# The conversion of glutamate by glutamine synthase in neocortical astrocytes from juvenile rat is important to limit glutamate spillover and peri/extrasynaptic activation of NMDA receptors

Yosra Trabelsi, Mohamed Amri, Hélène Becq, Florence Molinari, Laurent Aniksztejn

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Yosra Trabelsi, Mohamed Amri, Hélène Becq, Florence Molinari, Laurent Aniksztejn. The conversion of glutamate by glutamine synthase in neocortical astrocytes from juvenile rat is important to limit glutamate spillover and peri/extrasynaptic activation of NMDA receptors. Glia, 2017, 65 (2), pp.401-415. 10.1002/glia.23099. hal-01962551

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# The Conversion of Glutamate by Glutamine Synthase in Neocortical Astrocytes from Juvenile Rat Is Important to Limit Glutamate Spillover and Peri/Extrasynaptic Activation of NMDA Receptors

Yosra Trabelsi,<sup>1,2</sup> Mohamed Amri,<sup>3</sup> Hélène Becq,<sup>1,2</sup> Florence Molinari,<sup>1,2</sup> and Laurent Aniksztejn<sup>1,2</sup>

Glutamate transporters (EAATs) are important to maintain spatial and temporal specificity of synaptic transmission. Their efficiency to uptake and transport glutamate into the intracellular space depends on several parameters including the intracellular concentrations of Na<sup>+</sup> and glutamate, the elevations of which may slow down the cycling rate of EAATs. In astrocytes, glutamate is maintained at low concentration due to the presence of specific enzymes such as glutamine synthase (GS). GS inhibition results in cytosolic accumulation of glutamate suggesting that the conversion of glutamate by GS is important for EAATs operation. Here we recorded astrocytes from juvenile rat neocortical slices and analyzed the consequences of elevated intracellular glutamate concentrations and of GS inhibition on the time course of synaptically evoked transporter current (STC). In slices from rats treated with methionine sulfoximine (MSO), a GS inhibitor, STC evoked by short burst of high frequency stimulation (HFS; 100 Hz for 100 ms) but not by low frequency stimulation (LFS; 0.1 Hz) was twice slower than STC evoked from saline injected rats. Same results were obtained for astrocytes recorded with pipette containing 3–10 mM glutamate and compared with cells recorded with 0 or 1 mM glutamate in the patch pipette. We also showed that HFS elicited significantly larger NMDAR-excitatory postsynaptic currents (EPSCs) with a stronger peri/extrasynaptic component in pyramidal cells from MSO-treated compared with saline treated rats. Taken together our data demonstrate that the conversion of glutamate by GS is fundamental to ensure an efficient clearance of glutamate by EAATs and to prevent glutamate spillover.

GLIA 2016;00:000–000

**Key words:** glutamine synthase, glutamate transporters, astrocytes, NMDA receptors, glutamate spillover, neocortex, methionine sulfoximine

## Introduction

Glutamate is the main excitatory transmitter in the CNS and plays major roles in brain development and complex physiological functions, including neurogenesis, cell migration, synapse formation, learning, memory, plasticity. Dysregulation of glutamate metabolism or homeostasis has been involved in severe pathologies such as neurodegenerative diseases (amyotrophic lateral sclerosis, Alzheimer's disease, ...) and epilepsies (Danbolt, 2001; Maragakis and Rothstein,

2004; Vandenberg and Ryan, 2013). The influence exerted by glutamate in the brain is determined by the spatiotemporal distribution of its concentration in the extracellular space ([Glu]<sub>o</sub>). This parameter influences the number and subtype of glutamate receptors activated by the transmitter and so the amplitude and the duration of the synaptic signal. Regulation of [Glu]<sub>o</sub> is strongly controlled by excitatory amino acid transporters (EAATs). EAATs are located at the cell surface of neurons and glial cells and they bind and transport glutamate

View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.23099

Published online Month 00, 2016 in Wiley Online Library (wileyonlinelibrary.com). Received July 1, 2016, Accepted for publication Oct 26, 2016.

Address correspondence to: Laurent Aniksztejn, INMED-INSERM U901, Parc Scientifique de Luminy, 13273 Marseille cedex 09, France.
E-mail: laurent.aniksztejn@inserm.fr

From the ¹Institut de Neurobiologie de la Méditerranée (INMED), Aix-Marseille Université, Marseille 13009, France; ²INSERM, UMR_S 901, Marseille 13009, France; ³Laboratoire de Neurophysiologie Fonctionnelle et Pathologies, Tunis UR11ES09, Tunisie
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from extracellular to intracellular spaces (Cavelier et al., 2005; Danbolt, 2001; Huang and Bergles, 2004; Grewer and Rauen, 2005; Tzingounis and Wadiche, 2007; Vandenberg and Ryan, 2013). Through these actions, EAATs preserve the responsiveness of glutamate receptors and prevent against glutamate-induced excitotoxicity. EAATs also help to maintain spatio-temporal specificity of synaptic transmission by preventing against the diffusion of glutamate release out of active synapses. Moreover, EAATs contribute to maintain a low ambient concentration of glutamate (Arnth-Jensen et al., 2002; Asztely et al., 1997; Bergles et al., 1999; Diamond, 2001; Herman and Jahr, 2007; Huang and Bergles, 2004; Marcaggi and Attwell, 2004; Takahashi et al. 1997; Tzingounis and Wadiche, 2007). Five subtypes of EAATs have been identified so far: EAAT1/GLAST (humans/rodents) and EAAT2/GLT1 are expressed in glial cells; in the cortex GLT1 is also detected in neurons in presynaptic terminals. The three others (EAAT3/EAAC1; EAAT4; EAAT5) are expressed in neurons (Danbolt, 2001; Melone et al., 2009). In the cortex, neuronal glutamate transporters act primarily as a glutamate buffering system but the transport of glutamate is mainly achieved by glial cells (Diamond, 2001; Tzingounis and Wadiche, 2007; Scimemi et al., 2009). Indeed, glial cells are responsible for the absorption and metabolism of the major part of glutamate in the brain (Danbolt, 2001). This is due to: (i) high glutamate transporters expression on glial cells; (ii) their very hyperpolarized resting membrane potential and low input resistance that prevent against strong depolarization. Altogether this provides favorable conditions for the translocation of glutamate into the cells (Barbour et al., 1991; Brew and Attwell, 1987; Huang and Bergles, 2004). Another important parameter that is likely to play an important role is the intracellular glutamate concentration, which in the cytoplasm of astrocytes, is 10- to 20-fold lower than in neurons (Nedergaard et al., 2002; Zhou et al., 2014). Different studies have demonstrated that intracellular glutamate elevation, together with $Na^+$ intracellular elevation, decreased glutamate transporters current evoked by exogenous application of glutamate, and affected the net number of molecules of glutamate transported (Barbour et al., 1991; Otis and Jahr, 1998). The low intracellular glutamate concentration in astrocytes is due to several enzymes located in the cytosol and the mitochondria that rapidly catabolize the amino acid into different metabolites. In the cytosol the most important one is glutamate synthase (GS). This enzyme, which is specifically expressed by astrocytes, converts glutamate that has been taken up by glutamate transporters into glutamine in the presence of ATP and $NH_3$. Reduced expression or inactivation of GS is associated with seizures in both human and rodents (Cloix et al., 2010; Eid et al., 2004, 2008, 2013; Häberle et al., 2005, 2011). It was proposed that GS deficiency may

impact on glutamate transporters operation since its inhibition leads to an accumulation of glutamate in astrocytes (Coulter and Eid, 2012; Eid et al., 2013; Laake et al., 1995; Perez et al., 2012). Interestingly, we and others reported that a deficit in glutamate transport caused seizures *in vivo* and epileptical like activities *in vitro* in both hippocampus and neocortex suggesting a possible link between GS and EAATs (Cattani et al., 2007; Demarque et al., 2004; Milh et al., 2007; Molinari et al., 2012; Sepkuty et al., 2002; Tanaka et al., 1997). However, evidences that GS inactivation affected glutamate transporters efficiency have not yet been provided. In the present study, we investigated this issue by measuring the time course of synaptically activated transporter current (STC) in juvenile rat neocortical astrocytes and showed that inactivation of GS slowed the decay time constant of STC evoked by short burst of high frequency stimulation and facilitated activation of peri/extrasynaptic NMDA receptors.

## Materials and Methods

### Slices Preparation

Experiments were performed on astrocytes and pyramidal neurons located in layer IV/V of neocortical slices obtained from postnatal day 13-16 old Wistar rats (males/females) (INMED animal facilities).

Wistar rats were decapitated under chloral hydrate anesthesia (20-40 mg/kg). Brains were rapidly removed and placed in oxygenated ice-cooled artificial cerebrospinal fluid (ACSF) with the following composition (in mM): 126 $NaCl$, 3.5 $KCl$, 2 $CaCl_2$, 1.3 $MgCl_2$, 25 $NaHCO_3$, 1.2 $NaHPO_4$, and 10 glucose (95% $O_2$-5% $CO_2$, pH 7.4). Coronal neocortical slices (400 µm thick) were obtained with a vibratome (Leica VT1200S). Following sectioning, slices were incubated in oxygenated ACSF containing 1 µM sulforhodamine 101 (SR-101) for 20 min at 34°C followed by 10 min incubation in ACSF for 10 min at 34°C. This procedure described by Kafitz et al. (2008) allows identifying astrocytes in slices for electrophysiological recordings. Slices were kept in ACSF at room temperature at least 1 h before used. Individual slices were then transferred to the recording chamber where they were fully submerged and superfused with ACSF at 34-35°C at a rate of 4.5-5 mL/min.

### Electrophysiology

Astrocytes were recorded under visual control with a Zeiss Axioscope microscope using whole cell configuration. Patch electrodes were made from borosilicate glass capillaries (GC150F-15, Clark Electromedical Instruments). They had a resistance of 4-6 M Ohm and were filled with a solution of the following composition (in mM): 130 $KGlu$, 10 $EGTA$, 10 $HEPES-Na$, 3 $Mg^{2+}$-ATP, 0.3 $Na^+$-GTP, 10 phosphocreatinine. In some experiments L-glutamate at 1, 3, or 10 mM was added to the pipette solution.

Astrocytes were held at -80 mV and transporter currents were evoked by electrical stimulation (synaptically evoked transporter
current, STC) using a bipolar nickel-chrome electrode positioned
near the recorded astrocyte. The pipette solution used to record
astrocytes and the holding potential allow minimizing the uncoupled
EAAT anion current to STC. The stimulation protocol is close to
that described by Diamond and Jahr (2000) and consisted to evoke
STC by 5–10 stimuli at 0.1 Hz (60 µs duration, the intensity
adjusted to evoke a response of 150–250 pA) corresponding to low
frequency stimulation (LFS) followed by 10 stimuli at 100 Hz and
30 s latter by 11 stimuli at 100 Hz corresponding to high frequency
stimulations (HFS). Each series of stimulation (0.1 Hz followed by
100 Hz) were repeated maximum 10 times when possible with an
interval of 30 s. These series were numbered as serie #1, serie #2,...
serie#10. STC elicited by high frequency stimulation (HFS) was
extracted after subtracting transporter current evoked by 11 stimuli
from that evoked by 10 stimuli. The response is named STC<sub>HFS</sub>
whereas STC evoked at 0.1 Hz is named STC<sub>LFS</sub>. STC were
recorded in continuous presence of D-(-)-2-amino-5-phosphono-
pentaolic acid (D-AP5, 50 µM); 2,3-dioxo-6-nitro-1,2,3,4-tetrahydro-
benzo[f]quinoxaline-7-sulfonamide (NBQX, 20 µM); 6-imino-3-(4-
methoxyphenyl)-1(6H)-pyridazinebutanoic acid hydrobromide
(SR95531, Gabazine, 5 µM); (2S)-2-amino-2-[(1S,2S)-2-carboxy-
cycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495, 1 µM);
(2S)-3-[[[(1S)-1-(3,4-dichlorophenyl)ethyl] amino-2-hydroxy-
propyl] (phenylmethyl)phosphinicacid hydrochloride (CGP55845A,
10 µM); nipecotic acid (1 mM), BaCl₂ (200 µM). These drugs were
used to block ionotropic and metabotropic GABA and glutamate
receptors, GABA transporters, and Kir channels. All these drugs
were purchased from Tocris Bioscience.

Neurons were recorded in whole cell configuration with a
patch electrode filled with a solution of the following composition
(in mM): 130 CsGlu; 10 HEPES, 10 BAPTA, 3 Mg²⁺-ATP, 0.3
Na⁺-GTP, 10 phosphocreatinine. BAPTA was used to prevent syn-
aptic plasticity (long-term potentiation/depression). NMDAR-and
AMPAR-mediated excitatory postsynaptic currents were evoked
either at 0.1 Hz (NMDAR-EPSC<sub>LFS</sub> or AMPAR-EPSC<sub>LFS</sub>) or by 10
pulses at 100 Hz (NMDAR-EPSC<sub>HFS</sub> or AMPAR-EPSC<sub>HFS</sub>).
EPSCs were recorded in continuous presence of Gabazine (5 µM),
LY341495 (1 µM) and supplemented with NBQX (20 µM) when
NMDAR-EPSC were recorded or D-APV (50µM) when AMPAR-
EPSC were recorded. The extracellular solution contained also high
concentrations of Mg²⁺ and Ca²⁺ (6 and 4 mM, respectively) to
block polysynaptic activity.

All measurements were filtered at 3 kHz using an EPC-9
amplifier (HEKA Electronik), electrophysiological data were digitized
(1–2 kHz) with a Digidata (Axon Instruments) interface card to a
personal computer.

To measure the decay time constant, current traces were fitted
with a single or a double exponential function of the following
form: y = Afastexp(τ/τfast) + Aslowexp(τ/τslow) (where Afast and
Aslow are the fractions of the fast and slow component of the cur-
rent and τfast and τslow are the respective fast and slow time con-
stant). The time constant representing the weighted average of the
fast and slow components of current decay was calculated with the
following equation: τ = (τfastAfast + τslowAslow)/(Afast + Aslow).
Currents were analyzed using Origin 8.0 software.

Access resistance was measured from the peak of the current
transient elicited by a 2 mV test pulse of 50 ms duration and
applied before each EPSC<sub>LFS</sub> or STC<sub>LFS</sub> and was not compensated.
The access resistance ranged between 20 and 30 M Ohm. Experi-
ments were stopped when the resistance was increased by more than
20%. Membrane potentials of astrocytes and neurons were not cor-
rected for liquid junction potential.

**Injection of MSO**

L-methionine-DL-sulfoximine (MSO, Sigma Aldrich France) was dis-
solved in NaCl (0.09%) and administered intraperitoneally in a dose
of 170 mg/kg as previously described (Sellinger et al., 1968) the day
before electrophysiological recordings. Sham animals were injected
only with NaCl 0.09%.

**HPLC Analysis for Amino Acids Levels**

Twenty-four hours after the injection of MSO or NaCl, the brain
was removed and coronal cortical slices were obtained as described
earlier. After incubation for 1 h in ACSF at room temperature, slices
were freeze-dried, dialyzed with lysis buffer (50 mM TrisHCl, 1 mM
EDTA, 1% Triton; 10 µL/1 mg of tissue), sonicated and centrifuged
(5 min, 15,000g at 4°C). Supernatants were deproteinized with
perchloric acid (0.5 M, v/v) for 10 min at 4°C and precipitates were
spun down (10 min, 15,000g at 4°C). Supernatants were collected
and amino acids levels were determined using a Waters HPLC fluo-
rometric detection system (Waters, Milford, MA). Data were com-
puted with the Millenium software from Waters, identification and
quantification of peaks were achieved by comparison with standard
solutions.

**Statistical Analysis**

Values are indicated as mean ± SEM. Paired Student's *t*-test or a
one-way ANOVA with the Bonferroni *post hoc* test were used for
comparison. Statistical analysis was performed using Origin 8.0: ns:
not significant; \*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001. The number of
cells used for each experiment illustrated by a figure is indicated in
the legends.

**Results**

As already described, astrocytes can be visualized in slices
with the fluorescent marker sulforhodamine 101 (SR-101),
which allows to distinguish them from neurons (Kafitz et al.,
2008). Moreover, fluorescent cells displayed a linear current
response to incremental depolarizing voltage steps from –150
mV to +40 mV indicating that they are passive astrocytes
(Fig. 1A,B). In the presence of blockers of ionotropic, metab-
otropic GABA, and glutamate receptors, and of GABA trans-
porters and Kir channels (see materials and methods), low
frequency electrical stimulation (0.1 Hz) evoked fast inward
currents that were fully blocked by DL-TBOA (100 µM), a
specific blocker of glutamate transporters, indicating that they
were mediated by the activation of glutamate transporters
(Fig. 1B). This inward current is referred as synaptically
evoked transporter current (STC).
A

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B

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C

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D

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FIGURE 1: Low concentration of TBOA slows the time course of synaptically activated transporter current (STC). (A) Image of an astrocyte recorded in the deep layer of the neocortical slice with a pipette filled with a potassium gluconate (KGluc.) based solution and containing biocytin. (B) Left: Current response to 50 ms depolarizing voltage steps from -150 to 40 mV (10 mV increment) and showing the typical linear I/V relation characteristic of passive astrocytes (mean of 17 cells). Right: Traces showing transporter current evoked by electrical stimulation at 0.1 Hz and recorded in absence (black trace, mean of 5 events) and in presence of DL-TBOA (red trace, mean of 5 events 10 min after the installation of TBOA 100 µM). (C) Traces showing STC in the absence (black, control) or the presence of a low concentration of DL-TBOA (10 µM, red) and after the washout of the drug (green). The three traces were scaled to the control amplitude to show that TBOA slows STC time course. Each stimulus was preceded by a depolarizing step of 2 mV during 50 ms. Histograms represent the quantification of STC decay time constant before and after the installation of TBOA (n = 6 cells). (D) STC evoked by stimulus intensities ranging from 10 to 24 V. STC were scaled to the maximal amplitude to show that the time course is not dependent to current amplitude. Same results were obtained in two other cells. [Color figure can be viewed at wileyonlinelibrary.com]

## Partial Blockade of Glutamate Transporters Slows STC and NMDAR-EPSC Time Course

Our goal was to determine if the inactivation of GS could affect STC and evoked EPSCs. We first wondered how STC would be impacted when this uptake was artificially reduced using a low concentration of DL-TBOA (10 µM), close to the IC₅₀ of GLT1. We observed that TBOA reduced STC peak amplitude by 51 ± 5%. In addition, the decay time constant of STC was more than two times slower than STC recorded in the absence of TBOA (Fig. 1C). This prolonged time course did not result from the decrease in STC amplitude since in the absence of TBOA, the decay time was stable for STC evoked by a wide range of stimulus intensity (Fig. 1D). We next analyzed the consequences of TBOA on the kinetic and charge transfer of the EPSC evoked by NMDA activation in pyramidal neurons. We observed that both the time course and charge transfer of NMDAR-EPSC were increased after the application of TBOA (Fig. 2A,B). We performed same analysis on AMPAR-EPSC and did not find any effects of TBOA (10 µM) on the EPSC (Fig. 2C,D). These data are in keeping with other studies showing that NMDA but not AMPAR are impacted by the partial inhibition of EAATs unless the desensitization of AMPAR was relieved (Tzingounis and Wadiche, 2007). According to other studies, it is likely that the selective enlargement of NMDAR-

EPSC after TBOA is a consequence of an increase of glutamate diffusion and peri/extrasynaptic activation of NMDA receptors due to an insufficient uptake of glutamate by glial glutamate transporters (Arnth-Jensen et al., 2002; Diamond, 2001; Marcaggi and Attwell, 2004). We then focused our attention on decay kinetic in the next following experiments.

## Intracellular Elevation of Glutamate Concentration Affects STC Time Course Evoked by Short Burst of High Frequency Stimulation (HFS)

Immunogold electromicroscopic studies have shown that inactivation of GS with L-methionine-DL-sulfoximine (MSO) leads to intracellular accumulation of glutamate in astrocytes (Laake et al., 1995; Perez et al., 2012). In addition the presence of Na⁺ and glutamate in the cytosol may affect glutamate transport (Barbour et al., 1991; Otis and Jahr, 1998). We thus wondered if intracellular glutamate accumulation would change STC time course. For this purpose, we recorded astrocytes with patch pipettes filled with 1, 3, 10 mM glutamate or devoid of glutamate (0 glutamate) but which contained in all cases 10 mM Na⁺. First, we measured the input resistance of the cells and observed that intracellular elevation of glutamate did not have significant consequences on this parameter (Fig. 3F). Then, we analyzed the decay time constant of STC elicited at low frequency stimulation
A

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B

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C

AMPAR-EPSC

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D

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FIGURE 2: Low concentration of TBOA prolongs the decay of NMDAR but not of AMPAR-mediated EPSC. (A, C) NMDAR-EPSCs evoked at +40 mV (A) and AMPAR-EPSCs evoked at −70 mV (C) by electrical stimulation at 0.1 Hz and recorded with a pipette filled with a cesium gluconate (CsGluc.) based solution, in control (black), 10 min after the installation of TBOA (10 µM, red) and after the washout of the drug (green). Traces are scaled to the control amplitude for comparison of the decay. (B, D) Histograms represent the quantifications of the decay time constant (left) and the charge transfer (right) of NMDAR-EPSC (B, n = 9 cells) and AMPAR-EPSC (D, n = 8 cells) in control and after TBOA. [Color figure can be viewed at wileyonlinelibrary.com]

(0.1 Hz). No correlation between decay time constant of STC and the intracellular concentration of glutamate was observed. Indeed, the decay time of STC recorded with 1 mM glutamate in the patch pipette was similar to the one recorded with 10 mM (P = 0.34) but the 2 values were significantly smaller than the decay of STC recorded in absence or with 3 mM glutamate in the pipette (P < 0.001, Figs. 3E and 6C). We thus assumed that intracellular glutamate elevation in astrocytes had no major consequence on glutamate uptake when the transmitter is released at low frequency. We then wondered if such conclusion would also hold true when glutamate is released during high frequency stimulation. For this purpose, we used the same experimental procedure as described by Diamond and Jahr (2000) in which STC evoked by high frequency stimulation was extracted by subtracting transporter current evoked by 11 pulses at 100 Hz to the one evoked by 10 pulses at 100 Hz and the decay time of the current (STC<sub>HFS</sub>) compared with the one evoked at 0.1 Hz (STC<sub>LFS</sub>, Fig. 3A). We observed that the decay time constant was slightly increased in 0–1 mM glutamate recording conditions, leading in both cases to a τdecaySTC<sub>HFS</sub>/τdecaySTC<sub>LFS</sub> ratio of ~1.25 (Figs. 3B,E and 6C); this value is stronger

than the one reported by Diamond and Jahr in the CA1 region of the hippocampus (ratio of ~1). However, a much larger increase in STC decay time was observed in cells filled either with 3 or 10 mM glutamate, leading to a more important τdecaySTC<sub>HFS</sub>/τdecaySTC<sub>LFS</sub> ratio (Fig. 3C–E). These data suggested that glutamate accumulation in astrocytes may reduce glutamate uptake by the transporters particularly when the transmitter is released during a short burst of high frequency stimulation.

Glutamine Is Selectively Decreased in Cortical Slices After Intraperitoneal Injection of MSO

To determine the possible impact of GS on STC time course, MSO was administered intraperitoneally in rats 24 h before their sacrifice and the electrophysiological recordings of astrocytes in neocortical slices (see Materials and Methods section). First, to ensure that GS was inhibited, the concentration of glutamine, as well as other amino acids (glutamate, GABA, aspartate, alanine) in slices were determined by HPLC and their content (expressed in nmol/g of proteins) compared with slices from sham (saline injected) animals. As shown in Table 1, glutamine was decreased by ~three times in
A

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C

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E

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B

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D

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F

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FIGURE 3: Intracellular elevation of glutamate concentration in astrocytes slows STC time course. (A) Superimposed STCs evoked at 0.1 Hz (1) and at 100 Hz (10 and 11 pulses) in a cell recorded with a pipette filled with a KGluc. based solution and containing 10 mM Na⁺ without glutamate. (B) Left: Traces obtained from A. STC<sub>HFS</sub> (blue) is obtained after subtraction of the trace evoked by 11 pulses at 100 Hz from the one evoked by 10 pulses at 100 Hz. This current is superimposed on STC<sub>LFS</sub> evoked at 0.1 Hz (green). Right: STC<sub>HFS</sub> was scaled and aligned to the peak amplitude of STC<sub>LFS</sub> to compare the decays. Black lines are the exponential fits of the decays of both STC<sub>HFS</sub> and STC<sub>LFS</sub>. (C, D) Same as in B but in astrocytes recorded with pipettes containing 3 and 10 mM of L-glutamate (C and D, respectively). (E) Left: Histograms represent the quantification of the decay time constant (weight average) of both STC<sub>LFS</sub> (green) and STC<sub>HFS</sub> (blue) in astrocytes recorded in absence or in the presence of 3 and 10 mM glutamate in the patch pipette. Astrocytes recorded without glutamate in the pipette: *n* = 9 cells, 36 series of stimulation at 0.1 and 100 Hz. Astrocytes recorded with 3 mM glutamate in the pipette: *n* = 11 cells, 29 series of stimulation. Astrocytes recorded with 10 mM glutamate in the pipette: *n* = 8 cells, 37 series of stimulation. \*Comparison of the decay time of STC<sub>HFS</sub> with STC<sub>LFS</sub> for each conditions of recording; § comparison with the 0 glutamate condition. Right: histograms represent the ratio of the decay time constant of STC<sub>HFS</sub>/STC<sub>LFS</sub>. The ratio was calculated for each cell and the values were pooled together. (F) Histograms represent the mean input resistance of the three groups of cells. [Color figure can be viewed at wileyonlinelibrary.com]

neocortical slices from MSO treated animals as compared with slices from saline injected animals. This decrease was observed only for glutamine and the other amino acids were not significantly affected. We performed the same measurements in hippocampal slices and found that glutamine was also selectively decreased. Together, these data indicated that in vivo injection of MSO was efficient to inhibit GS, in agreement with previous studies in which the activity of GS was measured (Brusilow et al., 2010; Sellinger et al., 1968).

The inhibition of GS can be further demonstrated electrophysiologically through the analysis of tonic NMDAR-mediated current in pyramidal cells that is unmasked after complete inhibition of glutamate transporters with DL-TBOA (100 µM). This current, that is insensitive to voltage gated Na⁺ and Ca²⁺ channel blockers and to blocker of glutamate vesicular transporters or tetanus toxin, is proposed to be due to the leakage of glutamate from astrocytes (Cavelier and Attwell, 2005; Demarque et al., 2002; Jabaudon et al.,
<table><caption>TABLE 1: Concentrations (Expressed in nmol/g of Protein) of Glutamine, Glutamate, GABA, Aspartate and Alanine in Neocortical and Hippocampal Slices (400 µM Thick) Measured 24 h After the Intraperitoneal Injection of Saline Solution (0.09% of NaCl, n = 4 rats) or MSO (170 mg/kg, n = 4 rats)</caption><thead><tr><th></th><th>Glutamine</th><th>Glutamate</th><th>GABA</th><th>Aspartate</th><th>Alanine</th></tr></thead><tbody><tr><td><strong>Neocortex</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Saline</td><td>7.63 ± 1.29</td><td>87.25 ± 9.63</td><td>46.22 ± 6.53</td><td>36.67 ± 16.52</td><td>6.41 ± 1.25</td></tr><tr><td>MSO</td><td>2.65 ± 0.71*</td><td>85.4 ± 8.71</td><td>49.17 ± 7.84</td><td>33.68 ± 9.22</td><td>8.13 ± 2.59</td></tr><tr><td><strong>Hippocampus</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Saline</td><td>14.4 ± 3.17</td><td>119.26 ± 11.23</td><td>99.17 ± 21.7</td><td>41.52 ± 10.55</td><td>8.9 ± 1.73</td></tr><tr><td>MSO</td><td>3.11 ± 1.04*</td><td>101.4 ± 11.14</td><td>71.27 ± 20.2</td><td>27.74 ± 7.05</td><td>8.34 ± 2.28</td></tr></tbody></table>

MSO decreases significantly the amount of glutamine but has no effect on the other amino acids.

1999; Le Meur et al., 2007). Different studies have demonstrated that this current is increased in pyramidal neurons from cortical slices incubated with MSO (Cavelier and Attwell, 2005; Jabaudon et al., 1999; LeMeur et al., 2007). We tested if we can get same results in slices from MSO treated rats. In the continuous presence of TTX, and of AMPA and GABA<sub>A</sub>, receptors antagonists, application of DL-TBOA induced at +40 mV an outward current with ~3 times larger density in slices from MSO-treated animals than from sham animals (Fig. 4). This current was reversed by D-APV (50 µM, data not shown), indicating that it was mediated by NMDA receptors. Therefore, these data support that intraperitoneal injection of MSO had efficiently inhibited GS.

Another important control was also to evaluate if the inactivation of GS has any consequences on transmitter release with our protocol of stimulation. Notably it was important to determine if postsynaptic responses evoked by LFS and HFS (EPSC<sub>LFS</sub> and EPSC<sub>HFS</sub>) were stable with the iteration of the short burst at 100 Hz. Different studies have shown that GS activity is important to sustain synaptic

transmission (Marx et al., 2015; Tani et al., 2010, 2014). We recorded AMPAR-mediated EPSC and performed 10 series of stimulation each consisting of 5–10 stimuli at 0.1 Hz (LFS) followed by a stimulation at 100 Hz for 100 ms (HFS), with a delay of 30 s between each series; a stimulation protocol that is close to the one used for STC. The charge transfer of AMPAR-EPSC<sub>LFS</sub> and AMPAR-EPSC<sub>HFS</sub> were expressed as percentage of the respective response evoked by LFS and HFS of the first series. There was no major variation in the synaptic response evoked by LFS and HFS in slices from saline and MSO treated rats (Fig. 5A,B). Moreover, both the duration of the responses evoked by HFS and the ratio of the charge transfer of AMPAR-EPSC<sub>HFS</sub>/AMPAR-EPSC<sub>LFS</sub> were stable and not significantly different in slices from the 2 groups of animal (Fig. 5B). The decay time constant of AMPAR-EPSC<sub>LFS</sub> was also not affected in pyramidal cells from saline and MSO treated rats (saline: 5.16 ± 0.5 ms; MSO 5.1 ± 0.43 ms).

In separate experiments, we have also recorded spontaneous AMPAR-EPSC and did not find any differences in the

<figure><img src="image_18.png" /><figcaption>FIGURE 4: Endogenous NMDAR-mediated current unmasked after glutamate transporters inhibition is enhanced in animal treated with MSO. (A) Currents evoked at +40 mV in neocortical pyramidal cells after the application of DL-TBOA (100 µM) and recorded in presence of TTX (1 µM), NBQX (20 µM), LY341495 (1 µM), gabazine (5 µM), in slices obtained from rats 24 h after intraperitoneal injection of saline solution (0.09% NaCl) or MSO (170 mg/kg). (B) Mean current density evoked in neurons from the two groups of rat: Saline n = 14 cells; MSO: n = 14 cells.</figcaption></figure>
A

<figure><img src="image_19.png" /></figure>

B

<figure><img src="image_20.png" /></figure>

C

<figure><img src="image_21.png" /></figure>

FIGURE 5: Evoked and spontaneous AMPAR-mediated EPSC are not impacted by the inhibition of glutamine synthase. (A) AMPAR-EPSC evoked at low frequency stimulation (0.1 Hz, AMPAR-EPSC<sub>LFS</sub>) and high frequency stimulation (10 stimuli at 100 Hz, AMPAR-EPSC<sub>HFS</sub>) in slices from saline and MSO treated rats during the 1st and the 10th series of stimulation. Traces of the two series are superimposed and show that the iteration of the stimulation does not have major effects on EPSC<sub>LFS</sub> and EPSC<sub>HFS</sub>. (B) Left: graphs represent the mean charge transfer of EPSC<sub>LFS</sub> (top graph) and EPSC<sub>HFS</sub> (bottom graph) in the two groups of rat for the ten series of stimulation. For each experiment the values have been normalized to the charge transfer evoked during the first series of stimulation. In total the mean charge transfer of EPSC<sub>LFS</sub> and EPSC<sub>HFS</sub> was: 1.18 ± 0.03 pC and 9.22 ± 0.39 pC, respectively for saline treated rats and 1 ± 0.04 pC and 7.54 ± 0.22 pC for MSO treated rats. Right top graph: ratio of the charge transfer of EPSC<sub>HFS</sub>/EPSC<sub>LFS</sub> for the ten series of stimulation. Bottom graph: duration of the response evoked by HFS. Pyramidal neurons recorded from saline treated rats: n = 10 cells/3 rats. Pyramidal neurons recorded from MSO treated rats: n = 12 cells/3 rats. (C) Left: spontaneous AMPAR-mediated EPSC recorded in pyramidal neurons at -70 mV from saline and MSO treated rats. Right: histogram represents the amplitude distribution of spontaneous events (bin of 5 pA). The number of events is expressed as percentage of the maximal of events obtained in a bin. For the two groups of rat the most frequent events have amplitude ranging between 15 and 20 pA. The histograms corresponding to the pyramidal neurons of saline treated rats are from 21,357 events recorded in 15 cells/2 rats and in MSO treated rats from 39,775 events recorded in 16 cells/2 rats. (D) Histograms represent the mean amplitude and frequency of the AMPA events. [Color figure can be viewed at wileyonlinelibrary.com]
frequency or in events amplitude (Fig. 5C). The amplitude
distribution of the events (bin of 5 pA) was similar in both
groups of slice. Together our data indicated that inhibition of
GS had no major consequences on spontaneous and evoked
transmitter release with our stimulation protocol.

Inhibition of Glutamine Synthase by MSO Slows STC Time Course Evoked by HFS

We then determined whether GS inhibition had any conse-
quence on STC time course. For this purpose we recorded
STC with patch pipettes filled with 10 mM Na⁺ and 1 mM
glutamate, closed to the physiological concentrations (Chat-
ton et al., 2000; Lebon et al., 2002; Nedergaard et al., 2002;
Zhou et al., 2014). Decay time constant of both STC<sub>LFS</sub> and
STC<sub>HFS</sub> evoked during each series of stimulations were mea-
sured in neocortical astrocytes from saline and MSO treated
animals. First, as for cells loaded with glutamate, inhibition
of GS did not have significant consequences on input resis-
tance of astrocytes (Fig. 6E). Second, we observed that the
decay time constant of STC<sub>HFS</sub> but not of STC<sub>LFS</sub> was signif-
icantly affected by the inactivation of GS. Thus, the decay
time constant of STC<sub>HFS</sub> was about two times slower in
MSO treated rats leading to a large increase in
τdecaySTC<sub>HFS</sub>/τdecaySTC<sub>LFS</sub> ratio (Fig. 6A–C). In spite of
some variations this difference in current kinetic was main-
tained when stimulations were repeated. We have also mea-
sured the charge transfer of STC<sub>LFS</sub> and STC<sub>HFS</sub>. We
observed that for similar values of STC<sub>LFS</sub>, there was also no
difference in the charge transfer of STC<sub>HFS</sub> in astrocytes from
both groups of rat (Fig. 6D). Together, our data suggest that
same amount of glutamate is reuptake by glutamate trans-
porters but this takes longer time in astrocytes inactivated for
GS when glutamate is released by HFS.

Inhibition of GS Increases Peri/Extrasynaptic Activation of NMDA Receptors

The increase in STC<sub>HFS</sub> decay time constant in astrocytes inactivated for GS suggested that glutamate transporters close to synaptic sites are overwhelmed by glutamate released during HFS. This should facilitate the spillover of the transmitter out of the synaptic cleft and the activation of NMDA receptors located at peri/extrasynaptic sites leading to an amplification of NMDAR-mediated EPSC evoked by HFS. We tested this hypothesis and recorded NMDAR-mediated EPSC. We calculated the ratio of the charge transfer of NMDAR-EPSC<sub>HFS</sub>/NMDAR-EPSC<sub>LFS</sub> for each series of stimulation in pyramidal neurons from both MSO and saline injected rats as we did for AMPAR-EPSC. Figure 7A–C shows that the ratio was consistently and significantly stronger in pyramidal neurons from MSO treated rats. On average, the charge transfer of NMDAR-EPSC<sub>HFS</sub> was stronger in these

pyramidal neurons while NMDAR-EPSC<sub>LFS</sub> was even slightly
lower compared with the values of pyramidal neurons from
saline treated rats (Fig. 7C). Also, in line with the absence of
effects of GS inhibition on STC<sub>LFS</sub> decay time constant, the
time course of NMDAR-EPSC<sub>LFS</sub> was not significantly differ-
ent in pyramidal neurons from the 2 groups of rat (Fig. 7D).
We next examined the peri/extrasynaptic NMDA component
of NMDAR-EPSC<sub>HFS</sub> (Fig. 7E–G). This component was iso-
lated from the synaptic response using the irreversible
NMDA open channel blocker MK-801 (40 µM). After 5
min of stimulation at 0.1 Hz, MK-801 (40 µM) was installed
until the full abolishment of the response. MK-801 was then
washed out and HFS (same stimulation as above) was applied
every minutes. These stimulations consistently evoked a
response with a slow onset that was fully abolished by D-
APV (50 µM) indicating that they were mediated by peri/
extrasynaptic NMDA receptors (Fig. 7E). However, we can-
not fully exclude a contribution of some synaptic NMDA
receptors to the response because HFS may recruit additional
synaptic connections that were not active during application
of LFS in MK801. We calculated the charge transfer of the
NMDARs mediated response after MK801 (extrasynaptic
response) normalized to the charge transfer before the appli-
cation of the blocker (synaptic response) in pyramidal neu-
rons from the two groups of rat. The responses were
significantly stronger in MSO treated animal (Fig. 7F,G). On
an average, the responses evoked by extrasynaptic receptors
during the 10 series of HFS were ~45% larger in MSO
treated animals while synaptic NMDAR-EPSC<sub>LFS</sub> preceding
the application of MK-801 were not different in the two
groups of animal.

Discussion

Electron microscopy studies performed in the somatosensory
cortex demonstrated that most of glutamate transporters are
present in astrocytic processes and have a prominent perisy-
naptic localization that may contribute to restrict glutamate
spillover (Melone et al., 2009). Moreover, a recent study pro-
vided evidences that GLT1 in astrocytes are highly mobile
and that once bound by glutamate, they move away from the
synapse allowing their replacement by unbound transporters,
a process that may favor efficient clearance of glutamate from
synaptic cleft (Murphy-Royal et al., 2015). Here, we show
that these characteristics may be insufficient for a rapid clear-
ance of glutamate when glutamate catabolism in astrocytes is
altered. We provide evidences that GS activity regulates STC
time course when glutamate is released by short burst of high
frequency stimulation but not by low frequency stimulation.
This indicated that the conversion of glutamate by GS is a
fundamental process for proper glutamate transporters func-
tion. It is likely that the consequences of GS inactivation
<figure><img src="image_22.png" /></figure>

FIGURE 6: Inhibition of glutamine synthase increases STC time course. (A) Graph showing the decay time constant of both STC<sub>HFS</sub> (upper graph, blue) and STC<sub>LFS</sub> (middle graph, green) during 8 series of stimulation (see Materials and Methods section) in astrocytes from saline (triangle, n=12 cells/3 rats) and MSO treated rats (circle, n=10 cells/3 rats). (B) Left: superimposed STC<sub>LFS</sub> and STC<sub>HFS</sub> from saline (upper traces) and MSO (middle traces) treated rats corresponding to series #7 indicated by a letter in A. Right: STC<sub>HFS</sub> was scaled and aligned to the peak amplitude of STC<sub>LFS</sub> to compare the decays. Black lines are the exponential fits of the decays of both STC<sub>HFS</sub> and STC<sub>LFS</sub>. Bottom traces: STC<sub>LFS</sub> (left) and STC<sub>HFS</sub> (right) from saline and MSO treated rats were scaled and aligned to the peaks to compare the decays. The decay of STC<sub>HFS</sub> is slower in astrocytes from MSO compared with saline injected rats while the decays of STC<sub>LFS</sub> are similar. (C) Histograms represent the quantification of the decay time constant of both STC<sub>LFS</sub> (green) and STC<sub>HFS</sub> (blue) in astrocytes from saline and MSO treated rats (left histogram) obtained from pooled data depicted in B for the 8 series of stimulation. Right histograms represent the ratio of STC<sub>HFS</sub>/STC<sub>LFS</sub> decay time constant. The ratio was calculated for each cell and the values were averaged. (D) Same as in C but for the charge transfer of STC. (E) Mean input resistance of the cells from the two groups of rat. [Color figure can be viewed at wileyonlinelibrary.com]

result from an intracellular accumulation of glutamate occur-
ring with the stimulations, since the slowing of STC time
course was also observed in the same stimulation conditions
of astrocytes recorded with 3–10 *mM* glutamate. The tenden-
cy of glutamate to accumulate in astrocytes in the absence of
GS activity has been demonstrated in both hippocampal

astrocytes and Müller cells of the retina with immunogold
labelling (Barnett et al., 2000; Laake et al., 1995; Perez et al.,
2012), and with electrophysiological recordings of endoge-
nous NMDA current in CA1 pyramidal neurons unmasked
after inhibition of glutamate transporters (Cavelier and Att-
well, 2005; Demarque et al., 2002; Jabaudon et al., 1999; Le
A

<figure><img src="image_23.png" /></figure>

C

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

Sham
MSO

B

<figure><img src="image_26.png" /></figure>

D

<figure><img src="image_27.png" /></figure>

E

<figure><img src="image_28.png" /></figure>

Sham

<figure><img src="image_29.png" /></figure>

DL-APV

<figure><img src="image_30.png" /></figure>

MSO

25 pA
100 ms

F

<figure><img src="image_31.png" /></figure>

G

<figure><img src="image_32.png" /></figure>

(Synaptic) (Extrasynaptic)

FIGURE 7: Inhibition of glutamine synthase amplifies NMDAR-EPSC evoked by HFS. (A) Superimposed NMDAR-EPSC evoked at 0.1 Hz (LFS) and by 10 pulses at 100 Hz (HFS) recorded in a pyramidal neuron from saline (left traces, black) and MSO (right traces, purple) treated rats. (B) Ratio of the charge transfer of NMDAR-EPSC<sub>HFS</sub>/NMDAR-EPSC<sub>LFS</sub> for each series of stimulation in pyramidal cells from saline (n=16 cells/3 rats) and MSO treated rats (n=16 cells/2 rats). (C) Histograms represent the mean charge transfer of NMDAR-EPSC<sub>LFS</sub> and NMDAR-EPSC<sub>HFS</sub> in the two groups of rat. The respective values of NMDAR-EPSC evoked by LFS and HFS for the 10 series of stimulation have been pooled. (D) Graph showing the decay time constant of NMDA-EPSC evoked at 0.1 Hz in the two groups of rats for the 10 series of stimulation. (E) Traces showing NMDAR-EPSCs evoked at 0.1 Hz (LFS); 20 min after the installation of MK-801; by 10 pulses at 100 Hz (HFS) after MK-801 washout; after the addition of D-APV (50 µM) in both saline (black) and MSO (purple) treated rats. (F) Graph represents the charge transfer of NMDAR-EPSC for the two groups of rats normalized to the respective values evoked at 0.1 Hz before the installation of MK-801. Saline: n = 8 cells/3 rats; MSO: n = 9 cells/3 rats. (G) Histograms represent the mean charge transfer of synaptic NMDAR-EPSC (evoked by LFS) and peri/extrasynaptic stimulation NMDAR-EPSC (evoked by HFS) in the two groups of rats. The respective values of NMDAR-EPSC evoked by LFS and HFS for the 10 series of stimulation have been pooled. [Color figure can be viewed at wileyonlinelibrary.com]
Meur et al., 2007). Altogether, these data provide evidence that GS is a major enzyme involved in glutamate catabolism. In line with this, metabolic studies have shown that two-third of glutamate released and transported in astrocytes is converted by GS into glutamine whereas one-third is transported into mitochondria and converted to alpha-ketoglutarate (Hertz and Zielke, 2004).

The prolongation of STC time course observed after GS inhibition reflects a delayed clearance of extracellular glutamate and to a potent diffusion of the transmitter at distant sites as demonstrated by a larger peri/extrasynaptic NMDA response elicited by HFS in MSO compared with saline injected animals. Moreover, the observations that low concentration of TBOA prolonged the decay time constant of both STC<sub>LFS</sub> and NMDAR-EPSC<sub>LFS</sub> support the relationship between STC time course on one hand, and the kinetic of the NMDA response on the other hand, and indicate that glutamate transporters in astrocytes limit NMDA receptors activation at a juvenile stage (see also Hanson et al., 2015). We cannot exclude that neuronal glutamate transporters could also play an important role in shaping the NMDA response, as demonstrated in the CA1 region of the hippocampus (Diamond, 2001; Scimemi et al., 2009) but this was not investigated in the present study. Here, pyramidal cells were recorded at +40 mV a condition, which is not favorable for glutamate transport by neuronal glutamate transporters.

It has been suggested that intracellular ionic composition influences the cycling or turnover rate of glutamate transporters. As mentioned by Huang and Bergles (2004) and by Wadiche et al. (2006), the turnover rate of glutamate transporters of the same identity can vary from a preparation to another. Thus, the turnover rate of GLT1 in hippocampal astrocytes measured in outside-out patch configuration is about 11 ms at -90 mV (Bergles and Jahr, 1998) whereas intracellular recording of transporter current in oocytes expressing GLT1 and that contain 10 mM glutamate and 10 mM Na<sup>+</sup> showed that this rate was about 70 ms (Wadiche et al., 1995). It is therefore possible that GS inhibition through intracellular glutamate accumulation, and in presence of Na<sup>+</sup>, slow down the cycling rate of transporters and affects the capture efficiency i.e., the likelihood of glutamate to be translocated and released intracellularly as opposed to unbind extracellularly. Indeed, experimental data on the uncoupled anion transporter current and simulations suggest that before the final intracellular unbinding of glutamate and Na<sup>+</sup>, glutamate transporter seesaw between 2 states in which both ions are bind to the external face and to the internal face of the transporter, glutamate translocation forward and backward rates are almost equal for GLT1 (Bergles et al., 2002). In the presence of Na<sup>+</sup> and glutamate inside, the transporter has higher probability to keep bouncing between

these 2 states delaying the intracellular unbinding of glutamate and Na⁺, the reorientation of the transporter to the outside by the intracellular K⁺ ions and thus the possibility for the transporter to uptake another molecule of glutamate from the extracellular space. This mechanism could reduce the capacity of glutamate transporters near synaptic sites to rapidly uptake glutamate when released by HFS and favor diffusion of the transmitter to glutamate transporters at distant sites. This may explain the prolongation of STC<sub>HFS</sub> time course. Therefore, maintaining low glutamate concentration (and also Na⁺) in cytosol may be instrumental for astrocytes to efficiently clear glutamate released under certain circumstances of activation.

The actual resting concentration of glutamate in cortical astrocytes seems to range between 0.5 and 1 mM in rodent (Nedergaard et al., 2002; Zhou et al., 2014) and was estimated to be around 0.7 mM in human (Lebon et al., 2002). It is however not known how long it takes for glutamate after its transport in astrocytic process to reach back to this basal concentration. This may take longer time when GS is inhibited favoring glutamate accumulation. In physiological condition, the conversion of glutamate may be certainly fast enough since the decay time of STC<sub>HFS</sub> was close to that of STC<sub>LFS</sub> in astrocytes from saline injected rat during the different series of stimulation or in astrocytes recorded without glutamate in the patch pipette. However, we found that the ratio of the decay time constant of STC<sub>HFS</sub>/STC<sub>LFS</sub> was ~1.25. This value differs from what was observed in the CA1 region of the hippocampus from juvenile rats, where Diamond and Jahr (2000) found a ratio of ~1. This suggests that neocortical astrocytic glutamate transporters close to synaptic sites would be less efficient to uptake glutamate released by HFS than those from the hippocampus. Interestingly, it was recently shown that astrocyte glutamate transporters mature later in the neocortex than in the hippocampus (Hanson et al., 2015). Immunofluorescence studies showed that GLT1 and GLAST are ~40% less expressed in the neocortex than in the hippocampus from juvenile rats (Hanson et al., 2015). These authors provide also evidences that transporter current evoked by glutamate photolysis, decays slower in the neocortex than in the hippocampus until the third postnatal weeks of life. In keeping with this, the time course of STC recorded in the present study was also slower than the one previously reported in hippocampal astrocytes at the same developmental stage (Thomas et al., 2011). In addition, a study that has just been published while this article was revised showed that the efficiency of glutamate uptake in the adult mice neocortex was modulated by presynaptic activity but by a process that is independent of the amount of glutamate released. The clearance of glutamate is slowed when glutamate is released by burst of neuronal activity >30 Hz and particularly by 10
stimuli at 100 Hz (Armbruster et al., 2016). These observa-
tions, if this hold true in rat at juvenile stage, could also
explain the ratio >1 of the decay time constant of STC<sub>HFS</sub>/
STC<sub>LFS</sub> obtained here.

It has been shown in different preparations that the activity of GS is fundamental for synaptic transmission (Marx et al., 2015). This enzyme converts glutamate into glutamine, which is released by astrocytes and taken up by neurons where it is converted into glutamate by the phosphate activated glutaminase (Coulter and Eid, 2012; Marx et al., 2015). Our data show that synaptic transmission mediated by AMPA receptors are not impacted by the inactivation of GS. More precisely, the charge transfer of EPSC evoked by LFS and HFS as well as the ratio of EPSC<sub>HFS</sub>/EPSC<sub>LFS</sub> were relatively constant with the iteration of the 100 Hz stimulation and not different from the results obtained from saline treated rats. In addition, the mean charge transfer of both STC<sub>LFS</sub> and STC<sub>HFS</sub> were not different in the two groups of rat. These data suggest that GS activity is not necessary to sustain glutamate synaptic transmission in the neocortex. These results apparently disagree the conclusion of Tani et al. (2010, 2014) about the requirement of glutamate/glutamine cycle to sustain glutamate synaptic transmission in neocortex. The most likely explanation for this discrepancy is the experimental procedure and the situation of high demand of glutamate resulting from persistent epileptiform activity (Tani et al., 2010) or from the protocol of stimulation used by Tani et al. (2014) i.e., iteration of intermittent stimulation at 20 Hz for 40 s separated by stimulation at 0.2 Hz. Our procedure is certainly not sufficiently strong to deplete vesicles pools of glutamate as was the case in the experiments of Tani et al. (2014) and for which the synthesis of glutamate from glutamine may be important to replenish the pool of glutamate in presynaptic terminals. In our condition, the delay between 2 bursts of HFS and 2 series of stimulation were of 30 s. This delay may allow expression of alternatives mechanism of recycling via glutamine i.e., other metabolic pathways and presynaptic uptake of glutamate via GLT-1 (Marcaggi and Attwell, 2004; Marx et al., 2015; Danbolt et al., 2016). We cannot however fully exclude in our situation that the residual amount of glutamine in slices from MSO treated rats could also help maintaining synaptic transmission.

The importance of GS for cortical network activity and
brain development is highlighted by the deleterious effects of
mutations in the human *GLUL* gene that encodes GS, lead-
ing to a strong inactivation of GS and associated with severe
brain malformations, with seizures, and with early epileptic
encephalopathy (Häberle et al., 2005, 2011). In rodent, mice
lacking GS in astrocytes die rapidly after birth (He et al.,
2010). Moreover, different studies have shown that the reac-
tive astrocytes seen in a variety of pathological conditions

including mesial temporal lobe epilepsy, do not express GS
(Coulter and Eid, 2012; Eid et al., 2013). A defect in GS
activity may have various consequences due to an increase in
ammonia concentration in the brain and to the lack of gluta-
mine synthesis leading to a reduction of GABAergic inhibi-
tion (He et al., 2010; Kelly et al., 2009; Liang et al., 2006;
Ortinski et al., 2010), which may contribute to the genera-
tion of seizures. The delayed clearance and spillover of gluta-
mate described in the present study, following a pattern of
stimulation that could also happen during seizures, is likely
to contribute to the maintenance of a hyperexcitable state.
The possibility that this mechanism is actually operating in
epilepsy will be investigated in future studies in the appropri-
ate animal model and will help to clarify the electrophysiolog-
ical consequences of a dysregulation of glutamate catabolism
and homeostasis that is associated with some epileptic
disorders.

Acknowledgment

Grant sponsors: INSERM, Aix-Marseille University, and by
the Tunisian Ministry for Education and Research (Y.T.).

The authors would like to thank Christophe Melon at IBDML (Marseille) for the use of HPLC and his contribution in the analysis of amino acids content in slices. They are also grateful to Drs Paikan Marcaggi and Pierre Szepetowski for their helpful comments and criticism of the manuscript.

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