Phasic and tonic mGlu7 receptor activity modulates the thalamocortical network

ORIGINAL RESEARCH

published: 25 April 2016

doi: 10.3389/fnci r .2016.00031

Phasic and T o nic mGlu7 Receptor

Activity Modulates the

Thalamocortical Network

Valériane Tassin^1,2,3, Benoît Girard^1,2,3, Apolline Chotte^1,2,3, Pierre Fontanaud^1,2,3

Delphine Rigault⁴, Mikhail Kalinichev⁵, Julie Perroy^1,2,3, Francine Acher⁴,

,

Laurent Fagni^1,2,3and Federica Bertaso^1,2,3

*

¹CNRS, Institut de Génomique Fonctionnelle, UMR-5203, Montpellier, France, ²INSERM, U1191, Montpellier, France,

³UMR-5203, Université de Montpellier, Montpellier, France, ⁴CNRS, UMR-8601, Université Paris Descartes, Paris, France,

⁵IPSEN Innovation, Les Ulis, France

Mutation of the metabotropic glutamate receptor type 7 (mGlu7) induces absence-like

epileptic seizures, but its precise role in the somatosensory thalamocortical network

remains unknown. By combining electrophysiological recordings, optogenetics, and

pharmacology, we dissected the contribution of the mGlu7 receptor at mouse thalamic

synapses. We found that mGlu7 is functionally expressed at both glutamatergic and

GABAergic synapses, where it can inhibit neurotransmission and regulate short-term

plasticity. These effects depend on the PDZ-ligand of the receptor, as they are lost

in mutant mice. Interestingly, the very low afﬁnity of mGlu7 receptors for glutamate

raises the question of how it can be activated, namely at GABAergic synapses

and in basal conditions. Inactivation of the receptor activity with the mGlu7 negative

allosteric modulator (NAM), ADX71743, enhances thalamic synaptic transmission. In

vivo administration of the NAM induces a lethargic state with spindle and/or spike-

and-wave discharges accompanied by a behavioral arrest typical of absence epileptic

seizures. This provides evidence for mGlu7 receptor-mediated tonic modulation of

a physiological function in vivo preventing synchronous and potentially pathological

oscillations.

Edited by:

Franck Polleux,

Columbia University in the City

of New York, USA

Reviewed by:

Diasynou Fioravante,

University of California, Davis, USA

Colleen Niswender,

Vanderbilt University, USA

Keywords: glutamate, thalamic network, short-term plasticity, epilepsy, EEG

*Correspondence:

Federica Bertaso

federica.bertaso@igf.cnrs.fr

INTRODUCTION

Received: 13 January 2016

Accepted: 05 April 2016

Published: 25 April 2016

G protein-coupled receptors (GPCRs) represent the largest family of membrane receptors in

mammals and are involved in a big diversity of patho-physiological conditions. These receptors can

display constitutive (agonist-independent) activity, and mutations leading to constitutively active

GPCR receptors ex vivo have been linked to several human diseases (de Ligt et al., 2000). However,

only few reports exist on the role of constitutively active wild-type (WT) receptors in their natural

environment, in vitro and in vivo (Meye et al., 2014). For instance, constitutive activity of native H3

receptors can control rat histaminergic neurons in vivo (Morisset et al., 2000), while constitutively

active dopamine, opioid, and serotonin receptors are expressed in the brain reward system (Meye

et al., 2014).

Citation:

Tassin V, Girard B, Chotte A,

Fontanaud P, Rigault D, Kalinichev M,

Perroy J, Acher F, Fagni L

and Bertaso F (2016) Phasic

and Tonic mGlu7 Receptor Activity

Modulates the Thalamocortical

Network.

Among eight genes coding for metabotropic glutamate receptors (mGlu1-8), several studies

have investigated the eﬀects of negative allosteric modulation of WT mGlu1 and mGlu5 in neurons

Front. Neural Circuits 10:31.

doi: 10.3389/fnci r .2016.00031

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Tassin et al.

mGlu7 Modulates the Thalamic Network

(Prezeau et al., 1996; Ango et al., 2001) and in vivo (Rodriguez classical orthosteric agonist (D,L-AP4) and a mouse KI mutant

et al., 2010; Keck et al., 2012), while much less is known for mGlu7 (mGlu7a^AAAmouse), we revisited the function of

about the constitutive activity of the other mGluRs. In the this receptor in vitro and in vivo. We studied the functional

mammalian CNS, the mGlu7 receptor gene shows the highest distribution of the mGlu7a receptor in thalamic synapses in WT

degree of evolutionary conservation across species. It is the and mGlu7a^AAAmice, comparing electrically and optogenetically

most widely distributed in a broad range of synapses critical evoked postsynaptic responses, in vivo electroencephalography

for both normal CNS function as well as psychiatric and (EEG) and behavioral analysis. Our data provide evidence

neurological disorders including anxiety, depression, obsessive– for mGlu7 tonic activity in vivo, which occurs at speciﬁc

compulsive disorders, and schizophrenia (Hamilton, 2011; Shyn glutamatergic and GABAergic synapses of the thalamocortical

et al., 2011; Park et al., 2013). Diﬀerent splice mGlu7 variants (a– circuitry, resulting in persistent modulation of the network

e) exist, with mGlu7a being the predominantly expressed variant activity and animal vigilance state.

(Flor et al., 1997; Schulz et al., 2002). The mGlu7a receptor

is expressed at glutamatergic presynaptic terminals (Bradley

et al., 1998; Kinoshita et al., 1998), where its association to the

MATERIALS AND METHODS

PDZ-protein PICK1 mediates the inhibition of voltage-gated

calcium channels and, as a result, a decrease in neurotransmitter

ADX71743 Synthesis

release (Perroy et al., 2000, 2002; Millán et al., 2002). The

ADX71743 was synthesized according to the following scheme

activation of mGlu7 receptors remains a puzzling issue, due

(Contour et al., 2007):

to its very low aﬃnity for glutamate (in the millimolar range,

amongst the lowest for neurotransmitters). Such glutamate

concentration might be reached in the glutamatergic synaptic

cleft upon intense neuronal activity (Cartmell and Schoepp,

2000; Pelkey et al., 2005). Intriguingly, mGlu7 receptors are

also expressed at GABAergic presynaptic sites in hippocampus

(Somogyi et al., 2003), hypothalamus (Schrader and T a sker,

1997), and somatosensory cortex (Dalezios et al., 2002) where

the dearth of glutamate highlights the enigma of mGlu7 receptor

activation and function in physiological conditions.

The mGlu7 receptor knockout mouse displays impaired fear

response and higher susceptibility to alcohol and convulsants

(Sansig et al., 2001; Goddyn et al., 2008). The knock-in (KI)

AAA mutation of the exon coding for the mGlu7a C-terminal

PDZ ligand (here named mGlu7a^AAAmouse) suppresses the

receptor interaction with the PDZ protein PICK1. Blockade of

this interaction has consequences on the receptor downstream

signaling and results in loss of agonist-dependent inhibition of

synaptic transmission (Perroy et al., 2002). This is suﬃcient to

induce spatial working memory deﬁcits (Zhang et al., 2008) and

to trigger spontaneous absence-like epileptic seizures (Bertaso

et al., 2008), suggesting a crucial role of mGlu7a to control

neuronal network activity. We focused our attention on the

thalamic circuitry responsible for absence epileptic seizures,

characterized by a hypersynchronous oscillatory activity of

thalamic and cortical neurons, leading to typical spike-and-wave

discharges (SWD) of the thalamocortical (TC) loop in human

and animal models (Crunelli and Leresche, 2002). Seizures

involve reciprocal projections between two thalamic nuclei,

the GABAergic reticular nucleus (TRN) and the glutamatergic

ventro-postero-medial nucleus (VPM), and the somatosensory

(barrel) cortex (Figure 1A) (Crunelli and Leresche, 2002;

Steriade, 2006; Huguenard and McCormick, 2007; Beenhakker

and Huguenard, 2009).

(E)-4-(2,4-dimethylphenyl) but-3-en-2-one (1) (El-Batta et al.,

2007).

To

a solution of 2,4-dimethylbenzaldehyde (500 mg,

3.73 mmol, 1 eq) in methanol/water 1:1 (8 ml) was added

1-triphenyl-phosphoranylidene-2-propanone (1.2 g, 3.73 mmol,

1 eq) and the mixture was reﬂuxed for 2 h. After cooling, the

solvent was removed under reduced pressure. The residue

was extracted with dichloromethane (3^∗50 ml). The combined

organic layers were dried over magnesium sulfate, ﬁltered and

concentrated under vacuum. The crude was chromatographed

on a silicagel column with cyclohexane/ethyl acetate (85:15) to

aﬀord 1 as a slightly yellow oil (613.6 mg, 3.53 mmol, 94.5%).

TLC: Rf = 0.47 (cyclohexane/ethyl acetate 8:2, UV).

¹H NMR (500 MHz, CDCl₃): δ 2.21 (s, 3H), 2.25 (s, 3H),

2.28 (s, 3H), 6.50 (d, J = 16.2 Hz, 1H), 6.89 (s, 1H), 6.90 (d,

J = 8.2 Hz, 1H), 7.34 (d, J = 8.2 Hz, 1H), 7.67 (d, J = 16.2 Hz,

1H).

The lack of speciﬁc pharmacological tools has long been a

critical impairment for the study of mGlu7 receptor function.

Here, using a brain-penetrant negative allosteric modulator

(NAM), ADX7174, which displays high speciﬁcity and aﬃnity for

the mGlu7 receptor (Kalinichev et al., 2013), together with a more

¹³C NMR (126 MHz, CDCl₃): δ 19.2, 20.9, 27.2, 126.0, 126.6,

126.9, 130.1, 131.3, 137.4, 140.1, 140.2, 197.6.

MS (ESI): m/z 175.2 [M+H]⁺

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Tassin et al.

mGlu7 Modulates the Thalamic Network

5-(2,4-dimethylphenyl)cyclohexane-1,3-dione (2) (Courtney the crude was chromatographed on a silicagel column with

et al., 2008).

dichloromethane/ethyl acetate (98:2) to aﬀord a pale yellow solid

(ADX73741) (994 mg, 3.70 mmol, 44.2%).

To a solution of 1 (4.36 g, 25 mmol, 1 eq) in dry ethanol

(20 ml) was added NaOEt (21% in ethanol; 19.4 ml, 0.127 mol,

5 eq) and diethylmalonate (3.9 ml, 25 mmol, 1 eq) under

stirring. The mixture was stirred at reﬂuxed for 1 h 30 min. The

reaction mixture was cooled to ambient temperature. An aqueous

solution of sodium hydroxide (2 M, 20 ml) was added to the

reaction mixture, which was heated at 80^◦C for 3 h. Ethanol in

excess was removed by evaporation. The mixture was acidiﬁed

with concentrated HCl (20 ml) and the reaction mixture was

reﬂuxed for 3 h 30⁰and left to cool to ambient temperature. The

compound was extracted with ether (3^∗200 ml). The combined

organic layers were dried over magnesium sulfate, ﬁltered and

concentrated under vacuum. The crude was chromatographed

on a silicagel column with dichloromethane/ethyl acetate (6:4) to

aﬀord a pale yellow oil (2) (4.1 g, 19 mmol, 75.6%).

TLC: Rf = 0,23 (dichloromethane/ethyl acetate: 9:1, UV).

¹H NMR (500 MHz, CDCl₃): δ 1.41 (t, J = 7.6 Hz, 3H), 2.29

(s, 3H), 2.31 (s, 3H), 2.72 (dd, J = 16.4 and 3.9 Hz, 1H), 2.84

(d, J = 16.4 Hz, 1H), 2.85 (qu, J = 7.6 Hz, 2H), 3.10–3.13 (m,

2H), 3.83 (m, 1H), 7.06 (s, 1H), 7.07 (d, J = 7.8 Hz, 1H), 7.20

(d, J = 7.8 Hz, 1H).

¹³C NMR (126 MHz, CDCl₃): δ 11.1, 19.4, 21.0, 21.9, 29.5, 36.9,

44.9, 125.5, 127.4, 132.0, 134.3, 135.3, 137.0, 137.1, 163.5, 166.6,

190.5

HRMS calculated for [C₁₇H₂₀O₂N]⁺: 270.14940.

HRMS found for [C₁₇H₂₀O₂N]⁺: 270.14862.

Elementary analysis calculated, %: C 75.81; H 7.11; N 5.20.

Elementary analysis found, %: C 75.18; H 6.95; N 4.36.

TLC: Rf = 0,46 (dichloromethane/ethyl acetate 85:15, UV).

¹H NMR (250 MHz, CDCl₃): δ 2.29 (s, 6H), 2.56–2.63 (m,

1H), 2.75–2.83 (m, 3H), 3.34–3.45 (m, 1H), 3.50–3.57 (m, 1H),

4.09–4.30 (m, 1H), 7.00–7.03 (m, 3H)

HPLC: macherey nagel (125 mm × 4.0 mm, 5 µm). Flow rate:

0.7 ml/min et λ = 254 nm.

Solvent A (water/methanol/HCOOH: 900:100:1) and solvent B

(water/methanol/HCOOH 200:800:1) TR = 19.6 min.

¹³C NMR (126 MHz, CDCl₃): δ 19.5, 21.2, 37.2, 43.0, 103.4,

126.4, 127.9, 132.2, 136.3, 136.7, 140.6, 198.0.

MS (ESI): m/z 215.2 [M-H]⁻

Mice

All animal procedures were conducted in accordance with the

European Communities Council Directive (2010/63/EU) and

approved by the French Ministry for Agriculture. The generation

2-diazo-5-(2,4-dimethylphenyl)cyclohexane-1,3-dione

(Presset et al., 2011).

(3)

To a solution of 2 (100 mg, 0.46 mmol, 1 eq) in acetonitrile and characterization of the mutant mice are described elsewhere

(10 ml) was added a solution of p-toluenesulfonylazide (13% (mGluR7a^AAA/AAAmouse, herein named mGlu7a^AAAmice,

in toluene; 785 µl, 0.46 mmol, 1 eq) and K₂CO₃(76 mg, (Zhang et al., 2008).

0.55 mmol, 1.2 eq). The mixture was protected from light

Acute Thalamocortical Slices

Preparation

and stirred overnight at room temperature. The mixture was

then concentrated and the compound was extracted with

dichloromethane (3^∗50 ml). The combined organic layers were

dried over magnesium sulfate, ﬁltered and concentrated under

vacuum. The crude was chromatographed on a silicagel column

with dichloromethane/ethyl acetate (9:1) to aﬀord a white solid

(3) (78.2 mg, 0.32 mmol, 70.3%).

All chemicals used to prepare electrophysiological solutions

were purchased from Sigma–Aldrich (France). mGlu7^AAAor

WT littermates male and female mice aged P15–P21 were

deeply anesthetized by inhalation of isoﬂurane, and slices were

prepared with a vibratome (Campden Instruments, UK) as

previously described (350 µm-thick, 35^◦tilt from coronal,

Cruikshank et al., 2007) to preserve the thalamocortical network.

As intra-TRN projections are denser in the horizontal plan, we

prepared 230 µm-thick horizontal slices when studying intra-

TRN synapses. We then incubated slices at 32^◦C for 1 h and then

at 24–26^◦C, in artiﬁcial cerebrospinal ﬂuid (ACSF) containing (in

mM) 120 NaCl, 2.5 KCl, 1.6 NaH₂PO4, 1.3 MgCl₂, 2.5 CaCl₂, 27

NaHCO₃, and 22 glucose, equilibrated with 95% O₂and 5% CO₂,

pH 7.4.

TLC: Rf = 0,76 (dichloromethane/ethyl acetate 8:2, UV and

ninhydrin).

¹H NMR (500 MHz, CDCl₃): δ 2.27 (s, 3H), 2.28 (s, 3H), 2.63–

2.74 (m, 4H), 3.53–3.58 (m, 1H), 7.00–7.04 (m, 3H).

¹³C NMR (126 MHz, CDCl₃): δ 19.1, 20.8, 32.0, 43.7, 84.3,

124.7, 127.3, 131.8, 135.1, 136.4, 136.7, 189.5.

MS (ESI): m/z 243.11235 [M+H]⁺

6-(2,4-dimethylphenyl)-2-ethyl-6,7-dihydrobenzo[d]oxazol-

4(5H)-one (ADX71743) (Kallstrom et al., 2004).

To compound 3 (2.02 g, 8.35 mmol, 1 eq) was added recording chamber and superfused with ACSF (2 ml/min, 32^◦C).

propionitrile (2.3 g, 41.7 mmol, 5 eq) and rhodium (II)- For EPSC recording ACSF was supplemented with D-AP5

acetate dimer (6.88 mg, 16.7 µmol, 0.002 eq). The mixture (50 µM) and bicuculline (10 µM). For IPSC recording, ACSF

was heated at 60^◦C for 1 h, cooled to room temperature and was supplemented with CNQX (10 µM) and D-AP5 (50 µM).

Whole-Cell Patch-Clamp Recording

Following incubation, brain slices were transferred to the

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mGlu7 Modulates the Thalamic Network

All drugs were purchased from Tocris (France). Recordings were values for all cell type tested are reported in Supplementary

obtained from visually identiﬁed TRN, VPM, and cortical layer Figure 1. Averages of 10–20 trials were used to measure the

4 neurons using diﬀerential contrast optics with an upright amplitudes of ePSCs, from baseline to peak.

microscope (Olympus, France) and an infrared CCD video

We also tested the response plasticity by applying ﬁve

camera (COHU, SAIS, France). Recording electrodes made of pulses (0.1–1 mA, 100 µs duration) at 10 Hz, close to natural

borosilicate glass had a resistance of 4–7 M when ﬁlled whisker-input frequency into the thalamus during exploratory

with intracellular solution. For thalamic neurons, the internal whisking (Knutsen et al., 2008 ). Averaged 10 trials of 10 s

solution contained (in mM) 115 CsMeSO₃, 20 CsCl, 10 HEPES, inter-trials were used to measure the ﬁve peak amplitudes.

0.6 EGTA, 4 Na₂-ATP, 0.4 Na-GTP, 10 Na-phosphocreatine, Short-term plasticity depends on a presynaptic modulation

and 2.5 MgCl₂. For cortical layer 4 neurons, the internal of neurotransmitter release, which is reﬂected postsynaptically

solution contained (in mM) 125 K-gluconate, 10 KCl, 10 HEPES, by an increase (synaptic facilitation) or decrease (synaptic

0.2 EGTA, 4 Na₂-ATP, 0.3 Na-GTP, 14 Na-phosphocreatine, depression) of ePSCs along the stimulation train. Facilitation

and 1.8 MgCl₂. For both solution, pH was adjusted to 7.3 and depression ratios were calculated as the percentage of

with CsOH or KOH, respectively, and the osmolarity was the response amplitude to pulse 2 to 5 divided by the

300 mOsm. For all recording conditions, only cells with access response amplitude to the pulse 1. In experiments in which

resistance < 18 M and a change of resistance < 25% we added supplementary drugs in the perfusing solution, we

over the course of the experiment were analyzed. Data were repeated single-stimulation and train-stimuli protocols after

ﬁltered with a Hum Bug (Quest Scientiﬁc, Canada), digitized 7 min.

at 2 kHz (Digidata 1444A, Molecular Devices, Sunnyvale, CA,

USA), and acquired using Clampex 10.2 software (Molecular

Spontaneous Phasic and Tonic GABA_A

Currents Recordings

Thalamic reticular nucleus and VPM cells receiving TRN neurons

Devices).

Electrical Stimulation of VPM and TRN

inputs were voltage-clamped at −60 mV, and GABA_Areceptor-

Electrical stimulations were delivered via a bipolar stimulation

electrode (0.1–1 mA, 100 µs; FHC Inc.). To study the eﬀect

of the mGlu7 receptor at thalamic presynaptic sites, the

stimulation electrode was placed either in the VPM to evoke

excitatory postsynaptic currents (eEPSCs) in TRN neurons,

held at −60 mV (inward currents), or in the TRN to evoke

inhibitory postsynaptic currents (eIPSCs) within adjacent TRN

neurons or VPM neurons, held at 0 mV (outward currents).

The group III mGluRs agonist D,L-AP4 (1.2 mM, Tocris

Bioscience) and the mGlu7 receptor NAM ADX71743 (10 µM)

were applied and the eﬀects compared between the two

genotypes.

mediated currents were pharmacologically isolated with 10 µM

CNQX and 50 µM D-AP5. Miniature IPSCs (mIPSCs) were

obtained by adding 1 µM tetrodotoxin (TTX, Tocris) to block

action potentials. mIPSCs frequency was analyzed and compared

between WT and mGlu7a^AAAmice. Tonic GABA_Acurrents were

revealed as a shift in baseline current following the addition of the

GABA_Areceptor antagonist gabazine (SR-95531, Sigma–Aldrich)

to the perfusing solution.

Optogenetical Stimulation of

Thalamocortical Fibers

Adeno-associated virus carrying cDNA for channelrhodopsin

and the ﬂuorescent reporter mCherry (AAV2/1.CAG.hChR2

(H134R)-mCherry; Karl Deisseroth, Stanford University via the

UPenn VectorCore, PA, USA) were injected into the VPM of WT

or mGlu7^AAAmice at post-natal days 13 (stereotaxic coordinates

Minimal Evoked Monosynaptic

Responses: Single and Repetitive

Stimulation

For electrical and optogenetical stimulation experiments, single- from olfactive bulb junction were lateral, 2.10 mm; posterior,

stimulation and train-stimuli were applied on slices to activate 4.50 mm; and depth, 3.50 mm). The viral titer was 10¹¹IU/ml

small groups of neurons within TC loop regions, and we then and injection volume was 0.7 µl. After allowing 4–8 days for

recorded evoked responses in neurons receiving the input. ChR2 expression, acute somatosensory thalamocortical slices

Immediately after obtaining a whole cell recording, a few test were used for in vitro patch-clamp recording (see above). VPM

stimuli were delivered to check the synaptic responsiveness of synaptic terminals expressing hChR2-mCherry were stimulated

the neuron. Once monosynaptic response was clearly observed, with blue light (LED, 488 nm, 0.1 ms/pulse, 40–200 µW)

evoked excitatory (eEPSCs) or inhibitory postsynaptic currents shined in the barrel cortex layer IV. Cortical neurons were

(eIPSCs), respectively, were isolated by switching the perfusion identiﬁed as excitatory cells, Regular-Spiking (RS) or Fast-

solution from normal ACSF to one containing drugs (isolation Spiking (FS) interneurons, according to their electrophysiological

of AMPA currents with ionotropic GABA and NMDA receptor properties (see Supplementary Figure 2). In some cases, at the

blockers, 10 µM bicuculline and 50 µM D-AP5; isolation of end of the experiments TC slices were incubated overnight in

GABA currents with ionotropic glutamate receptor blockers, phosphate buﬀer saline solution (PBS) containing 4% PFA at

10 µM CNQX and 50 µM D-AP5; Tocris Bioscience). After 4^◦C and mounted between microscope slides and coverslips

7 min drugs perfusion, test stimuli of increasing intensity were using Mowiol to image the correct localization of virus-induced

applied to determine the minimal excitability threshold evoking expression of hChR2-mCherry with an AxioImager Z1 apotome

monosynaptic postsynaptic currents. Stimulation vs. amplitude and AxioVision software (Carl Zeiss, France).

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mGlu7 Modulates the Thalamic Network

in triplicate) in 96-well sterile, black microplates in DMEM

supplemented with 10% FCS. After 24 h, DMEM medium

was replaced by Glutamax^TM(GIBCO, Thermo Fisher, France)

2 h before performing TR-FRET experiments. Notice that

the expression of EAAC1 as well as Glutamax^TMallowed

obtaining a minimal glutamate extracellular concentration, to

avoid glutamate-mediated activation of mGlu7a receptors.

Surgery and Electroencephalography

(EEG) Recordings

Wild-type or mGlu7^AAAmale mice were anesthetized with

2 ml/kg of a saline solution containing 40% (v/v) ketamine

(Imalgene 500) and 20% (v/v) xylazine (Rompun 2%) and placed

in a stereotaxic frame using the David Kopf mouse adaptor.

A male microconnector (Pinnacle Technology Inc., Lawrence,

KS, USA) was ﬁxed by four extradural screws, two on each

parietal bone. The microconnector was then ﬁxed with dental

acrylic cement. After surgery, animals were individually housed

and maintained in a 12 h light/dark cycle with food and water ad

libitum for 1 week of recovery.

Inositol Phosphate 1 Measurement by

Time-Resolved Fluorescence Resonance

Energy Transfer (TR-FRET) Assay

The production of IP1 by HEK293 cells was tested using

TR-FRET assays, based on Time-Resolved Resonance Energy

Transfer. TR-FRET assays were performed in presence of LiCl

to block IP1 degradation, using anti-IP1 mouse antibody labeled

with Terbium cryptate-conjugate (Tb, Tb-Ab) that will be the

donor of energy, and d2-conjugated IP1 (d2, d2-IP1) that will

be the acceptor of energy (Cisbio Bioassays, France). After

30 min incubation at 37^◦C with stimulation buﬀer alone (ꢁ

Base ꢂ) or supplemented with a group III mGluRs agonist (L-

AP4, 600 µM) or antagonist (CPPG, 100 µM, Tocris Bioscience,

France) or the mGlu7-speciﬁc NAM ADX71743 (10 µM), d2-

IP1 acceptor and Tb-Ab donor were added and cells incubated

for 1 h at room temperature. The anti-IP1 antibody competes

with native IP1 produced by cells and D2-labeled IP1 from the

kit. The ﬂuorescence of Tb and d2 were measured, respectively,

at 620 and 665 nm (after 50 µs delay) and divided by the

337 nm excitation signal using a RUBYstar plate reader (BMG,

Germany). The resulting signal is inversely proportional to

the concentration of IP1 in samples, determined by using a

standard curve construction, since the donor–acceptor pair

gives a high transfer of energy signal that decreases upon

competition with IP1 produced by the cells (Garbison et al.,

2012).

For recordings, animals were put into individual Plexiglas

boxes, and their micro connectors were plugged to an

EEG preampliﬁer circuit and to the EEG ampliﬁer (Pinnacle

Technology Inc., Lawrence, KS, USA). The electrical activity

recorded by extradural electrodes was ﬁltered at 50 Hz, sampled

at 400 Hz and recorded by a computer equipped with Sirenia^ꢀR

software (Pinnacle Technology Inc.). A 30 min period of baseline

recording before ADX71743 injection (i.p.; volume of injection:

100 µl/10 g mouse weight of a 100 mg/kg suspension in 50% v/v

H₂0 and hydroxyl-propyl-β-cyclodextrin) allowed detection of

EEG abnormalities independent of experimental manipulations.

Control animals were injected with vehicle alone. EEG recordings

were performed together with monitoring animal behavior.

The symptoms developing after injection were classiﬁed into

diﬀerent phases as described by (Weiergraber et al., 2006)):

phase 0 corresponds to normal exploration and posture; phase

1 corresponds to an “absence-like” non-convulsive state, with

reduced motility and typical prostrated position of the animal;

phase 2 shows partial clonus of head, vibrissae and/or forelimbs;

phase 3 consists of a generalized clonus of the extremities, the

tail, and sometimes vocalization; this phase can develop into

typical “running ﬁts”; phase 4 corresponds to a generalized

tonic-clonic seizure with extension of the four paws; this phase

is occasionally followed by death by respiratory failure. We

deﬁned “lethargy” as a continuous period of immobility or

slow movement of the mouse limited to less than one body-

length of the mouse. Observations were made by a blind

observer.

Data Analysis

Whole-cell recordings were analyzed using Clampﬁt 10.2

software (Molecular Devices). Response amplitudes, short-term

plasticity ratios, mIPSCs properties and tonic current amplitudes

were compared between WT and mGlu7a^AAAmice using the

Mann-Whitney test or repeated measures ANOVA (^∗P < 0.05,

HEK293 Transfection

In order to enhance the read-out of our pharmacological tests, ^∗∗P < 0.01, ^∗∗∗P < 0.001). Normalized IP1 concentrations

we used the chimeric Gq/io protein that is able to bind all GPCRs obtained from TR-FRET experiments were compared using an

(GqTOP). Stimulation of the mGlu7/GqTOP pathway activates unpaired t-student test (^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001). All

phospholipase C (PLC), which hydrolyzes phosphatidylinositol averaged data are presented as mean ± SEM.

bisphosphate (PIP2) to form two second messengers, inositol

Eelectroencephalography signals were analyzed using Matlab

1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 is very 2013b software (Matworks Inc.). Short time Fourier transform

rapidly hydrolyzed to IP2 then to IP1, and ﬁnally to inositol analysis was realized using the Signal Processing toolbox

by a series of enzymatic reactions (Berridge and T a ylor, 1988) (Spectrogram function) and applied to the EEG data in order to

blocked by lithium chloride (LiCl; Garbison et al., 2012). HEK293 obtain the energy of the signal as a function of frequency and time

cells were transfected by electroporation (250 V–500 µF for (Freeman and Quiroga, 2013). Bartlett-type window was used to

5.10⁶cells) with plasmids expressing the GqTOP protein and the avoid leakage.

glutamate transporter EAAC1, associated with vehicle plasmids

We extracted a qualitative measure for each of the EEG

(control, MOCK) or mGlu7a-expressing plasmid (Perroy et al., frequency bands i (i = δ, θ, α, β, γ), where δ = 2 – 4 Hz, θ = 4 –

2000). Cells were plated at 150,000 per well (each condition 8 Hz, α = 8 – 14 Hz, β = 14 – 30 Hz, and γ = 30 – 70. This

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mGlu7 Modulates the Thalamic Network

measurement is deﬁned as:

mice (31.2 ± 9.8%; n = 15), highlighting a tonic activity of the

receptor. The eﬀect of the NAM was much smaller in mGlu7a^AAA

mice (1.9 ± 9.2%; n = 13; p = 0.0111; Figures 1 F ,G). These results

suggest the existence of an mGlu7a receptor PDZ-dependent

inhibition of glutamatergic VPM projections within the TRN in

WT mice.

As previously described (Mistry et al., 2008 ), a 10 Hz 5-pulse

stimulation protocol applied to VPM-TRN synapses induced

either synaptic depression or synaptic facilitation, in both WT

and mGlu7a^AAAmice (Figure 2). Depression ratios were non-

signiﬁcantly modiﬁed in mutant mice (Figures 2A,B), while

facilitation ratios were signiﬁcantly higher compared to WT

mice (Figures 2C,D). Moreover, the proportion of neurons

showing a facilitating response was signiﬁcantly increased in

mGlu7a^AAAmice (Figure 2E). These data indicate that an

active mGlu7a receptor PDZ-dependent pathway promotes

Z

f_mⁱ_ax

Iⁱ(t) =

I(f , t)dﬁ = δ, θ, α, β, γ

(1)

f_mⁱ_in

where (f_mⁱ_in, f_mⁱ_ax) are the frequency limits for the band i, and I(f,t)

is the power spectrum as a function of frequency f and time t.

The time windows used 256 points (0.64 s at a sampling rate of

400 Hz).

RESULTS

Modulation of Glutamatergic Thalamic

Synapses by mGlu7a Receptor

PDZ-Dependent Signaling

We aimed at identifying the thalamic synapses functionally the low-pass ﬁltering of information from the VPM to

expressing mGlu7a receptors, by comparing the response of WT the TRN by increasing synaptic depression and lessening

mice to mGlu7a^AAAmice, which are deﬁcient in controlling facilitation in basal conditions. Interestingly, treatment with

neurotransmitter release (Zhang et al., 2008).

ADX7173 did not change the short-term plasticity (data not

Intra-thalamic transmission properties were ﬁrst tested by shown), likely a consequence of a long-term absence of

electrically stimulating VPM neurons and recording evoked mGlu7a C-terminal signaling. The lower EPSCs amplitudes in

excitatory postsynaptic currents (EPSCs) in the TRN on in vitro mGlu7a^AAAmice seen in Figure 1C might reﬂect presynaptic

TC slices (Figure 1B). Neurons from mGlu7a^AAAmice showed fatigue due to the over-excitation of synaptic terminals. The

smaller EPSCs than WT neurons (WT: −124.3 ± 17.9 pA, tonic activity of mGlu7a receptors might lead to an inhibition

n = 48; mGlu7a^AAA: −68.0 ± 15.2 pA, n = 28, p = 0.050; of voltage-gated calcium channels, which would decrease Ca²⁺

Figure 1C). This observation was not due to a change in input entry into WT synapse terminals. Another possibility would

resistance, which was similar in WT and mGlu7a^AAATRN be that postsynaptic glutamate receptors desensitize upon

neurons (261 ± 90 MOhm for WT, 272 ± 43 MOhm for sustained activation. This would be consistent with our results

mGlu7a^AAA, p = 0.9088). The non-selective mGlu7 receptor showing that the synaptic facilitation is favored and depression

agonist, D,L-AP4 (1.2 mM), induced the inhibition of evoked is reduced in mutant mice, in the absence of exogenous

EPSC. This was signiﬁcantly smaller in mGlu7a^AAA(32.6 ± 5.3%, agonists.

n = 15) than in WT neurons (53.0 ± 3.8%, n = 32; p = 0.0034;

Figures 1D,E). The diﬀerence between the two values represents

Modulation of GABAergic Synaptic

Transmission within and from Thalamic

Reticular Neurons by mGlu7 Receptors

the mGlu7a-PDZ ligand-dependent pathway activation. The

residual inhibition induced by D,L-AP4 likely resulted from

action of the drug on mGlu4 and/or mGlu8 receptors, or PDZ

ligand-independent mGlu7 receptor signaling. This applies to Thalamic reticular nucleus neurons innervate adjacent reticular

all the synapses we will analyze in the following experiments. neurons as well as VPM neurons and constitute the only

Analysis of amplitude response as a function of stimulation inhibitory input in these two nuclei in mice (Pinault and

intensity is given in Supplementary Figure 1. For neurons Deschenes, 1998). We investigated the synaptic function of

requiring low stimulation intensity, the responses of WT and the mGlu7 receptor on synaptic transmission and short-term

mGlu7a^AAAsynapses were similar, suggesting that the properties plasticity by electrically stimulating TRN neurons, and recording

of postsynaptic receptors are not involved. However, for neurons the evoked IPSCs into either TRN or VPM nuclei in TC slices

requiring higher stimulation intensities, which require more (Figures 3 and 4).

available vesicles, WT synapses respond more strongly than

Amplitudes of intra-TRN IPSCs evoked by single stimulations

mGlu7a^AAAsynapses, suggesting that the pool of vesicles in were similar in WT and mGlu7a^AAAslices (Figure 3B). D,L-AP4

the mGlu7a^AAAneurons is limiting. This is in keeping with a inhibited intra-TRN evoked IPSCs and this eﬀect was reduced in

greater proportion of neurons presenting a facilitating response mGlu7a^AAAmice (14.7 ± 2.6%, n = 21) as compared to WT mice

in mGlu7a^AAAVPM to TRN synapses, as smaller initial responses (31.6 ± 3.6%, n = 23; p < 0.001; Figures 3C,D). Interestingly,

have a greater potential to facilitate (see for example Zucker we found that the amplitudes of eIPSCs were increased by

and Regehr, 2002). Another possibility is that a lower number ADX71743 in WT mice (31.2 ± 9.7%; n = 12). In this context

of ﬁbers is recruited upon stimulation in mGlu7a^AAAmice the contribution of endogenous glutamate release to activation

as a consequence of developmental modiﬁcations of network of the receptor should be limited, suggesting that the mGlu7

excitability.

receptor constitutively inhibits GABA release by TRN neurons.

We also found that eEPSCs were increased by the negative Conversely, the eﬀect of the NAM was minimal in mGlu7^AAA

allosteric mGlu7 modulator (NAM) ADX71743 (10 µM) in WT mice (0.56 ± 10.8%; n = 13; p = 0.0297; Figures 3E,F), implying

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similar in WT and mGlu7a^AAAmice (Figures 4C,D), implying a

non-contribution of mGlu7a receptor PDZ-dependent pathway

in the control of those synapses. However, we found that eIPSCs

were smaller in mGlu7a^AAAmice (Figure 4B), a phenomenon

that might be due to indirect fatigue following overactivation of

the intra-TRN pathway.

In synthesis, the PDZ-dependent pathway of mGlu7a receptor

modulates evoked inhibitory synaptic transmission only in intra-

TRN, but not in TRN–VPM synapses.

The above results showed that mGlu7a receptors control

evoked synaptic activity in the thalamus. Here, we investigated

whether the mGlu7a receptor could also aﬀect the spontaneous

synaptic activity of reticular neurons. We ﬁrst examined phasic

that this constitutive activity is dependent on mGlu7 PDZ ligand-

binding site.

In accordance with previous studies (Reichova and Sherman,

2004), we found that repetitive stimulation induced depression

of intra-TRN evoked IPSCs. This eﬀect was signiﬁcantly reduced

in mGlu7a^AAAmice (Figures 3G,H), thus reﬂecting a reduced

plasticity of GABA synapses.

mGlu7a Receptors Reduce Tonic GABA

Currents in the VPM

At TRN–VPM synapses (Figure 4A), we found that phasic,

D,L-AP4-induced inhibition as well as synaptic depression were

FIGURE 1 | The mGlu7 receptor modulates glutamatergic transmission at VPM synapses onto TRN neurons. (A) Schematic representation of the

thalamocortical and intra-thalamic connections between glutamatergic and GABAergic neurons in the somatosensory cortex (S1), the thalamic reticular nucleus

(TRN) composed exclusively of GABAergic neurons, and the ventro-postero-medial nucleus (VPM), devoid of interneurons. FS, Fast-Spiking interneurons; RS,

Regular-Spiking interneurons; Exc, excitatory glutamatergic neurons. (B) Experimental conﬁguration, including the positions of the electrical stimulation into the VPM

and the recording electrode into the TRN. (C) Single and average amplitudes of evoked EPSCs in WT and mGlu7a^AAAmice. (D,F) Representative evoked EPSCs

recorded from TRN neurons in response to VPM stimulation of WT (left) and mGlu7a^AAA(right) mice and effect of D,L-AP4 (1.2 mM, D) and ADX71743 (10 µM, F).

Black arrows indicate electrical stimulations. (E) Summary of D,L-AP4-induced inhibition of EPSCs. (G) Summary of ADX71743-induced increase of EPSCs.

^∗P < 0.05, ^∗∗P < 0.01, Mann-and-Whitney test. Error bars indicate SEM.

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GABA_Ainhibition in neurons from WT and mGlu7^AAATC 35.23 ± 7.96, n = 14; mGlu7a^AAA: 60.20 ± 6.83, n = 18; p < 0.01;

slices. mIPSC were studied in both the TRN and VPM neurons, Figures 5G,H), suggesting that extra-synaptic GABA level in the

which receive GABAergic inputs exclusively from TRN neurons. VPM is higher in mGlu7^AAAthan in control mice and reinforcing

The frequency of mIPSCs was higher in both TRN (WT: the idea of an essential basal modulation of TRN activity by the

1.30 ± 0.10 Hz, n = 12; mGlu7^AAA: 3.09 ± 0.27 Hz, n = 14; mGlu7 receptor. Indeed, application of ADX71743 (10 µM) on

p < 0.0001; Figures 5A,B) and VPM neurons (WT: 10.92 ± 1.23, WT VPM neurons increased the gabazine-sensitive current to

n = 18; mGlu7^AAA: 18.34 ± 1.05, n = 20; p < 0.0001; levels comparable to those found in mGlu7a^AAAmice and had

Figures 5D,E) of mGlu7^AAAthan in WT mice, while mIPSCs no eﬀect on mGlu7a^AAAmice (Figure 5H). These results suggest

amplitudes were identical in both genotypes (Figures 5C,F). that mGlu7 receptors tonically inhibit GABA release on VPM

Enhanced mIPSCs frequency could be due to long-term change neurons. Conversely, tonic GABA_Acurrent was absent in TRN

of the release machinery and/or diﬀerential presynaptic bouton neurons of both WT and mGlu7a^AAAmice (data not shown),

handling of calcium levels.

which is in accordance with the absence of extra-synaptic GABA_A

Altogether these results suggest the implication of the mGlu7a receptors in this nucleus (Jia et al., 2005).

receptor PDZ-dependent pathway in spontaneous GABA release

from TRN neurons.

mGlu7 Receptors Depress VPM to

Cortical Layer 4 Fast-spiking Interneuron

Synaptically released GABA can also diﬀuse outside the

synapse and activate extra-synaptic GABA_Areceptors, leading

to a tonic GABA_Acurrent (Mody, 2001 ) that can be

speciﬁcally blocked by gabazine (Cope et al., 2005 ). Using this

pharmacological tool, we found that tonic GABA_Acurrent

amplitude was increased in the VPM of mGluR7a^AAAslices (WT:

Synapses

Thalamic neurons relay sensory information to the barrel

cortex, and VPM axon terminals project predominantly onto

cortical layer 4, where three main classes of neurons are

FIGURE 2 | Short-term synaptic plasticity in VPM to TRN synapses. (A,C) Representative current traces from TRN neurons responding to ﬁve stimuli at 10 Hz

(black arrows) in WT (left) and mGlu7^AAA(right) mice. VPM afferents display either short-term synaptic depression (A) or facilitation (C) in both genotypes.

(B,D) Short-term synaptic depression ratios show no signiﬁcant differences between the two genotypes, while facilitation ratios are strongly increased in mGlu7^AAA

mice. (E) Proportion of neurons displaying either short-term synaptic depression or facilitation in WT (left) or mGlu7a^AAA(right) mice. ^∗P < 0.05, Mann-and-Whitney

test. Error bars indicate SEM.

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FIGURE 3 | The mGlu7 receptor modulates GABAergic transmission and short-term synaptic plasticity in intra-TRN synapses. (A) Experimental

conﬁguration, including positions of electrical stimulation into the TRN and recording in adjacent reticular neurons. (B) Evoked IPSC amplitudes show no difference in

responsiveness between WT and mGlu7^AAAmice. (C,E) Representative IPSC recorded from TRN neurons in response to TRN single stimulation in WT (left) and

mGlu7a^AAA(right) mice before and after application of D,L-AP4 (C) or ADX71743 (E). Black arrows indicate electrical stimulations. (D) D,L-AP4-induced IPSC

inhibition. (F) ADX71743-induced EPSC increase. (G) Representative traces in response to 5 × 10 Hz stimuli and (H) short-term synaptic depression ratios.

^∗P < 0.05, ^∗∗∗P < 0.001, Mann-and-Whitney test. Error bars indicate SEM.

found: glutamatergic neurons (i.e., pyramidal and spiny stellate VPM neurons by AAV-mediated viral infection and eEPSCs were

neurons), and the RS and FS GABAergic interneurons. They are induced in cortical layer 4 neurons by shining blue light (488 nm)

so-called on the basis of their ﬁring pattern in response to current in this area (Figure 6A).

injection, as conﬁrmed by our characterization (Supplementary

Figure 2).

The inhibition of evoked EPSCs by D,L-AP4 in FS

interneurons was smaller in mutant mGlu7a^AAAthan in

As thalamo-cortical and cortico-thalamic ﬁbers follow the WT mice (26.4 ± 3.4% vs. 39.0 ± 3.5%; p = 0.023; Figures 6B,C).

same path, we used an optogenetic approach to selectively VPM-FS synapses showed depression upon repetitive stimulation

stimulate thalamocortical aﬀerents in brain slices (Cruikshank (Figures 6D,E), as described in previous studies (Cruikshank

et al., 2010). Channelrhodopsin 2 (hChR2) was expressed in et al., 2010), but depression ratios were signiﬁcantly smaller in

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FIGURE 4 | The mGlu7 receptors modulate TRN neurons-mediated tonic GABA_Ainhibition in the VPM. (A) Experimental conﬁguration, including positions

of recording electrode into the VPM and electrical stimulus on TRN. (B) Single and average IPSC amplitudes in WT and mGlu7a^AAAmice. (C,E) Representative IPSC

traces from VPM neurons responding to TRN single (C) or 5 × 10 Hz (E) stimulation of WT (left) and mGlu7a^AAA(right) mice. Black arrows indicate electrical

stimulations. (D,F) D,L-AP4-induced IPSC inhibition and short-term synaptic depression ratios are similar in both genotypes. ^∗∗P < 0.01, Mann-and-Whitney test.

Error bars indicate SEM.

mGlu7a^AAAthan in control mice FS interneurons. These data

suggest a regulation of cortical layer 4 FS interneuron activity by

VPM aﬀerents involving an mGlu7a receptor PDZ-dependent

pathway.

We then investigated the eﬀects of negative allosteric

modulation of the mGlu7 receptor-mediated inhibition of FS

interneurons EPSCs in TC slices from WT mice. ADX71743

increased FS interneuron EPSC amplitude evoked by single

stimulation of VPM axons (Figures 6 F ,G).

On the contrary, inhibition of single evoked EPSCs as

well as synaptic depression ratio upon repetitive VPM aﬀerent

stimulations (Figures 6H,I) recorded from cortical layer 4

glutamatergic neurons and RS interneurons were similar in

both mice genotypes. Taken together, these results indicate an

mGlu7a receptor PDZ dependent-pathway mediated inhibition

of VPM glutamatergic contacts innervating cortical layer 4 FS

interneurons, but not other studied cortical cell types. They also

suggest that mGlu7 receptors tonically inhibit excitation of FS

interneurons by VPM aﬀerents.

we investigated whether the mGlu7 receptor NAM ADX71743

displayed a true inverse agonist activity in the virtual absence

of glutamate. Inverse agonists are a class of pharmacological

compounds that block receptor constitutive activity. We

transfected HEK cells with diﬀerent amounts of mGlu7a receptor

cDNA and measured receptor activity as inositol production

by using a chimeric G protein-based time-resolved FRET

assay (TR-FRET, see Section “Materials and Methods” for

details). Importantly, co-transfection of the excitatory amino-

acid transporter EAAC1 allowed lowering the extracellular

glutamate concentration to a sub-micromolar range, unable

to activate mGlu7a receptors (P. Rondard, IGF Montpellier,

personal communication). We remarked a higher receptor

activity as compared to the control transfection, which was

increasingly proportional to the quantity of mGlu7a receptor

expressed (Figure 7A). This suggested a constitutive activity

of the recombinant receptor. We then compared the eﬀect of

the orthosteric non-selective mGlu7a receptor agonist L-AP4

(600 µM), and antagonist CPPG (100 µM), as well as the

selective mGlu7 receptor NAM ADX71743 (10 µM) on the

production of inositol phosphates (IP) in mGlu7a receptor-

transfected HEK cells (Figure 7B). In mGlu7a-transfected cells,

IP1 production increased in the presence of L-AP4, conﬁrming

Potential Constitutive Activity of mGlu7

Receptors

The lack of mGlu7-speciﬁc orthosteric antagonist prevented us that the recombinant receptor was functionally expressed in

to deﬁnitively conclude on whether mGlu7 has a constitutive, HEK cells. The basal production of IP1 in HEK cells expressing

agonist-independent activity in acute TC slices. Nevertheless mGlu7a receptors was not modiﬁed by CPPG, as expected from

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FIGURE 5 | The mGlu7a receptor decreases TRN neurons-mediated tonic and phasic GABA_Ainhibition in VPM and TRN. (A) Representative current

traces of spontaneous mIPSCs recorded from WT and mGlu7^AAATRN neurons in the presence of TTX (1 µM). (B,C) Mean mIPSCs frequency (B) and amplitude (C)

in TRN neurons. (D,E,F) Same legend as in (A–C), respectively, in VPM neurons. (G) Example traces of tonic GABA_Acurrent recorded in WT and mGlu7^AAATRN

neurons upon application of 50 µM gabazine (top traces, black and blue) or gabazine plus ADX71743 (10 µM, bottom traces, orange and red). The baseline shift

reﬂects the inhibition of tonic GABA_Acurrent. (H) Average tonic GABA_Acurrent amplitude in WT and mGlu7^AAAneurons with and without co-application of

ADX71743 (10 µM). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, Mann-and-Whitney test. Error bars indicate SEM.

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FIGURE 6 | The mGlu7 receptor modulates glutamatergic transmission between VPM neurons and cortical layer 4 Fast-Spiking interneurons.

(A) Experimental conﬁguration and example of a thalamocortical slice expressing hChR2-mCherry in VPM thalamic neurons and their axons targeting cortical layer 4.

LED stimuli (blue light, 488 nm) were directed on layer 4 thalamocortical hChR2-expressing terminals where postsynaptic responses of cortical neurons were

recorded. (B,D) Representative current traces recorded from a Fast-Spiking interneuron in response to VPM single (B) or 5 × 10 Hz (D) stimulations, in WT (left) and

mGlu7a^AAA(right) mice. Blue arrows indicate light stimulations. (C) D,L-AP4-induced EPSC amplitude inhibition (n = 17 for WT, n = 19 for mGlu7a^AAAmice).

(E) Synaptic depression ratios in WT (left) and mGlu7a^AAAmice. Please note the decrease in synaptic depression in the mGlu7^AAAmice. (F) Representative current

traces showing the increase in EPSC by ADX71743. (G) Summary of the effects of ADX71743 (10 µM) on EPSCs. (H,I) Average EPSC inhibition induced by D,L-AP4

and short-term synaptic depression ratios in excitatory neurons and Regular-Spiking interneurons, respectively. Please note the absence of difference between WT

and mGlu7a^AAAmice. ^∗P < 0.05, Mann-and-Whitney test. Error bars indicate SEM.

a classical orthosteric antagonist in the absence of agonist. On the eﬀects of ADX71743 (100 mg/kg i.p.) or vehicle alone

the contrary, ADX71743 inhibited basal IP1 production in the (β-cyclodextrin, CD) on brain activity and behavior in WT

absence of agonist, likely resulting from constitutive activity of and mGlu7a^AAAmice, where neurotransmitter release inhibition

the recombinant mGlu7a receptor.

mediated by mGlu7a receptor is lost. CD-injected (control)

mice continued normal exploratory activity and grooming,

and showed no modiﬁcation of their EEG pattern. The

majority of WT mice (31 out of 36 mice) injected with

ADX71743 showed a net decrease in exploratory activity and

reactivity, entering a phase of prostration (Figure 8A). Such

a lethargic status appeared 5 ± 0.5 min after injection and

lasted 43.8 ± 3.7 min (Figure 8G). The mGlu7a^AAAmutant

In Vivo Induction of Absence Epileptic

Seizures and Lethargy by the mGlu7

Receptor Inverse Agonist

What is the physiological role of the mGlu7 receptor tonic

activity? We examined this issue in vivo by comparing

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FIGURE 7 | Constitutive activity of mGlu7a receptors in the absence of extracellular glutamate. (A) Normalized IP1 production in HEK cells transfected or

not (Mock) with increasing amount of mGlu7a receptor cDNA (n = 3). Note that transfection of mGlu7a receptor in the absence of agonist induces IP1 synthesis.

(B) IP1 formation measured in the absence (Base) and presence of the indicated compounds in HEK cells transfected with either wild-type (WT) or a control empty

vector (n = 5). ^∗P < 0.05, ^∗∗∗P < 0.001, Mann-and-Whitney test. Error bars indicate SEM.

mice showed the typical absence-like behavior with repeated in TRN synapses. Furthermore, we demonstrate the involvement

episodes of exploratory activity arrest and facial myoclonus of the mGlu7a receptor PDZ-dependent pathway in short-term

previously reported (Bertaso et al., 2008). Administration of plasticity enhancement, reinforcing depression, and reducing

ADX71743 did not worsen this phenotype (31 out of 32, facilitation in WT mice. Mutation of the C-terminal PDZ ligand

Figure 8A).

motif changes the balance between facilitation and depression

The analysis of EEG in WT mice treated with the ADX71743 in synapses onto TRN neurons and cortical FS interneurons in

revealed an increase in the power of the 0–4 Hz delta band mGlu7a^AAAmice, an eﬀect that might result from the long-

and 4–8 theta band (Niedermeyer and Lopes Da Silva, 1993), term absence of mGlu7 in the TC network. Previous work has

with appearance of slow-wave spindles (2–8 Hz) with recurrent shown that expression of the closely related mGlu4 receptor is

spikes typical of K-complexes of non-REM sleep compared not changed in the mGlu7a^AAAmouse, nor is the localization of

to control mice (Figures 8B,D, n = 21 mice). In four cases mGlu7 (Zhang et al., 2008). However, the results were not speciﬁc

out of 21, on top of the described sleep-related activity, to the thalamocortical circuit. Also, the possibility that other

we observed SWD with an intrinsic frequency of 8–10 Hz proteins involved in the mGlu7/PICK1 signaling pathway (e.g.,

(Figure 8C), similar to those spont aneously arising in non- vesicular proteins, calcium channels) may be changed warrants

treated mGlu7a^AAAmice (Figure 8E and Bertaso et al., 2008). further investigation.

Administration of ADX71743 to mGlu7a^AAAmice did not

These data suggest that the receptor exerts a tonic modulation

increase the shape, number or duration of SWD (Figure 8F). of basal synaptic transmission by constantly and speciﬁcally

More details on frequency band analysis can be found in shaping low frequency excitatory and inhibitory postsynaptic

Supplementary Figure 3. These data show that pharmacological responses. This novel mode of action of the receptor takes place

blockade of mGlu7 receptor tonic activity in vivo induces a in near-physiological conditions, in addition to its classical low-

lethargic state accompanied by abnormal sleep-related behavior pass ﬁlter action during enhanced synaptic activity. The receptor

and absence epileptic seizures.

modulates both GABAergic and glutamatergic transmission in

the thalamus, and its inhibition has consequences on animal

behavior.

DISCUSSION

ADX71743 Reveals the Potential

Constitutive Activity of the mGlu7

Receptor

Despite its wide distribution and conservation among species

(Flor et al., 1997; Kinoshita et al., 1998), the function of

mGlu7 receptor in the brain remains poorly understood. The

homology with the other mGluRs has to date hindered the Our work indicates that endogenous WT mGlu7 receptors

design of type-speciﬁc orthosteric agonists and antagonists. Here, are tonically active. Due to the limitations brought by the

we took advantage of the combination of a genetic mouse available pharmacology we cannot exclude a contribution of

model and a new inverse agonist to challenge the classical view elicited glutamate release to the activity of the receptor. However,

of mGlu7 as a receptor exclusively activated when glutamate we may deduce that at least part of the eﬀect was due to

concentration rises high. Our results show that mGlu7 receptors an agonist-independent activity. Several arguments favor the

are functionally expressed at synapses formed onto and between existence of a constitutive activity of mGlu7. First, our results

TRN neurons, as well as VPM synapses onto fast-spiking in HEK cells show that, in an isolated system where the

interneurons of the somatosensory cortical layer 4. We uncover receptor expression and glutamate concentration are tightly

the modulation of the tonic GABA current by mGlu7 receptors controlled, mGlu7 receptors can act in an agonist-independent

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FIGURE 8 | EEG activity induced by treatment with the mGlu7 receptor NAM. (A) Behavioral response of mice WT and mGlu7^AAAmice treated with the either

vehicle or mGlu7 NAM (100 mg/kg i.p.) represented as percentage of total animals tested (n for each score group is indicated on the histogram bars). The scoring

scale is given on the right. (B–F) Example of EEG traces and corresponding short-time Fourier transform representation (frequency over time) are shown for WT mice

injected with either the vehicle alone (B) or the mGlu7 NAM (C,D) and for representative mGlu7^AAAmice showing spontaneous SWD when treated with the vehicle

(E) or with the mGlu7 NAM (F). Traces in (C,D) show, respectively, SWD-like and low frequency activity with recurrent spikes corresponding to K-complexes. The

level of wavelet power is represented using the color scale. (G) Latency and duration of the lethargic state observed in WT mice upon ADX71743 administration.

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FIGURE 9 | Summary of ﬁndings on functional expression of mGlu7 receptors in the thalamocortical network and alterations brought by the loss of

mGlu7 constitutive activity or by the mGlu7a^AAAmutation.

manner. Furthermore, the glutamate concentration in GABA PDZ-dependent pathway. We found that in the TC network,

synapses is bound to be well below the EC50 of mGlu7 mGlu7a receptor-expressing synapses display higher short-term

receptors (500 µM–1 mM, Schoepp et al., 1999) and yet mGlu7 synaptic depression than mGlu7a^AAAmutant synapses. Pelkey

responded to the application of the NAM in the TRN (phasic et al. (2008) showed that the combination of mGlu7a receptor

current) and VPM (tonic current). Interestingly, Turner and activation and mossy ﬁbers high frequency stimulation induces

Salt (1999) showed that two orthosteric mGluR antagonists, a PICK1-dependent long-term depression in hippocampus. It

MAP4 (acting on mGlu4, mGlu7, and mGlu8) and LY341495 thus appears that the mGlu7a receptor-PDZ ligand signaling

(acting on mGlu2, mGlu3, mGlu4, mGlu7, and mGlu8), had participates in both short-term and long-term plasticity, the

no eﬀect on evoked EPSP amplitude in thalamocortical slices, neural basis of many cognitive and emotional processes

suggesting the absence of endogenous glutamate-dependent (O’Connor et al., 2010). The PDZ interaction of mGlu7a with

activity. We previously proposed the existence of a tonic mGlu7a PICK1 is required for the receptor intracellular signaling, which

receptor-mediated inhibition of voltage-gated calcium channels could explain these eﬀects, but whether or not control of the

in cultured cerebellar granule cells, antagonized by binding of receptor tonic activity by PDZ interaction is required remains

the protein kinase C substrate MacMARCKS to the receptor open.

C-terminal tail (Bertaso et al., 2006). Recent data in dissociated

Our data suggest an excessive activation of VPM → TRN,

superior cervical ganglia neurons showed that transfection of VPM → cortical layer 4 FS interneurons and intra-TRN

mGlu7a receptor is suﬃcient to induce spontaneous receptor synapses in mGlu7^AAAmice, with several consequences on

intracellular signaling, and to inhibit voltage-dependent calcium thalamocortical network physiology. A tentative hypothesis is

channels (Kammermeier, 2015). However, the inverse agonist that such a tonic modulation of synaptic activities probably

used in the study, MMPIP, seems to have a diﬀerent eﬀect results from tonic activity of mGlu7 receptors. This may

depending on the cellular context (Niswender et al., 2010). The have the following consequences on the TC network functions

future discovery of a true, mGlu7-speciﬁc negative orthosteric (summarized in Figure 9):

antagonist will allow to conﬁrm these results in a clear-cut way.

(1) Over-activation of the TRN neurons by VPM should

induce hyper-excitation of reticular neurons, and increase

Tonically Active mGlu7a Receptors as

Gatekeepers against Aberrant TC

Oscillations

What could be the physiological role of the mGlu7a receptor

PDZ-dependent tonic activity? Genetic mouse models allowed a

deeper understanding of the mGlu7 receptor synaptic functions.

For example, the mGlu7 receptor KO and mGlu7a^AAAKI mice

display diﬀerent phenotypes (Sansig et al., 2001; Zhang et al.,

2008), suggesting speciﬁc synaptic functions of mGlu7a receptor

the inhibitory feedback of reticular neurons toward VPM

nucleus via a recurrent pathway. This eﬀect is ampliﬁed

by the reticular neurons-mediated spontaneous phasic

and tonic hyper-inhibition of thalamic cells. Interestingly,

increased TRN neurons-mediated tonic inhibition has been

observed in all the other absence epilepsy models (Cope and

Di Giovanni, 2009). Hyperpolarization of VPM neurons is

able to switch their ﬁring pattern from tonic to burst mode,

typically observed during absence seizures (Huguenard

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Tassin et al.

mGlu7 Modulates the Thalamic Network

and McCormick, 2007). This would be more likely in synapses onto interneurons expressing a postsynaptic protein,

mGlu7a^AAAas opposed to WT mice, where tonic activity of Elfn1 (Tomioka et al., 2014). Recently, Klar et al. (2015)

mGlu7 would provide a permanent control of those bursts. have found that mGlu7 receptors control the feed-forward

inhibition onto CA1 pyramidal neurons, acting on GABAergic

(2) Intra-TRN inhibition is able to desynchronize reticular

parvalbumin-expressing presynapses. Incidentally, the lack of

neurons activity (Beenhakker and Huguenard, 2009).

mGlu7-dependent inhibition observed in the VPM is in

Our results show that intra-TRN inhibition is increased

agreement with previous immunohistochemical data showing the

in the young mGlu7a^AAAmice used in the present

absence of mGlu7 receptor in mouse VPM (Kinoshita et al.,

studied. This eﬀect mediated by mGlu7a receptor PDZ

1998).

interaction should therefore prevent synchronization

An interesting question is raised by the consistently smaller

of the TRN neurons and protect against absence

response in some mGlu7a^AAAsynapses compared to WT.

seizures. Intra-TRN connections are known to slowly

These synapses displayed high EPSC or IPSC amplitudes

disappear during cerebral maturation in mice (Pinault,

(>50 pA) in WT mice, while no diﬀerence was observed in

2004). Therefore this protective eﬀect would be lost

intra-TRN low amplitude postsynaptic currents. This could

in adult mice, which may support the diﬀerence in

reﬂect a variety of mechanisms. First, the lack of mGlu7

the age at which absence seizures appear in rodent

receptor-mediated voltage-dependent Ca²⁺channels inhibition

models compared to human (Crunelli and Leresche,

in mutant mice might induce a sustained glutamate and

2002).

GABA release by VPM and TRN axon terminals, respectively.

Indeed, we showed that spontaneous GABA release by

TRN neurons is increased in mutant mice. As TRN and

cortical L4 neurons receive other glutamatergic inputs in

addition to VPM aﬀerents, we could not examine the VPM

neuron spontaneous activity. Ca²⁺accumulation into mGlu7

receptor-expressing axon terminals may also induce higher

facilitation and decreased depression, as observed in mutant

mice synapses. These eﬀects might deplete the releasable

pool of neurotransmitter in presynaptic sites, leading to

a synaptic depression. Alternative explanations could be

the internalization of postsynaptic receptors after sustained

neurotransmitter release or a decrease in thalamic ﬁber

excitability.

(3) Layer 4 FS interneurons are known to mediate fast and

powerful perisomatic IPSCs, and are responsible for

the main feed-forward inhibition in the TC network

(Porter et al., 2001; Cruikshank et al., 2007). Our

experiments in mGlu7a^AAATC slices suggest that mGlu7

receptor would prevent over-activation of VPM-FS

interneuron synapses in cortical layer 4, and hyper-

synchronization mediated by these interneurons (Swadlow

et al., 1998).

(4) Several studies have highlighted the impact of tonic GABA_A

current on the control of neuronal excitability, with

implications in diﬀerent pathologies such as epilepsies,

stress or depression-related paradigms (Glykys and Mody,

2007). We demonstrate here a negative feedback provided

by the mGlu7 receptor on tonic thalamic GABA_Acurrent.

Similarly, Errington et al. (2011) showed an inhibition of

the tonic GABA_Acurrent by mGlu receptors in thalamic

neurons, but the concentrations of L-AP4 used in the

study were too low to fully activate the mGlu7 receptor

subtype. Given the low aﬃnity of the receptor for glutamate,

it is possible that in physiological conditions the mGlu7

receptor acts mainly via its tonic rather than phasic

agonist-dependent activity, at least at GABAergic synapses

where the concentration of glutamate is expected to be

low.

mGlu7 Receptors at the Borders of Sleep

and Epilepsy

A few studies have highlighted the link of mGlu receptors

with absence seizures (Snead and Banerjee, 2000; Bertaso et al.,

2008). In vivo administration of ADX71743 enhanced thalamic

oscillations in association with drowsiness and behavioral arrest

in WT mice. In vitro activation of mGlu7 by L-AP4 in rats reduces

spindle oscillations, which typically occur during the early stage

of sleep or during active phases of slow-wave-sleep oscillations

(Kyuyoung and Huguenard, 2014).

As both sleep and absence seizures are supported by reciprocal

connections between glutamatergic VPM and GABAergic TRN

All these data highlight the fact that the physiological neurons, and somatosensory cortex (Steriade, 2006; Huguenard

function of the mGlu7 receptor seems of particular importance and McCormick, 2007), our data suggest that tonic activity

to modulate inputs onto GABAergic neurons. The phenotypic of the mGlu7 receptor prevents unbalanced activity of the

abnormalities found in mGlu7 receptor mutant models reﬂect network that would lead to abnormal sleep-related behavior

the strength of these synaptic controls in WT CNS. It or seizures. The border between the lethargic eﬀect and

is also interesting to underline the emerging concept that absence seizures seems blurry, as in a few animals the EEG

mGlu7 is capable to inhibit synaptic transmission in a revealed SWDs typical of epileptic seizures. The lack of eﬀect

target-speciﬁc manner, as shown by the diﬀerential eﬀect of ADX71743 in mGlu7a^AAAmice suggests the PDZ ligand-

on synapses harboring from VPM neurons to either the dependent tonic activity as essential for receptor functions.

cortical layer 4 excitatory neurons (no eﬀect of mGlu7) Alternatively, one could imagine an occlusion mechanism

or FS interneurons (inhibition). Interestingly, synapse-speciﬁc between the mutation of the receptor PDZ ligand motif and the

expression of mGlu7 has been found in hippocampal CA1 action of ADX71743.

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April 2016 | Volume 10 | Article 31

Tassin et al.

mGlu7 Modulates the Thalamic Network

Overall, it appears that the mGlu7 receptor modulates the

global thalamic state of excitability, reducing its propensity to

switch from the tonic to the oscillatory mode.

FUNDING

VT was supported by grants from the French MRT and the

In human, GRM7 gene polymorphisms have recently been Fondation pour la Recherche Médicale (FDT20130928376).

associated with autism (Y a ng and Pan, 2013), schizophrenia The study was supported by funding from the INSB (ITMM,

(Ohtsuki et al., 2008), attention deﬁcits and hyperactivity (Park Innovation Technologique pour les Maladies Mentales).

et al., 2013), age-dependent auditive deﬁcits (Friedman et al.,

2009), although not yet to epilepsies (Goodwin et al., 2000). By

acting directly at crucial nodes of oscillatory networks, the mGlu7

receptor could provide a new therapeutic target against epilepsies,

and more generally neuropathologies resulting from unbalanced

excitation/inhibition inputs.

ACKNOWLEDGMENTS

We thank the IGF/IGH RAM animal facility, N. Marchi and the

IPAM platform for help with the EEG recording and analysis, the

ARPEGE platform at IGF for advice on TR-FRET experiments,

K. Deisseroth for the hChR2-mCherry construct, H. Adesnik, M.

Bagnall, L. Ceolin, S. Dadak, F. De Bock, R. Lambert, and J. Ster

for useful discussion.

AUTHOR CONTRIBUTIONS

VT, JP, LF, and FB designed the study and wrote the manuscript;

VT, FB, and BG performed the electrophysiology, optogenetics,

and pharmacology experiments; AC and BG performed the

in vivo EEG recordings; PF designed the EEG analysis routine;

SUPPLEMENTARY MATERIAL

DR and FA designed the synthesis and synthesized the The Supplementary Material for this article can be found online

ADX71743.

at: http://journal.frontiersin.org/article/10.3389/fncir.2016.00031

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Cruikshank, S. J., Urabe, H., Nurmikko, A. V., and Connors, B. W. (2010).

Pathway-speciﬁc feedforward circuits between thalamus and neocortex

revealed by selective optical stimulation of axons. Neuron 65, 230–245. doi:

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Crunelli, V., and Leresche, N. (2002). Childhood absence epilepsy: genes, channels,

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Dalezios, Y., Luján, R., Shigemoto, R., Roberts, J. D. B., and Somogyi, P. (2002).

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GABAergic terminals on interneurons in the rat somatosensory cortex. Cereb.

Cortex 12, 961–974. doi: 10.1093/cercor/12.9.961

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Conﬂict of Interest Statement: The authors declare that the research was

conducted in the absence of any commercial or ﬁnancial relationships that could

be construed as a potential conﬂict of interest.

Acher, Fagni and Bertaso. This is an open-access article distributed under the terms

of the Creative Commons Attribution License (CC BY). The use, distribution or

reproduction in other forums is permitted, provided the original author(s) or licensor

are credited and that the original publication in this journal is cited, in accordance

with accepted academic practice. No use, distribution or reproduction is permitted

which does not comply with these terms.

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Article Doi

Phasic and tonic mGlu7 receptor activity modulates the thalamocortical network

DOI: 10.3389/fncir.2016.00031

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Article abstract of DOI:10.3389/fncir.2016.00031

Full text of DOI:10.3389/fncir.2016.00031

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