GluN2D-containing NMDA receptors inhibit neurotransmission in the mouse striatum through a cholinergic mechanism: implication for Parkinson's disease

Authors

  • Xiaoqun Zhang,

    1. Department of Physiology and Pharmacology, Section of Molecular Neurophysiology, The Karolinska Institute, Stockholm, Sweden
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  • Ze-Jun Feng,

    1. Department of Physiology and Pharmacology, Section of Molecular Neurophysiology, The Karolinska Institute, Stockholm, Sweden
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  • Karima Chergui

    Corresponding author
    1. Department of Physiology and Pharmacology, Section of Molecular Neurophysiology, The Karolinska Institute, Stockholm, Sweden
    • Address correspondence and reprint requests to Karima Chergui, Department of Physiology and Pharmacology, Section of Molecular Neurophysiology, the Karolinska Institute, Von Eulers väg 8, 171 77 Stockholm, Sweden. E-mail: karima.chergui@ki.se

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Abstract

The GluN2 subunits that compose NMDA receptors (NMDARs) determine functional and pharmacological properties of the receptor. In the striatum, functions and potential dysfunctions of NMDARs attributed to specific GluN2 subunits have not been clearly elucidated, although NMDARs play critical roles in the interactions between glutamate and dopamine. Through the use of amperometry and field potential recordings in mouse brain slices, we found that NMDARs that contain the GluN2D subunit contribute to NMDA-induced inhibition of evoked dopamine release and of glutamatergic neurotransmission in the striatum of control mice. Inhibition is likely mediated through increased firing in cholinergic interneurons, which were shown to express GluN2D. Indeed, NMDA-induced inhibition of both dopamine release and glutamatergic neurotransmission is reduced in the presence of muscarinic receptor antagonists and is mimicked by a muscarinic receptor agonist. We have also examined whether this function of GluN2D-containing NMDARs is altered in a mouse model of Parkinson's disease. We found that the inhibitory role of GluN2D-containing NMDARs on glutamatergic neurotransmission is impaired in the 6-hydroxydopamine lesioned striatum. These results identify a role for GluN2D-containing NMDARs and adaptive changes in experimental Parkinsonism. GluN2D might constitute an attractive target for the development of novel pharmacological tools for therapeutic intervention in Parkinson's disease.

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We have examined the role of NMDA receptors composed of the GluN2D subunit in the mouse striatum. These receptors inhibit the release of dopamine and of glutamate through a mechanism that involves activation of cholinergic interneurons. This inhibitory role is impaired in the dopamine-depleted striatum. This study identifies GluN2D as a potential target for therapeutic intervention in Parkinson's disease.

Abbreviations 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

The main input nucleus of the basal ganglia, the striatum, receives convergent afferents from dopaminergic neurons whose cell bodies are located in the substantia nigra, and glutamatergic afferents that originate in the cortex and thalamus (David et al. 2005). Interactions between glutamate and dopamine in the striatum play critical roles in health and disease. The complex interactions between dopamine receptors and the NMDA type of glutamate receptors (NMDARs) have been extensively examined (Cepeda and Levine 2012). However, the mechanisms by which glutamate acting on NMDARs modulates dopamine release at the terminals as well as glutamatergic neurotransmission are still unresolved (Zhang and Sulzer 2012). Early studies have suggested that NMDARs with different sensitivities for Mg2+ modulate the release of dopamine in the striatum through direct and indirect mechanisms (Krebs et al. 1991; Ohta et al. 1994; Iravani and Kruk 1996; Cheramy et al. 1998). NMDARs with distinct subunit compositions might control neurotransmitter release. Indeed, functional and pharmacological properties of NMDARs are closely dependent on the subunit composition of these receptors, and in particular on the GluN2 subunits they contain. NMDARs are heterotetrameric assemblies of GluN1, GluN2 (A–D), and GluN3 (A, B) subunits (Paoletti et al. 2013). GluN2B is the most abundant GluN2 subunit in the striatum and is also expressed in dopaminergic neurons (Landwehrmeyer et al. 1995; Standaert et al. 1999; Jones and Gibb 2005). Interestingly, we found that GluN2B-containing NMDARs do not contribute significantly to NMDA-induced inhibition of evoked, action potential-dependent, dopamine release and of glutamatergic synaptic transmission in mouse corticostriatal and striatal brain slices (Schotanus and Chergui 2008). Our previous findings demonstrated a significant contribution of GluN2A-containing NMDARs, but also suggested that NMDARs-containing subunits other than GluN2A and GluN2B control dopamine and glutamate release, directly or indirectly (Schotanus and Chergui 2008).

GluN2D forms functional NMDARs in midbrain dopaminergic neurons (Standaert et al. 1994; Jones and Gibb 2005; Brothwell et al. 2008) and is expressed in striatal interneurons, in particular, large cholinergic interneurons (Standaert et al. 1996; Bloomfield et al. 2007). Although these interneurons represent less than 2% of the total neuronal population in the striatum, they are likely to control neurotransmission in the striatum because of their extensive axonal branching (Pisani et al. 2007; Bonsi et al. 2011; Goldberg et al. 2012). GluN2D-containing NMDARs localized at dopaminergic axon terminals in the striatum and/or in cholinergic interneurons might thus play a role in the control of dopamine release and glutamatergic neurotransmission.

The GluN2 subunits that compose NMDARs are attractive drug targets for therapeutic intervention in several neurological and psychiatric disorders which are associated with dysfunctional neurotransmission mediated by glutamate and/or dopamine (Loftis and Janowsky 2003; Gogas 2006). Moreover, altered expression of GluN2B in the striatum of animal models of Parkinson's disease (PD) is suggested to contribute to L-DOPA-induced dyskinesia (Dunah et al. 2000; Gardoni et al. 2006; Paille et al. 2010). Whether the functions of GluN2D-containing NMDARs are altered in experimental Parkinsonism has not been examined. The aim of this study was to investigate whether GluN2D-containing NMDARs contribute to NMDA-induced modulation of dopamine release and of glutamatergic synaptic transmission in corticostriatal mouse brain slices. We determined whether striatal cholinergic interneurons contributed to the observed modulation 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 results were presented as a meeting abstract (Zhang and Chergui 2011).

Materials and methods

Animals and brain slice preparation

All efforts were made to minimize animal suffering and to reduce the number of animals used. Experiments were approved by our local ethical committee (Stockholms norra djurförsöksetiska nämnd), followed the ARRIVE guidelines, and were performed as described previously (Chergui et al. 2004; Schotanus and Chergui 2008; Zhang et al. 2008; Chergui 2011). We used male C57BL/6 mice aged 4–9 weeks (Harlan Laboratories, The Netherlands). Mice were maintained on a 12:12 h light/dark cycle and had free access to food and water. A group of mice underwent unilateral stereotaxic injection of the toxin 6-OHDA to lesion dopaminergic neurons in the substantia nigra pars compacta. These mice were anesthetized with 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 μg of 6-OHDA in 0.01% ascorbic acid into the substantia nigra pars compacta of the right hemisphere. The coordinates for injection were AP, −3 mm; ML, −1.1 mm; and DV, −4.5 mm relative to bregma and the dural surface (Paxinos and Franklin 2001). Mice underwent cervical dislocation followed by decapitation (for lesioned mice, this was done 1–3 weeks following surgery). Their brains were rapidly removed and brain slices (coronal and sagittal, 400 μm thick) containing the striatum and the overlying cortex were prepared with a microslicer (VT 1000S; Leica Microsystem, Heppenheim, Germany). Slices were incubated, for at least 1 h, at 32°C in oxygenated (95% O2 + 5% CO2) artificial cerebrospinal 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 (Warner Instruments, Hamden, CT, USA; recording chamber from Scientifica Ltd., Uckfield, UK) mounted on an upright microscope (Olympus, Solna, Sweden and Scientifica Ltd.) and were continuously perfused with oxygenated aCSF at 28°C.

Amperometry in brain slices

Amperometric detection of dopamine release was performed with carbon fiber electrodes (10 μm diameter, World Precision Instruments Europe) which had an active part of 100 μm that was positioned within the dorsal striatum in the brain slice. A constant voltage of + 500 mV was applied to the carbon fiber via an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA) and currents were recorded with the same amplifier. A stimulating electrode (patch electrode filled with aCSF) was placed on the slice surface, in the vicinity of the carbon fiber electrode. Stimulations consisted of single pulses (0.1 ms, 8–14 μA) applied every minute, which evoked a response corresponding to oxidation of 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 micropipette filled with aCSF positioned on the slice surface. These synaptic responses were evoked by stimulation pulses applied every 15 s to the brain slice through a concentric bipolar stimulating electrode (FHC, Bowdoinham, ME, USA) placed near the recording electrode on the surface of the slice (Schotanus et al. 2006). Single stimuli (0.1 ms duration) were applied at an intensity yielding 50–60% maximal response as assessed by a stimulus/response curve established, for each slice, at the beginning of the recording session, by measuring the amplitude of the field excitatory post-synaptic potentials/population spikes (fEPSP/PSs) evoked by increasing stimulation intensities. Paired-pulse stimulations consisted in two stimulation pulses separated by a 20-ms interval. Signals were amplified 500 or 1000 times via an Axopatch 200B or a GeneClamp 500B amplifier (Axon Instruments), acquired at 10 kHz, and filtered at 2 kHz.

Cell-attached and whole-cell patch-clamp recordings of cholinergic interneurons in the dorsal striatum were made with the help of infrared-differential interference contrast video microscopy. Cholinergic interneurons were identified by their morphological and electrophysiological properties which include a large soma, spontaneous firing, pronounced long-lasting spike after hyperpolarization, resting membrane potential around −60 mV (Kawaguchi 1993). Patch electrodes were filled with a solution containing, in mM: 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. Whole-cell membrane currents and potentials were recorded with a MultiClamp 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 10 software (Axon Instruments). Numerical values are shown as means with SEM, with n indicating the number of slices or neurons tested. For dopamine release and fEPSP/PS, data are expressed as percent of the baseline response measured for each slice during the 5–10 min preceding start of perfusion with NMDA or oxotremorine-M. Statistical significance of the results was assessed by using the Student's t-test for paired and unpaired observations or one-way anova followed by Bonferroni's multiple comparison test.

Chemicals and drugs

Chemicals and drugs were purchased from Sigma-Aldrich (Stockholm, Sweden), Tocris Bioscience (Bristol, UK), and Abcam Biochemicals (Cambridge, UK). All compounds were prepared in stock solutions, diluted in aCSF to the desired final concentration, and applied in the perfusion solution. The following compounds were used (final concentrations in μM): AF-DX 116 (0.1), J 104129 fumarate (0.01), NMDA (20), oxotremorine-M (0.1 and 0.3), PD 102807 (0.5), pirenzepine dihydrochloride (1), cis-PPDA ((2S*,3R*)-1-(Phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic acid) (0.5), and UBP141 (3-6). We used the competitive GluN2C/GluN2D-preferring antagonists UBP141 and PPDA which display 5- to 10-fold selectivity for GluN2C/GluN2D-containing NMDARs over GluN2A/GluN2B-containing NMDARs, with UBP141 displaying higher selectivity than PPDA (Feng et al. 2004; Costa et al. 2009). The concentrations of these compounds used in our study were previously shown to inhibit synaptic and extrasynaptic NMDAR-mediated currents in hippocampal and midbrain slices with minimal effect on receptors containing GluN2A or GluN2B (Brothwell et al. 2008; Harney et al. 2008; Costa et al. 2009; Harney and Anwyl 2012). Because GluN2C is absent from the striatum (Bloomfield et al. 2007), UBP141 and PPDA likely antagonize the action of NMDA on GluN2D-containing NMDARs.

Western blotting

Western blots were performed to confirm and quantify the loss of tyrosine hydroxylase (TH) following 6-OHDA lesioning in the slices that were used for electrophysiological experiments. The slices were frozen and stored at −20°C until processed. The samples were sonicated in 1% sodium dodecyl sulfate and boiled for 10 min. Protein concentration was determined in each sample with a bicinchoninic acid protein assay (BCA-kit, Pierce, Rockford, USA). Equal amounts of protein (30 μg) were resuspended in sample buffer and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using a 10% running gel and transferred to an Immobilon-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 against total TH (Millipore, Billerica, MA, USA) in 5% dry milk dissolved in TBS-Tween 20. The membranes were washed three times with TBS-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 the incubation, membranes were washed six times with TBS-Tween 20 and immunoreactive bands were detected by enhanced chemiluminescence (Perkin Elmer, Waltham, MA, USA). The autoradiograms were scanned and quantified with the NIH Image 1.63 software (National Institute of Health, Bethesda, MD, USA). Data were analyzed with two-tailed unpaired Student's t-test to evaluate statistical differences.

Results

GluN2D-containing NMDARs contribute to NMDA-induced inhibition of evoked dopamine release

We first evaluated the effect of NMDA on stimulation-evoked release of dopamine in corticostriatal slices, as done previously (Schotanus et al. 2006; Schotanus and Chergui 2008). We found that NMDA (20 μM), applied in the perfusion solution for 3 min, reversibly depressed the peak current amplitude, corresponding to released dopamine, evoked in the dorsal striatum (to 53.0 ± 6.2% of baseline, n = 8, Fig. 1). In the presence of the GluN2D antagonist UBP141, the inhibitory effect of NMDA on dopamine release was significantly reduced (to 74.7 ± 4.4% of baseline, n = 8; Fig. 1c), as compared with control slices (p < 0.05). A similar reduction in NMDA-induced depression of dopamine release was observed in the presence of another GluN2D antagonist, PPDA (n = 8, Fig. 1c). These results demonstrate that GluN2D-containing NMDARs participate in NMDA-induced depression of evoked dopamine release in the striatum. Given that cholinergic striatal interneurons express GluN2D (Landwehrmeyer et al. 1995), and because acetylcholine acting on muscarinic receptors exerts a powerful inhibitory control on dopamine release (Threlfell et al. 2010), we examined whether the mechanism by which GluN2D-containing NMDARs inhibit dopamine release included activation of cholinergic interneurons. We first tested the effects of muscarinic receptor antagonists on the depressant action of NMDA. We found that muscarinic receptor antagonists acting on M1 (pirenzepine), M2 (AF-DX 116), and M4 (PD 102807) receptors decreased NMDA-induced inhibition of evoked dopamine release, to the same extent as GluN2D antagonists (Fig. 1c). NMDA-induced inhibition was not significantly altered in the presence of the M3 receptor antagonist, J 104129. Interestingly, pirenzepine, but not the other muscarinic receptor antagonists, significantly increased dopamine release when applied alone (to 115.3 ± 6.3% of baseline, n = 9; p < 0.05, data not shown). This observation demonstrates a tonic control of dopamine release in the striatum by ambient acetylcholine acting on muscarinic M1 receptors. We then demonstrated that the muscarinic receptor agonist oxotremorine-M inhibited evoked dopamine release at a concentration as low as 0.1 μM (Fig. 2), demonstrating that activation of muscarinic receptors mimics the effect of NMDA. We also recorded, in the cell-attached configuration, action potential firing in cholinergic interneurons and applied NMDA (20 μM) in the perfusion solution for 3 min, as for dopamine release experiments. We found that NMDA increased the firing rate of these interneurons with a similar time course as for the inhibitory action of NMDA on dopamine release (n = 11, Fig. 3). Finally, we found that cholinergic interneurons express functional GluN2D-containing NMDARs because NMDA-induced whole-cell depolarizations 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). Taken together, these results suggest that activation of GluN2D-containing NMDARs by bath applied NMDA increases the firing rate of cholinergic interneurons, and that subsequent release of acetylcholine inhibits dopamine release through muscarinic receptors.

Figure 1.

GluN2D-containing NMDA receptors (NMDARs) and muscarinic receptors contribute to NMDA-induced inhibition of evoked dopamine release in the mouse striatum. (a) Representative amperometric traces from one slice, at the time points indicated in (b), before (1) and after (2) bath application of NMDA (20 μM). (b) Time course of the effect of NMDA (20 μM), applied in the perfusion solution at the time indicated by the black bar (3 min duration), on evoked dopamine release (n = 8). (c) Average magnitude of NMDA-induced inhibition (maximal effect in individual slices) in control slices (n = 8), in slices perfused with GluN2D antagonists (UBP141, n = 8; and PPDA, n = 8), and with M1–M4 antagonists (M1: pirenzepine, n = 11; M2: AF-DX 116, n = 9; M3: J 104129, n = 7; and M4: PD 102807, n = 9). **p < 0.01, *p < 0.05 compared with control slices.

Figure 2.

The muscarinic receptor agonist oxotremorine-M inhibits evoked dopamine release. (a) Representative amperometric traces from one slice, at the time points indicated in (b), before (1), and during (2) perfusion with oxotremorine-M (0.3 μM). (a) Time course of the effect of oxotremorine-M (0.1 μM, n = 9; 0.3 μM, n = 8) on evoked dopamine release.

Figure 3.

NMDA increases the firing of cholinergic interneurons. (a) Firing in a cholinergic interneuron, measured in somatic cell-attached mode, before and after perfusion with NMDA (20 μM, for 3 min). (b) Time histogram of the firing in the neuron presented in (a). NMDA was applied in the perfusion solution at the time indicated by the black bar. (c) Average firing in cholinergic interneurons before and after bath application of NMDA (n = 11). ***p < 0.001 compared with baseline firing in the same neurons. (d) NMDA-induced whole-cell membrane depolarization and firing in control conditions are reduced in the presence of PPDA.

GluN2D-containing NMDARs contribute to NMDA-induced depression of glutamatergic neurotransmission

We previously demonstrated that NMDA depressed glutamatergic neurotransmission through an intrastriatal, GABA-independent, mechanism (Schotanus et al. 2006; Schotanus and Chergui 2008). In this study, we investigated whether GluN2D-containing NMDARs contributed to this synaptic depression. In control slices, NMDA (20 μM, bath applied for 3 min) produced a reversible, short-lasting, reduction in the amplitude of the fEPSP/PS (to 75.5 ± 2.3% of baseline, n = 18, Fig. 4a–c). This depression was accompanied by a reversible increase in the ratio between the second and the first fEPSP/PS in a paired-pulse stimulation protocol in the 11 slices examined (Fig. 4d), suggesting a pre-synaptic mechanism. We found that synaptic depression was significantly reduced in the presence of the GluN2D antagonists UBP141 (n = 9) and PPDA (n = 8) (Fig. 4c). Cholinergic interneurons were likely involved in part of the effect of NMDA because synaptic depression was reduced in the presence of muscarinic receptor antagonists acting on M1 receptors (pirenzepine, n = 11), M2 receptors (AF-DX 116, n = 9), M3 receptors (J 104129, n = 8), and M4 receptors (PD 102807, n = 8) (Fig. 4c). As for dopamine release, we confirmed that the muscarinic receptor agonist oxotremorine-M pre-synaptically inhibited glutamatergic neurotransmission in our experimental conditions (Fig. 5). These results demonstrate that NMDA-induced synaptic depression is in part mediated by NMDARs that contain GluN2D and by muscarinic M1–M4 receptors.

Figure 4.

GluN2D-containing NMDA receptors (NMDARs) and muscarinic receptors mediate NMDA-induced depression of glutamatergic neurotransmission in the striatum of control mice. (a) Representative records 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 the effect of NMDA (3). (b) Time course of the effect of NMDA (20 μM, 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-DX 116, 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 first fEPSP/PS in a paired-pulse stimulation protocol (20 ms interstimulus interval). The results on the first fEPSP/PS from these paired-pulse experiments (n = 11) are included in the graph presented in (b).

Figure 5.

The muscarinic receptor agonist oxotremorine-M pre-synaptically inhibits glutamatergic neurotransmission. (a) Representative field excitatory post-synaptic potentials/population spikes (fEPSP/PSs) obtained from one slice during a paired-pulse stimulation protocol, at the time points indicated in (b), before (1), and during (2) perfusion with oxotremorine-M (0.3 μM), and during the washout of the effect of oxotremorine-M (3). (b) Time course of the effect of oxotremorine-M (0.3 μM) on the amplitude of the first fEPSP/PS in paired-pulse stimulation experiments (n = 9). (c) Time course of the effect of oxotremorine-M (0.3 μM) on the ratio between the second and the first fEPSP/PS in paired-pulse stimulation experiments (n = 9, same slices as in (b)).

The contribution of GluN2D-containing NMDARs to NMDA-induced synaptic depression is lost in the dopamine-depleted striatum

We then examined whether the ability of GluN2D-containing NMDARs to depress glutamatergic neurotransmission was altered in the 6-OHDA-lesioned mouse model of PD. In the dopamine-depleted striatum, NMDA-induced depression of the fEPSP/PS amplitude was significantly reduced, but not abolished (85.8 ± 1.9% of baseline, n = 16) as compared with the intact striatum (78.5 ± 1.8% of baseline, n = 15, p < 0.05, Fig. 6). In contrast to the observation made in control mice, the depression in the dopamine-depleted striatum did not involve GluN2D-containing NMDARs or muscarinic M1 receptors. Indeed, in the dopamine-depleted striatum, neither UBP141 nor pirenzepine affected synaptic depression as compared with control slices (Fig. 6b). We confirmed the 6-OHDA-induced lesion of dopaminergic neurons by measuring the levels of TH with western blot analyses of the slices used in the electrophysiological experiments presented in Fig. 6. The levels of TH were dramatically reduced in the injected hemisphere as compared with the intact hemisphere (p < 0.001; n = 11 mice; Fig. 6c).

Figure 6.

NMDA-induced depression of glutamatergic neurotransmission is reduced in the dopamine-depleted striatum. (a) Time course of the effect of NMDA (20 μM) on the amplitude of the field excitatory post-synaptic potentials/population spikes (fEPSP/PS) measured in the intact striatum (open squares, n = 15) and in the dopamine-depleted striatum [6-hydroxydopamine (6-OHDA), filled squares, n = 16]. (b) Average magnitude of NMDA-induced synaptic depression 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 tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of dopamine, from intact and lesioned hemispheres of the same mice. Total protein amounts are expressed as percentage of intact hemisphere from individual animals (n = 11). Blots above graphs are representative examples from intact (left) and dopamine-depleted (right) hemispheres. ***< 0.001 compared with intact hemisphere.

Discussion

This study identifies a role for GluN2D-containing NMDARs in the control of dopaminergic and glutamatergic neurotransmission in the dorsal striatum, and demonstrates dysfunction of these receptors in a mouse model of PD. We found that GluN2D-containing NMDARs inhibit both dopamine and glutamate release through the action of acetylcholine released by cholinergic interneurons in the intact striatum. In the dopamine-depleted striatum, the control of glutamate release by GluN2D-containing NMDARs is lost (Fig. 7).

Figure 7.

Role and dysfunction of GluN2D-containing NMDA receptors (NMDARs) in the striatum. Activation of GluN2D-containing NMDARs located in cholinergic interneurons inhibits dopamine and glutamate release in the intact striatum. This function is impaired in the dopamine-depleted striatum.

We previously found that the depressant action of NMDA on dopamine release and fEPSP/PS amplitude involved at least in part GluN2A-, but not GluN2B-, containing NMDARs (Schotanus and Chergui 2008). This study shows that GluN2D-containing NMDARs also contribute to NMDA-induced depression. Given that functional NMDARs in midbrain dopaminergic neurons are composed of GluN2D, in addition to GluN2B (Jones and Gibb 2005; Brothwell et al. 2008), direct pre-synaptic activation of GluN2D-containing NMDARs located on dopaminergic terminals might contribute to the depressant action of NMDA on dopamine release. Although controversial, the presence of pre-synaptic NMDARs on dopaminergic terminals has been suggested in earlier studies (Krebs et al. 1991; Ohta et al. 1994; Iravani and Kruk 1996; Cheramy et al. 1998; David et al. 2005; Zhang and Sulzer 2012). The presence of GluN2D on glutamatergic terminals in the striatum has not been examined, but is unlikely given the preferential expression of this subunit in cortical interneurons (Standaert et al. 1996). Nevertheless, the effect of NMDA on the fEPSP/PS amplitude involves a pre-synaptic mechanism because we found that the paired-pulse ratio increases concomitantly with a decrease in the fEPSP/PS amplitude. NMDA-induced depression of glutamate release, and also dopamine release, might involve indirect mechanisms. This possibility is supported by previous observations that the modulation of dopamine and glutamate release following activation of post-synaptic NMDARs involves a diffusible retrograde messenger such as H2O2 or adenosine or another neurotransmitter 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 dopamine release and fEPSP/PS amplitude because these neurons, but not medium spiny projection neurons, express GluN2D (Landwehrmeyer et al. 1995). Our results suggest that GluN2D-containing NMDARs located in cholinergic interneurons contribute to NMDA-induced depression of dopamine and glutamate release. Several observations support this possibility. First, the depressant action of NMDA was correlated with an increased firing in cholinergic interneurons. The ability of a low concentration of NMDA to induce action potential firing in cholinergic interneurons and not in projection neurons (data not shown) is likely attributed to the low sensitivity to Mg2+ block of channels made of GluN2D, combined with a depolarized resting membrane potential in cholinergic interneurons. Second, the muscarinic receptor agonist oxotremorine-M inhibited evoked dopamine release and the fEPSP/PS through a pre-synaptic mechanism. Third, we found that there is a small, but significant, inhibitory action of ambient acetylcholine acting on M1 receptors on dopamine release. Spontaneous activity in cholinergic interneurons in the brain slice preparation likely produces this endogenous tone of acetylcholine. Fourth, muscarinic receptor antagonists counteracted, to some degree, the depressant effect of NMDA. The involvement of multiple muscarinic receptors might be attributable to different mechanisms, described in earlier studies (Pisani et al. 2007; Goldberg et al. 2012), that may contribute to NMDA-induced depression of dopamine and glutamate release. Previous studies found that dopamine release is under the control of M2 and M4 receptors in cholinergic interneurons and nicotinic receptors likely localized in dopaminergic axon terminals (Threlfell et al. 2010). In addition to these receptors, we found that M1, but not M3, receptors mediate NMDA-induced depressant action on dopamine release, suggesting that additional mechanisms control dopamine release. For glutamate release, several mechanisms could contribute to NMDA-induced inhibition through activation of cholinergic interneurons and muscarinic M1–M4 receptors. Indeed, M1 receptors are suggested to have a post-synaptic location on medium spiny striatal neurons, M2/M3 receptors were shown to be present on glutamatergic terminals, and M4 receptors are expressed in cholinergic interneurons axon terminals where they regulate the release of acetylcholine, as well as on medium spiny neurons (Pisani et al. 2007).

Taken together, these results suggest that GluN2D-containing NMDARs in cholinergic interneurons contribute to the pre-synaptic control of dopamine and glutamate release in the striatum. Our results further confirm the potent inhibitory role of cholinergic interneurons in the regulation of dopamine release in the striatum (Threlfell et al. 2010). Our observations are consistent with the demonstration that agonists at most heteroreceptors, except for nicotinic acetylcholine receptors, inhibit dopamine release (Rice et al. 2011; Zhang and Sulzer 2012). Our study identifies another receptor whose activation inhibits dopamine release, further contributing to signaling salient contextual stimuli, as suggested in earlier studies (Zhang and Sulzer 2012). Our results also extend the observations made for dopamine release to glutamatergic neurotransmission, demonstrating that both neurotransmitter systems are regulated by striatal cholinergic interneurons.

Several lines of evidence indicate that the expression of NMDAR subunits, in particular GluN2B, is altered in the striatum of animal models of PD. Down-regulation of GluN2B expression might not have a dramatic impact on the ability of NMDARs to depress synaptic transmission given the lack of involvement of GluN2B-containing NMDARs in NMDA-induced synaptic depression in the striatum of control mice (Schotanus and Chergui 2008). We found, however, that the ability of a GluN2D antagonist or a muscarinic receptor antagonist to reduce NMDA-induced synaptic depression is lost in the dopamine-depleted striatum. Although cholinergic neurotransmission is altered in experimental Parkinsonism (Pisani et al. 2007), it is unlikely that the concentration of pirenzepine (1 μM) used in this study was not high enough to block the effect of acetylcholine on muscarinic M1 receptors in the dopamine-depleted striatum. Indeed, the expression of M1 receptor mRNA is unaffected in the striatum of 6-OHDA-lesioned mice (Kayadjanian et al. 1999) and the effect of pirenzepine on glutamatergic synaptic transmission was shown to be similar in the intact and in the dopamine-depleted striatum (Tozzi et al. 2011). However, we found that the functions of GluN2D-containing NMDARs in cholinergic interneurons are impaired in the dopamine-depleted striatum (unpublished results). Thus, it is likely that the contribution of these receptors, and of cholinergic interneurons, to NMDA-induced synaptic depression is reduced as compared with that in the intact striatum. Taken together, our results suggest that neurophysiological alterations that occur in PD include dysfunction or loss of GluN2D-containing NMDARs in cholinergic interneurons. The reduced inhibitory control of GluN2D-containing NMDARs could contribute to the increased glutamatergic neurotransmission 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, and an impairment of this function in the dopamine-depleted striatum. This study proposes GluN2D as a potential drug target for the development of novel pharmacological tools for therapeutic intervention in PD. GluN2D-selective compounds might also be useful in the management of psychosis where hypofunction of NMDARs is associated with increased dopaminergic transmission in subcortical brain regions (Moghaddam and Javitt 2012), as well as in drug addiction (Ma et al. 2009).

Acknowledgments and conflict of interest disclosure

This study was supported by the Swedish Research Council (grants 2008-2636 and 2011-2770), the Loo and Hans Ostermans Foundation for Geriatric Research, Parkinsonfonden, Stiftelse Lars Hiertas Minne. X.Z. was a recipient of a post-doctoral fellowship from the Swedish Society for Medical Research (SSMF).

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

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