Experimental Neurology 255 (2014) 154–160

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Experimental Neurology

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Regular Article

Allosteric modulation of NMDA receptors alters neurotransmission in thestriatum of a mouse model of Parkinson's disease

Ze-Jun Feng, Xiaoqun Zhang, Karima Chergui ⁎The Karolinska Institute, Department of Physiology and Pharmacology, Section of Molecular Neurophysiology, Von Eulers väg 8, 171 77 Stockholm, Sweden

Abbreviations:6-OHDA, 6-hydroxydopamine; aCSF, armedium spiny neuron; NMDAR, N-methyl-D-aspartate reminiature excitatory postsynaptic current; sIPSC, spontacurrent; TH, tyrosine hydroxylase.⁎ Corresponding author.

E-mail addresses: ze-jun.feng@ki.se (Z.-J. Feng), xiaoqukarima.chergui@ki.se (K. Chergui).

http://dx.doi.org/10.1016/j.expneurol.2014.03.0010014-4886/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 January 2014Revised 3 March 2014Accepted 5 March 2014Available online 12 March 2014

Keywords:Parkinson's diseaseStriatumAllosteric modulationNMDA receptorsGlutamateGABAAcetylcholine

TheGluN2 subunits that composeN-methyl-D-aspartate receptors (NMDARs) are attractive drug targets for ther-apeutic intervention in several diseases, in particular Parkinson's disease (PD). The precise roles and possible dys-functions of NMDARs attributed to specific GluN2 subunits are however unresolved. Through the use of CIQ, anovel positive allosteric modulator of GluN2C/GluN2D-containing NMDARs, we have examined the functionsand dysfunctions of NMDARs made of GluN2D in the striatum of control mice and of the 6-hydroxydopamine(6-OHDA)-lesioned mouse model of PD. We found that CIQ (20 μM), applied to corticostriatal brain slices, in-creased the firing rate of spontaneously active cholinergic interneurons in the striatum of control mice and inthe intact striatum of 6-OHDA-lesioned mice. CIQ also presynaptically depressed GABAergic neurotransmissionthrough a cholinergic mechanism, but had no effect on glutamatergic neurotransmission, in medium spiny pro-jection neurons (MSNs) of control and intact striatum. In the dopamine-depleted striatum, the effect of CIQ onthe firing of cholinergic interneurons and GABAergic neurotransmission was lost. However, CIQ increased gluta-matergic neurotransmission in MSNs. We also found that the protein levels of GluN2D were increased in thedopamine-depleted striatum as compared to the intact striatum. However, the contribution of GluN2D-containing NMDARs to whole-cell NMDA currents was reduced in cholinergic interneurons and increased inMSNs. These results demonstrate an impaired modulatory role of GluN2D-containing NMDARs on the activityof cholinergic interneurons and inhibitory transmission in the dopamine-depleted striatum. However, potentia-tion of excitatory neurotransmission occurs upon activation of these receptors. Thus, altered functions ofGluN2D-containing NMDARs might contribute to adaptive changes in experimental Parkinsonism.

© 2014 Elsevier Inc. All rights reserved.

Introduction

Physiological and pathophysiological processes involving theN-methyl-D-aspartate type of glutamate receptor (NMDAR) are depen-dent on the subunit composition of these receptors. NMDARs areheterotetrameric assemblies of two GluN1 subunits and other GluN2(A-D) and GluN3 (A, B) subunits (Cull-Candy et al., 2001; Kohr, 2006;Paoletti, 2011; Paoletti and Neyton, 2007). The GluN2 subunits are at-tractive drug targets for therapeutic intervention in Parkinson's disease(PD) (Gardoni et al., 2010; Hallett and Standaert, 2004). GluN2B is themost abundant GluN2 subunit in the striatum, and antagonists actingon GluN2B-containing NMDARs have been investigated as possibletherapeutic agents for treatment of PDwith reduced propensity to elicitside effects as compared to broad spectrumNMDAR antagonists (Gogas,

tificial cerebrospinal fluid;MSN,ceptor; s/m EPSC, spontaneous/neous inhibitory postsynaptic

n.zhang@ki.se (X. Zhang),

2006; Loftis and Janowsky, 2003). Unfortunately, clinical trials withGluN2B-selective antagonists failed to provide clear benefit in PDpatients (Addy et al., 2009). The possibility that subunit-specific com-pounds acting on GluN2 subunits other than GluN2B might have benefi-cial properties in the treatment of PD has not been extensivelyexamined. Among the other GluN2 subunits present in the striatum thatcould play significant roles in the physiology of this brain region and inthe pathophysiology of PD, GluN2D is a potential candidate. Indeed,GluN2D-containing NMDARs have a low sensitivity to magnesium block-ade, as compared to NMDARs made of GluN2A and GluN2B, and are thuseasily activated under physiological conditions (Cull-Candy et al., 2001;Misra et al., 2000; Monyer et al., 1994; Paoletti, 2011). GluN2D is notexpressed in medium spiny projection neurons (MSNs) in the striatumbut is present in large aspiny cholinergic interneurons (Standaert et al.,1996). These interneurons play major roles in the physiology of the stria-tum and probably also in the pathophysiology of PD (Pisani et al., 2007).

Positive and negative allosteric modulators of ionotropic andG-protein coupled receptors offer the possibility to decrease or enhancethe function of the targeted receptors when stimulated by the endoge-nous neurotransmitter. Such compounds have been suggested as thera-peutic tools for intervention in several diseases, including PD. The newly

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developed GluN2C/GluN2D-selective positive allosteric modulator CIQwas recently shown to potentiate NMDA responses in neurons of thesubthalamic nucleus, which express GluN2D (Mullasseril et al., 2010).The effects of CIQ on neurotransmission have not been described inany brain region and therefore need to be identified in health and dis-ease. The aim of our study was to use CIQ as a tool to identify the physio-logical roles played by GluN2D-containing NMDARs in the healthystriatum and to determine if these roles are altered in the dopamine-depleted striatum. Given the absence of GluN2C in the striatum(Bloomfield et al., 2007), it is anticipated that CIQ will potentiate the ac-tion of glutamate on GluN2D-containing NMDARs in this brain region.

Materials and methods

Animals and brain slice preparation

All effortsweremade tominimize animal suffering and to reduce thenumber of animals used. Experiments were approved by our local ethi-cal committee (Stockholms norra djurförsöksetiska nämnd) and wereperformed using methods previously described (Chergui, 2011; Zhanget al., 2008). We used male C57BL/6 mice aged 4–9 weeks (HarlanLaboratories, The Netherlands). Mice were maintained on a 12:12 hlight/dark cycle and had free access to food and water. Control micedid not undergo surgery prior to electrophysiological experiments. An-other group of mice underwent unilateral stereotaxic injection of thetoxin 6-hydroxydopamine (6-OHDA) in the substantia nigra parscompacta to produce degeneration of dopaminergic neurons, and totallesion of the dopaminergic innervation of the striatum. These micewere anesthetized with intraperitoneal (i.p.) injection of 80 mg/kg ke-tamine and 5 mg/kg xylazine, placed in a stereotaxic frame, andinjected, over 2 min, with 3 μg of 6-OHDA in 0.01% ascorbate into thesubstantia nigra pars compacta of the right hemisphere. The coordinatesfor injection were AP,−3 mm; ML,−1.1 mm; and DV,−4.5 mm rela-tive to bregma and the dural surface (Paxinos and Franklin, 2001). Miceunderwent cervical dislocation followed by decapitation (for le-sioned mice, this was done 1–3 weeks following surgery). Theirbrains were rapidly removed and coronal brain slices (400 μmthick) containing the striatum were prepared with a microslicer(VT 1000S, Leica Microsystem, Heppenheim, Germany). We usedthe intact and the dopamine-depleted striatum from the same le-sioned mouse. Slices were incubated, for at least 1 h, at 32 °C in ox-ygenated (95% O2 + 5% CO2) artificial cerebrospinal fluid (aCSF)containing (in mM): 126 NaCl, 2.5 KCl, 1.2 NaH2PO4, 1.3 MgCl2, 2.4CaCl2, 10 glucose and 26 NaHCO3, pH 7.4. Slices were transferredto a recording chamber mounted on an upright microscope (ScientificaLtd., Uckfield, UK) and were continuously perfused with oxygenatedaCSF at 28 °C.

Electrophysiology

Whole cell recordings of MSNs and cholinergic interneurons in thedorsal striatum were made with the help of infrared-differential inter-ference contrast video microscopy. Striatal neurons were identified bytheir morphological and electrophysiological properties which include,for Ach interneurons, a large soma, spontaneous firing, pronouncedlong lasting spike after hyperpolarization, resting membrane potentialaround −60 mV (Kawaguchi, 1993). Patch electrodes were filled witha solution containing, in mM: either 140 CsCl, 2 MgCl2, 1 CaCl2, 10HEPES, 10 EGTA, 2 MgATP, 0.3 Na3GTP, pH adjusted to 7.3 with CsOH;or 120 D-gluconic acid, 20 KCl, 2 MgCl2, 1 CaCl2, 10 HEPES, 10 EGTA,2 MgATP, 0.3 Na3GTP, pH adjusted to 7.3 with KOH. The membrane po-tential of MSNs was held at −80 mV. Whole-cell membrane currentsand potentials were recorded with a MultiClamp 700B (Axon Instru-ments, Foster City CA, USA).

Data analysis and statistical methods

Data were acquired and analyzed with the pClamp 10 software(Axon Instruments, Foster City CA, USA). Action potential firing and fre-quency and amplitude of EPSCs and IPSCs were calculated using theMiniAnalysis program (Synaptosoft, Leonia, NJ). Numerical values areshown as means ± S.E.M, with n indicating the number of neurons(or mouse for Western blotting) examined. Data are expressed as per-cent of the baseline response measured for each neuron during the 5min preceding the start of perfusion with a compound, except forwhole-cell NMDA currents which were measured before and 10 minafter perfusion with UBP141. Statistical significance of the results wasassessed byusing the two-tailed Student's t-test for paired and unpairedobservations as well as a two-way ANOVA test for multiple measures(Fig. 5A). Graphs and statistical tests were made with the GraphPadPrism software.

Chemicals

Chemicals and drugs were purchased from Sigma-Aldrich(Stockholm, Sweden), Tocris Bioscience (Bristol, UK) and AbcamBiochemicals (Cambridge, UK). All compounds were prepared instock solutions, diluted in aCSF to the desired final concentrationand applied in the perfusion solution. The following compoundswere used (final concentrations in μM): bicuculline methiodide (BIC,10), (3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (CIQ, 20), CNQX disodium(10), DL-AP5 (APV, 50), NMDA (20), oxotremorine-M (OXO, 0.3), scopol-amine hydrobromide (2), tetrodotoxin citrate (0.5) and UBP141 (6).

Western blotting

Western blots were performed to confirm loss of tyrosine hydroxy-lase (TH) following 6-OHDA lesioning and to examine the expressionof GluN2D in the striatal slices that were used for electrophysiologicalexperiments. The striatum was dissected from the slices, frozen andstored at−20 °C until processed. The samples were sonicated in 1% so-diumdodecyl sulfate (SDS) and boiled for 10min. Protein concentrationwas determined in each sample with a bicinchoninic acid protein assay(BCA-kit, Pierce, Rockford, US). Equal amounts of protein (30 or 60 μg)were re-suspended in sample buffer and separated by SDS-polyacryl-amide gel electrophoresis using a 10% running gel and transferred toan Immobilon-P (Polyvinylidene Difluoride) transfer membrane(Sigma-Aldrich, Stockholm, Sweden). The membranes were incubatedfor 1 h at room temperature with 5% (w/v) dry milk in TBS–Tween20.Immunoblotting was carried out with antibodies against total GluN2D(Sigma-Aldrich), TH (Millipore, Billerica, USA) and β-actin (Sigma-Aldrich) in 5% dry milk dissolved in TBS–Tween 20. The membraneswere washed three times with TBS–Tween20 and incubated with sec-ondary horseradish peroxidase-linked Anti-Rabbit IgG (H+L) (ThermoScientific; 1:6000 dilution) for 1 h at room temperature. At the end of theincubation, membranes were washed six times with TBS–Tween20and immunoreactive bands were detected by enhanced chemilumi-nescence (PerkinElmer, Massachusetts, US). The autoradiogramswere scanned and quantified with the NIH Image 1.63 software.The levels of protein were normalized for the value of β-actin. Datawere analyzed with two-tailed unpaired Student's t-test to evaluate sta-tistical differences.

Results

Effect of CIQ in the striatum of control mice

Because GluN2D is expressed in cholinergic interneurons (Bloomfieldet al., 2007; Standaert et al., 1996), we hypothesized that CIQ could affectaction potential firing in these interneurons through a glutamatergic tone

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in brain slices.We applied CIQ in the perfusion solution at a concentration(20 μM) which was demonstrated to increase NMDA-activated currentsin neurons of the subthalamic nucleus in brain slices (Mullasseril et al.,2010).We found that CIQ reversibly increased thefiring rate of spontane-ously active cholinergic interneurons, measured in cell-attachedmode, inthe striatumof controlmice (n=9 cholinergic interneurons from6mice,Figs. 1A, B). CIQ failed to modulate the firing of these interneurons inthe presence of the non-subunit selective NMDAR antagonist APV(50 μM, n = 6 cholinergic interneurons from 3 mice, Fig. 1C) andin the presence of the GluN2D-preferring antagonist UBP141(6 μM, n = 7 cholinergic interneurons from 4 mice, Fig. 1D). Whenapplied alone, UBP141 did not affect the spontaneous firing of cholinergicinterneurons (1.63 ± 1.01 Hz before UBP141 and 1.69 ± 0.91 Hz duringUBP141, n = 4 cholinergic interneurons from 3 mice, not shown).These results demonstrate that CIQ has an excitatory action on the firingof cholinergic interneurons in the striatum of control mice, and that thiseffect is mediated through GluN2D-containing NMDARs.

MSNs are not spontaneously active in the brain slice. However, localinhibitory circuits play important roles in shaping striatal output, andexcitatory inputs, arising from the cortex and thalamus, are critical fordetermining the firing activity of these neurons in vivo. GABAergic andglutamatergic inputs provide, respectively, an inhibitory and excitatorytone to MSNs even in brain slices. We tested the possibility that CIQcould affect neurotransmission in MSNs when applied alone. We there-fore measured spontaneous inhibitory and excitatory postsynaptic cur-rents (sIPSCs and sEPSCs) in MSNs recorded in voltage-clamp mode.sIPSCs were recorded in the presence of CNQX (10 μM) to inhibit trans-mission mediated by AMPA receptors while sEPSCs were recorded inthe presence of bicuculline methiodide (10 μM) to inhibit transmissionmediated by GABAA receptors. In some cases, tetrodotoxin (0.5 μM)wasadded in the perfusion solution to record miniature EPSCs (mEPSCs).

Fig. 1. CIQ increases the firing of cholinergic interneurons in the striatum of control mice.(A) Firing in a cholinergic interneuron from the striatum of a control mouse, measured insomatic cell-attached mode, before (baseline) and during (CIQ) perfusion with CIQ(20 μM). (B–D) Average firing (number of action potentials (AP) per 30 s) in cholinergicinterneurons from the striatum of control mice before and during bath application ofCIQ in control conditions (B, n = 9), in the presence of APV (50 μM) (C, n = 6) and inthe presence of UBP141 (6 μM) (D, n = 7). *P b 0.05 compared with baseline firing inthe same neurons, n.s.: not significant.

We found that CIQ decreased the frequency, but did not affect the am-plitude, of sIPSCs in MSNs of control mice (n = 7 MSNs from 5 mice,Figs. 2A–C). CIQ had no effect on sIPSC frequency in the presence ofAPV (50 μM, n = 6 MSNs from 3 mice, Fig. 2D). These results demon-strate that CIQ depresses GABAergic neurotransmission in the striatumthrough NMDARs.

We then determined whether the increased firing of cholinergic in-terneurons could contribute to the depressant action of CIQ on sIPSC fre-quency.We found that CIQ failed to affect the frequency of sIPSCs in thepresence of themuscarinic receptor antagonist, scopolamine (2 μM, n=5 MSNs from 3 mice, Fig. 2D). In addition, the muscarinic receptor ago-nist oxotremorine-M (0.3 μM) depressed the frequency, but did not af-fect the amplitude, of sIPSCs in MSNs of control mice (n = 12 MSNsfrom 8mice, Figs. 3A–C). Scopolamine (2 μM) blocked the inhibitory ef-fect of oxotremorine-M on sIPSC frequency (n = 5 MSNs from 3 mice,Fig. 3D), demonstrating the efficacy of this antagonist at the concentra-tion used in this study. Moreover, the effect of oxotremorine-M did notinvolve the activation of GluN2D-containing NMDARs because it wasnot affected by UBP141 (6 μM, n = 6 MSNs from 4 mice, Fig. 3D).Thus, activation of muscarinic receptors likely follows the activation ofGluN2D-containing NMDARs. Together, these results demonstrate thatCIQ depresses GABAergic synaptic transmission in MSNs from the stria-tum of control mice through amechanism that involves increased firingof cholinergic interneurons, release of acetylcholine and activation ofmuscarinic receptors.

We also found that CIQ did not affect glutamatergic neurotrans-mission because it did not change the frequency or amplitude ofsEPSCs in MSNs from the striatum of control mice (n = 6 MSNsfrom 4 mice, Figs. 4A–C). However, oxotremorine-M depressedthe frequency, and did not affect the amplitude, of mEPSCs inMSNs in the striatum of control mice (n = 11 MSNs from 7 mice,Figs. 4D–F).

Effect of CIQ in the striatum of 6-OHDA-lesioned mice

We then sought to investigate whether the effects of CIQ, andthus the functions attributed to GluN2D-containing NMDARs, wereaffected in the striatum of the unilaterally 6-OHDA-lesionedmouse model of PD. As shown in an earlier study (Dehorter et al.,2009), the baseline firing of cholinergic interneurons was similarin the dopamine-depleted striatum as compared with the intact stri-atum (n = 11 and n = 13, respectively, Figs. 5A). As seen in controlmice (Figs. 1A, B), we found that CIQ increased the firing of sponta-neously active cholinergic interneurons in the intact striatum (n =11 cholinergic interneurons from 8 mice, Fig. 5A). This effect waslost in the dopamine-depleted striatum (n = 13 cholinergic inter-neurons from 8 mice, Fig. 5A). The depressant action of CIQ on sIPSCfrequencywas also lost inMSNs from the dopamine-depleted striatum(n= 9MSNs from 6mice Fig. 5B). CIQwas however able to depress in-hibitory neurotransmission in MSNs from the intact striatum (n = 9MSNs from 7 mice Fig. 5B).

As observed in control mice, CIQ did not affect the frequency oramplitude of sEPSCs in MSNs from the intact striatum (n = 10MSNs from 7 mice, Fig. 5C). The frequency, but not amplitude, ofsEPSCs was however increased in MSNs of the dopamine-depletedstriatum during bath application of CIQ (n = 11 MSNs from 7mice, Fig. 5C). This effect was mediated by activation of NMDARs be-cause it was blocked in the presence of APV (50 μM, n = 6 MSNsfrom 3 mice, Fig. 5C). Furthermore, the potentiation of glutamater-gic neurotransmission by CIQ was action potential-independent be-cause CIQ increased mEPSC frequency, and did not affect mEPSCamplitude, in MSNs from the dopamine-depleted striatum (frequency:2.32 ± 0.31 Hz before and 2.53 ± 0.36 Hz after CIQ, P b 0.05; amplitude:12.78 ± 0.76 pA before and 12.93 ± 0.71 pA after CIQ P N 0.05, n = 10MSNs from 7 mice, not shown). CIQ did not affect mEPSC frequency oramplitude in MSNs from the intact striatum (frequency: 2.38 ± 0.38 Hz

Fig. 2. CIQ depresses GABAergic transmission inMSNs from control mice. (A) Example sIPSCs measured in oneMSN, in the voltage-clampmode (−80mV), from control striatum, before(baseline) and during (CIQ) perfusion with CIQ (20 μM). The blockade of the sIPSCs by the GABAA receptor antagonist bicuculline (BIC, 10 μM) confirmed that the events measured weremediated by GABA. (B) Cumulative plot of sIPSC inter-event intervals in the neuron presented in (A), before and during perfusion with CIQ. The cumulative distribution of inter-eventintervals was shifted to the right, indicative of a decreased frequency in sIPSCs during bath application of CIQ. (C) Average frequency (left) and amplitude (right) of sIPSCs measured inn = 7 MSNs, before (white bars) and during (black bars) CIQ. **P b 0.01, n.s.: not significant. (D) Average frequency of sIPSCs measured MSNs, before (white bars) and during (blackbars) CIQ in the presence of APV (50 μM, n = 6, left) and scopolamine (2 μM, n = 5, right). n.s.: not significant.

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before and 2.36 ± 0.42 Hz after CIQ, P N 0.05; amplitude: 12.43 ±0.58 pA before and 12.18 ± 0.47 pA after CIQ, P N 0.05, n = 8MSNs from 5 mice, not shown). These results demonstrate a

Fig. 3. The muscarinic receptor agonist oxotremorine-M depresses GABAergic transmission inmode (−80mV), from control striatum, before and during perfusionwith oxotremorine-M (0.3in the neuron presented in (A), before and during perfusion with oxotremorine-M. The cumulafrequency in sIPSCs duringbath application oxotremorine-M. (C) Average frequency (left) and abars) oxotremorine-M. *P b 0.05, n.s.: not significant. (D) Average frequency of sIPSCs measurscopolamine (2 μM, n = 5, left) and UBP141 (6 μM, n = 6, right). **P b 0.01, n.s.: not significan

decreased control of inhibitory transmission by GluN2D-containingNMDARs, and a potentiating role of these receptors on excitatory in-puts in the dopamine-depleted striatum.

MSNs from control mice. (A) Example sIPSCs measured in one MSN, in the voltage-clampμM, OXO) andwith bicuculline (10 μM). (B) Cumulative plot of sIPSC inter-event intervalstive distribution of inter-event intervals was shifted to the right, indicative of a decreasedmplitude (right) of sIPSCsmeasured inn=12MSNs, before (white bars) and during (blacked MSNs, before (white bars) and during (black bars) oxotremorine-M in the presence oft.

Fig. 4. Effect of CIQ and of the muscarinic receptor agonist oxotremorine-M on glutamatergic transmission in MSNs from control mice. (A) Example sEPSCs measured in one MSN, in thevoltage-clampmode (−80mV), from control striatum, before and during perfusionwith CIQ (20 μM). The blockade of the sEPSCs by the ionotropic receptor antagonists CNQX (for AMPAreceptors) and APV (for NMDARs) confirmed that the events measured were mediated by glutamate. (B) Cumulative plot of sEPSC inter-event intervals in the neuron presented in (A),before and during perfusion with CIQ. (C) Average frequency (left) and amplitude (right) of sEPSCs measured in n = 6 MSNs, before (white bars) and during (black bars) CIQ. n.s.: notsignificant. (D) Example mEPSCs measured in one MSN from control striatum, before and during perfusion with oxotremorine-M (0.3 μM) and with CNQX+ APV. (E) Cumulative plotof mEPSC inter-event intervals in the neuron presented in (D), before and during perfusion with oxotremorine-M. The cumulative distribution of inter-event intervals was shifted tothe right, indicative of a decreased frequency in mEPSCs during bath application of oxotremorine-M. (F) Average frequency (left) and amplitude (right) of mEPSCs measured in n =11 MSNs, before (white bars) and during (black bars) oxotremorine-M. **P b 0.01, n.s.: not significant.

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Altered protein level, and contribution to NMDA currents, of GluN2D in thedopamine-depleted striatum

To determine whether the observed neurophysiological changes inthe dopamine-depleted striatum could be due to an altered GluN2Dprotein level, we processed the slices used in electrophysiological ex-periments for Western blotting. We determined the extent of the 6-OHDA lesion with the expression of tyrosine hydroxylase (TH), therate limiting enzyme in the synthesis of dopamine. The amount of THwas dramatically reduced in the lesioned hemisphere as comparedwith the intact side of the same mice (n= 38 mice, Fig. 6A), indicatingloss of striatal dopaminergic terminals. Interestingly, we found thatGluN2D protein levels were increased in the dopamine-depleted stria-tum as compared with the intact striatum (n = 11 mice, Fig. 6B). Inan attempt to determine the cellular location of this altered GluN2Dprotein level, we examined the effect of UBP141 onwhole-cell currents,activated by bath application of NMDA (20 μM), measured in choliner-gic interneurons and in MSNs in the presence of CNQX, bicucullineand TTX. UBP141 (6 μM) inhibited NMDA-currents in cholinergic inter-neurons from the intact striatum (n= 5 cholinergic interneurons from4mice, Fig. 6C), but failed to affect NMDA-currents in interneurons fromthe dopamine-depleted striatum (n = 5 cholinergic interneurons from4 mice, Fig. 6C). Conversely, UBP141 (6 μM) did not affect NMDA-

currents in MSNs from the intact striatum (n = 7 MSNs from 5 mice,Fig. 6D), but depressed NMDA-currents in MSNs from the dopamine-depleted striatum (n = 6 MSNs from 5 mice, Fig. 6D). These resultsdemonstrate the presence of functional GluN2D-containing NMDARsin cholinergic interneurons, and the absence of these receptors inMSNs, in the intact striatum. These results are in accordancewith previ-ously published histochemical reports (Bloomfield et al., 2007;Landwehrmeyer et al., 1995; Standaert et al., 1996). These results alsodemonstrate the loss of functional GluN2D-containing NMDARs in cho-linergic interneurons, and the up-regulation of these receptors inMSNs,in the dopamine-depleted striatum.

Discussion

Through the use of the newly developed GluN2C/GluN2D-selectivepositive allosteric modulator CIQ, we have investigated the roles anddysfunctions of GluN2D-containing NMDARs in the striatum of controlmice and of the 6-OHDA-lesion mouse model of PD. We found thatCIQ increased the firing of cholinergic interneurons and presynapticallydepressed GABAergic neurotransmission in MSNs, while glutamatergicneurotransmission in these projection neurons remained unaffected.In the dopamine-depleted striatum, the effect of CIQ on the firing of

Fig. 5. Effect of CIQ on the firing of cholinergic interneurons and on neurotransmission inMSNs from intact and dopamine-depleted striatum. (A) CIQ (20 μM) increases thefiring ofcholinergic interneurons in the intact striatum, but not in the dopamine-depleted(6-OHDA) striatum. *: P b 0.05. (B) Average CIQ-induced percent change in the frequencyand amplitude of sIPSCs in MSNs from intact (white bars, n = 9) and dopamine-depleted(6-OHDA, black bars, n = 9) striatum. *: P b 0.05 compared to baseline in individualneurons. (C) Average CIQ-induced percent change in the frequency and amplitude ofsEPSCs in MSNs from intact (white bars, n = 10) and dopamine-depleted (6-OHDA) stri-atum in the absence (black bars, n=11) and in the presence of APV (hatched bars, n=6).*: P b 0.05 compared to baseline in individual neurons.

Fig. 6.Altered protein levels of GluN2D and contribution of GluN2D to NMDA currents in thedopamine-depleted striatum. (A, B)Westernblots of tyrosinehydroxylase (TH, n=38mice)(A), the rate-limiting enzyme in the synthesis of dopamine, and of GluN2D (n = 11 mice)(B) from intact and lesioned striatum. Total protein amounts are expressed as percentageof intact striatum. Blots above graphs are representative examples from intact (left) anddopamine-depleted (right) hemispheres for TH, GluN2D and β-actin. *** P b 0.001,** P b 0.01 compared with intact hemisphere. (C, D) Effect of UBP141 (6 μM) onNMDA-activated whole-cell currents in cholinergic interneurons (C) andMSNs (D) from in-tact (n=5and n=7, respectively) and dopamine-depleted (n=5 and n=6, respectively)striatum. Currents in UBP141 are expressed as percentage of control NMDA-current in thesame neurons. *P b 0.05 compared with baseline.

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cholinergic interneurons and on GABAergic neurotransmission inMSNswas lost, but excitatory neurotransmission in MSNs was increased.

We recently found that NMDA applied in the perfusion solution in-duces a presynaptic inhibition of glutamatergic synaptic transmission,measured with field excitatory postsynaptic potentials, in control micethrough the activation of a type of striatal interneuron that expressesGluN2D, i.e. the large cholinergic interneuron (Zhang et al., 2014). In ac-cordance with these findings, we found that CIQ increased the firing ofcholinergic interneurons, although to a lower degree than NMDA,through the activation of GluN2D-containing NMDARs. The ability ofCIQ to modulate NMDAR functions in the slice preparation likely relieson an endogenous tone of glutamate and glycine. We hypothesizedthat the CIQ-induced increase in the firing of cholinergic interneuronscould affect GABAergic neurotransmission in MSNs. Indeed, activationof cholinergic interneurons was shown to increase GABAergic eventsonto MSNs through a disynaptic pathway which involved nicotinic re-ceptors in different classes of GABAergic interneurons (English et al.,2012; Luo et al., 2013). We found that CIQ depressed GABAergic neuro-transmission in MSNs in the striatum of control mice. In addition, the

depressant action of CIQ on sIPSC frequencywas blocked by amuscarin-ic receptor antagonist and mimicked by a muscarinic receptor agonist.The possibility exists that CIQ has a direct, inhibitory, action onGABAergicinterneurons. Alternatively, the presynaptic inhibitory action of ace-tylcholine acting on muscarinic receptors might dominate over theincreased GABAergic events mediated by nicotinic receptors. How-ever, CIQ did not affect glutamatergic transmission in MSNs in con-trol striatum, suggesting that muscarinic receptors might be morepotent in inhibiting GABAergic than glutamatergic neurotransmission.Nevertheless, our observations demonstrate that GluN2D-containingNMDARs contribute to functional NMDARs in cholinergic interneurons,and not in MSNs, in the striatum of control mice. These results are inagreement with the demonstrated expression of GluN2D in cholinergicinterneurons and the absence of this subunit in MSNs (Bloomfield et al.,2007; Landwehrmeyer et al., 1995; Standaert et al., 1996).

Several studies, including unpublished observations from our labo-ratory, have shown that the functions of GluN2B-containing NMDARsare altered in the striatum of animal models of PD (Calabresi et al.,2013; Gardoni et al., 2010; Paille et al., 2010). Furthermore, we have re-cently demonstrated that the depressant action of NMDA applied in theperfusion solution on evoked glutamatergic neurotransmission is re-duced in the striatum of the 6-OHDA mouse model of PD suggesting areduced function of GluN2D-containing NMDARs in cholinergic inter-neurons (Zhang et al., 2014). The present results further demonstratethat GluN2D-containingNMDARs are dysfunctional in cholinergic inter-neurons in the dopamine-depleted striatum. Indeed, the ability of CIQ toincrease the firing rate of cholinergic interneurons was lost, and the

160 Z.-J. Feng et al. / Experimental Neurology 255 (2014) 154–160

contribution of GluN2D-containing NMDARs to whole-cell NMDA cur-rents was reduced. The impaired depressant action of CIQ on GABAergicneurotransmission in MSNs likely results from a reduced functionof GluN2D-containing NMDARs in cholinergic interneurons in thedopamine-depleted striatum. These results further support a close asso-ciation between cholinergic activity and GABAergic neurotransmissionin control striatum. In addition, the increased spontaneous glutamaterelease induced by CIQ in the dopamine-depleted striatummight resultfrom a reduced inhibitory function of GluN2D-containing NMDARsexpressed in cholinergic interneurons (Zhang et al., 2014) and an in-creased function of GluN2D-containing NMDARs in MSNs. Indeed, theincreased level of GluN2D in the dopamine-depleted striatum likely oc-curs in MSNs. Further studies will determine whether this change un-derlies the potentiating effect of CIQ on spontaneous glutamatergicneurotransmission. Nevertheless, in the absence of dopamine, activa-tion of GluN2D-containing NMDARs might promote glutamatergicneurotransmission.

Conclusions

This study identifies functions of GluN2D-containing NMDARs andadaptive alterations involving these receptors in a mouse model of PD.Taken togetherwith the observations that glutamatergic andGABAergicinputs are affected in PD (Bagetta et al., 2010; Taverna et al., 2008), ourfindings indicate that pharmacological agents thatmodulate the activityof GluN2D-containing NMDARs might rescue or exacerbate synapticdysfunctions observed in animal models of PD.

Acknowledgments

This study was supported by the Swedish Research Council (grant2011-2770).

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