glun2d-containing nmda receptors inhibit neurotransmission in the mouse striatum through a...

Department of Physiology and Pharmacology, Section of Molecular Neurophysiology, The Karolinska

Institute, Stockholm, Sweden

AbstractThe GluN2 subunits that compose NMDA receptors(NMDARs) determine functional and pharmacological proper-ties of the receptor. In the striatum, functions and potentialdysfunctions of NMDARs attributed to specific GluN2 subunitshave not been clearly elucidated, although NMDARs playcritical roles in the interactions between glutamate anddopamine. Through the use of amperometry and field potentialrecordings in mouse brain slices, we found that NMDARs thatcontain the GluN2D subunit contribute to NMDA-inducedinhibition of evoked dopamine release and of glutamatergicneurotransmission in the striatum of control mice. Inhibition islikely mediated through increased firing in cholinergic inter-neurons, which were shown to express GluN2D. Indeed,NMDA-induced inhibition of both dopamine release and

glutamatergic neurotransmission is reduced in the presenceof muscarinic receptor antagonists and is mimicked by amuscarinic receptor agonist. We have also examined whetherthis function of GluN2D-containing NMDARs is altered in amouse model of Parkinson’s disease. We found that theinhibitory role of GluN2D-containing NMDARs on glutamater-gic neurotransmission is impaired in the 6-hydroxydopaminelesioned striatum. These results identify a role for GluN2D-containing NMDARs and adaptive changes in experimentalParkinsonism. GluN2D might constitute an attractive target forthe development of novel pharmacological tools for therapeu-tic intervention in Parkinson’s disease.Keywords: acetylcholine, dopamine, GluN2D, glutamate,NMDA receptor, Parkinson’s disease.J. Neurochem. (2014) 129, 581–590.

The main input nucleus of the basal ganglia, the striatum,receives convergent afferents from dopaminergic neuronswhose cell bodies are located in the substantia nigra, andglutamatergic afferents that originate in the cortex andthalamus (David et al. 2005). Interactions between glutamateand dopamine in the striatum play critical roles in health anddisease. The complex interactions between dopamine recep-tors and the NMDA type of glutamate receptors (NMDARs)have been extensively examined (Cepeda and Levine 2012).However, the mechanisms by which glutamate acting onNMDARs modulates dopamine release at the terminals aswell as glutamatergic neurotransmission are still unresolved(Zhang and Sulzer 2012). Early studies have suggested thatNMDARs with different sensitivities for Mg2+ modulate therelease of dopamine in the striatum through direct andindirect mechanisms (Krebs et al. 1991; Ohta et al. 1994;Iravani and Kruk 1996; Cheramy et al. 1998). NMDARswith distinct subunit compositions might control neurotransmitter

release. Indeed, functional and pharmacological properties ofNMDARs are closely dependent on the subunit compositionof these receptors, and in particular on the GluN2 subunitsthey contain. NMDARs are heterotetrameric assemblies ofGluN1, GluN2 (A–D), and GluN3 (A, B) subunits (Paolettiet al. 2013). GluN2B is the most abundant GluN2 subunit inthe striatum and is also expressed in dopaminergic neurons(Landwehrmeyer et al. 1995; Standaert et al. 1999;

Received October 18, 2013; revised manuscript received December 19,2013; accepted January 9, 2014.Address correspondence and reprint requests to Karima Chergui,

Department of Physiology and Pharmacology, Section of MolecularNeurophysiology, the Karolinska Institute, Von Eulers v€ag 8, 171 77Stockholm, Sweden. E-mail: [email protected] used: 6-OHDA, 6-hydroxydopamine; aCSF, artificial

cerebrospinal fluid; fEPSP/PSs, field excitatory post-synaptic potentials/population spikes; NMDAR, NMDA receptor; PD, Parkinson’s disease;TH, tyrosine hydroxylase.

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 129, 581--590 581

JOURNAL OF NEUROCHEMISTRY | 2014 | 129 | 581–590 doi: 10.1111/jnc.12658

Jones and Gibb 2005). Interestingly, we found that GluN2B-containing NMDARs do not contribute significantly toNMDA-induced inhibition of evoked, action potential-dependent, dopamine release and of glutamatergic synaptictransmission in mouse corticostriatal and striatal brain slices(Schotanus and Chergui 2008). Our previous findingsdemonstrated a significant contribution of GluN2A-contain-ing NMDARs, but also suggested that NMDARs-containingsubunits other than GluN2A and GluN2B control dopamineand glutamate release, directly or indirectly (Schotanus andChergui 2008).GluN2D forms functional NMDARs in midbrain dopami-

nergic neurons (Standaert et al. 1994; Jones and Gibb 2005;Brothwell et al. 2008) and is expressed in striatal interneu-rons, in particular, large cholinergic interneurons (Standaertet al. 1996; Bloomfield et al. 2007). Although these inter-neurons represent less than 2% of the total neuronalpopulation in the striatum, they are likely to controlneurotransmission in the striatum because of their extensiveaxonal branching (Pisani et al. 2007; Bonsi et al. 2011;Goldberg et al. 2012). GluN2D-containing NMDARs local-ized at dopaminergic axon terminals in the striatum and/or incholinergic interneurons might thus play a role in the controlof dopamine release and glutamatergic neurotransmission.The GluN2 subunits that compose NMDARs are attractive

drug targets for therapeutic intervention in several neurolog-ical and psychiatric disorders which are associated withdysfunctional neurotransmission mediated by glutamate and/or dopamine (Loftis and Janowsky 2003; Gogas 2006).Moreover, altered expression of GluN2B in the striatum ofanimal models of Parkinson’s disease (PD) is suggested tocontribute to L-DOPA-induced dyskinesia (Dunah et al.2000; Gardoni et al. 2006; Paille et al. 2010). Whether thefunctions of GluN2D-containing NMDARs are altered inexperimental Parkinsonism has not been examined. The aimof this study was to investigate whether GluN2D-containingNMDARs contribute to NMDA-induced modulation ofdopamine release and of glutamatergic synaptic transmissionin corticostriatal mouse brain slices. We determined whetherstriatal cholinergic interneurons contributed to the observedmodulation and we examined if the functions of GluN2D-containing NMDARs were altered in the 6-hydroxydopamine(6-OHDA)-lesion mouse model of PD. Parts of the resultswere presented as a meeting abstract (Zhang and Chergui2011).

Materials and methods

Animals and brain slice preparation

All efforts were made to minimize animal suffering and to reducethe number of animals used. Experiments were approved by ourlocal ethical committee (Stockholms norra djurf€ors€oksetiskan€amnd), followed the ARRIVE guidelines, and were performed asdescribed previously (Chergui et al. 2004; Schotanus and Chergui

2008; Zhang et al. 2008; Chergui 2011). We used male C57BL/6mice aged 4–9 weeks (Harlan Laboratories, The Netherlands). Micewere maintained on a 12:12 h light/dark cycle and had free access tofood and water. A group of mice underwent unilateral stereotaxicinjection of the toxin 6-OHDA to lesion dopaminergic neurons inthe substantia nigra pars compacta. These mice were anesthetizedwith intraperitoneal (i.p.) injection of 80 mg/kg ketamine and 5 mg/kg xylazine, placed in a stereotaxic frame, and injected, over 2 min,with 3 lg of 6-OHDA in 0.01% ascorbic acid into the substantianigra pars compacta of the right hemisphere. The coordinates forinjection were AP, �3 mm; ML, �1.1 mm; and DV, �4.5 mmrelative to bregma and the dural surface (Paxinos and Franklin2001). Mice underwent cervical dislocation followed by decapita-tion (for lesioned mice, this was done 1–3 weeks followingsurgery). Their brains were rapidly removed and brain slices(coronal and sagittal, 400 lm thick) containing the striatum and theoverlying cortex were prepared with a microslicer (VT 1000S; LeicaMicrosystem, Heppenheim, Germany). Slices were incubated, for atleast 1 h, at 32°C in oxygenated (95% O2 + 5% CO2) artificialcerebrospinal fluid (aCSF) containing (in mM): 126 NaCl, 2.5 KCl,1.2 NaH2PO4, 1.3 MgCl2, 2.4 CaCl2, 10 glucose, and 26 NaHCO3,pH 7.4. Slices were transferred to a recording chamber (WarnerInstruments, Hamden, CT, USA; recording chamber from Scientif-ica Ltd., Uckfield, UK) mounted on an upright microscope(Olympus, Solna, Sweden and Scientifica Ltd.) and were continu-ously perfused with oxygenated aCSF at 28°C.

Amperometry in brain slices

Amperometric detection of dopamine release was performed withcarbon fiber electrodes (10 lm diameter, World Precision Instru-ments Europe) which had an active part of 100 lm that waspositioned within the dorsal striatum in the brain slice. A constantvoltage of + 500 mV was applied to the carbon fiber via anAxopatch 200B amplifier (Axon Instruments, Foster City, CA,USA) and currents were recorded with the same amplifier. Astimulating electrode (patch electrode filled with aCSF) was placedon the slice surface, in the vicinity of the carbon fiber electrode.Stimulations consisted of single pulses (0.1 ms, 8–14 lA) appliedevery minute, which evoked a response corresponding to oxidationof dopamine at the surface of the electrode, as described previously(Chergui et al. 2004).

Electrophysiology in brain slices

Extracellular field potentials were recorded using a glass micropipettefilled with aCSF positioned on the slice surface. These synapticresponses were evoked by stimulation pulses applied every 15 s to thebrain slice through a concentric bipolar stimulating electrode (FHC,Bowdoinham, ME, USA) placed near the recording electrode on thesurface of the slice (Schotanus et al. 2006). Single stimuli (0.1 msduration) were applied at an intensity yielding 50–60% maximalresponse as assessed by a stimulus/response curve established, foreach slice, at the beginning of the recording session, by measuring theamplitude of the field excitatory post-synaptic potentials/populationspikes (fEPSP/PSs) evoked by increasing stimulation intensities.Paired-pulse stimulations consisted in two stimulation pulses sepa-rated by a 20-ms interval. Signals were amplified 500 or 1000 timesvia an Axopatch 200B or a GeneClamp 500B amplifier (AxonInstruments), acquired at 10 kHz, and filtered at 2 kHz.

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 129, 581--590

582 X. Zhang et al.

Cell-attached and whole-cell patch-clamp recordings of cholin-ergic interneurons in the dorsal striatum were made with the help ofinfrared-differential interference contrast video microscopy. Cho-linergic interneurons were identified by their morphological andelectrophysiological properties which include a large soma, spon-taneous firing, pronounced long-lasting spike after hyperpolariza-tion, resting membrane potential around �60 mV (Kawaguchi1993). Patch electrodes were filled with a solution containing, inmM: 120 D-gluconic acid, 20 KCl, 2 MgCl2, 1 CaCl2, 10 HEPES, 10EGTA, 2 MgATP, 0.3 Na3GTP, pH adjusted to 7.3 with KOH.Whole-cell membrane currents and potentials were recorded with aMultiClamp 700B and an Axopatch 200B (Axon Instruments),acquired at 10 kHz, and filtered at 2 kHz.

Data acquisition and analysis

Data were acquired and analyzed with the pClamp 9 or pClamp 10software (Axon Instruments). Numerical values are shown as meanswith SEM, with n indicating the number of slices or neurons tested.For dopamine release and fEPSP/PS, data are expressed as percentof the baseline response measured for each slice during the5–10 min preceding start of perfusion with NMDA or oxotremo-rine-M. Statistical significance of the results was assessed by usingthe Student’s t-test for paired and unpaired observations or one-wayANOVA followed by Bonferroni’s multiple comparison test.

Chemicals and drugs

Chemicals and drugs were purchased from Sigma-Aldrich (Stock-holm, Sweden), Tocris Bioscience (Bristol, UK), and AbcamBiochemicals (Cambridge, UK). All compounds were prepared instock solutions, diluted in aCSF to the desired final concentration,and applied in the perfusion solution. The following compoundswere used (final concentrations in lM): AF-DX 116 (0.1), J104129 fumarate (0.01), NMDA (20), oxotremorine-M (0.1 and0.3), PD 102807 (0.5), pirenzepine dihydrochloride (1), cis-PPDA((2S*,3R*)-1-(Phenanthren-2-carbonyl)piperazine-2,3-dicarboxylicacid) (0.5), and UBP141 (3-6). We used the competitive GluN2C/GluN2D-preferring antagonists UBP141 and PPDA which dis-play 5- to 10-fold selectivity for GluN2C/GluN2D-containingNMDARs over GluN2A/GluN2B-containing NMDARs, withUBP141 displaying higher selectivity than PPDA (Feng et al.2004; Costa et al. 2009). The concentrations of these compoundsused in our study were previously shown to inhibit synaptic andextrasynaptic NMDAR-mediated currents in hippocampal andmidbrain slices with minimal effect on receptors containingGluN2A or GluN2B (Brothwell et al. 2008; Harney et al. 2008;Costa et al. 2009; Harney and Anwyl 2012). Because GluN2C isabsent from the striatum (Bloomfield et al. 2007), UBP141 andPPDA likely antagonize the action of NMDA on GluN2D-containing NMDARs.

Western blotting

Western blots were performed to confirm and quantify the loss oftyrosine hydroxylase (TH) following 6-OHDA lesioning in theslices that were used for electrophysiological experiments. Theslices were frozen and stored at �20°C until processed. The sampleswere sonicated in 1% sodium dodecyl sulfate and boiled for 10 min.Protein concentration was determined in each sample with abicinchoninic acid protein assay (BCA-kit, Pierce, Rockford,

USA). Equal amounts of protein (30 lg) were resuspended in samplebuffer and separated by sodium dodecyl sulfate–polyacrylamidegel electrophoresis using a 10% running gel and transferred to anImmobilon-P (Polyvinylidene Difluoride) transfer membrane(Sigma-Aldrich). The membranes were incubated for 1 h at� 22�C with 5% (w/v) dry milk in Tris-buffered saline (TBS)-Tween20. Immunoblotting was carried out with an antibody againsttotal TH (Millipore, Billerica, MA, USA) in 5% dry milk dissolved inTBS-Tween 20. The membranes were washed three times withTBS-Tween20 and incubated with secondary horseradish peroxidase-linked Anti-Rabbit IgG (H+L) (1 : 6000 dilution; Thermo Scientific,Philadelphia, PA, USA) for 1 h at � 22�C. At the end of theincubation, membranes were washed six times with TBS-Tween 20and immunoreactive bands were detected by enhanced chemilumi-nescence (Perkin Elmer, Waltham, MA, USA). The autoradiogramswere scanned and quantified with the NIH Image 1.63 software(National Institute of Health, Bethesda, MD, USA). Data wereanalyzed with two-tailed unpaired Student’s t-test to evaluatestatistical differences.

Results

GluN2D-containing NMDARs contribute to NMDA-induced

inhibition of evoked dopamine releaseWe first evaluated the effect of NMDA on stimulation-evokedrelease of dopamine in corticostriatal slices, as done previ-ously (Schotanus et al. 2006; Schotanus and Chergui 2008).We found that NMDA (20 lM), applied in the perfusionsolution for 3 min, reversibly depressed the peak currentamplitude, corresponding to released dopamine, evoked in thedorsal striatum (to 53.0 � 6.2% of baseline, n = 8, Fig. 1). Inthe presence of the GluN2D antagonist UBP141, the inhib-itory effect of NMDA on dopamine release was significantlyreduced (to 74.7 � 4.4% of baseline, n = 8; Fig. 1c), ascompared with control slices (p < 0.05). A similar reductionin NMDA-induced depression of dopamine release wasobserved in the presence of another GluN2D antagonist,PPDA (n = 8, Fig. 1c). These results demonstrate thatGluN2D-containing NMDARs participate in NMDA-induceddepression of evoked dopamine release in the striatum. Giventhat cholinergic striatal interneurons express GluN2D (Land-wehrmeyer et al. 1995), and because acetylcholine acting onmuscarinic receptors exerts a powerful inhibitory control ondopamine release (Threlfell et al. 2010), we examinedwhether the mechanism by which GluN2D-containingNMDARs inhibit dopamine release included activationof cholinergic interneurons. We first tested the effects ofmuscarinic receptor antagonists on the depressant actionof NMDA. We found that muscarinic receptor antagonistsacting on M1 (pirenzepine), M2 (AF-DX 116), and M4 (PD102807) receptors decreased NMDA-induced inhibition ofevoked dopamine release, to the same extent as GluN2Dantagonists (Fig. 1c). NMDA-induced inhibition was notsignificantly altered in the presence of the M3 receptorantagonist, J 104129. Interestingly, pirenzepine, but not the other


Role of GluN2D-containing NMDARs in the striatum 583

muscarinic receptor antagonists, significantly increased dopa-mine release when applied alone (to 115.3 � 6.3% ofbaseline, n = 9; p < 0.05, data not shown). This observation

demonstrates a tonic control of dopamine release in thestriatum by ambient acetylcholine acting on muscarinic M1receptors. We then demonstrated that the muscarinic receptoragonist oxotremorine-M inhibited evoked dopamine release ata concentration as low as 0.1 lM (Fig. 2), demonstrating thatactivation of muscarinic receptors mimics the effect ofNMDA. We also recorded, in the cell-attached configuration,action potential firing in cholinergic interneurons and appliedNMDA (20 lM) in the perfusion solution for 3 min, as fordopamine release experiments. We found that NMDAincreased the firing rate of these interneurons with a similartime course as for the inhibitory action of NMDA ondopamine release (n = 11, Fig. 3). Finally, we found thatcholinergic interneurons express functional GluN2D-contain-ing NMDARs because NMDA-induced whole-celldepolarizations were reduced in the presence of PPDA(12.2 � 3.1 mV in control solution, n = 4; and 3.5 �1.1 mV in the presence of PPDA, n = 6; Fig. 3d). Takentogether, these results suggest that activation of GluN2D-containing NMDARs by bath applied NMDA increases thefiring rate of cholinergic interneurons, and that subsequentrelease of acetylcholine inhibits dopamine release throughmuscarinic receptors.

GluN2D-containing NMDARs contribute to NMDA-induced

depression of glutamatergic neurotransmission

We previously demonstrated that NMDA depressed gluta-matergic neurotransmission through an intrastriatal, GABA-independent, mechanism (Schotanus et al. 2006; Schotanusand Chergui 2008). In this study, we investigated whetherGluN2D-containing NMDARs contributed to this synapticdepression. In control slices, NMDA (20 lM, bath appliedfor 3 min) produced a reversible, short-lasting, reduction inthe amplitude of the fEPSP/PS (to 75.5 � 2.3% ofbaseline, n = 18, Fig. 4a–c). This depression was accom-panied by a reversible increase in the ratio between thesecond and the first fEPSP/PS in a paired-pulse stimulationprotocol in the 11 slices examined (Fig. 4d), suggesting apre-synaptic mechanism. We found that synaptic depres-sion was significantly reduced in the presence of theGluN2D antagonists UBP141 (n = 9) and PPDA (n = 8)(Fig. 4c). Cholinergic interneurons were likely involved inpart of the effect of NMDA because synaptic depressionwas reduced in the presence of muscarinic receptorantagonists acting on M1 receptors (pirenzepine, n = 11),M2 receptors (AF-DX 116, n = 9), M3 receptors (J104129, n = 8), and M4 receptors (PD 102807, n = 8)(Fig. 4c). As for dopamine release, we confirmed that themuscarinic receptor agonist oxotremorine-M pre-synapticallyinhibited glutamatergic neurotransmission in our experi-mental conditions (Fig. 5). These results demonstrate thatNMDA-induced synaptic depression is in part mediated byNMDARs that contain GluN2D and by muscarinic M1–M4receptors.

(a)

(b)

(c)

Fig. 1 GluN2D-containing NMDA receptors (NMDARs) and musca-rinic receptors contribute to NMDA-induced inhibition of evokeddopamine release in the mouse striatum. (a) Representative amper-

ometric traces from one slice, at the time points indicated in (b), before(1) and after (2) bath application of NMDA (20 lM). (b) Time course ofthe effect of NMDA (20 lM), applied in the perfusion solution at the

time indicated by the black bar (3 min duration), on evoked dopaminerelease (n = 8). (c) Average magnitude of NMDA-induced inhibition(maximal effect in individual slices) in control slices (n = 8), in slicesperfused with GluN2D antagonists (UBP141, n = 8; and PPDA, n = 8),

and with M1–M4 antagonists (M1: pirenzepine, n = 11; M2: AF-DX116, n = 9; M3: J 104129, n = 7; and M4: PD 102807, n = 9).**p < 0.01, *p < 0.05 compared with control slices.


584 X. Zhang et al.

The contribution of GluN2D-containing NMDARs toNMDA-induced synaptic depression is lost in the dopamine-

depleted striatum

We then examined whether the ability of GluN2D-containingNMDARs to depress glutamatergic neurotransmission wasaltered in the 6-OHDA-lesioned mouse model of PD. In the

dopamine-depleted striatum, NMDA-induced depression ofthe fEPSP/PS amplitude was significantly reduced, but notabolished (85.8 � 1.9% of baseline, n = 16) as comparedwith the intact striatum (78.5 � 1.8% of baseline, n = 15,p < 0.05, Fig. 6). In contrast to the observation made incontrol mice, the depression in the dopamine-depletedstriatum did not involve GluN2D-containing NMDARs ormuscarinic M1 receptors. Indeed, in the dopamine-depletedstriatum, neither UBP141 nor pirenzepine affected synapticdepression as compared with control slices (Fig. 6b). Weconfirmed the 6-OHDA-induced lesion of dopaminergicneurons by measuring the levels of TH with western blotanalyses of the slices used in the electrophysiologicalexperiments presented in Fig. 6. The levels of TH weredramatically reduced in the injected hemisphere as comparedwith the intact hemisphere (p < 0.001; n = 11 mice;Fig. 6c).

Discussion

This study identifies a role for GluN2D-containing NMDARsin the control of dopaminergic and glutamatergic neurotrans-mission in the dorsal striatum, and demonstrates dysfunctionof these receptors in a mouse model of PD. We found thatGluN2D-containing NMDARs inhibit both dopamine andglutamate release through the action of acetylcholine releasedby cholinergic interneurons in the intact striatum. In thedopamine-depleted striatum, the control of glutamate releaseby GluN2D-containing NMDARs is lost (Fig. 7).We previously found that the depressant action of NMDA

on dopamine release and fEPSP/PS amplitude involved atleast in part GluN2A-, but not GluN2B-, containingNMDARs (Schotanus and Chergui 2008). This study showsthat GluN2D-containing NMDARs also contribute toNMDA-induced depression. Given that functional NMDARsin midbrain dopaminergic neurons are composed of GluN2D,

Oxotremorine-M

(a)

(b)

Fig. 2 The muscarinic receptor agonist oxotremorine-M inhibitsevoked dopamine release. (a) Representative amperometric tracesfrom one slice, at the time points indicated in (b), before (1), and during(2) perfusion with oxotremorine-M (0.3 lM). (a) Time course of the

effect of oxotremorine-M (0.1 lM, n = 9; 0.3 lM, n = 8) on evokeddopamine release.

(a)

(d)

(b) (c)

Fig. 3 NMDA increases the firing of cholinergicinterneurons. (a) Firing in a cholinergic inter-neuron, measured in somatic cell-attached mode,before and after perfusion with NMDA (20 lM, for

3 min). (b) Time histogram of the firing in theneuron presented in (a). NMDA was applied in theperfusion solution at the time indicated by the black

bar. (c) Average firing in cholinergic interneuronsbefore and after bath application of NMDA(n = 11). ***p < 0.001 compared with baseline

firing in the same neurons. (d) NMDA-inducedwhole-cell membrane depolarization and firing incontrol conditions are reduced in the presence of

PPDA.



in addition to GluN2B (Jones and Gibb 2005; Brothwellet al. 2008), direct pre-synaptic activation of GluN2D-containing NMDARs located on dopaminergic terminalsmight contribute to the depressant action of NMDA ondopamine release. Although controversial, the presence ofpre-synaptic NMDARs on dopaminergic terminals has beensuggested in earlier studies (Krebs et al. 1991; Ohta et al.1994; Iravani and Kruk 1996; Cheramy et al. 1998; Davidet al. 2005; Zhang and Sulzer 2012). The presence ofGluN2D on glutamatergic terminals in the striatum has notbeen examined, but is unlikely given the preferentialexpression of this subunit in cortical interneurons (Standaertet al. 1996). Nevertheless, the effect of NMDA on thefEPSP/PS amplitude involves a pre-synaptic mechanismbecause we found that the paired-pulse ratio increasesconcomitantly with a decrease in the fEPSP/PS amplitude.NMDA-induced depression of glutamate release, and alsodopamine release, might involve indirect mechanisms. This

possibility is supported by previous observations that themodulation of dopamine and glutamate release followingactivation of post-synaptic NMDARs involves a diffusibleretrograde messenger such as H2O2 or adenosine or anotherneurotransmitter such as acetylcholine (Cheramy et al. 1998;Avshalumov et al. 2003; Schotanus et al. 2006).In this study, we investigated the role of cholinergic

interneurons in NMDA-induced inhibition of dopaminerelease and fEPSP/PS amplitude because these neurons, butnot medium spiny projection neurons, express GluN2D(Landwehrmeyer et al. 1995). Our results suggest thatGluN2D-containing NMDARs located in cholinergic inter-neurons contribute to NMDA-induced depression of dopa-mine and glutamate release. Several observations support thispossibility. First, the depressant action of NMDA was corre-lated with an increased firing in cholinergic interneurons. Theability of a low concentration of NMDA to induce actionpotential firing in cholinergic interneurons and not in

Baseline (1) NMDA (2) Washout (3)

(a) (b)

(c) (d)

Fig. 4 GluN2D-containing NMDA receptors (NMDARs) and musca-

rinic receptors mediate NMDA-induced depression of glutamatergicneurotransmission in the striatum of control mice. (a) Representativerecords of field excitatory post-synaptic potentials/population spikes(fEPSP/PSs), obtained from a control slice during a paired-pulse

stimulation protocol, at the time points indicated in (b), i.e., before (1)and after (2) bath application of NMDA, and during washout of theeffect of NMDA (3). (b) Time course of the effect of NMDA (20 lM, for

3 min) on the amplitude of the fEPSP/PS in control slices (n = 18).(c) Average magnitude of NMDA-induced synaptic depression

(maximal effect in individual slices) in control slices (n = 18), in slices

perfused with GluN2D antagonists (UBP141, n = 9; and PPDA, n = 8),and with M1–M4 antagonists (M1: pirenzepine, n = 11; M2: AF-DX116, n = 9; M3: J 104129, n = 8; and M4: PD 102807, n = 8).*p < 0.05, **p < 0.01 compared with control slices. (d) Time course of

the effect of NMDA on the ratio between the second and the firstfEPSP/PS in a paired-pulse stimulation protocol (20 ms interstimulusinterval). The results on the first fEPSP/PS from these paired-pulse

experiments (n = 11) are included in the graph presented in (b).


586 X. Zhang et al.

projection neurons (data not shown) is likely attributed to thelow sensitivity to Mg2+ block of channels made of GluN2D,combined with a depolarized resting membrane potential incholinergic interneurons. Second, the muscarinic receptoragonist oxotremorine-M inhibited evoked dopamine releaseand the fEPSP/PS through a pre-synaptic mechanism. Third,we found that there is a small, but significant, inhibitoryaction of ambient acetylcholine acting on M1 receptors ondopamine release. Spontaneous activity in cholinergic

Oxotremorine-M

Oxotremorine-M

Baseline (1) oxo. (2) Washout (3)(a)

(b)

(c)

Fig. 5 The muscarinic receptor agonist oxotremorine-M pre-synaptic-ally inhibits glutamatergic neurotransmission. (a) Representative field

excitatory post-synaptic potentials/population spikes (fEPSP/PSs)obtained from one slice during a paired-pulse stimulation protocol, atthe time points indicated in (b), before (1), and during (2) perfusion with

oxotremorine-M (0.3 lM), and during the washout of the effect ofoxotremorine-M (3). (b) Time course of the effect of oxotremorine-M(0.3 lM) on the amplitude of the first fEPSP/PS in paired-pulse

stimulation experiments (n = 9). (c) Time course of the effect ofoxotremorine-M (0.3 lM) on the ratio between the second and the firstfEPSP/PS in paired-pulse stimulation experiments (n = 9, same slices

as in (b)).

(a)

(b)

(c)

Fig. 6 NMDA-induced depression of glutamatergic neurotransmis-sion is reduced in the dopamine-depleted striatum. (a) Time course

of the effect of NMDA (20 lM) on the amplitude of the fieldexcitatory post-synaptic potentials/population spikes (fEPSP/PS)measured in the intact striatum (open squares, n = 15) and in thedopamine-depleted striatum [6-hydroxydopamine (6-OHDA), filled

squares, n = 16]. (b) Average magnitude of NMDA-induced synapticdepression in control conditions (intact, open bar; and 6-OHDA,filled bar) and with UBP141 (n = 8) or pirenzepine (n = 7) in the

dopamine-depleted striatum. *p < 0.05. (c) Western blots of tyrosinehydroxylase (TH), the rate-limiting enzyme in the synthesis ofdopamine, from intact and lesioned hemispheres of the same mice.

Total protein amounts are expressed as percentage of intacthemisphere from individual animals (n = 11). Blots above graphsare representative examples from intact (left) and dopamine-

depleted (right) hemispheres. ***p < 0.001 compared with intacthemisphere.



interneurons in the brain slice preparation likely producesthis endogenous tone of acetylcholine. Fourth, muscarinicreceptor antagonists counteracted, to some degree, thedepressant effect of NMDA. The involvement of multiplemuscarinic receptors might be attributable to differentmechanisms, described in earlier studies (Pisani et al.2007; Goldberg et al. 2012), that may contribute toNMDA-induced depression of dopamine and glutamaterelease. Previous studies found that dopamine release isunder the control of M2 and M4 receptors in cholinergicinterneurons and nicotinic receptors likely localized indopaminergic axon terminals (Threlfell et al. 2010). Inaddition to these receptors, we found that M1, but not M3,receptors mediate NMDA-induced depressant action ondopamine release, suggesting that additional mechanismscontrol dopamine release. For glutamate release, severalmechanisms could contribute to NMDA-induced inhibitionthrough activation of cholinergic interneurons and musca-rinic M1–M4 receptors. Indeed, M1 receptors are suggestedto have a post-synaptic location on medium spiny striatalneurons, M2/M3 receptors were shown to be present onglutamatergic terminals, and M4 receptors are expressed incholinergic interneurons axon terminals where they regulatethe release of acetylcholine, as well as on medium spinyneurons (Pisani et al. 2007).Taken together, these results suggest that GluN2D-

containing NMDARs in cholinergic interneurons contributeto the pre-synaptic control of dopamine and glutamaterelease in the striatum. Our results further confirm the potentinhibitory role of cholinergic interneurons in the regulationof dopamine release in the striatum (Threlfell et al. 2010).Our observations are consistent with the demonstration thatagonists at most heteroreceptors, except for nicotinic acetyl-choline receptors, inhibit dopamine release (Rice et al. 2011;

Zhang and Sulzer 2012). Our study identifies anotherreceptor whose activation inhibits dopamine release, furthercontributing to signaling salient contextual stimuli, assuggested in earlier studies (Zhang and Sulzer 2012). Ourresults also extend the observations made for dopaminerelease to glutamatergic neurotransmission, demonstratingthat both neurotransmitter systems are regulated by striatalcholinergic interneurons.Several lines of evidence indicate that the expression of

NMDAR subunits, in particular GluN2B, is altered in thestriatum of animal models of PD. Down-regulation ofGluN2B expression might not have a dramatic impact onthe ability of NMDARs to depress synaptic transmissiongiven the lack of involvement of GluN2B-containingNMDARs in NMDA-induced synaptic depression in thestriatum of control mice (Schotanus and Chergui 2008). Wefound, however, that the ability of a GluN2D antagonist or amuscarinic receptor antagonist to reduce NMDA-inducedsynaptic depression is lost in the dopamine-depleted striatum.Although cholinergic neurotransmission is altered in exper-imental Parkinsonism (Pisani et al. 2007), it is unlikely thatthe concentration of pirenzepine (1 lM) used in this studywas not high enough to block the effect of acetylcholine onmuscarinic M1 receptors in the dopamine-depleted striatum.Indeed, the expression of M1 receptor mRNA is unaffectedin the striatum of 6-OHDA-lesioned mice (Kayadjanianet al. 1999) and the effect of pirenzepine on glutamatergicsynaptic transmission was shown to be similar in the intactand in the dopamine-depleted striatum (Tozzi et al. 2011).However, we found that the functions of GluN2D-containingNMDARs in cholinergic interneurons are impaired in thedopamine-depleted striatum (unpublished results). Thus, it islikely that the contribution of these receptors, and ofcholinergic interneurons, to NMDA-induced synaptic depres-sion is reduced as compared with that in the intact striatum.Taken together, our results suggest that neurophysiologicalalterations that occur in PD include dysfunction or loss ofGluN2D-containing NMDARs in cholinergic interneurons.The reduced inhibitory control of GluN2D-containingNMDARs could contribute to the increased glutamatergicneurotransmission observed in experimental Parkinsonism(Bagetta et al. 2010).

Conclusions

This study demonstrates an inhibitory role for GluN2D-containing NMDARs, mediated by cholinergic interneurons,on dopamine and glutamate release in the intact striatum, andan impairment of this function in the dopamine-depletedstriatum. This study proposes GluN2D as a potential drugtarget for the development of novel pharmacological toolsfor therapeutic intervention in PD. GluN2D-selectivecompounds might also be useful in the management ofpsychosis where hypofunction of NMDARs is associated

Fig. 7 Role and dysfunction of GluN2D-containing NMDA receptors(NMDARs) in the striatum. Activation of GluN2D-containing NMDARslocated in cholinergic interneurons inhibits dopamine and glutamate

release in the intact striatum. This function is impaired in the dopamine-depleted striatum.


588 X. Zhang et al.

with increased dopaminergic transmission in subcorticalbrain regions (Moghaddam and Javitt 2012), as well as indrug addiction (Ma et al. 2009).

Acknowledgments and conflict of interestdisclosure

This study was supported by the Swedish Research Council (grants2008-2636 and 2011-2770), the Loo and Hans Ostermans Founda-tion for Geriatric Research, Parkinsonfonden, Stiftelse Lars HiertasMinne. X.Z. was a recipient of a post-doctoral fellowship from theSwedish Society for Medical Research (SSMF).

All experiments were conducted in compliance with the ARRIVEguidelines. The authors have no conflict of interest to declare.

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glun2d-containing nmda receptors inhibit neurotransmission in the mouse striatum through a...

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