Glutamatergic alterations in the cortex of genetic absence epilepsy rats.

Abstract

In absence epilepsy, the neuronal hyper-excitation and hyper-synchronization, which induce spike and wave discharges in a cortico-thalamic loop are suspected to be due to an imbalance between GABA and glutamate (GLU) neurotransmission. In order to elucidate the role played by GLU in disease outcome, we measured cortical and thalamic extracellular levels of GLU and GABA. We used an in vivo quantitative microdialysis approach (no-net-flux method) in an animal model of absence epilepsy (GAERS). In addition, by infusing labelled glutamate through the microdialysis probe, we studied in vivo glutamate uptake in the cortex and thalamus in GAERS and non-epileptic control (NEC) rats. Expression of the vesicular glutamate transporters VGLUT1 and VGLUT2 and a synaptic component, synaptophysin, was also measured.
Although extracellular concentrations of GABA and GLU in the cortex and thalamus were not significantly different between GAERS and NEC rats, cortical GLU uptake was significantly decreased in unrestrained awake GAERS. Expression of VGLUT2 and synaptophysin was increased in the cortex of GAERS compared to NEC rats, but no changes were observed in the thalamus.
The specific decrease in GLU uptake in the cortex of GAERS linked to synaptic changes suggests impairment of the glutamatergic terminal network. These data support the idea that a change in glutamatergic neurotransmission in the cortex could contribute to hyperexcitability in absence epilepsy.

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BioMed Central
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BMC Neuroscience
Open Access
Research article
Glutamatergic alterations in the cortex of genetic absence epilepsy
rats
Monique Touret*1, Sandrine Parrot2, Luc Denoroy3, Marie-Françoise Belin1
and Marianne Didier-Bazes1
Address: 1INSERM, U842, Lyon; Université de Lyon, Lyon1, Faculté de Médecine Laennec, UMR-S842, Lyon, F-69372, France, 2Neurochem,
Université de Lyon, Lyon1, Faculté de Pharmacie, Lyon 1 France and 3CNRS FRE 3006, Lyon; Université de Lyon, Lyon1, Faculté de Pharmacie,
Lyon 1 France
Email: Monique Touret* - touret@univ-lyon1.fr; Sandrine Parrot - sparrot@sante.univ-lyon1.fr; Luc Denoroy - denoroy@sante.univ-lyon1.fr;
Marie-Françoise Belin - belin@laennec.univ-lyon1.fr; Marianne Didier-Bazes - bazes@msn.com
* Corresponding author
Abstract
Background: In absence epilepsy, the neuronal hyper-excitation and hyper-synchronization,
which induce spike and wave discharges in a cortico-thalamic loop are suspected to be due to an
imbalance between GABA and glutamate (GLU) neurotransmission. In order to elucidate the role
played by GLU in disease outcome, we measured cortical and thalamic extracellular levels of GLU
and GABA. We used an in vivo quantitative microdialysis approach (no-net-flux method) in an
animal model of absence epilepsy (GAERS). In addition, by infusing labelled glutamate through the
microdialysis probe, we studied in vivo glutamate uptake in the cortex and thalamus in GAERS and
non-epileptic control (NEC) rats. Expression of the vesicular glutamate transporters VGLUT1 and
VGLUT2 and a synaptic component, synaptophysin, was also measured.
Results: Although extracellular concentrations of GABA and GLU in the cortex and thalamus
were not significantly different between GAERS and NEC rats, cortical GLU uptake was significantly
decreased in unrestrained awake GAERS. Expression of VGLUT2 and synaptophysin was increased
in the cortex of GAERS compared to NEC rats, but no changes were observed in the thalamus.
Conclusion: The specific decrease in GLU uptake in the cortex of GAERS linked to synaptic
changes suggests impairment of the glutamatergic terminal network. These data support the idea
that a change in glutamatergic neurotransmission in the cortex could contribute to
hyperexcitability in absence epilepsy.
Background
Generalized absence seizures, mainly seen in childhood,
are characterized by loss of consciousness associated with
bursts of bilaterally synchronous spike and wave dis-
charges (SWDs). The Genetic Absence Epilepsy Rat from
Strasbourg (GAERS) [1,2] is recognized as a valid tool for
investigating the human disorder. These rats develop
spontaneous absence seizures at about postnatal day 40
that persist throughout life. As in the human disease, the
SWDs in GAERS are generated in a cortico-thalamic loop
involving reciprocal glutamatergic projections and
gamma amino butyric acid (GABA) inter-neurons [2].
Several electrophysiological and pharmacological studies
have suggested the involvement of GABA and glutamate
Published: 29 August 2007
BMC Neuroscience 2007, 8:69doi:10.1186/1471-2202-8-69
Received: 19 December 2006
Accepted: 29 August 2007
This article is available from: http://www.biomedcentral.com/1471-2202/8/69
© 2007 Touret et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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(GLU) in the initiation and spreading of SWDs [2], with a
predominant role of GABA. Although no obvious ana-
tomical differences in these two systems have been
reported in GAERS compared to non-epileptic control
(NEC) rats, increased extracellular GABA levels in the tha-
lamus and cortex [3] and reduced GABA uptake in tha-
lamic synaptosomes [4] have been reported in GAERS. As
regards the glutamatergic system, we previously observed
deregulation of the metabolism of GLU and its membrane
transporters in the cortex and/or thalamus of young
GAERS before the occurrence of seizures [5,6], suggesting
an early impairment of the GLU neuro-circuitry, which
may be involved in the genesis of the pathology. In adult
rats, electrophysiological studies have demonstrated an
increased response to NMDA and non-NMDA glutamater-
gic receptor activation in the cortex of GAERS compared to
NEC rats [7], which may play a major role in the control
of absence seizures in the thalamus [8]. Furthermore, we,
and others found deregulation of GLU metabolism [9,5]
and perturbation of membrane transporter gene expres-
sion [6] in the cortex. However, the in vivo consequences
of these perturbations on basal levels of GLU and its clear-
ance from the extracellular space are not known.
In order to understand the involvement of GLU in the
genesis of SWDs, we measured basal extracellular GLU
and GABA levels in GAERS and NEC rats. We also evalu-
ated GLU transport capacity using an in vivo microdialysis
approach in awake animals. Furthermore, we investigated
possible alterations in glutamatergic terminals in the
GAERS cortex and thalamus by studying the expression of
the neuronal GLU vesicular transporters VGLUT1 and
VGLUT2 [10], and of synaptophysin, one of the most
abundant proteins in synaptic vesicles, used as a marker of
terminals [11,12].
Results
Microdialysis experiments
The microdialysate values (Cdial) obtained when no exog-
enous GLU or GABA was perfused through the probe did
not differ between NEC rats and GAERS in either the tha-
lamus or cortex (Table 1). The true extracellular concen-
trations (Cext), which reflect in vivo release and uptake
processes, were also determined using the no-net flux
method. In the two structures studied no difference
between NEC rats and GAERS was seen for GLU and
GABA (Table 1). However, a difference in the variance
(F37.1, p = 0.041) of GLU values have been observed in
the cortex between GAERS, and NEC, suggesting a possi-
ble instability of excitation only present in the cortex of
epileptic rats.
Estimation of GLU uptake using exogenous radioactive
GLU
In the cortex, GLU uptake was lower in GAERS than in
NEC rats (Student's t test, *p < 0.05) when a mixed solu-
tion of labeled MAN and GLU was perfused (Figure 1A).
In the presence of 20 mmol/L THA, a GLU uptake blocker,
GLU uptake was reduced and the difference between the
two strains disappeared (Figure 1B). When the same
experiment was carried out in the thalamus, no difference
was seen between GAERS and NEC rats either in the pres-
ence or absence of uptake inhibitor (Figure 1C,D).
Western blot analysis
VGLUT1 expression
Quantification of the immunolabeled band at the
expected molecular weight of about 59 kDa revealed no
difference in protein levels in either the thalamus or cortex
between GAERS and NEC rats (Figure 2).
VGLUT2 expression
When the immunolabeled band at the expected molecular
weight of about 62 kDa was quantified, an increase in pro-
tein levels (37% P < 0.01) was seen in the cortex, but not
the in thalamus, in GAERS compared to NEC rats (Figure
2).
Synaptophysin expression
When the immunolabeled band found at the expected
molecular weight of about 38 kDa was quantified, a sig-
nificant increase (21%, p < 0.05) was seen in the cortex,
but not in the thalamus, in GAERS compared to NEC rats
(Figure 3).
Discussion and conclusion
The identification of the biochemical and anatomical tar-
gets impaired in absence epilepsy is crucial to the under-
standing of the genesis of the disease and for focusing
therapy. The present work provides biochemical data sug-
gesting an alteration in GLU transmission, restricted to the
cortex of GAERS. These data are in accordance with previ-
ous studies demonstrating: a) a primary role of corticoth-
alamic neurons in the synchronized excitation of thalamic
relay and reticular neurons in GAERS [13], b) initiation of
the paroxysmal oscillation by a cortical focus in WAG/Rij
rats, another model of absence epilepsy [14], and, c)
immediate cessation of SWD on applying the anti-absence
drug, ethosuximide, to a focalized zone in the cortex, but
not the thalamus, in GAERS [15].
Using an in vivo approach, we demonstrated decreased
GLU uptake in the cortex of unrestrained awake adult
GAERS. In contrast, quantitative in vivo microdialysis
failed to show any significant change in basal GABA or
GLU extracellular levels in GAERS in the two structures
involved in SWD (thalamus and cortex). However, the

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variances for the GLU values were larger in the cortex of
GAERS, but not in the thalamus, suggesting, an instability
in glutamate excitation restricted to the cortex in epileptic
rats. However, SWDs were present in some of the time
intervals in which the microdialysis samples were col-
lected, but not in others, which might increase the varia-
bility of the GLU. Further studies taking into account this
experimental aspect by performing sub-minute sampling
during brain microdialysis will be of interest to more pre-
cisely describe alterations in extracellular GLU in the cor-
tex of GAERS.
The lower in vivo GLU uptake in the cortex of GAERS may
be due to an impairment of GLU transporters in astro-
cytes. These cells are a major contributor to the clearance
of extracellular GLU via two main glial transporters,
Table 1: Comparison of extracellular levels (Cext, i.e. Cin = Cout), microdialysate concentrations (Cdial, i.e. Cin = 0 mol/L), and in vivo
extraction efficiency (Ed) i.e. slope of Cin-Cout vs Cin) of GLU and GABA determined by the "no net flux" method (for details, see
Methods) in absence epilepsy rats (GAERS strain) and non-epileptic Wistar (NEC) rats
GLU (µmol/L)GABA (nmol/L)
GAERS NECGAERS NEC
Cortex
Cext
Cdial
Ed
Thalamus
Cext
Cdial
Ed
(5)(5) (5) (5)
1.67 ± 0.44
0.78 ± 0.10
0.59 ± 0.18
(5)
3.31 ± 146
1.94 ± 0.78
0.63 ± 0.1
0.91 ± 0.01
0.68 ± 0.1
0.76 ± 0.10
(6)
4.82 ± 1.90
2.26 ± 0.96
0.47 ± 0.08
18.1 ± 8.1
10 ± 3.0
0.67 ± 0.12
(5)
18.5 ± 4.4
15.0 ± 3.3
0.83 ± 0.05
16 ± 6.1
12.5 ± 4.2
0.82 ± 0.05
(6)
14.1 ± 1.9
12.5 ± 2.4
0.87 ± 0.09
Number of rats per group in brackets
Uptake of exogenous radioactive GLU in the cortex of GAERS (n = 6) and NEC rats (n = 6) and thalamus of GAERS (n = 6) and NEC rats (n = 6)
Figure 1
Uptake of exogenous radioactive GLU in the cortex of GAERS (n = 6) and NEC rats (n = 6) and thalamus of GAERS (n = 6)
and NEC rats (n = 6). 250 µM 3H-GLU and 120 µM 14C-MAN were infused for 10 min with or without 20 mM THA and
microdialysis samples collected every 2 min. The results, expressed as the mean ± SEM, are plotted as the extraction fraction
EL-GLU, versus time

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GLAST and GLT1, which are situated in the vicinity of the
synaptic cleft and play a crucial physiological role in the
normal functioning of neurons [16,17]. In a previous
study [6], we found decreased levels of GLAST mRNA in
the cortex of adult GAERS, without a similar change in
protein expression. This could reflect impairment of the
transcription and/or turnover of GLAST protein, leading
to the lower level of GLU uptake seen in the present study
by in vivo microdialysis. From a physiological point of
view, this lack of GLU reuptake could favor spillover of
the neurotransmitter outside the synaptic cleft and this
could be involved in the neuronal synchronization and
firing amplification giving rise to SWDs.
The second main finding of the present work was
increased VGLUT2 expression in the cortex of adult
GAERS, but not in the thalamus, while VGLUT1 expres-
sion appeared normal. Interestingly in normal adult rat
brain, VGLUT2 protein expression is mainly restricted to
lamina IV of the neo-cortex, while its mRNA is found in
the thalamus [10,18,19]. In contrast, VGLUT1 is expressed
in all layers [20] and probably originates from cortico-cor-
tical projections, as pyramidal neurons express VGLUT1
mRNA [21]. Thus, the increased VGLUT2 expression in
the cortex of GAERS strengthens the idea of a glutamater-
gic alteration in the cortex and suggests that this may
involve a glutamatergic thalamocortical input. Finally,
since VGLUT2 levels are high at birth and rapidly reach
adult values [22,23], one may hypothesize that the higher
VGLUT2 expression seen in the cortex in adult GAERS,
together with the higher levels of synaptophysin, a marker
of synaptic plasticity, might correspond to a developmen-
tal process that leads to functional synaptic alterations.
However, further studies to determine changes in VGLUT
expression during the development of GAERS, especially
in young animals before the onset of SWD, are needed to
explore this hypothesis. Such work will be of interest
given the important role of GLU in the regulation of CNS
development [24], especially in the induction or elimina-
tion of synapses and synapse plasticity [25,26], and the
Expression of synaptophysin protein in cortex and thalamus of adult GAERS (grey bars) compared to controls (NEC) (hatched bars)
Figure 3
Expression of synaptophysin protein in cortex and thalamus
of adult GAERS (grey bars) compared to controls (NEC)
(hatched bars). Sample homogenates were subjected to SDS-
PAGE and immuno-blotted with anti-synaptophysin antibod-
ies. The relative quantification was performed by a fluorim-
ager. The results are represented as a mean percentage of
control rats ± SEM. GAERS values (n = 6) were compared to
control rat values (n = 6) with a two-tailed Student's T test
(*p < 0,05)
Expression of vesicular GLU transporter proteins in the cor-tex and thalamus of adult GAERS (grey bars) and NEC con- trols (hatched bars)
Figure 2
Expression of vesicular GLU transporter proteins in the cor-
tex and thalamus of adult GAERS (grey bars) and NEC con-
trols (hatched bars). Homogenates were subjected to SDS-
PAGE and immunoblotted with anti-VGLUT1 (A) or anti-
VGLUT2 (B) antibodies. The relative quantification was per-
formed using a fluorimager. The results are represented as a
percentage of the value in NEC rats (mean ± SEM; n = 6).
Values were compared to control rat values with a two-
tailed Student's t test (*p < 0.05)

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developmental
described in the WAG/Rij epilepsy model [27].
cortical morphological alterations
Together, these observations of alterations in the cortical
glutamatergic system in adult GAERS linked to impair-
ment of cortical astrocytic function in terms of GLU
reuptake and metabolism are in line with the cortical
focus theory of absence epilepsy.
Methods
Animals
Male Wistar rats from the Genetic Absence Epilepsy Rats
from Strasbourg (GAERS) strain and non-epileptic control
(NEC) Wistars were used in this study. GAERS display
recurrent generalized absence seizures characterized by
bilateral and synchronous SWDs on the EEG, concomi-
tant with behavioral arrest. The animals were kept and
used in experiments according to the European Commu-
nities Council Directives of November 24, 1986 (86/609/
EEC). For microdialysis studies, the rats (250–300 g) were
anaesthetized with chloral hydrate (400 mg/kg/i.p.) and
placed in a stereotaxic frame (David Kopf, USA) with the
body temperature maintained close to 37.5°C using a
heated under-blanket (Harvard Instruments, USA). The
skull was exposed and, after drilling appropriate holes, a
cannula-guide (CMA12, CMA, Sweden) was implanted
either i) in the cortex at a 30° angle (coordinates relative
to the bregma: AP -3.3 mm, L +2.3 mm, V -2.5 mm below
the brain surface) or ii) in the ventrobasal thalamus (coor-
dinates relative to the bregma: AP -3.5 mm, L +2 mm, V -
6 mm below the brain surface) according to the atlas of
Paxinos & Watson [28], and secured with stainless steel
screws and dental cement. After surgery, the rats were
housed in individual cages with food and water ad libitum
for 10 days when the microdialysis experiment was per-
formed.
Microdialysis studies
Concentric microdialysis probes were constructed from
regenerated cellulose dialysis tubing (MWCO 6000 Da,
225 mm o.d., 2 mm active dialysis length) and fused-silica
capillary tubing, the body of the probe being made of a 3
cm 26 G stainless steel tube, which was glued on a flat
probe holder (Harvard, USA). Before implantation, the
probes were perfused at a rate of 1 µl/min with artificial
cerebrospinal fluid (aCSF) (149 mM NaCl, 2.80 mM KCl,
1.2 mM MgCl2, 1.2 mM CaCl2, 2.78 mM phosphate
buffer, pH 7.4). A probe was implanted into the guide-
cannula of a freely moving animal placed in the experi-
ment cylinder, the inlet and outlet of the probe being con-
nected to a liquid swivel (mouse model, Instech Solomon
USA). The rate of infusion was 1 µl/min for the in vivo
evaluation of GLU uptake and 2 µl/min for the "no net
flux" experiment. For each dialysis experiments, at least 3
hours was allowed to elapse after microdialysis probe
implantation before collection of basal samples. At the
end of the experiment, Evans blue was injected via the
probe, the rats were sacrificed with a lethal dose of pento-
barbital, and the placement of the cannula was verified on
the frozen brain [29].
"No net flux" studies
The basal extracellular concentrations of GLU and GABA
were determined by the "no net flux" quantitative micro-
dialysis method [30]. Four different concentrations of
GLU (0, 0.5, 1, and 5 µmol/L) or GABA (0, 10, 50, and
100 nanomoles/L) (Cin), chosen to bracket the expected
extracellular concentration, were passed through the
microdialysis membrane (each for 16 min, separated by
perfusion with aCSF for washing); no sample was col-
lected during the first 10 min of GLU perfusion, allowing
a period of equilibration, then three 2-min dialysis sam-
ples were collected and stored at -40°C until analyzed for
amino acid content.
The concentrations of neurotransmitter in the dialysate
samples (Cout) obtained during perfusion with the various
concentrations of neurotransmitter (Cin) were used to
construct a linear regression plot of the net change in neu-
rotransmitter (∆C = Cin - Cout) against Cin for each animal.
The true extracellular levels and the extraction fraction of
the probe were determined as described by Parsons and
Justice [31]. The true extracellular concentration is equal
to Cin when ∆C = 0 and the extraction fraction (Ed) is
determined from the slope of the linear regression. Differ-
ences in extracellular concentrations or extraction frac-
tions between GAERS and control rats were tested using
the F test followed by either Welch's test or Student's t test,
as appropriate.
Measurement of amino acid content
On the day of analysis, 4 µL of sample and 4 µL of stand-
ard solutions were derivatized at room temperature by
adding 1.6 µL of a mixture (1:2:1 v/v/v) of (i) internal
standard (10-4 mol/L cysteic acid in 0.117 mol/L perchlo-
ric acid), (ii) a borate/NaCN solution (100:20 v/v mixture
of 500 mmol/L borate buffer, pH 8.7, and 87 mmol/L
NaCN in water), and (iii) a 2.925 mmol/L solution of
naphthalene-2,3-dicarboxaldehyde in acetonitrile/water
(50:50 v/v). The samples were then analyzed for amino
acid content using an automatic capillary zone electro-
phoresis P/ACE™ MDQ system (Beckman, USA) equipped
with a ZETALIF laser-induced fluorescence detector (Pico-
metrics, France). Excitation was performed using a He-Cd
laser (Liconix, USA) at a wavelength of 442 nm, the emis-
sion wavelength being 490 nm. Separations were carried
out on a 63 cm × 50 µm i.d. fused-silica capillary (Com-
posite Metal Services, Worcester, England) with an effec-
tive length of 52 cm. Each day, before the analyses, the
capillary was sequentially flushed with 0.25 mol/L NaOH