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The Journal of Neuroscience, February 15, 2001, 21(4):1378-1384
Contribution of GABAA and GABAB Receptors
to Thalamic Neuronal Activity during Spontaneous Absence Seizures in
Rats
Rainer
Staak and
Hans-Christian
Pape
Institute of Physiology, Medical School, Otto-von-Guericke
University, 39120 Magdeburg, Germany
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ABSTRACT |
The contribution of GABAergic mechanisms in thalamic relay nuclei
to spike and wave discharges (SWDs) during spontaneous seizures was
assessed using the WAG/Rij strain of rats, an established genetic model of absence epilepsy, in combination with single-unit recordings and microiontophoretic techniques in the ventrobasal thalamic complex in vivo. Spontaneous SWDs
occurring on the electroencephalogram at 5-9 Hz were associated with
burst firing in thalamocortical neurons, which was phase-locked with
the spike component. Microiontophoretic application of the
GABAA receptor antagonist bicuculline significantly increased the magnitude of SWD-related firing in all tested cells. Application of the GABAB receptor antagonist CGP 55845A
exerted a statistically insignificant modulatory effect on neuronal
activity during spontaneous SWDs but significantly attenuated the
bicuculline-evoked aggravation of SWD-related firing. The data indicate
that, in thalamocortical neurons, (1) GABAA
receptor-mediated events are recruited with each SWD, (2) SWD-related
activity can be evoked with no significant contribution of
GABAB receptors, and (3) blockade of GABAA
receptors potentiates SWD-related activity, presumably through an
indirect effect mediated through GABAB receptors. These results vote against a predominant or even exclusive contribution of
GABAB receptors to spontaneous SWDs in thalamic relay
nuclei in the WAG/Rij strain, but rather point to a critical role of GABAA receptor activation. This conclusion is in support of
the view that the two subtypes of GABA receptors play a differential role in fast (5-10 Hz) and slow (3 Hz) spike-wave paroxysms observed during absence seizures.
Key words:
absence epilepsy; spike and wave discharges; thalamus; GABAA; GABAB; microiontophoresis; GAERS; WAG/Rij
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INTRODUCTION |
The electrophysiological hallmark of
absence seizures are bilaterally synchronous spike and wave discharges
(SWDs) on the electroencephalogram (EEG), typically occurring at three
cycles per second (Gloor and Fariello, 1988 ). Previous results from
humans as well as from experimental models have demonstrated that
cortical and thalamic networks, which generate and maintain certain
sleep rhythms, are also critically involved in the production of SWDs (Snead, 1995). Rhythmogenesis during sleep involves mutually
interconnected thalamocortical neurons and GABAergic neurons of the
reticular thalamic nucleus (NRT) (Steriade et al., 1993). These
mechanisms in the thalamus, and the GABAergic interactions in
particular, seem to be crucially involved also in the pathophysiology
of absence epilepsy. The function of GABAergic systems is generally
preserved in absence epilepsy, and an increase in GABAergic inhibition
has been found to potentiate clinical and experimental seizure activity (Snead, 1995). Two established genetic models of absence epilepsy are
the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) (Danober et
al., 1998) and the WAG/Rij strain of rats (van Luijtelaar and Coenen,
1997). In GAERS and WAG/Rij, SWD duration was exacerbated upon
injection of the GABAA receptor agonist muscimol
(Peeters et al., 1989; Liu et al., 1991). SWDs were similarly
potentiated through GABAA agonists in a number of
models, although not blocked in all models by
GABAA antagonists such as bicuculline (Snead, 1995). Moreover, injection of agonists and antagonists to the GABAB receptor subtype potentiated and dampened
SWDs, respectively (Hosford et al., 1992; Liu et al., 1992; Snead,
1992; Vergnes et al., 1997). These findings have led to the hypothesis
that an increase in GABAB receptor influence,
particularly in thalamocortical neurons, contributes to the
pathophysiological events associated with SWD generation (Crunelli and
Leresche, 1991; Snead, 1992). Studies in slice preparations of the
ferret visual thalamus in vitro have indeed demonstrated
that an increase in GABAB can shift spindle-like
activity patterns of thalamocortical neurons toward slower rhythms
resembling 3 Hz SWDs (Bal et al., 1995; Kim et al., 1997; Bal et al.,
2000; Blumenfeld and McCormick, 2000). Results from in vivo
studies in GAERS, however, have challenged this view: no evidence was
found that binding properties, density, or affinity of
GABAB receptors in the thalamus are altered
compared with nonepileptic control rats (Knight and Bowery, 1992;
Mathivet et al., 1994) or that rhythmic inhibitory potentials
suggestive of GABAB responses are generated in
thalamocortical neurons during SWDs (Pinault et al., 1998; Charpier et
al., 1999).
Therefore, the present study was undertaken to investigate the
recruitment of GABAA and
GABAB receptors during spontaneously occurring
SWDs, making use of the established WAG/Rij model of absence epilepsy
combined with electrophysiological and microiontophoretic in
vivo techniques.
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MATERIALS AND METHODS |
Acute experiments were performed in 19 male WAG/Rij rats, aged
between 180 and 300 d (210-420 gm). All experimental procedures were approved by the Animal Care and Use Committee
(53a-42502/2-028/5). Operative procedures were performed under
pentobarbital anesthesia (40-50 mg/kg, i.p.). Wounds and pressure
edges were infiltrated with xylocaine cream (2%). The rat was
positioned into a stereotaxic instrument with bregma and lambda in a
horizontal plane. Body temperature was kept constant at 36-38°C.
Epidural EEG was bilaterally monitored [anteroposterior (AP), +2.0 mm;
lateral (L), 3.6-3.8 mm; from bregma].
Recordings were performed under neurolept anesthesia (0.102 ± 0.006 mg · kg 1 ·hr1
fentanyl; CuraMed, Karlsruhe, Germany; 6.120 ± 0.307 mg · kg1 ·hr1
dehydrobenzperidol; Janssen-Cilag, Neuss, Germany). The level of
anesthesia was assessed by monitoring of the EEG and by limb withdrawal
in response to tactile stimuli. Five-barrel glass electrodes (140 5316;
Hilgenberg, Malsfeld, Germany) were used for simultaneous single-unit
recording and microiontophoresis. The recording barrel was filled with
0.5 M sodium acetate. One barrel was filled with 0.5 M sodium acetate containing 2-6% Chicago sky blue and was used for current balancing and labeling of the recording site. The
remaining barrels were filled with CGP 55845A (6 mM in 165 mM NaCl, pH 3.5, in general two barrels), bicuculline
methiodide (5 mM in 165 mM NaCl, pH 3.0), and,
in some experiments, either GABA (0.5 M, pH 3.0) or
baclofen (15 mM, pH 3.5). The impedance (measured at 1 kHz)
of the electrodes was 10-20 M . Retaining, ejection, and balance
currents were controlled through a NeuroPhore BH-2 (Medical Systems
Corporation, Greenvale, NY). Ejection and retention currents were +5 to
+50 nA, and  10 to 20 nA, respectively. Drugs were obtained from
Sigma (St. Louis, MO), except for CGP 55845A, which was kindly provided
by Novartis (Basel, Switzerland). Electrodes were positioned at AP
3.3 mm, L 3.0 mm (with reference to bregma) and lowered into the
ventrobasal thalamic complex (VB) (depth, 5.3-6.3 mm) using a
micropositioner (model 650; David Kopf Instruments, Tujunga, CA).
Single-unit activity of VB neurons was recorded with an EXT-20F
amplifier (NPI, Tamm, Germany). Recordings were high- and low-pass
filtered at 0.5 and 10 kHz, respectively. Selectivity and effectiveness
of the microiontophoretically applied substances were controlled in the
following manner. For GABAA receptors, the
GABAA receptor antagonist bicuculline and GABA were separately applied, and their effects on SWD-related unit activity
were tested (Fig. 1A).
Next, bicuculline was applied, followed by application of GABA during
maintained bicuculline ejection, termination of GABA, and, finally,
termination of bicuculline application (Fig. 1B).
Similarly, for GABAB receptors, the
GABAB receptor agonist baclofen was
microiontophoretically applied and its effects on SWDs were monitored,
followed by application of the GABAB receptor
antagonist CGP 55845A during maintained baclofen ejection, termination
of CGP 55845A, and termination of baclofen (Fig. 1C). In all
cells tested that way (n = 24), the effects of GABA
receptor agonists were reliably and reversibly antagonized during
simultaneous application of the specific receptor antagonist. Examples
are illustrated in Figure 1. It is important to note that application
of GABA readily and completely suppressed unit activity in the
thalamus, even at low ejection strengths.
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Figure 1.
Effectiveness of microiontophoretically applied
GABA antagonists on SWD-related thalamic unit activity.
A, Effects of the GABAA receptor antagonist
bicuculline (BICU) and GABA on burst firing of a
VB neuron (top trace) related to SWDs on the EEG
(bottom trace). Note the increase in SWD-related unit
activity by bicuculline and the complete blockade of unit activity by
GABA. B, Aggravating effect of bicuculline on
SWD-related burst firing of a VB neuron is abolished by simultaneous
application of GABA. Effects of bicuculline and GABA on seizure-related
burst firing are reversible. C, Depressing effect of the
GABAB receptor agonist baclofen on SWD-related burst firing
is antagonized by the GABAB receptor antagonist CGP 55845A.
Effects created by baclofen and CGP 55845A on seizure-related activity
are reversible.
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Analog data were fed into a personal computer via an
analog-to-digital interface (1401plus; Cambridge Electronics Design, Cambridge, UK). In parallel, all recordings were stored on analog tape
for off-line analysis. Data were analyzed using the SPIKE 2 software
(Cambridge Electronics Design). Single-unit activity was discriminated
from noise using a level-time function of SPIKE 2. Using the maximum
peak of the spike component of a given SWD on the EEG as a trigger,
peri-event time (PT) histograms were constructed to determine
SWD-related neuronal activity (EEG spike-triggered analysis). The
counts were calculated in 3 msec bins in a time range 80 msec before
and after the spike peak on the EEG. The average number of discharges
per each SWD were calculated from the number of spikes in PT histograms
divided by the respective number of SWDs. For evaluation of drug
effects, SWDs were monitored within intervals of 30 sec duration. When
no average change in SWD frequency was observed in at least two
subsequent intervals (control period), drug application commenced, and
the effect on SWD-related activity was monitored in five subsequent
intervals (overall duration of 210 sec). EEG-triggered PT histograms
were calculated from these intervals, each containing the same number of SWD-correlated trials. Thereafter, drug application was interrupted (recovery period), or an additional drug was simultaneously applied and
its effects were monitored in intervals as before. Data from different
cells were averaged using this protocol for each cell. Numerical values
are expressed as mean ± SEM. Statistical analysis was performed
through a two-tailed paired Student's t test (including F test) and the Wilcoxon test, and by linear regression
analysis. t test and Wilcoxon test yielded the same results
with respect to significant differences between data. p
values in text and figures refer to the t test. Differences
of p  0.05 were considered statistically significant.
Animals were killed by an overdose of pentobarbital (150 mg/kg,
i.p.), and the brain was fixed in 4% phosphate-buffered
paraformaldehyde, pH 7.4. Chicago sky blue injections were identified
in frozen frontal sections of 40 µm, counterstained with cresyl
violet. Only cells from recordings proven to be situated in the VB were
included in the analysis.
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RESULTS |
Effects of bicuculline on SWD-related firing
All rats of the WAG/Rij strain investigated under neurolept
anesthesia in the present study spontaneously developed bilaterally synchronized SWDs at 5-9 Hz on the epidural EEG, which started and
ended abruptly on a normal background pattern, as has been shown
previously for this and the GAERS strains (Inoue et al., 1993, 1994;
Seidenbecher et al., 1998). Single-unit activity in the VB during these
states was characterized by high-frequency burst-like discharges, with
each burst consisting of two to several action potentials (Fig.
2A). PT histograms
calculated from EEG spike-triggered analysis revealed the phase-locking
of unit activity in the VB with the spike component on the EEG (Fig.
2B) (Inoue et al., 1993; Seidenbecher et al.,
1998). The spike-correlated initial burst discharge was followed by a
second discharge of one to several action potentials, temporally
correlating with the wave component on the EEG, in 11.3 ± 4.2%
of SWDs (n = 2291) (Fig. 2). No silent relay cells were
observed during SWDs; in only one relay cell, relatively rare burst
discharges occurred, which, however, were temporally correlated with
the spike component on the EEG (data not shown).
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Figure 2.
Increase in SWD-related burst firing in the VB
upon microiontophoretic application of bicuculline. A,
Single-unit activity in the right VB (top trace;
calibration is 200 µV), phase-locked with the spike component of
bilaterally synchronous SWDs on the EEG (middle and
bottom traces; calibration is 500 µV), before
(CONTROL), during, and after
(RECOVERY) application of bicuculline
(BICU). Examples of single-unit burst firing are
shown at an enlarged time scale as indicated. Note the occurrence of an
afterdischarge after the initial spike-correlated burst.
B, PT histograms (3 msec bins) of activity from the
neuron in A, triggered by the spike component on the EEG
(as shown by dashed lines in A and
C), averaged from 126 trials
(C).
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Microiontophoretic application of the GABAA
receptor antagonist bicuculline evoked a significant increase in
SWD-related activity in all VB neurons tested (n = 23).
An example is illustrated in Figure 2. Typically, both the
spike-correlated initial burst discharge and the delayed afterdischarge
were potentiated. The time course of bicuculline effects and the
influence on the temporal pattern of SWD-related discharges were
investigated more quantitatively in a sample of 12 VB neurons. During
local application of bicuculline (see Materials and Methods), the
number of action potentials fired by a single VB neuron during one SWD
on the EEG steadily and significantly increased from an average of
1.9 ± 0.5 spikes to an average of 4.0 ± 0.6 spikes within
~3 min (p 0.0005). Partial recovery was
obtained within 50 sec after cessation of drug application (Fig.
3A). PT histograms revealed
that bicuculline increased the maximum and duration of the SWD-related
firing, but not the latency of onset with respect to occurrence of the
spike component on the EEG (Fig. 3B). In addition, the
occurrence of a secondary discharge after the initial burst
discharge during an SWD was not significantly different before and
during action of bicuculline (11.3 ± 4.2 vs 12.7 ± 3.7%;
n = 2291; p = 0.47). During prolonged ejection of bicuculline, single-unit discharges per SWD maintained at
this increased level, with no indication of a fading of bicuculline action for as long as 12 min (longest period of time tested;
n = 2-6) (Fig. 3C).
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Figure 3.
Time course of bicuculline effect and influence on
SWD-related single-unit activity. A, Average number of
single-unit discharges in the VB associated with one SWD on the EEG,
within intervals before (CONTROL), during, and after
(RECOVERY) microiontophoretic application of
bicuculline (BICU). Data were obtained from
EEG-triggered PT histograms, averaged from recordings in 7-12 neurons.
See Materials and Methods for details. An example at the time period
indicated is shown in B. Dashed line is
control, and solid line is bicuculline. Bin width is 3 msec,
141.9 ± 5.2 trials averaged per neuron. Asterisks
in A indicate significant differences from control
(*p < 0.05; **p < 0.02).
C, Lack of fading of bicuculline action during prolonged
ejection. Number of single-unit discharges per one SWD, under control
conditions and action of bicuculline, averaged within 3 min periods
over a total time period of 12 min. Data were obtained from
EEG-triggered PT histograms, averaged from recordings in two to six
neurons (139.5 ± 12.3 trials averaged per neuron). Number of
neurons are given in parenthesis. Asterisks indicate
significant differences (*p < 0.05;
**p < 0.02).
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Effects of CGP 55845A
The effect of the GABAB antagonist CGP
55845A on SWD-correlated single-unit activity was investigated in 16 VB
neurons. Microiontophoretic application of CGP 55845A exerted a
continuum of effects, ranging from a decrease by 35% to an increase to
133% of SWD-correlated activity in different cells. Two examples are
illustrated in Figure 4, A and
B. Linear regression analyses revealed that the maximum effect created by CGP 55845A was not dependent on (1) the number of SWD
complexes generated during a 30 sec control period before application,
(2) the change in SWD frequency occurring within two subsequent
intervals of 30 sec duration before application, (3) the overall spike
activity or SWD-correlated spike firing of a given thalamic neuron, or
(4) the size of the ejection current (data not shown). Analyzing the
effects of CGP 55845A over the whole application period in the
population of cells that was investigated revealed that the number of
action potentials associated with one SWD on the EEG was not
significantly altered (Fig. 4C).
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Figure 4.
Effects of CGP 55845A on SWD-related burst firing
in the VB. Examples in A and B illustrate
burst firing in two single VB units (top traces;
calibration is 100 µV) phase-locked with the spike component on the
EEG (bottom traces; calibration is 500 µV) before
(CONTROL) and during application of CGP 55845A. Note the
slight facilitatory (A) and disfacilitatory
(B) effect of CGP 55845A. C,
Average number of single-unit discharges associated with one SWD on the
EEG, within intervals before (CONTR.), during, and after
(RECOV.) application of CGP 55845A. Data were
obtained from EEG-triggered PT histograms, averaged from recordings in
9-16 neurons. See Materials and Methods for details. An example at the
time period indicated is shown in D. Bin width is 3 msec, 136.5 ± 11.5 trials averaged per neuron.
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Blockade of GABAA and GABAB receptors
In nine VB neurons, the maximum effects created by CGP
55845A were investigated during maintained action of bicuculline. An example is shown in Figure 5,
A and B. In these neurons, bicuculline induced a
maximal increase in seizure-related neuronal activity from an average
of 4.1 ± 0.9 to 7.9 ± 1.3 discharges per SWD
(p 0.002). It is important to add that the
addition of CGP 55845A was started within 499.9 ± 72.5 sec after
the commencement of the ejection of bicuculline, i.e., during constant
action of bicuculline (Fig. 3C). The addition of CGP 55845A
resulted in a reduction in seizure-related burst firing in all nine
tested cells (Fig. 5A,B). The
SWD-related number of discharges significantly
(p < 0.001) decreased from an average of
7.9 ± 1.3 to 6.3 ± 1.3 during CGP 55845A application within
218.9 ± 43.6 sec in the tested population of cells (Fig.
5C). Linear regression analysis revealed that the maximal
effects by CGP 55845A were not dependent on the absolute increase in
SWD-related firing caused by bicuculline or the duration of CGP 55845A
application until the maximum effect occurred (data not shown). These
effects of CGP 55845A were not or only partially reversible after
cessation of CGP 55845A application within the tested period of
time.
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Figure 5.
Effects of CGP 55845A during maintained action of
bicuculline. A, Examples of single-unit burst firing in
the VB (top trace; calibration is 200 µV) phase-locked
with the spike component on the EEG (bottom trace;
calibration is 250 µV) under control conditions, during application
of bicuculline, addition of CGP 55845A during maximal effects of
bicuculline, and removal of CGP 55845A. B, PT histograms
(3 msec bins) of activity from the neuron in A,
triggered by the spike component on the EEG, averaged from 234 trials
(3 msec bins). C, Average number of single-unit
discharges in the VB associated with one SWD on the EEG, under control
conditions, during maximal action of bicuculline
(BICU), bicuculline and CGP 55845A
(CGP), and after removal of CGP 55845A. Data were
obtained from EEG-triggered PT histograms, averaged from recordings in
nine neurons (151.7 ± 16.7 trials averaged per neuron).
Asterisks indicate significant differences
(**p < 0.005).
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DISCUSSION |
Compared with experimental models of absence epilepsy that use
pharmacologically active substances to produce epileptiform activity,
the present results were obtained in the WAG/Rij strain in
vivo, which bears the advantage of studying spontaneously
occurring SWDs in the thalamocortical synaptic network. Furthermore,
neuroactive substances were microiontophoretically applied during
single-unit recording, thereby allowing to study the contribution of
local synaptic mechanisms to SWD-correlated activity in single thalamic neurons without altering the principle characteristics of the seizures
or seizure generation. The experiments were performed under light
neurolept anesthesia, which reportedly facilitates the generation of
SWDs without altering the characteristics of paroxysmal discharges or
associated behavioral traits (Inoue et al., 1994). The present results
indicate that, in thalamocortical neurons during absence seizures, (1)
GABAA-mediated events are recruited with each
SWD, (2) SWD-related activity can be evoked with no significant
contribution of GABAB receptors, and (3) blockade of GABAA receptors potentiates SWD-related
activity, presumably through an indirect effect mediated through
GABAB receptors.
These results confirm the involvement of GABAergic mechanisms in the
thalamus during SWD generation. The thalamocortical circuits involved
in SWD generation are those that normally sustain spindle waves, which
appear on the EEG as synchronized waves of electrical activity at 7-14
Hz during early stages of slow-wave sleep (Steriade et al., 1993,
1994). Important mechanisms of spindling are reciprocal interactions
between GABAergic NRT neurons and thalamocortical neurons (McCormick
and Bal, 1997). The release of GABA from NRT neurons onto
thalamocortical neurons results in a membrane hyperpolarization and
associated removal of inactivation from a T-type calcium current, which, in turn, activates upon repolarization and triggers a rebound burst of fast action potentials. The transfer of this burst activity via excitatory synaptic connections to NRT neurons results in correlated bursting and GABA release, and the cycle starts again. Rhythmic burst activity spreads as a propagating wave through recruitment of neurons in the thalamocortical network, resulting in
spindle waves on the EEG (Contreras et al., 1996; McCormick and Bal,
1997). The shift from spindle waves to SWDs is associated with an
increase in the degree of synchronization in the thalamus and a shift
in the predominant frequency from 7-14 to ~3 Hz (Steriade et al.,
1994). Although this scenario seems to be generally accepted, an as yet
unresolved question relates to the involvement of
GABAA and GABAB receptors
in thalamocortical neurons. One line of evidence, mostly derived from
in vitro models, indicates that 7-14 Hz spindle-like rhythmic activity in thalamic networks is associated with a predominant activation of GABAA receptors in thalamocortical
neurons, whereas an increased or exclusive contribution of
GABAB receptors results in ~3 Hz oscillations
indicative of paroxysmal discharges (Crunelli and Leresche, 1991;
McCormick and Bal, 1997). The mechanistic basis being that activation
of GABAB receptors mediates hyperpolarizing membrane responses (through an increase in potassium conductance), whose amplitude and duration are increased compared with those upon
GABAA activation (associated with an increase in
chloride conductance). This then results in an increased
de-inactivation of the T-type calcium current and facilitated
production of rebound burst activity at intervals of ~300 msec,
similar to intervals of paroxysmal oscillations (Crunelli and Leresche,
1991). Studies in slice preparations of the ferret visual thalamus
in vitro have indeed demonstrated that enhanced burst firing
in cortical or NRT inputs can result in an increase in
GABAB responses and associated transition from
spindle-like to paroxysmal-like oscillations (Kim et al., 1997; Bal et
al., 2000; Blumenfeld and McCormick, 2000).
The present findings confirm these conclusions, in that a
bicuculline-sensitive, most likely GABAA
receptor-mediated component of discharges was recruited during each
SWD. The present data do not support, however, the hypothesis of a
significant or even exclusive contribution of
GABAB receptors to spontaneous SWDs in
thalamocortical neurons in this model, because the
GABAB receptor antagonist CGP 55845A had no
significant effect on SWD-related burst firing. In agreement with this
conclusion are recent intracellular studies in GAERS, which obtained
evidence for the occurrence of rhythmic inhibitory potentials
suggestive of GABAA but not
GABAB receptor activation during SWDs in
thalamocortical neurons (Pinault et al., 1998; Charpier et al., 1999).
Previous studies involving systemic application or microinjection into
the thalamus of GABAA or
GABAB antagonists and/or agonists in GAERS
or WAG/Rij (Liu et al., 1991, 1992; Snead, 1992; Vergnes et al., 1997)
are difficult to compare with the present one, because the basic
activity of large populations of cells may have been affected,
regardless of the mechanisms by which single neurons are recruited
during each SWD. In fact, the number, affinity, and expression of
GABAA and GABAB receptors
are not altered in the thalamus of epileptic compared with control rats
(Knight and Bowery, 1992; Snead et al., 1992; Mathivet et al., 1994),
arguing against an imbalance in receptor populations. That
GABAB receptors are operative (and pharmacologically responsive) under the present experimental conditions is demonstrated during pharmacological blockade of
GABAA receptors, which resulted in a CGP
55845A-sensitive enhancement of SWD-related burst firing. It seems
reasonable to speculate that these effects reflect the enhancement of
GABAB-mediated inhibitory potentials upon
blockade of GABAA receptors, as observed in
thalamocortical neurons in vitro (Crunelli and Leresche,
1991) and which has indeed been observed to facilitate paroxysmal-like
oscillations in thalamic networks in vitro (Kim et al.,
1997). The underlying mechanistic basis is presumably a reduction in
shunting effect of the GABAA chloride conductance
(Crunelli and Leresche, 1991) and/or transsynaptic effects mediated via
GABAB receptors (Mody et al., 1994) during blockade of GABAA receptors.
It is important to note that SWDs in WAG/Rij occur at a range of 5-10
Hz (Inoue et al., 1994), thereby differing from the "classical"
~3 Hz SWDs seen in the EEG of human petit mal patients (Malafosse et
al., 1994) and various experimental models, including monkeys
(Steriade, 1974) and cats (Gloor and Fariello, 1988). These differences
may indicate the involvement of multiple mechanisms in the generation
of different forms of SWDs. For instance, a computational model
suggested that the two different frequency ranges, 2-4 and 5-10 Hz,
reflect the predominant influence of GABAA and
GABAB receptors in thalamocortical relay cells,
respectively (Destexhe, 1999). The present study provides experimental
evidence in support of this model in that GABAA
receptors were found to be activated upon 5-9 Hz spike-wave
oscillations in thalamocortical neurons during spontaneous seizures,
whereas GABAB receptors did not significantly
contribute. In line with these results are the findings by
Castro-Alamancos (1999) that infusion of GABAA
receptor antagonists into the thalamus induced two forms of
epileptiform discharges in neocortex, namely 3 Hz discharges blocked by
thalamic infusion of GABAB antagonists and 12 Hz
discharges insensitive to thalamic infusion of
GABAB antagonists. Following from that and the
present study is the conclusion that activation of
GABAA receptors in thalamocortical neurons plays
a critical role during generation of the "fast" 5-10 Hz spike-wave
paroxysms and that blockade of the GABAA
receptors can unmask a GABAB receptor-mediated component during SWDs. That a shift in the major frequency of burst
discharges from 5-10 to ~3 Hz, as typically occurs upon blockade of
GABAA receptors in vitro or widespread
infusion of GABAA antagonists in vivo,
has not been observed in the present study is most likely
attributable to the very localized application of the receptor
antagonist through microiontophoresis, which cannot be expected to
change the general characteristics of a spontaneous SWD. In any case,
the conclusion of a significant contribution of
GABAA receptor activation in thalamic relay
neurons to the production of spike-wave paroxysms may relate to the
clinical observation that phenobarbital can worsen absence seizures
(Malafosse et al., 1994). Similar conclusions have been reached in the
lethargic mouse model, in which the production of 5-6 Hz epileptiform
spike bursts on the EEG was facilitated upon microinjection of low
concentrations of muscimol or phenobarbital into thalamic relay nuclei
(Hosford et al., 1997). Absence seizures in that model have been
reported to be also regulated by GABAB receptors
in the thalamus, although by a receptor subpopulation restricted to
particular thalamic structures not involving prototypical relay nuclei
(Hosford et al., 1995). In fact, GABAB responses
were unaltered in VB neurons of lethargic compared with control mice
(Caddick and Hosford, 1996). That GABAB receptors
may play a role for modulating SWD patterns also in thalamic relay
nuclei of the rat models under study cannot be ruled out. For instance,
the presence of a long-lasting hyperpolarizing response enveloping
SWD-related paroxysmal oscillations in thalamocortical neurons in GAERS
has been suggested to represent functional GABAB
receptors (Pinault et al., 1998), which may contribute to the rather
variable (albeit statistically not significant) effect of CGP 55845A
observed in the present study.
In summary, experimental as well as computational models of absence
epilepsies suggest that GABA receptors in thalamic relay nuclei are
differentially recruited during the production of thalamocortical oscillations related to seizures, with GABAA
receptors predominating during fast (5-10 Hz) and
GABAB receptors predominating during slow (2-4
Hz) spike-wave paroxysms, respectively. However, available evidence
seems to be difficult to reconcile with the hypothesis that an
imbalance of GABAergic neurotransmission within thalamic relay nuclei
per se is critically involved in SWD generation during spontaneous
seizures in genetic rat models (Danober et al., 1998). The increase in
extracellular GABA concentration in thalamic relay nuclei in epileptic
compared with control rats (Richards et al., 1995) with no associated
change in the number of GABAergic neurons (Spreafico et al., 1993) may
not necessarily relate to an alteration of GABAergic mechanisms within
these nuclei but rather to a potentiated burst firing of GABAergic NRT
neurons resulting from an increased expression of T-type calcium
channels (Tsakiridou et al., 1995; Talley et al., 2000) and/or an
indirect effect mediated via recurrent inputs from a hyperexcitable
cortex (Bal et al., 2000; Blumenfeld and McCormick, 2000).
| |
FOOTNOTES |
Received July 26, 2000; revised Nov. 14, 2000; accepted Nov. 27, 2000.
This research was supported by Kultusministerium des Landes
Sachsen-Anhalt Grant FKZ 1906A/2587B. We thank Sieglinde Staak for
expert technical assistance and Anja Reupsch for help with histological procedures.
Correspondence should be addressed to Hans-Christian Pape, Institute of
Physiology, Medical School, Otto-von-Guericke University, Leipziger
Strasse 44, D-39120 Magdeburg, Germany. E-mail:
hans-christian.pape{at}medizin.uni-magdeburg.de.
| |
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ABSTRACT
INTRODUCTION
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