { 5.0% (n Å 10) in control
cultures to 9.3 { 3.2% (n Å 6) in hippocampal cultures 10
days after low Mg 2/ pretreatment.
Picrotoxin induction of epileptic discharges
Additional experiments were conducted to determine the
potential relationship between the reduction in GABAergic
efficacy associated with epileptogenesis in epileptic cultures
and the actual mechanisms responsible for generation of
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FIG . 3. g-Aminobutyric acid (GABA)
responses in control and 2 h after low Mg 2/
pretreatment hippocampal neurons. A:
traces of control and 2 h after low Mg 2/
illustrating decrement in efficacy of GABA
in these low Mg 2/ pretreated cells relative
to controls. B: GABA concentration/response curves of control and 2 h after low
Mg 2/ neurons. Note that concentration response curve for 2-h after low Mg 2/ neurons (n Å 6) shows an Ç50% decrease
in efficacy of GABA in relation to control
neurons (n Å 8).
ALTERED GABA RECEPTORS IN EPILEPTIC LIMBIC CULTURES
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the epileptic discharges. Induction of the epileptic state was
associated with a significant reduction (50–65% at GABA
1 mM) in the amplitude of GABA-evoked responses (Figs.
3–5). Experiments were designed to examine whether a
similar or smaller level of reduction in GABAergic efficacy
in normal cultures also would be associated with the occurrence of recurrent spontaneous seizurelike events, as might
be expected if the GABAergic alterations were wholly or
partially responsible for the spontaneous epileptiform activity. To do this, a noncompetitive GABAA antagonist, picrotoxin, was chosen for study, because the efficacy of this
drug is independent of the amount of GABA present in the
cultures, and the level of GABA blockade by picrotoxin
therefore could be controlled rigorously. A concentration/
response relationship for picrotoxin inhibition of GABA re-
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sponses was first determined in the hippocampal cultures
under study (Fig. 8). This is an important study to conduct,
because agonist and antagonist effects on the GABA receptor
are determined to a great extent by the subunit composition
of the GABA receptor (e.g., Lüddens and Korpi 1995; reviewed in Macdonald and Olsen 1994), which can vary
dramatically in different brain areas and under different culture conditions. Kinetic fits of the picrotoxin concentration
response relationship provided a picrotoxin IC50 of 14 mM
(n Å 7) in cultured hippocampal neurons under the present
experimental conditions. Application of picrotoxin at half
this concentration (7 mM) was sufficient to block 30% of
the GABA response in hippocampal cultures (Fig. 8). This
concentration of picrotoxin also was found to be sufficient
to trigger spontaneous recurrent epileptiform activity indis-
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FIG . 4. GABA responses in control and
1–3 and 8 days after low Mg 2/ pretreatment hippocampal neurons. A: traces of
GABA responses in a control and 1–3 and
8 days after low Mg 2/ treatment neurons
illustrating persistent decrement in efficacy
of GABA in these ‘‘epileptic’’ cells relative
to controls, when GABA is applied at concentrations of 1–1,000 mM. B: GABA concentration/response curves of control and
1–3 and 8 days after low Mg 2/ pretreatment neurons. Note that concentration response curve for 1–3 and 8 days after low
Mg 2/ neurons show a 50–65% reduction
in efficacy of GABA, with no difference in
potency in relation to control neurons.
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the reduction of postsynaptic GABAA inhibition associated
with epileptogenesis in the low Mg 2/ model could be a
decrease in the functional expression of GABAA receptor
subunits. Decreases in the mRNA levels of GABA receptor
subunits, in particular the a2 and a5 subunits, previously
have been seen acutely with the induction of epileptogenesis
in a hippocampal slice model exposed to low Mg 2/ (Vick
et al. 1996), chronically in a pilocarpine model of chronic
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FIG . 5. Time series of GABA current density illustrating decrease induced by 3-h pretreatment with a low Mg 2/ media. Note that there was no
effect after application of low Mg 2/ for 10 min (r ). Three-hour low Mg 2/
pretreament induced an Ç50–65% decrease in GABA current density as
soon as 2 h after treatment (*P õ 0.001). Reduced GABA current density
was sustained for life of culture (measured out to 8 days after treatment).
tinguishable from that seen in low Mg 2/ pretreated epileptic
cultures (Fig. 9). Therefore, the 50–65% reduction of
GABAergic efficacy measured in these cultures was sufficient to explain the occurrence of spontaneous seizurelike
activity, although other processes certainly could contribute
to the generation of these discharges.
DISCUSSION
In this study, whole cell voltage-clamp techniques were
employed to analyze the potential role of alterations in postsynaptic GABAA currents in epileptogenesis in cultured hippocampal neurons transformed to a chronic epileptic state by
pretreatment with low Mg 2/ extracellular media. Cultured
hippocampal neurons exposed to normal and low Mg 2/ extracellular levels appeared similar in morphology and cellular density and showed identical sensitivities and EC50s to
exogenous GABA application. However, neurons recorded
from epileptic cultures showed a marked reduction in the
current density of functional GABAA receptors and a diminished modulation of GABA responses by clonazepam. The
reduction in postsynaptic functional GABAA receptor current
density and altered GABAergic pharmacology induced by
an epileptogenic treatment could have resulted from several
possible mechanisms, including alterations in GABAA subunit expression, GABA receptor downregulation, or posttranslational modification of the GABAA receptor complex,
with none of these possibilities being mutually exclusive.
One important possible mechanism that could account for
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2/
FIG . 6. GABA concentration response curves of control and low Mg
pretreated neurons. A: GABA concentration response curves in control and
2 h after low Mg 2/ treated neurons, normalized to GABA response evoked
by 1 mM GABA (maximal response). Note that there was no difference
in EC50 between 2 groups of neurons. B: GABA concentration response
curves in control, 2-, 3-, and 8-day after low Mg 2/ treated neurons, normalized as in A. Note there was no difference in EC50 between controls and
treatment groups of neurons (P ú 0.05; ALLFIT).
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ALTERED GABA RECEPTORS IN EPILEPTIC LIMBIC CULTURES
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temporal lobe epilepsy (Houser et al. 1995; Rice et al. 1996),
and chronically in cultured hippocampal neurons pre-exposed to low Mg 2/ media (Blair et al. 1996). In the pilocarpine model, a2 and a5 subunit mRNA levels remained permanently downregulated in the CA1 region of the hippocampus for as long as the rats manifested recurrent seizure
discharge activity, but no changes were seen in a1 , b2 , and
g2 subunit expression (Rice et al. 1996). Importantly, the
selective decreases in mRNA expression did not correlate
with neuronal cellular loss in the pilocarpine model (Houser
et al. 1995; Rice et al. 1996). These findings suggest that
epileptogenesis may cause a selective decrease in the genetic
expression of specific GABAA receptor subunits. Additional
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data suggest that this GABA downregulation may be an
important mechanism responsible at least in part for triggering the epileptic activity evident in these cultures. In the
hippocampal culture model, a selective knockdown of the
a2 subunit with antisense oligonucleotides induced recurrent
epileptic seizure discharge activity, whereas removal of the
antisense a2 oligonucleotide resulted in a gradual decline of
the seizure discharge activity (Jakoi et al. 1996). A similar
induction of recurrent epileptic seizure discharge activity has
been obtained with a5 antisense oligonucleotide knockdown
(E. R. Jakoi, S. Sombati, and R. J. DeLorenzo, unpublished
data). The association of the decrease of GABAA receptor
subunit mRNA and the subsequent loss of the inhibition in
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FIG . 7. Traces and clonazepam (CNZ)GABA augmentation curves of control and
low Mg 2/ pretreated neurons. A: traces illustrating modulation of GABA 10 mM current by varying concentrations of CNZ in
control and 10 days after low Mg 2/ pretreatment neurons. Note lack of efficacy of
CNZ in neurons pretreated with low Mg 2/
media. B: GABA-CNZ augmentation curve
illustrating reduced efficacy of CNZ in augmenting GABA responses induced by a low
Mg 2/ pretreatment and induction of an
‘‘epileptic’’ state.
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J. W. GIBBS, S. SOMBATI, R. J. DELORENZO, AND D. A. COULTER
the low Mg 2/ model implies that decreased receptor availability may contribute to the loss of inhibition, which could
in turn contribute to increased excitability, triggering the
epileptiform discharges as observed in the this model of
epilepsy.
Other possible mechanisms could be hypothesized potentially underlying the reduced GABA current density and
altered pharmacology evident in neurons recorded after low
Mg 2/ treatment. If this treatment induces either prolonged
cell swelling or significant neurotoxicity, then the potential
exists that the composition of the medium to large pyramidal
cell populations recorded before and after treatment were
fundamentally different. The pyramidal cell population that
was recorded before treatment could have been depleted
significantly due to cell death, and a different population of
cells sampled posttreatment, with this latter population having different GABA receptors. A second possibility is that
prolonged cell swelling may have occurred after low Mg 2/
treatment, which would reduce artifactually the recorded
GABA current density by increasing cell capacitance in the
presence of unchanging numbers of GABA receptors. One
would expect that if either of these mechanisms were oc-
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curring, significant cell death or capacitance changes should
be evident in the treatment cultures compared with controls.
Only modest levels of cell death were evident in treated
cultures (6.8% on average), and no significant differences
in cell capacitance were recorded at any time point after low
Mg 2/ treatment, even at the 10-min time point, where these
cell swelling effects would be expected to be maximal. So,
although the potential for these kinds of ‘‘anatomic’’ shifts
in the recorded populations still must be considered, the
probability that these types of effects account fully for the
large amplitude shifts in both GABA current density and
benzodiazepine pharmacology alterations seem unlikely.
The EC50s for GABA activation of GABAA currents in
cultures that were exposed to normal or low Mg 2/ media
showed no statistically significant difference. This differs
from previous findings in other studies of altered GABA
responses associated with seizures, where decreases in both
GABAergic potency and efficacy were seen in acutely isolated CA1 neurons as an acute consequence of pilocarpineinduced status epilepticus compared with control rats (Kapur
and Coulter 1996) and as a consequence of prolonged epilepsy partialis continua in human cortical neurons isolated
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FIG . 8. Traces and GABA-picrotoxin
inhibition curve in control hippocampal
neurons. A: traces illustrating block of
GABA (100 mM)-evoked currents by noncompetitive GABAA antagonist, picrotoxin
(PTX; 1–300 mM). Note that increasing
concentrations of PTX produced an increase in levels of block of GABA-evoked
current. B: kinetic fits of GABA-picrotoxin
inhibition curve determined IC50 of this
drug to be 14 mM in hippocampal culture
neurons using experimental conditions of
present study (n Å 7).
ALTERED GABA RECEPTORS IN EPILEPTIC LIMBIC CULTURES
2149
from a patient with Rasmussen’s Encephalitis (Gibbs et al.
1996b) compared with control GABA responses from nonepileptic (i.e., nonfocal) human cortical neurons (Gibbs et
al. 1996c). In contrast to the lack of effect of low Mg 2/
pretreatment on the potency of GABA, reductions of functional GABAA receptor current density were evident as soon
as 2 h after low Mg 2/ pretreatment (Figs. 3–5). This low
Mg 2/ pretreatment induces continuous epileptiform highfrequency discharges, which could be equated with status
epilepticus (Sombati and DeLorenzo 1995). GABA levels
in the in vivo pilocarpine model have been shown to rise
early and to remain elevated in the late stages of pilocarpineinduced status epilepticus (Walton et al. 1990). If a similar
increase in GABA levels occurs in the cultures as a consequence of low-Mg 2/ -induced status epilepticus, one possibility is that increased levels of endogenous extracellular
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GABA in the hippocampal cultures resulting from the low
Mg 2/ status epilepticus discharges could have induced a
downregulation in GABAA receptors or subunits, which in
turn could result in a functional reduction of the postsynaptic
current density of GABAA receptors in the low Mg 2/ treated
cultures. Alternatively, acute posttranslational modulation of
the GABAA receptor may occur during prolonged epileptic
events to reduce the number of available GABAA receptors.
Elevated intracellular calcium concentrations induced by the
prolonged seizure discharges occurring as a result of low
Mg 2/ treatment could activate phosphatases or inhibit selected protein kinases and alter the phosphorylation state of
GABA receptors in the hippocampal cultures. Decreased
phosphorylation of GABAA receptors has been shown to
lead to a reduction in GABAergic function (Stelzer et al.
1988). This alteration in phosphorylation state could under-
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FIG . 9. Current clamp recordings in control and picrotoxin-treated neurons. A: an intracellular recording from a control
neuron showing EPSPs and IPSPs and occasional spontaneous action potentials. An expansion at a faster sweep speed
illustrates spontaneous synaptic activity associated with control cultures. B: a recording from a neuron treated with bath
applied picrotoxin 7 mM illustrating induction of spontaneous epileptiform bursting activity. Note that this concentration of
picrotoxin (7 mM) was sufficient to reduced GABA-evoked currents by 30% (see Fig. 8), which was less than GABA
reductions induced by the low Mg 2/ treatment ( ú50%, Figs. 3–5). Thirty percent reduction in GABA current was sufficient
to induce spontaneous epileptiform activity indistinguishable from that seen in low-Mg 2/ pretreated ‘‘epileptic’’ cultures.
Individual complex burst firing events are shown at a faster sweep speed to further illustrate morphology associated with
epileptiform activity induced by picrotoxin medium.
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functional deafferentation of interneurons in CA1 of hippocampus may occur as a long-term consequence of kindling,
resulting in decreases in feedforward and feedback inhibition
in this hippocampal subfield independent of alterations in
postsynaptic GABA receptors (Bekenstein and Lothman
1993). The low Mg 2/ culture model demonstrates not only
an acute attenuation but also a chronic, prolonged decrease in
functional GABAergic inhibition that has not been reported
previously in a model of epilepsy. The noncompetitive GABAA receptor blocker, picrotoxin, was used to mimic the
effects observed after treatment with a low Mg 2/ media
to examine the potential role of epileptogenesis associated
reductions in GABA-mediated transmission in generation of
the electrophysiological epileptic behavior of the hippocampal culture model. Bath application of picrotoxin 7 mM was
sufficient to induce spontaneous epileptiform activity indistinguishable from that seen in the epileptic cultures (Fig.
9). This concentration of picrotoxin blocked only 30% of
the GABA current (Fig. 8), whereas there was a 50–65%
reduction of the GABA-evoked responses in the epileptic
cultures (Figs. 3–5). This suggests that the level of reduction of GABA responses evident in the epileptic cultures
was more than sufficient to underlie the generation of spontaneous epileptiform activity.
Cultured hippocampal neurons exposed to a low Mg 2/
pretreatment also exhibited diminished GABAergic modulation by clonazepam. Reductions in GABAA modulation by
benzodiazepines as a result of the induction of spontaneous,
epileptiform discharge activity has important clinical implications, because loss of GABAergic modulation by anticonvulsants such as clonazepam could change clinical treatment
strategies. A decrease in the functional expression of the
g subunit, which confers benzodiazepine sensitivity to the
GABAA receptor complex (reviewed in Macdonald and
Olsen 1994), or the a subunit, to which the benzodiazepine
binds and the nature of which determines the pharmacology
of the benzodiazepine response, could be downregulated,
resulting in a decreased pharmacological efficacy to benzodiazepines (Prichett et al. 1989). Decreased efficacy of benzodiazepines has been reported as soon as 35 min after onset
of seizures in status epilepticus (Walton and Treiman 1988).
In another epilepsy model, decreased benzodiazepine binding has been demonstrated 1 mo after initial kindling in the
CA1 and CA2 regions of hippocampus (Shin et al. 1985;
Titulaer et al. 1995a,b). In the pilocarpine model of epilepsy,
a decrease in the efficacy of benzodiazepines was observed
in CA1 neurons (Gibbs et al. 1997). In the low Mg 2/ culture
model, decreased expression of the a2 and a5 subunits were
observed (Blair et al. 1995). The presence of these subunits
within a GABA receptor pharmacologically confer type II
benzodiazepine characteristics (reviewed in Macdonald and
Olsen 1994). A decrease in the overall number of GABAA
receptors or function brought about by subunit downregulation after periods of intense and prolonged epileptiform activity, such as status epilepticus, would render benzodiazepines less efficacious. This could help explain the increased
difficulty seen in the treatment of prolonged human status
epilepticus, where insensitivity to benzodiazepines is often
seen (Treiman et al. 1990). In human neurons acutely isolated from a patient with Rasmussen’s Encephalitis, efficacy
of clonazepam (100 nM) in modulating GABA responses
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lie some of the acute effects of low Mg 2/ treatment on
functional GABA receptor levels (e.g., the reduction seen 2
h after treatment, Figs. 3 and 5). Whether this could account
for long-term changes ( ú1 wk) in GABA receptor levels
remains to be determined.
The concept of an epilepsy-associated decrease in inhibition forms the basis of the ‘‘GABA hypothesis of epilepsy,’’
which posits that decreases in the strength of GABA-mediated inhibitory neurotransmission associated with epilepsy
leads to an imbalance favoring excitatory transmission and
results in the occurrence of epileptic seizure discharge activity in the brain (reviewed in De Deyn et al. 1990). Although
this hypothesis is supported by the fact that blockade of
GABA transmission by antagonists such as bicuculline and
picrotoxin can induce acute pathological burst firing behavior and epileptic discharges, that GABA agonists or GABA
metabolism inhibitors are clinically effective anticonvulsants, and that some indicators of levels of GABA function
indicate potential impairments in patients with temporal lobe
epilepsy (De Deyn et al. 1990; During et al. 1995; Johnson
et al. 1992; Savic et al. 1988), there has been a paucity of
data amassed to date in chronic epilepsy models (i.e., models
in which spontaneous recurrent seizure discharges are generated in normal medium), which demonstrate a sustained
attenuation of inhibitory transmission underlying and generating epileptogenesis. In addition, models exist that do not
manipulate the GABAergic system (e.g., low Mg 2/ perfusion, high K / perfusion) but still result in pathological epileptiform activity (e.g., Coulter and Lee 1993; Traynelis and
Dingledine 1988; Walther et al. 1986; Wilson et al. 1990;
Zhang and Coulter 1996; Zhang et al. 1996a,b).
Few models of epilepsy to date have unequivocally and
convincingly demonstrated both acute and long-term decreases in GABAergic inhibition that could underlie development of a permanent epileptic state. In the kindling model,
mRNA levels of several GABA receptor subunits vary
acutely during the kindling process in the dentate gyrus, but
return to control levels within a few days, suggesting that
reductions in the genetic expression of GABA receptors are
not involved in maintenance of the epileptic condition (Kamphius et al. 1995; Kokia et al. 1994). In fact, long-term
elevated levels of expression of both a3 and g2 GABA subunit mRNA were evident in the dentate gyrus of kindled
rats, suggesting, if altered at all, GABAergic inhibition actually might be enhanced in the dentate gyrus by kindling
(Kamphius et al. 1995). Recordings of spontaneous inhibitory postsynaptic currents in dentate gyrus of kindled rats
have shown these events to be larger in epileptic animals
than in controls, and this amplitude increase was attributed
to increased numbers of postsynaptic GABA receptors present in granule cells of epileptic animals (Otis et al. 1994).
Further electrophysiological evidence for alterations in the
functional composition of dentate gyrus GABA receptors has
suggested that this kindling-associated elevation in inhibition
may collapse due to enhanced zinc sensitivity of GABA
responses in kindled animals. It is hypothesized that zinc
release during sustained synaptic activation will act dynamically to block inhibition during pathological excitation in
the dentate gyrus of kindled animals (Buhl et al. 1996) and
recently in the pilocarpine model of epilepsy (Gibbs et al.
1997). Evidence also has been presented suggesting that
ALTERED GABA RECEPTORS IN EPILEPTIC LIMBIC CULTURES
We thank G. Berkow Schroder for technical assistance.
This work was supported by National Institute of Neurological Disorders
and Stroke Grants RO1-NS32403 and PO1-NS25630 to D. A. Coulter and
RO1-NS23350 and PO1-NS25630 to R. J. DeLorenzo, the Medical College
of Virginia MD/PhD program for J. W. Gibbs, III, and the Sophie and
Nathan Gumenick Neuroscience and Alzheimer’s Research Fund.
Address for reprint requests: D. A. Coulter, Dept. of Neurology, P.O.
Box 980599, Medical College of Virginia, Richmond, VA 23298-0599.
Received 22 April 1996; accepted in final form 9 December 1996.
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