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Dopamine depletion of the striatum causes a cell-type specific reorganization of GluN2B- and GluN2D-containing NMDA receptors
Neuropharmacology 92 (2015) 108e115
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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
Dopamine depletion of the striatum causes a cell-type specific reorganization of GluN2B- and GluN2D-containing NMDA receptors Xiaoqun Zhang, Karima Chergui* €g 8, 171 77 Stockholm, Sweden The Karolinska Institute, Department of Physiology and Pharmacology, Section of Molecular Neurophysiology, Von Eulers Va
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 August 2014 Received in revised form 9 January 2015 Accepted 10 January 2015 Available online 23 January 2015
The GluN2B subunit of NMDA receptors (NMDARs) is an attractive drug target for therapeutic intervention in Parkinson's disease (PD). We have used whole-cell patch clamp recordings in brain slices to examine the contribution of GluN2B and GluN2D to functional NMDARs in the striatum of the unilateral 6-hydroxydopamine-lesioned mouse model of PD. We found that current/voltage relationships of NMDAR-mediated excitatory post synaptic currents were altered in a population of medium spiny projection neurons (MSNs) in the dopamine-depleted striatum. Using antagonists for GluN2B- and GluN2D-containing NMDARs, we found that GluN2B contributes to functional NMDARs in MSNs in the intact striatum and in the striatum of control mice. The function of GluN2B-containing NMDARs is however reduced in MSNs from the dopamine-depleted striatum. GluN2D is absent in MSNs from intact striatum and from control mice, but the contribution of this subunit to functional NMDARs is increased in the dopamine-depleted striatum. These changes in the subunit composition of NMDARs are associated with a decreased protein level of GluN2B and an increased level of GluN2D in the dopamine-depleted striatum. In cholinergic interneurons from the intact striatum and control mice, both GluN2B and GluN2D contribute to functional NMDARs. The functions of GluN2D, and to some extent GluN2B, are reduced in the dopamine-depleted striatum. Our findings demonstrate a cell-type specific reorganization of GluN2B and GluN2D in a mouse model of PD and suggest GluN2D as a potential target for the management of the disease. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Parkinson's disease NMDA receptor GluN2B GluN2D Striatum 6-Hydroxydopamine lesion
1. Introduction Parkinson's disease (PD) is associated with the loss of dopaminergic neurons in the substantia nigra pars compacta and a resultant dramatic reduction in dopamine contents in the striatum, a major recipient of dopaminergic innervation. The glutamatergic neurotransmitter system is also affected in the dopamine-depleted striatum (Bagetta et al., 2010). Broad spectrum NMDAR antagonists alleviate some of the motor symptoms of PD but these beneficial effects are accompanied by unwanted side effects. Because physiological and pathophysiological processes involving NMDARs are dependent on the subunit composition of these receptors, subunitspecific antagonists have been investigated as possible therapeutic agents for treatment of PD with reduced propensity to elicit side effects. NMDARs are heterotetrameric assemblies of two GluN1
* Corresponding author. Tel.: þ46 8 524 86882. E-mail addresses: [email protected] (X. Zhang), (K. Chergui). http://dx.doi.org/10.1016/j.neuropharm.2015.01.007 0028-3908/© 2015 Elsevier Ltd. All rights reserved.
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subunits and other GluN2 (AeD) and GluN3 (A, B) subunits (Kohr, 2006; Paoletti, 2011; Paoletti et al., 2013). In the striatum of humans and rodents, the expression of GluN2A is moderate and that of GluN2B is high, especially in medium spiny projection neurons (MSNs), which represent 95% of the total striatal population. GluN2C is scarce and GluN2D is expressed in some classes of interneurons, in particular the large aspiny cholinergic interneurons (Bloomfield et al., 2007; Landwehrmeyer et al., 1995; Standaert et al., 1996). Although cholinergic interneurons represent <2% of the total neuronal population of the striatum, these interneurons are key players in the physiology of the striatum, and probably also in the pathophysiology of PD, due to their extensive axonal branching (Goldberg et al., 2012; Pisani et al., 2007). In the striatum of animal models of PD the expression of GluN2A is unaffected but that of GluN2B is decreased (Dunah et al., 2000; Landwehrmeyer et al., 1995; Paille et al., 2010; Standaert et al., 1999). Although GluN2B is an attractive drug target for therapeutic intervention in PD (Loftis and Janowsky, 2003), the physiological implication of the decreased expression of this subunit in the
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dopamine-depleted striatum has not been clearly established. In addition, the properties of GluN2D-containing NMDARs have not been investigated, and it is not known whether the expression and functions of these receptors are altered in experimental Parkinsonism. Therefore, in the present study, we have examined whether GluN2B- and GluN2D-containing NMDARs are functional at glutamatergic synapses in the striatum of control mice and whether these receptors are dysfunctional in the 6-hydroxydopamine (6OHDA)-lesioned mouse model of PD. Our results identify celltype specific dysfunctions of GluN2B- and GluN2D-containing NMDARs. In particular, we demonstrate a switch between GluN2B and GluN2D in MSNs and a loss of GluN2D in cholinergic interneurons in the dopamine-depleted striatum.
2. Materials and methods
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2.3. Data analysis and statistical methods Data were acquired and analyzed with the pClamp 9 or pClamp 10 software (Axon Instruments, Foster City CA, USA). Numerical values are shown as means ± SEM, with n indicating the number of neurons tested. For NMDAR-EPSCs, data are expressed as percent of the baseline response measured for each neuron during the 5 min preceding the start of perfusion with a compound. Statistical significance of the results was assessed by using the two-tailed Student's t-test for paired and unpaired observations or one way ANOVA followed by Bonferroni's multiple comparison test.
2.4. Chemicals Chemicals and drugs were purchased from SigmaeAldrich (Stockholm, Sweden), Tocris Bioscience (Bristol, UK) and AbcamBiochemicals (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 mM): DL-AP5 (50), bicuculline methiodide (10), CNQX disodium (10), NMDA (25), cis-PPDA (0.5), Ro 25-6981 hydrochloride (0.6 and 2), TCN201 (10), tetrodotoxin citrate (0.5) and UBP141 (6e10).
2.1. 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 (Stock€rso €ksetiska na €mnd). Experiments were performed using holms norra djurfo methods previously described (Chergui, 2011). We used male C57BL/6 mice aged 4e9 weeks (Harlan Laboratories, The Netherlands). Mice were maintained on a 12:12 h light/dark cycle and had free access to food and water. Control mice did not undergo surgery prior to electrophysiological experiments. Another group of mice underwent unilateral stereotaxic injection of the toxin 6-OHDA. These mice were anesthetized with intraperitoneal (i.p.) injection of 80 mg/kg ketamine and 5 mg/kg xylazine, pretreated with 25 mg/kg desipramine (i.p.) and 5 mg/kg pargyline (i.p.), placed in a stereotaxic frame, and injected, over 2 min, with 3 mg 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 1e3 weeks following surgery. This time frame was chosen to allow maximal dopamine depletion, to avoid compensatory adaptations which occur during the first week following injection of 6-OHDA and also to avoid neurochemical changes in the intact striatum which have been shown to occur a few weeks after lesioning dopaminergic pathways (Labandeira-Garcia et al., 1996; Schlachetzki et al., 2014; Schwarting and Huston, 1996a, b). The brains of control and lesioned mice were rapidly removed and coronal brain slices (400 mm thick) containing the striatum were prepared with a microslicer (VT 1000S, Leica Microsystem, Heppenheim, Germany). We used the intact and the dopamine-depleted striatum from the same lesioned mouse. Slices were incubated, for at least 1 h, at 32 C in oxygenated (95% O2 þ 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) mounted on an upright microscope (Olympus, Solna, Sweden) and were continuously perfused with oxygenated aCSF at 28 C.
2.2. Electrophysiology Whole cell voltage-clamp recordings of medium-sized neurons and cholinergic interneurons in the dorsal striatum were made with the help of infrared-differential interference contrast video microscopy. Striatal neurons were identified by their morphological and electrophysiological properties which include, for cholinergic interneurons, 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: either 140 CsCl, 2 MgCl2, 1 CaCl2, 10 HEPES, 10 EGTA, 2 MgATP, 0.3 Na3GTP, pH adjusted to 7.3 with CsOH, for synaptic current recordings; 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, for current-clamp and whole-cell NMDA-evoked current recordings. Whole-cell membrane currents and potentials were recorded with an Axopatch 200B (Axon Instruments, Foster City CA, USA). Errors due to liquid junction potential were not corrected. Excitatory post synaptic currents (EPSCs) were evoked every 15 sec by electrical stimulation of the slice using a patch electrode filled with aCSF positioned on the slice surface in the vicinity of the recorded neuron. NMDAR-EPSCs were recorded in the presence of CNQX and bicuculline methiodide to inhibit AMPAREPSCs and GABAA inhibitory post synaptic currents, respectively. Whole-cell currents evoked by bath application of NMDA were recorded in the presence of CNQX, bicuculline methiodide and tetrodotoxin. Stimulation-evoked NMDAR-EPSCs were examined to analyze synaptic NMDARs, while NMDA-currents evoked by bath application of NMDA were studied to examine both synaptic and extrasynaptic NMDARs.
2.5. Western blotting Western blots were performed to confirm loss of tyrosine hydroxylase (TH) following 6-OHDA lesioning and to examine the levels of GluN2B and GluN2D 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 (SDS) and boiled for 10 min. Protein concentration was determined in each sample with a bicinchoninic acid protein assay (BCA-kit, Pierce, Rockford, US). Equal amounts of protein (30 or 60 mg) were re-suspended in sample buffer and separated by SDSepolyacrylamide gel electrophoresis using a 10% running gel and transferred to an Immobilon-P (Polyvinylidene Difluoride) transfer membrane (SigmaeAldrich, Stockholm, Sweden). The membranes were incubated for 1 h at room temperature with 5% (w/v) dry milk in TBS-Tween 20. Immunoblotting was carried out with antibodies against total GluN2B (Calbiochem, Merck, Darmstadt, Germany), GluN2D (SigmaeAldrich, SAB4501307; and AbcamBiochemicals, ab35448, Cambridge, UK), TH (Millipore, Billerica, USA) and b-actin (SigmaeAldrich) in 5% dry milk dissolved in TBS-Tween 20. The membranes were washed three times with TBS-Tween 20 and incubated with secondary horseradish peroxidase-linked Anti-Rabbit IgG (H þ L) (Thermo Scientific; 1:6000 dilution) for 1 h at room temperature. At the end of the incubation, membranes were washed six times with TBSTween 20 and immunoreactive bands were detected by enhanced chemiluminescence (Perkin Elmer, Massachusetts, US). The scanned blots were quantified with the NIH Image 1.63 software. The levels of protein were normalized for the value of b-actin. Data were analyzed with the ManneWhitney test to evaluate statistical differences between intact and lesioned striatum.
3. Results 3.1. Altered current/voltage relationship in medium spiny projection neurons We performed whole-cell patch clamp recordings of MSNs in brain slices to determine the contribution of GluN2B and GluN2D to synaptic NMDARs. We first examined current/voltage (I/V) relationships of NMDAR-mediated excitatory post synaptic currents (NMDAR-EPSCs) evoked in MSNs. In control mice and in the intact striatum of 6-OHDA lesioned mice, I/V curves showed a rectification index (RI: ratio between EPSC amplitude at 80 mV and that at 40 mV) < 0.9, indicative of Mg2þ block at hyperpolarized membrane potentials (Fig. 1A, C). Of the 38 MSNs examined in the dopamine-depleted striatum, 15 displayed a RI < 0.9, and 23 had a RI > 0.9 (Fig. 1B, C). At depolarized membrane potentials (þ60 mV), all MSNs in the dopamine-depleted striatum displayed reduced NMDAR-EPSC amplitude as compared to MSNs in intact striatum and in control mice (Fig. 1A, B, D). These altered I/V relationships suggest a change in the subunit composition of synaptic NMDARs in the dopamine-depleted striatum, e.g. incorporation of a subunit with low sensitivity for voltage-dependent Mg2þ blockade and low conductance, such as GluN2D (Cull-Candy et al., 2001; Misra et al., 2000; Monyer et al., 1994; Paoletti, 2011; Paoletti et al., 2013). We tested this possibility by determining the contribution of GluN2D and of GluN2B to NMDAR-EPSCs in MSNs.
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Fig. 1. Change in current/voltage relationships in medium spiny projection neurons (A, B) Current/voltage (I/V) relationships of NMDAR-EPSC amplitude normalized by 40 mV in MSNs from control mice (n ¼ 9, A) and from the intact (n ¼ 13, A) and dopamine-depleted (n ¼ 15 for RI < 0.9 and n ¼ 23 for RI > 0.9, B) striatum of 6-OHDA-lesioned mice. RI: Rectification index (ratio between the peak amplitude of the NMDAR-EPSC evoked at 80 mV and that evoked at 40 mV). Example traces of NMDAR-EPSCs recorded in a MSN from a control mouse (A) and in a MSN from a dopamine-depleted striatum (with a RI > 0.9, B) at holding potentials of 80 mV and 40 mV. Scale bars represent 50 pA and 40 ms for both neurons. (C, D) Distribution of the RIs (C) and distribution of the ratio between the peak amplitude of the NMDAR-EPSC evoked at þ60 mV and that evoked at 40 mV (D) in MSNs from the striatum of control mice and from the intact and dopamine-depleted striatum of 6-OHDA-lesioned mice in the same neurons as in (A, B). (n ¼ 8e23 in each group). ***p < 0.001 (compared to the other groups in graph C); *p < 0.05 (compared to control and intact in graph D).
3.2. Reorganization of GluN2B and GluN2D in medium spiny projection neurons We applied Ro 25-6981, a selective antagonist of NMDARs that contain GluN1 and GluN2B, in the perfusion solution while monitoring NMDAR-EPSCs. Ro 25-6981 (0.6 mM) depressed the NMDAREPSC amplitude in MSNs from control mice and from the intact striatum of 6-OHDA treated mice to 41.2 ± 3.0 and 47.7 ± 5.3% of baseline (n ¼ 7 and n ¼ 7, respectively, Fig. 2A, B). In the dopaminedepleted striatum, this effect was reduced in MSNs with a RI < 0.9 (62.5 ± 6.6%, n ¼ 4) and inverted in MSNs with a RI > 0.9 (116.1 ± 2.7%, n ¼ 7, Fig. 2A, B). We also found that a higher concentration of Ro 25-6981 (2 mM) failed to inhibit NMDAR-EPSCs in MSNs with a RI > 0.9 (94.1 ± 2.2%, n ¼ 4) but inhibited the NMDAREPSC in one MSN with a RI < 0.9 (to 54.8% of baseline). This observation shows that the reduced or lack of effect of Ro 25-6981 on NMDAR-EPSCs in the dopamine-depleted striatum is not due to the low concentration used (0.6 mM). These results demonstrate a diminished contribution of GluN2B to synaptic NMDARs, which was pronounced in a group of MSNs, in the dopamine-depleted striatum. We also examined whole-cell currents activated by bath applied NMDA, which stimulates both synaptic and extrasynaptic NMDARs. We found that these currents have a significantly smaller amplitude in MSNs from the dopamine-depleted striatum (79.2 ± 5.2 pA,
n ¼ 34) as compared to the intact striatum (120.2 ± 12.5 pA, n ¼ 40, p < 0.01, Fig. 2C), suggesting an overall reduced NMDAR function in these neurons, and/or a switch to low conductance-channel. As seen with synaptic NMDARs, Ro 25-6981 inhibited NMDA-evoked whole-cell currents in MSNs from the intact striatum but did not affect NMDA-evoked currents in the dopamine-depleted striatum (Fig. 2D). This result suggests that extrasynaptic NMDARs also lose GluN2B-containing NMDARs. We then examined the effect of the GluN2D-prefering antagonist, UBP141. This antagonist did not affect NMDAR-EPSC amplitude in MSNs from control (n ¼ 8) and intact (n ¼ 7) striatum. However, in the dopamine-depleted striatum, UBP141 depressed the NMDAR-EPSC amplitude to 73.9 ± 6.3% of baseline in MSNs with a RI < 0.9 (n ¼ 7, p > 0.05) and to 51.2 ± 4.0% of baseline in MSNs with a RI > 0.9 (n ¼ 5, p < 0.05, Fig. 3). A similar observation was made with a second GluN2D-prefering antagonist, PPDA (0.5 mM), which inhibited NMDAR-EPSCs to 44.9 ± 2.0% of baseline in MSNs with a RI > 0.9 (n ¼ 4) and did not block NMDAR-EPSCs in MSNs from control mice (% of baseline: 87.7 ± 13.2, n ¼ 4). Thus, GluN2D is absent at glutamatergic synapses onto MSNs in control and intact striatum, but contributes to NMDAR-EPSCs in MSNs with a RI > 0.9 and to some extent, although not significantly, in MSNs with a RI < 0.9 in the dopamine-depleted striatum. GluN2D was shown to be functional at glutamatergic synapses onto hippocampal interneurons (Harney and Anwyl, 2012) and was
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Fig. 2. Reduced GluN2B in medium spiny projection neurons (A) Graphs show the time course of the effect of the GluN1/GluN2B antagonist Ro 25-6981 (0.6 mM), applied at the time indicated by the black bar, on the amplitude of the NMDAR-EPSC expressed as % of baseline (mean ± SEM) in MSNs from intact (left; n ¼ 7) and dopamine-depleted (right; 6OHDA; n ¼ 7 MSNs with an RI > 0.9) striatum. Records of NMDAR-EPSCs from two different MSNs at þ40 mV, before and during perfusion with Ro 25-6981, are shown above the graphs. Scale bars represent 50 pA and 40 ms. (B) Average magnitude of the effect of Ro 25-6981 on NMDAR-EPSC amplitude in control (n ¼ 7), intact (n ¼ 7) and dopaminedepleted (n ¼ 4 and 7) striatum. #p < 0.05 compared to control and intact; ***p < 0.001 compared to the other three groups in Ro 25-6981. (C) Average amplitude of wholecell currents, evoked by NMDA (25 mM), in MSNs from intact (n ¼ 40) and dopamine-depleted (n ¼ 34) striatum. **p < 0.01. (D) Effect of Ro 25-6981 on NMDA-evoked wholecell currents in MSNs from intact (n ¼ 5) and dopamine-depleted (n ¼ 4) striatum. Currents in Ro 25-6981 are expressed as percentage of control NMDA-evoked current in the same neurons. *p < 0.05.
previously shown to have an extrasynaptic location in principal neurons in the cerebellum and hippocampus (Brickley et al., 2003; Harney et al., 2008; Kohr, 2006; Misra et al., 2000). As shown in a recent study (Feng et al., 2014), we found that GluN2D-containing NMDARs contributed to whole-cell NMDA-evoked currents in MSNs from the dopamine-depleted striatum, but not from the intact striatum (not shown). These results demonstrate a switch between GluN2B-containing and GluN2D-containing NMDARs at glutamatergic synapses onto MSNs, and at extrasynaptic sites, following lesion of midbrain dopaminergic neurons. 3.3. Downregulation of GluN2B and upregulation of GluN2D in the dopamine-depleted striatum To determine whether the altered subunit composition of NMDARs was associated with a change in the protein levels of GluN2B and GluN2D in the dopamine-depleted striatum, we processed the slices used in electrophysiological experiments for Western blotting. The levels of tyrosine hydroxylase (TH), the rate limiting enzyme in the synthesis of dopamine, were dramatically reduced in the samples examined (to 14.6 ± 2.6%, n ¼ 37, p < 0.001, Fig. 4A), as compared with the intact side of the same mice,
indicating loss of dopaminergic terminals in the striatum. As shown previously (Dunah et al., 2000; Paille et al., 2010) the levels of GluN2B were decreased (to 67.3 ± 9.5%, n ¼ 37, p < 0.01, Fig. 4C) in the dopamine-depleted hemisphere. The levels of GluN2D were however increased in the dopamine-depleted hemisphere as compared to the intact hemisphere (to 161.8 ± 17.3%, n ¼ 37, p < 0.001, Fig. 4D, Sigma antibody; and to 150.5 ± 16.5%, n ¼ 33, p < 0.01, AbcamBiochemicals antibody). These results suggest that in MSNs from the dopamine-depleted striatum the decreased contribution of GluN2B-containing NMDARs to NMDAR-EPSCs and whole-cell NMDA-evoked currents is associated with a reduced protein level of GluN2B. In addition, the increased level of GluN2D might contribute to the increased function of GluN2D-containing NMDARs in MSNs. 3.4. Reduced function of GluN2D in cholinergic interneurons We then examined whether the changes in the subunit composition of NMDARs were restricted to MSNs and we performed similar experiments in cholinergic interneurons. We found that both GluN2B and GluN2D contributed to functional synaptic NMDARs in cholinergic interneurons from control and intact
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Fig. 3. Increased function of GluN2D in medium spiny neurons (A) Graphs show the time course of the effect of the GluN2D antagonist UBP141 (6e10 mM), applied at the time indicated by the black bar, on the amplitude of the NMDAR-EPSC expressed as % of baseline (mean ± SEM) in MSNs from intact striatum (left; n ¼ 7) and dopamine-depleted striatum (right; 6-OHDA; n ¼ 12 MSNs; data pooled from MSNs with an RI > 0.9 and MSNs with an RI < 0.9). Records of NMDAR-EPSCs from two different MSNs at þ40 mV, before and during perfusion with UBP141, are shown above the graphs. Scale bars represent 50 pA and 40 ms. (B) Average magnitude of the effect of UBP141 on NMDAR-EPSC amplitude in control (n ¼ 8), intact (n ¼ 7) and dopamine-depleted (n ¼ 7 and 5) striatum. *p < 0.05 compared to the other three groups in UBP141.
striatum (Fig. 5A, B). Indeed, Ro 25-6981 reduced the amplitude of the EPSC evoked at þ40 mV to 74.1 ± 8.6 and to 71.7 ± 9.1% of baseline in cholinergic interneurons from control (n ¼ 7) and intact (n ¼ 7) striatum, respectively. UBP141 reduced the amplitude of the EPSC to 72.6 ± 9.5 and to 73.5 ± 6.7% of baseline in cholinergic interneurons from control (n ¼ 8) and intact (n ¼ 8) striatum, respectively. We also found that the GluN2A-containing NMDAR antagonist TCN201 (10 mM) did not significantly affect the EPSC measured in cholinergic interneurons of the intact striatum (% of baseline: 108.0 ± 19.5, n ¼ 4). This result demonstrates that GluN2A does not contribute to functional NMDARs in these interneurons and suggests that the inhibitory effects of UBP141 on NMDAREPSCs are not due to antagonism of NMDARs containing GluN2A. In the dopamine-depleted striatum, the contribution of GluN2D to synaptic NMDARs, and to some extent that of GluN2B, was reduced or lost (Fig. 5A, B). The non-subunit selective antagonist DL-AP5 reduced to 39.4 ± 4.5% of baseline the EPSC measured in cholinergic interneurons in control, intact and dopamine-depleted striatum (n ¼ 13, not shown), demonstrating that these EPSCs were mostly, but not entirely, mediated by NMDARs. As seen with MSNs, whole-cell NMDA-evoked currents were significantly reduced in the dopamine-depleted striatum
(68.1 ± 5.7 pA, n ¼ 28) as compared to the intact striatum (92.1 ± 8.6 pA, n ¼ 32, p < 0.05, Fig. 5C). In addition, the contribution of GluN2B to whole-cell NMDA-evoked currents in these interneurons was decreased in the dopamine-depleted striatum (Fig. 5D). We also found that the contribution of GluN2D to wholecell NMDA-evoked currents was lost (not shown), as demonstrated previously (Feng et al., 2014). The observation that neither Ro 256981 nor UBP141 inhibited NMDA-evoked currents in the dopaminedepleted striatum suggests the possibility that these antagonists failed to totally block NMDARs, as described earlier (Neyton and Paoletti, 2006). Alternatively, triheteromeric receptors (containing GluN1/GluN2B/GluN2D), or receptors containing a subunit other than GluN2B and GluN2D, could contribute to functional NMDARs in cholinergic interneurons in the dopamine-depleted striatum. 4. Discussion Our study identifies novel neuroadaptations in the striatum of a mouse model of PD. We found that synaptic transmission mediated by GluN2B-containing NMDARs is impaired in MSNs of the dopamine-depleted striatum. This impairment is however balanced by an increased function of GluN2D in these neurons
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Fig. 4. Changes in the protein levels of GluN2B and GluN2D in the dopamine-depleted striatum Western blots of tyrosine hydroxylase (TH, A), the rate-limiting enzyme in the synthesis of dopamine, b-actin (B), GluN2B (C) and GluN2D (D) from intact and lesioned hemispheres of the same mice (n ¼ 37). Total protein amounts are expressed as percentage of intact hemisphere from individual animals. Blots above graphs are representative examples from intact (left) and dopamine-depleted (right) hemispheres. Protein levels are normalized by the value of b-actin. **p < 0.01, ***p < 0.001 compared with intact hemisphere.
which contributes to the maintenance of NMDAR-mediated synaptic transmission. This switch is cell-type specific, because cholinergic interneurons lose GluN2D and to some extent GluN2B in the dopamine-depleted striatum.
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Fig. 5. Reorganization of GluN2B and GluN2D in cholinergic interneurons (A) Records of EPSCs from four cholinergic interneurons held at þ40 mV, before and during perfusion with Ro 25-6981 or UBP141. Scale bars represent 50 pA and 40 ms. (B) Effect of Ro 25-6981 and UBP141 on the EPSC amplitude in control (n ¼ 7 and 8), intact (n ¼ 7 and 8) and dopamine-depleted (n ¼ 8 and 9) striatum. **p < 0.01 compared with control and intact in UBP141. (C) Average amplitude of whole-cell currents, evoked by NMDA (25 mM), in cholinergic interneurons from intact (n ¼ 32) and dopaminedepleted (n ¼ 28) striatum. *p < 0.05. (D) Effect of Ro 25-6981 on NMDA-evoked whole-cell currents in cholinergic interneurons from intact (n ¼ 8) and dopaminedepleted (n ¼ 4) striatum. Currents in Ro 25-6981 are expressed as percentage of control NMDA-evoked currents in the same neurons. *p < 0.05.
4.1. GluN2B-containing NMDARs We found that in the striatum of control mice the GluN2 subunits that compose functional NMDARs at glutamatergic synapses in MSNs include, for at least 50%, GluN2B. This agrees well with the high level of this subunit in the striatum, particularly in MSNs (Landwehrmeyer et al., 1995; Standaert et al., 1999). Our findings demonstrate that the functions of GluN2B-containing NMDARs are impaired in the dopamine-depleted striatum. Indeed, we found a decreased synaptic function of GluN2B-containing NMDARs in a subpopulation of MSNs, and to some extent in cholinergic interneurons. We found that this loss correlates with a decreased protein levels of GluN2B, in accordance with previous studies (Dunah et al., 2000; Paille et al., 2010). Future experiments will determine whether the two populations of MSNs, which display different RIs and respond differently to Ro 25-6981, belong to the direct and indirect pathway or whether these differences are due to
variable levels of dopamine depletion. At the synapse, however, GluN2B is replaced by GluN2D in MSNs, allowing maintenance of NMDAR-mediated synaptic transmission. Our findings have potential clinical implications. Indeed, antagonists acting on GluN2B-containing NMDARs have been investigated as possible therapeutic agents for treatment of PD with reduced propensity to elicit side effects as compared to broad spectrum NMDAR antagonists (Gogas, 2006; Loftis and Janowsky, 2003). Unfortunately, clinical trials with GluN2B-selective antagonists failed to provide clear benefit in PD patients (Addy et al., 2009). Taken together with our observations, it is probable that the locus of action of GluN2B-antagonists, in the behavioral improvement in animal models of PD, is not in MSNs. Alternatively, the beneficial effects of GluN2B antagonists could be related to their ability to potentiate, rather than inhibit, NMDAR-mediated responses in a subpopulation of MSNs.
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4.2. GluN2D-containing NMDARs
References
Several lines of evidence indicate that GluN2D-containing NMDARs are targeted to extrasynaptic sites and are absent from synapses in principal neurons of the hippocampus and cerebellum of healthy rodents (Brickley et al., 2003; Harney and Anwyl, 2012; Harney et al., 2008; Misra et al., 2000). These receptors are however functional at glutamatergic synapses onto hippocampal interneurons (Harney and Anwyl, 2012). The presence of functional synaptic GluN2D-containing receptors in cholinergic interneurons in the striatum, but not in MSNs, further supports the suggestion that these receptors contribute to synaptic transmission specifically in interneurons. We recently found that extrasynaptic GluN2Dcontaining NMDARs in cholinergic interneurons, and the modulatory role of these receptors, are reduced in the striatum in experimental Parkinsonism (Feng et al., 2014; Zhang et al., 2014). The present study clearly identifies a loss of synaptic e and confirms loss of extrasynaptic e function of GluN2D-containing NMDARs in cholinergic interneurons in the dopamine-depleted striatum. In contrast, the contribution of GluN2D to functional NMDARs is enhanced in MSNs. However, because the conductance of these receptors is low and because GluN2B is down-regulated, it is likely that the overall functions of NMDARs are reduced in the dopaminedepleted striatum. Together with our observation that the protein levels of GluN2D are increased in the dopamine-depleted striatum, our findings suggest that GluN2D might be expressed, albeit in low amounts, in MSNs of control mice. This hypothesis is supported by the observation that a population of MSNs expresses low levels of GluN2D in the human caudateeputamen (Kuppenbender et al., 2000). Furthermore, low levels of GluN2D are detected in the striatum of very young rats but this subunit is down-regulated, to undetectable levels, in the striatum of adult rodents (Dehorter et al., 2011; Dunah et al., 1996; Landwehrmeyer et al., 1995; Logan et al., 2007; Tong and Gibb, 2008). Nevertheless, the protein level changes identified here likely contribute to altered functions of NMDARs. The loss of GluN2D in cholinergic interneurons is likely masked by the increased level of this subunit in MSNs, which comprise around 95% of the total neuronal population in the striatum.
Addy, C., Assaid, C., Hreniuk, D., Stroh, M., Xu, Y., Herring, W.J., Ellenbogen, A., Jinnah, H.A., Kirby, L., Leibowitz, M.T., Stewart, R.M., Tarsy, D., Tetrud, J., Stoch, S.A., Gottesdiener, K., Wagner, J., 2009. Single-dose administration of MK0657, an NR2B-selective NMDA antagonist, does not result in clinically meaningful improvement in motor function in patients with moderate Parkinson's disease. J. Clin. Pharmacol. 49, 856e864. Bagetta, V., Ghiglieri, V., Sgobio, C., Calabresi, P., Picconi, B., 2010. Synaptic dysfunction in Parkinson's disease. Biochem. Soc. Trans. 38, 493e497. Bloomfield, C., O'Donnell, P., French, S.J., Totterdell, S., 2007. Cholinergic neurons of the adult rat striatum are immunoreactive for glutamatergic N-methyl-d-aspartate 2D but not N-methyl-d-aspartate 2C receptor subunits. Neuroscience 150, 639e646. Brickley, S.G., Misra, C., Mok, M.H.S., Mishina, M., Cull-Candy, S.G., 2003. NR2B and NR2D subunits coassemble in cerebellar golgi cells to form a distinct NMDA receptor subtype restricted to extrasynaptic sites. J. Neurosci. 23, 4958e4966. Chergui, K., 2011. Dopamine induces a GluN2A-dependent form of long-term depression of NMDA synaptic responses in the nucleus accumbens. Neuropharmacology 60, 975e981. Cull-Candy, S., Brickley, S., Farrant, M., 2001. NMDA receptor subunits: diversity, development and disease. Curr. Opin. Neurobiol. 11, 327e335. Dehorter, N., Michel, F.J., Marissal, T., Rotrou, Y., Matrot, B., Lopez, C., Humphries, M.D., Hammond, C., 2011. Onset of pup locomotion coincides with loss of NR2C/D-mediated cortico-striatal EPSCs and dampening of striatal network immature activity. Front. Cell. Neurosci. 5. Dunah, A.W., Wang, Y., Yasuda, R.P., Kameyama, K., Huganir, R.L., Wolfe, B.B., Standaert, D.G., 2000. Alterations in subunit expression, composition, and phosphorylation of striatal N-methyl-D-aspartate glutamate receptors in a rat 6hydroxydopamine model of Parkinson's disease. Mol. Pharmacol. 57, 342e352. Dunah, A.W., Yasuda, R.P., Wang, Y.-h., Luo, J., Davila-Garcia, M.I., Gbadegesin, M., Vicini, S., Wolfe, B.B., 1996. Regional and ontogenic expression of the NMDA receptor subunit NR2D protein in rat brain using a subunit-specific antibody. J. Neurochem. 67, 2335e2345. Feng, Z.J., Zhang, X., Chergui, K., 2014. Allosteric modulation of NMDA receptors alters neurotransmission in the striatum of a mouse model of Parkinson's disease. Exp. Neurol. 255, 154e160. Gogas, K.R., 2006. Glutamate-based therapeutic approaches: NR2B receptor antagonists. Curr. Opin. Pharmacol. 6, 68e74. Goldberg, J.A., Ding, J.B., Surmeier, D.J., 2012. Muscarinic modulation of striatal function and circuitry. In: Fryer, A.D., Christopoulos, A., Nathanson, N.M. (Eds.), Muscarinic Receptors. Springer, Berlin Heidelberg, pp. 223e241. Harney, S.C., Anwyl, R., 2012. Plasticity of NMDA receptor-mediated excitatory postsynaptic currents at perforant path inputs to dendrite-targeting interneurons. J. Physiol. 590, 3771e3786. Harney, S.C., Jane, D.E., Anwyl, R., 2008. Extrasynaptic NR2D-containing NMDARs are recruited to the synapse during LTP of NMDAR-EPSCs. J. Neurosci. 28, 11685e11694. Kawaguchi, Y., 1993. Physiological, morphological, and histochemical characterization of 3 classes of interneurons in rat neostriatum. J. Neurosci. 13, 4908e4923. Kohr, G., 2006. NMDA receptor function: subunit composition versus spatial distribution. Cell Tissue Res. 326, 439e446. Kuppenbender, K.D., Standaert, D.G., Feuerstein, T.J., Penney Jr., J.B., Young, A.B., Landwehrmeyer, G.B., 2000. Expression of NMDA receptor subunit mRNAs in neurochemically identified projection and interneurons in the human striatum. J. Comp. Neurol. 419, 407e421. Labandeira-Garcia, J.L., Rozas, G., Lopez-Martin, E., Liste, I., Guerra, M.J., 1996. Time course of striatal changes induced by 6-hydroxydopamine lesion of the nigrostriatal pathway, as studied by combined evaluation of rotational behaviour and striatal fos expression. Exp. Brain Res. 108, 69e84. Landwehrmeyer, G.B., Standaert, D.G., Testa, C.M., Penney Jr., J.B., Young, A.B., 1995. NMDA receptor subunit mRNA expression by projection neurons and interneurons in rat striatum. J. Neurosci. 15, 5297e5307. Loftis, J.M., Janowsky, A., 2003. The N-methyl-D-aspartate receptor subunit NR2B: localization, functional properties, regulation, and clinical implications. Pharmacol. Ther. 97, 55e85. Logan, S.M., Partridge, J.G., Matta, J.A., Buonanno, A., Vicini, S., 2007. Long-lasting NMDA receptor-mediated EPSCs in mouse striatal medium spiny neurons. J. Neurophysiol. 98, 2693e2704. Misra, C., Brickley, S.G., Farrant, M., Cull-Candy, S.G., 2000. Identification of subunits contributing to synaptic and extrasynaptic NMDA receptors in golgi cells of the rat cerebellum. J. Physiol. 524, 147e162. Monyer, H., Burnashev, N., Laurie, D.J., Sakmann, B., Seeburg, P.H., 1994. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12, 529e540. Neyton, J., Paoletti, P., 2006. Relating NMDA receptor function to receptor subunit composition: limitations of the pharmacological approach. J. Neurosci. 26, 1331e1333. Paille, V., Picconi, B., Bagetta, V., Ghiglieri, V., Sgobio, C., Di Filippo, M., Viscomi, M.T., Giampa, C., Fusco, F.R., Gardoni, F., Bernardi, G., Greengard, P., Di Luca, M., Calabresi, P., 2010. Distinct levels of dopamine denervation differentially alter striatal synaptic plasticity and NMDA receptor subunit composition. J. Neurosci. 30, 14182e14193. Paoletti, P., 2011. Molecular basis of NMDA receptor functional diversity. Eur. J. Neurosci. 33, 1351e1365.
5. Conclusions Our study provides evidence for cell-type specific alterations in the functions and subunit composition of NMDARs in the striatum of the 6-OHDA lesioned mouse model of PD. Thus, one of the consequences of dopamine depletion includes a switch, in various amounts, between GluN2B and GluN2D in MSNs NMDARs and a reduced function of GluN2D-containing NMDARs in cholinergic interneurons. Our results propose GluN2D as a potential novel target for the development of antiparkinsonian compounds. Further studies will determine whether manipulations of GluN2Dcontaining NMDARs could be of therapeutic benefit in the management of symptoms of PD.
Acknowledgments This study was supported by the Swedish Research Council (grants 2008e2636 and 2011e2770), the Loo and Hans Ostermans foundation for geriatric research, Parkinsonfonden, Stiftelse Lars €r medicinsk forskning, Hiertas Minne, Tore Nilsons Stiftelse fo €kares€ Svenska La allskapet and the Karolinska Institute. X.Z. was a recipient of a postdoctoral fellowship from the Swedish Society for Medical Research (SSMF).
X. Zhang, K. Chergui / Neuropharmacology 92 (2015) 108e115 Paoletti, P., Bellone, C., Zhou, Q., 2013. NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. Nat. Rev. Neurosci. 14, 383e400. Paxinos, G., Franklin, K.B.J., 2001. The Mouse Brain in Stereotaxic Coordinates. Academic, San Diego. Pisani, A., Bernardi, G., Ding, J., Surmeier, D.J., 2007. Re-emergence of striatal cholinergic interneurons in movement disorders. Trends Neurosci. 30, 545e553. Schlachetzki, J.C.M., Marxreiter, F., Regensburger, M., Kulinich, A., Winner, B., Winkler, J., 2014. Increased tyrosine hydroxylase expression accompanied by glial changes within the non-lesioned hemisphere in the 6-hydroxydopamine model of Parkinson's disease. Restor. Neurol. Neurosci. 32, 447e462. Schwarting, R.K.W., Huston, J.P., 1996a. The unilateral 6-hydroxydopamine lesion model in behavioral brain research. Analysis of functional deficits, recovery and treatments. Prog. Neurobiol. 50, 275e331. Schwarting, R.K.W., Huston, J.P., 1996b. Unilateral 6-hydroxydopamine lesions of meso-striatal dopamine neurons and their physiological sequelae. Prog. Neurobiol. 49, 215e266.
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Standaert, D.G., Friberg, I.K., Landwehrmeyer, G.B., Young, A.B., Penney, J.B., 1999. Expression of NMDA glutamate receptor subunit mRNAs in neurochemically identified projection and interneurons in the striatum of the rat. Mol. Brain Res. 64, 11e23. Standaert, D.G., Landwehrmeyer, G.B., Kerner, J.A., Penney Jr., J.B., Young, A.B., 1996. Expression of NMDAR2D glutamate receptor subunit mRNA in neurochemically identified interneurons in the rat neostriatum, neocortex and hippocampus. Brain Res. Mol. Brain Res. 42, 89e102. Tong, H., Gibb, A.J., 2008. Dopamine D1 receptor inhibition of NMDA receptor currents mediated by tyrosine kinase-dependent receptor trafficking in neonatal rat striatum. J. Physiol. 586, 4693e4707. Zhang, X., Feng, Z.J., Chergui, K., 2014. GluN2D-containing NMDA receptors inhibit neurotransmission in the mouse striatum through a cholinergic mechanism: implication for Parkinson's disease. J. Neurochem. 129, 581e590.
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Dopamine depletion of the striatum causes a cell-type specific reorganization of GluN2B- and GluN2D-containing NMDA receptors Xiaoqun Zhang, Karima Chergui* €g 8, 171 77 Stockholm, Sweden The Karolinska Institute, Department of Physiology and Pharmacology, Section of Molecular Neurophysiology, Von Eulers Va
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 August 2014 Received in revised form 9 January 2015 Accepted 10 January 2015 Available online 23 January 2015
The GluN2B subunit of NMDA receptors (NMDARs) is an attractive drug target for therapeutic intervention in Parkinson's disease (PD). We have used whole-cell patch clamp recordings in brain slices to examine the contribution of GluN2B and GluN2D to functional NMDARs in the striatum of the unilateral 6-hydroxydopamine-lesioned mouse model of PD. We found that current/voltage relationships of NMDAR-mediated excitatory post synaptic currents were altered in a population of medium spiny projection neurons (MSNs) in the dopamine-depleted striatum. Using antagonists for GluN2B- and GluN2D-containing NMDARs, we found that GluN2B contributes to functional NMDARs in MSNs in the intact striatum and in the striatum of control mice. The function of GluN2B-containing NMDARs is however reduced in MSNs from the dopamine-depleted striatum. GluN2D is absent in MSNs from intact striatum and from control mice, but the contribution of this subunit to functional NMDARs is increased in the dopamine-depleted striatum. These changes in the subunit composition of NMDARs are associated with a decreased protein level of GluN2B and an increased level of GluN2D in the dopamine-depleted striatum. In cholinergic interneurons from the intact striatum and control mice, both GluN2B and GluN2D contribute to functional NMDARs. The functions of GluN2D, and to some extent GluN2B, are reduced in the dopamine-depleted striatum. Our findings demonstrate a cell-type specific reorganization of GluN2B and GluN2D in a mouse model of PD and suggest GluN2D as a potential target for the management of the disease. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Parkinson's disease NMDA receptor GluN2B GluN2D Striatum 6-Hydroxydopamine lesion
1. Introduction Parkinson's disease (PD) is associated with the loss of dopaminergic neurons in the substantia nigra pars compacta and a resultant dramatic reduction in dopamine contents in the striatum, a major recipient of dopaminergic innervation. The glutamatergic neurotransmitter system is also affected in the dopamine-depleted striatum (Bagetta et al., 2010). Broad spectrum NMDAR antagonists alleviate some of the motor symptoms of PD but these beneficial effects are accompanied by unwanted side effects. Because physiological and pathophysiological processes involving NMDARs are dependent on the subunit composition of these receptors, subunitspecific antagonists have been investigated as possible therapeutic agents for treatment of PD with reduced propensity to elicit side effects. NMDARs are heterotetrameric assemblies of two GluN1
* Corresponding author. Tel.: þ46 8 524 86882. E-mail addresses: [email protected] (X. Zhang), (K. Chergui). http://dx.doi.org/10.1016/j.neuropharm.2015.01.007 0028-3908/© 2015 Elsevier Ltd. All rights reserved.
[email protected]
subunits and other GluN2 (AeD) and GluN3 (A, B) subunits (Kohr, 2006; Paoletti, 2011; Paoletti et al., 2013). In the striatum of humans and rodents, the expression of GluN2A is moderate and that of GluN2B is high, especially in medium spiny projection neurons (MSNs), which represent 95% of the total striatal population. GluN2C is scarce and GluN2D is expressed in some classes of interneurons, in particular the large aspiny cholinergic interneurons (Bloomfield et al., 2007; Landwehrmeyer et al., 1995; Standaert et al., 1996). Although cholinergic interneurons represent <2% of the total neuronal population of the striatum, these interneurons are key players in the physiology of the striatum, and probably also in the pathophysiology of PD, due to their extensive axonal branching (Goldberg et al., 2012; Pisani et al., 2007). In the striatum of animal models of PD the expression of GluN2A is unaffected but that of GluN2B is decreased (Dunah et al., 2000; Landwehrmeyer et al., 1995; Paille et al., 2010; Standaert et al., 1999). Although GluN2B is an attractive drug target for therapeutic intervention in PD (Loftis and Janowsky, 2003), the physiological implication of the decreased expression of this subunit in the
X. Zhang, K. Chergui / Neuropharmacology 92 (2015) 108e115
dopamine-depleted striatum has not been clearly established. In addition, the properties of GluN2D-containing NMDARs have not been investigated, and it is not known whether the expression and functions of these receptors are altered in experimental Parkinsonism. Therefore, in the present study, we have examined whether GluN2B- and GluN2D-containing NMDARs are functional at glutamatergic synapses in the striatum of control mice and whether these receptors are dysfunctional in the 6-hydroxydopamine (6OHDA)-lesioned mouse model of PD. Our results identify celltype specific dysfunctions of GluN2B- and GluN2D-containing NMDARs. In particular, we demonstrate a switch between GluN2B and GluN2D in MSNs and a loss of GluN2D in cholinergic interneurons in the dopamine-depleted striatum.
2. Materials and methods
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2.3. Data analysis and statistical methods Data were acquired and analyzed with the pClamp 9 or pClamp 10 software (Axon Instruments, Foster City CA, USA). Numerical values are shown as means ± SEM, with n indicating the number of neurons tested. For NMDAR-EPSCs, data are expressed as percent of the baseline response measured for each neuron during the 5 min preceding the start of perfusion with a compound. Statistical significance of the results was assessed by using the two-tailed Student's t-test for paired and unpaired observations or one way ANOVA followed by Bonferroni's multiple comparison test.
2.4. Chemicals Chemicals and drugs were purchased from SigmaeAldrich (Stockholm, Sweden), Tocris Bioscience (Bristol, UK) and AbcamBiochemicals (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 mM): DL-AP5 (50), bicuculline methiodide (10), CNQX disodium (10), NMDA (25), cis-PPDA (0.5), Ro 25-6981 hydrochloride (0.6 and 2), TCN201 (10), tetrodotoxin citrate (0.5) and UBP141 (6e10).
2.1. 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 (Stock€rso €ksetiska na €mnd). Experiments were performed using holms norra djurfo methods previously described (Chergui, 2011). We used male C57BL/6 mice aged 4e9 weeks (Harlan Laboratories, The Netherlands). Mice were maintained on a 12:12 h light/dark cycle and had free access to food and water. Control mice did not undergo surgery prior to electrophysiological experiments. Another group of mice underwent unilateral stereotaxic injection of the toxin 6-OHDA. These mice were anesthetized with intraperitoneal (i.p.) injection of 80 mg/kg ketamine and 5 mg/kg xylazine, pretreated with 25 mg/kg desipramine (i.p.) and 5 mg/kg pargyline (i.p.), placed in a stereotaxic frame, and injected, over 2 min, with 3 mg 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 1e3 weeks following surgery. This time frame was chosen to allow maximal dopamine depletion, to avoid compensatory adaptations which occur during the first week following injection of 6-OHDA and also to avoid neurochemical changes in the intact striatum which have been shown to occur a few weeks after lesioning dopaminergic pathways (Labandeira-Garcia et al., 1996; Schlachetzki et al., 2014; Schwarting and Huston, 1996a, b). The brains of control and lesioned mice were rapidly removed and coronal brain slices (400 mm thick) containing the striatum were prepared with a microslicer (VT 1000S, Leica Microsystem, Heppenheim, Germany). We used the intact and the dopamine-depleted striatum from the same lesioned mouse. Slices were incubated, for at least 1 h, at 32 C in oxygenated (95% O2 þ 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) mounted on an upright microscope (Olympus, Solna, Sweden) and were continuously perfused with oxygenated aCSF at 28 C.
2.2. Electrophysiology Whole cell voltage-clamp recordings of medium-sized neurons and cholinergic interneurons in the dorsal striatum were made with the help of infrared-differential interference contrast video microscopy. Striatal neurons were identified by their morphological and electrophysiological properties which include, for cholinergic interneurons, 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: either 140 CsCl, 2 MgCl2, 1 CaCl2, 10 HEPES, 10 EGTA, 2 MgATP, 0.3 Na3GTP, pH adjusted to 7.3 with CsOH, for synaptic current recordings; 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, for current-clamp and whole-cell NMDA-evoked current recordings. Whole-cell membrane currents and potentials were recorded with an Axopatch 200B (Axon Instruments, Foster City CA, USA). Errors due to liquid junction potential were not corrected. Excitatory post synaptic currents (EPSCs) were evoked every 15 sec by electrical stimulation of the slice using a patch electrode filled with aCSF positioned on the slice surface in the vicinity of the recorded neuron. NMDAR-EPSCs were recorded in the presence of CNQX and bicuculline methiodide to inhibit AMPAREPSCs and GABAA inhibitory post synaptic currents, respectively. Whole-cell currents evoked by bath application of NMDA were recorded in the presence of CNQX, bicuculline methiodide and tetrodotoxin. Stimulation-evoked NMDAR-EPSCs were examined to analyze synaptic NMDARs, while NMDA-currents evoked by bath application of NMDA were studied to examine both synaptic and extrasynaptic NMDARs.
2.5. Western blotting Western blots were performed to confirm loss of tyrosine hydroxylase (TH) following 6-OHDA lesioning and to examine the levels of GluN2B and GluN2D 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 (SDS) and boiled for 10 min. Protein concentration was determined in each sample with a bicinchoninic acid protein assay (BCA-kit, Pierce, Rockford, US). Equal amounts of protein (30 or 60 mg) were re-suspended in sample buffer and separated by SDSepolyacrylamide gel electrophoresis using a 10% running gel and transferred to an Immobilon-P (Polyvinylidene Difluoride) transfer membrane (SigmaeAldrich, Stockholm, Sweden). The membranes were incubated for 1 h at room temperature with 5% (w/v) dry milk in TBS-Tween 20. Immunoblotting was carried out with antibodies against total GluN2B (Calbiochem, Merck, Darmstadt, Germany), GluN2D (SigmaeAldrich, SAB4501307; and AbcamBiochemicals, ab35448, Cambridge, UK), TH (Millipore, Billerica, USA) and b-actin (SigmaeAldrich) in 5% dry milk dissolved in TBS-Tween 20. The membranes were washed three times with TBS-Tween 20 and incubated with secondary horseradish peroxidase-linked Anti-Rabbit IgG (H þ L) (Thermo Scientific; 1:6000 dilution) for 1 h at room temperature. At the end of the incubation, membranes were washed six times with TBSTween 20 and immunoreactive bands were detected by enhanced chemiluminescence (Perkin Elmer, Massachusetts, US). The scanned blots were quantified with the NIH Image 1.63 software. The levels of protein were normalized for the value of b-actin. Data were analyzed with the ManneWhitney test to evaluate statistical differences between intact and lesioned striatum.
3. Results 3.1. Altered current/voltage relationship in medium spiny projection neurons We performed whole-cell patch clamp recordings of MSNs in brain slices to determine the contribution of GluN2B and GluN2D to synaptic NMDARs. We first examined current/voltage (I/V) relationships of NMDAR-mediated excitatory post synaptic currents (NMDAR-EPSCs) evoked in MSNs. In control mice and in the intact striatum of 6-OHDA lesioned mice, I/V curves showed a rectification index (RI: ratio between EPSC amplitude at 80 mV and that at 40 mV) < 0.9, indicative of Mg2þ block at hyperpolarized membrane potentials (Fig. 1A, C). Of the 38 MSNs examined in the dopamine-depleted striatum, 15 displayed a RI < 0.9, and 23 had a RI > 0.9 (Fig. 1B, C). At depolarized membrane potentials (þ60 mV), all MSNs in the dopamine-depleted striatum displayed reduced NMDAR-EPSC amplitude as compared to MSNs in intact striatum and in control mice (Fig. 1A, B, D). These altered I/V relationships suggest a change in the subunit composition of synaptic NMDARs in the dopamine-depleted striatum, e.g. incorporation of a subunit with low sensitivity for voltage-dependent Mg2þ blockade and low conductance, such as GluN2D (Cull-Candy et al., 2001; Misra et al., 2000; Monyer et al., 1994; Paoletti, 2011; Paoletti et al., 2013). We tested this possibility by determining the contribution of GluN2D and of GluN2B to NMDAR-EPSCs in MSNs.
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Fig. 1. Change in current/voltage relationships in medium spiny projection neurons (A, B) Current/voltage (I/V) relationships of NMDAR-EPSC amplitude normalized by 40 mV in MSNs from control mice (n ¼ 9, A) and from the intact (n ¼ 13, A) and dopamine-depleted (n ¼ 15 for RI < 0.9 and n ¼ 23 for RI > 0.9, B) striatum of 6-OHDA-lesioned mice. RI: Rectification index (ratio between the peak amplitude of the NMDAR-EPSC evoked at 80 mV and that evoked at 40 mV). Example traces of NMDAR-EPSCs recorded in a MSN from a control mouse (A) and in a MSN from a dopamine-depleted striatum (with a RI > 0.9, B) at holding potentials of 80 mV and 40 mV. Scale bars represent 50 pA and 40 ms for both neurons. (C, D) Distribution of the RIs (C) and distribution of the ratio between the peak amplitude of the NMDAR-EPSC evoked at þ60 mV and that evoked at 40 mV (D) in MSNs from the striatum of control mice and from the intact and dopamine-depleted striatum of 6-OHDA-lesioned mice in the same neurons as in (A, B). (n ¼ 8e23 in each group). ***p < 0.001 (compared to the other groups in graph C); *p < 0.05 (compared to control and intact in graph D).
3.2. Reorganization of GluN2B and GluN2D in medium spiny projection neurons We applied Ro 25-6981, a selective antagonist of NMDARs that contain GluN1 and GluN2B, in the perfusion solution while monitoring NMDAR-EPSCs. Ro 25-6981 (0.6 mM) depressed the NMDAREPSC amplitude in MSNs from control mice and from the intact striatum of 6-OHDA treated mice to 41.2 ± 3.0 and 47.7 ± 5.3% of baseline (n ¼ 7 and n ¼ 7, respectively, Fig. 2A, B). In the dopaminedepleted striatum, this effect was reduced in MSNs with a RI < 0.9 (62.5 ± 6.6%, n ¼ 4) and inverted in MSNs with a RI > 0.9 (116.1 ± 2.7%, n ¼ 7, Fig. 2A, B). We also found that a higher concentration of Ro 25-6981 (2 mM) failed to inhibit NMDAR-EPSCs in MSNs with a RI > 0.9 (94.1 ± 2.2%, n ¼ 4) but inhibited the NMDAREPSC in one MSN with a RI < 0.9 (to 54.8% of baseline). This observation shows that the reduced or lack of effect of Ro 25-6981 on NMDAR-EPSCs in the dopamine-depleted striatum is not due to the low concentration used (0.6 mM). These results demonstrate a diminished contribution of GluN2B to synaptic NMDARs, which was pronounced in a group of MSNs, in the dopamine-depleted striatum. We also examined whole-cell currents activated by bath applied NMDA, which stimulates both synaptic and extrasynaptic NMDARs. We found that these currents have a significantly smaller amplitude in MSNs from the dopamine-depleted striatum (79.2 ± 5.2 pA,
n ¼ 34) as compared to the intact striatum (120.2 ± 12.5 pA, n ¼ 40, p < 0.01, Fig. 2C), suggesting an overall reduced NMDAR function in these neurons, and/or a switch to low conductance-channel. As seen with synaptic NMDARs, Ro 25-6981 inhibited NMDA-evoked whole-cell currents in MSNs from the intact striatum but did not affect NMDA-evoked currents in the dopamine-depleted striatum (Fig. 2D). This result suggests that extrasynaptic NMDARs also lose GluN2B-containing NMDARs. We then examined the effect of the GluN2D-prefering antagonist, UBP141. This antagonist did not affect NMDAR-EPSC amplitude in MSNs from control (n ¼ 8) and intact (n ¼ 7) striatum. However, in the dopamine-depleted striatum, UBP141 depressed the NMDAR-EPSC amplitude to 73.9 ± 6.3% of baseline in MSNs with a RI < 0.9 (n ¼ 7, p > 0.05) and to 51.2 ± 4.0% of baseline in MSNs with a RI > 0.9 (n ¼ 5, p < 0.05, Fig. 3). A similar observation was made with a second GluN2D-prefering antagonist, PPDA (0.5 mM), which inhibited NMDAR-EPSCs to 44.9 ± 2.0% of baseline in MSNs with a RI > 0.9 (n ¼ 4) and did not block NMDAR-EPSCs in MSNs from control mice (% of baseline: 87.7 ± 13.2, n ¼ 4). Thus, GluN2D is absent at glutamatergic synapses onto MSNs in control and intact striatum, but contributes to NMDAR-EPSCs in MSNs with a RI > 0.9 and to some extent, although not significantly, in MSNs with a RI < 0.9 in the dopamine-depleted striatum. GluN2D was shown to be functional at glutamatergic synapses onto hippocampal interneurons (Harney and Anwyl, 2012) and was
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Fig. 2. Reduced GluN2B in medium spiny projection neurons (A) Graphs show the time course of the effect of the GluN1/GluN2B antagonist Ro 25-6981 (0.6 mM), applied at the time indicated by the black bar, on the amplitude of the NMDAR-EPSC expressed as % of baseline (mean ± SEM) in MSNs from intact (left; n ¼ 7) and dopamine-depleted (right; 6OHDA; n ¼ 7 MSNs with an RI > 0.9) striatum. Records of NMDAR-EPSCs from two different MSNs at þ40 mV, before and during perfusion with Ro 25-6981, are shown above the graphs. Scale bars represent 50 pA and 40 ms. (B) Average magnitude of the effect of Ro 25-6981 on NMDAR-EPSC amplitude in control (n ¼ 7), intact (n ¼ 7) and dopaminedepleted (n ¼ 4 and 7) striatum. #p < 0.05 compared to control and intact; ***p < 0.001 compared to the other three groups in Ro 25-6981. (C) Average amplitude of wholecell currents, evoked by NMDA (25 mM), in MSNs from intact (n ¼ 40) and dopamine-depleted (n ¼ 34) striatum. **p < 0.01. (D) Effect of Ro 25-6981 on NMDA-evoked wholecell currents in MSNs from intact (n ¼ 5) and dopamine-depleted (n ¼ 4) striatum. Currents in Ro 25-6981 are expressed as percentage of control NMDA-evoked current in the same neurons. *p < 0.05.
previously shown to have an extrasynaptic location in principal neurons in the cerebellum and hippocampus (Brickley et al., 2003; Harney et al., 2008; Kohr, 2006; Misra et al., 2000). As shown in a recent study (Feng et al., 2014), we found that GluN2D-containing NMDARs contributed to whole-cell NMDA-evoked currents in MSNs from the dopamine-depleted striatum, but not from the intact striatum (not shown). These results demonstrate a switch between GluN2B-containing and GluN2D-containing NMDARs at glutamatergic synapses onto MSNs, and at extrasynaptic sites, following lesion of midbrain dopaminergic neurons. 3.3. Downregulation of GluN2B and upregulation of GluN2D in the dopamine-depleted striatum To determine whether the altered subunit composition of NMDARs was associated with a change in the protein levels of GluN2B and GluN2D in the dopamine-depleted striatum, we processed the slices used in electrophysiological experiments for Western blotting. The levels of tyrosine hydroxylase (TH), the rate limiting enzyme in the synthesis of dopamine, were dramatically reduced in the samples examined (to 14.6 ± 2.6%, n ¼ 37, p < 0.001, Fig. 4A), as compared with the intact side of the same mice,
indicating loss of dopaminergic terminals in the striatum. As shown previously (Dunah et al., 2000; Paille et al., 2010) the levels of GluN2B were decreased (to 67.3 ± 9.5%, n ¼ 37, p < 0.01, Fig. 4C) in the dopamine-depleted hemisphere. The levels of GluN2D were however increased in the dopamine-depleted hemisphere as compared to the intact hemisphere (to 161.8 ± 17.3%, n ¼ 37, p < 0.001, Fig. 4D, Sigma antibody; and to 150.5 ± 16.5%, n ¼ 33, p < 0.01, AbcamBiochemicals antibody). These results suggest that in MSNs from the dopamine-depleted striatum the decreased contribution of GluN2B-containing NMDARs to NMDAR-EPSCs and whole-cell NMDA-evoked currents is associated with a reduced protein level of GluN2B. In addition, the increased level of GluN2D might contribute to the increased function of GluN2D-containing NMDARs in MSNs. 3.4. Reduced function of GluN2D in cholinergic interneurons We then examined whether the changes in the subunit composition of NMDARs were restricted to MSNs and we performed similar experiments in cholinergic interneurons. We found that both GluN2B and GluN2D contributed to functional synaptic NMDARs in cholinergic interneurons from control and intact
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Fig. 3. Increased function of GluN2D in medium spiny neurons (A) Graphs show the time course of the effect of the GluN2D antagonist UBP141 (6e10 mM), applied at the time indicated by the black bar, on the amplitude of the NMDAR-EPSC expressed as % of baseline (mean ± SEM) in MSNs from intact striatum (left; n ¼ 7) and dopamine-depleted striatum (right; 6-OHDA; n ¼ 12 MSNs; data pooled from MSNs with an RI > 0.9 and MSNs with an RI < 0.9). Records of NMDAR-EPSCs from two different MSNs at þ40 mV, before and during perfusion with UBP141, are shown above the graphs. Scale bars represent 50 pA and 40 ms. (B) Average magnitude of the effect of UBP141 on NMDAR-EPSC amplitude in control (n ¼ 8), intact (n ¼ 7) and dopamine-depleted (n ¼ 7 and 5) striatum. *p < 0.05 compared to the other three groups in UBP141.
striatum (Fig. 5A, B). Indeed, Ro 25-6981 reduced the amplitude of the EPSC evoked at þ40 mV to 74.1 ± 8.6 and to 71.7 ± 9.1% of baseline in cholinergic interneurons from control (n ¼ 7) and intact (n ¼ 7) striatum, respectively. UBP141 reduced the amplitude of the EPSC to 72.6 ± 9.5 and to 73.5 ± 6.7% of baseline in cholinergic interneurons from control (n ¼ 8) and intact (n ¼ 8) striatum, respectively. We also found that the GluN2A-containing NMDAR antagonist TCN201 (10 mM) did not significantly affect the EPSC measured in cholinergic interneurons of the intact striatum (% of baseline: 108.0 ± 19.5, n ¼ 4). This result demonstrates that GluN2A does not contribute to functional NMDARs in these interneurons and suggests that the inhibitory effects of UBP141 on NMDAREPSCs are not due to antagonism of NMDARs containing GluN2A. In the dopamine-depleted striatum, the contribution of GluN2D to synaptic NMDARs, and to some extent that of GluN2B, was reduced or lost (Fig. 5A, B). The non-subunit selective antagonist DL-AP5 reduced to 39.4 ± 4.5% of baseline the EPSC measured in cholinergic interneurons in control, intact and dopamine-depleted striatum (n ¼ 13, not shown), demonstrating that these EPSCs were mostly, but not entirely, mediated by NMDARs. As seen with MSNs, whole-cell NMDA-evoked currents were significantly reduced in the dopamine-depleted striatum
(68.1 ± 5.7 pA, n ¼ 28) as compared to the intact striatum (92.1 ± 8.6 pA, n ¼ 32, p < 0.05, Fig. 5C). In addition, the contribution of GluN2B to whole-cell NMDA-evoked currents in these interneurons was decreased in the dopamine-depleted striatum (Fig. 5D). We also found that the contribution of GluN2D to wholecell NMDA-evoked currents was lost (not shown), as demonstrated previously (Feng et al., 2014). The observation that neither Ro 256981 nor UBP141 inhibited NMDA-evoked currents in the dopaminedepleted striatum suggests the possibility that these antagonists failed to totally block NMDARs, as described earlier (Neyton and Paoletti, 2006). Alternatively, triheteromeric receptors (containing GluN1/GluN2B/GluN2D), or receptors containing a subunit other than GluN2B and GluN2D, could contribute to functional NMDARs in cholinergic interneurons in the dopamine-depleted striatum. 4. Discussion Our study identifies novel neuroadaptations in the striatum of a mouse model of PD. We found that synaptic transmission mediated by GluN2B-containing NMDARs is impaired in MSNs of the dopamine-depleted striatum. This impairment is however balanced by an increased function of GluN2D in these neurons
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Fig. 4. Changes in the protein levels of GluN2B and GluN2D in the dopamine-depleted striatum Western blots of tyrosine hydroxylase (TH, A), the rate-limiting enzyme in the synthesis of dopamine, b-actin (B), GluN2B (C) and GluN2D (D) from intact and lesioned hemispheres of the same mice (n ¼ 37). Total protein amounts are expressed as percentage of intact hemisphere from individual animals. Blots above graphs are representative examples from intact (left) and dopamine-depleted (right) hemispheres. Protein levels are normalized by the value of b-actin. **p < 0.01, ***p < 0.001 compared with intact hemisphere.
which contributes to the maintenance of NMDAR-mediated synaptic transmission. This switch is cell-type specific, because cholinergic interneurons lose GluN2D and to some extent GluN2B in the dopamine-depleted striatum.
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Fig. 5. Reorganization of GluN2B and GluN2D in cholinergic interneurons (A) Records of EPSCs from four cholinergic interneurons held at þ40 mV, before and during perfusion with Ro 25-6981 or UBP141. Scale bars represent 50 pA and 40 ms. (B) Effect of Ro 25-6981 and UBP141 on the EPSC amplitude in control (n ¼ 7 and 8), intact (n ¼ 7 and 8) and dopamine-depleted (n ¼ 8 and 9) striatum. **p < 0.01 compared with control and intact in UBP141. (C) Average amplitude of whole-cell currents, evoked by NMDA (25 mM), in cholinergic interneurons from intact (n ¼ 32) and dopaminedepleted (n ¼ 28) striatum. *p < 0.05. (D) Effect of Ro 25-6981 on NMDA-evoked whole-cell currents in cholinergic interneurons from intact (n ¼ 8) and dopaminedepleted (n ¼ 4) striatum. Currents in Ro 25-6981 are expressed as percentage of control NMDA-evoked currents in the same neurons. *p < 0.05.
4.1. GluN2B-containing NMDARs We found that in the striatum of control mice the GluN2 subunits that compose functional NMDARs at glutamatergic synapses in MSNs include, for at least 50%, GluN2B. This agrees well with the high level of this subunit in the striatum, particularly in MSNs (Landwehrmeyer et al., 1995; Standaert et al., 1999). Our findings demonstrate that the functions of GluN2B-containing NMDARs are impaired in the dopamine-depleted striatum. Indeed, we found a decreased synaptic function of GluN2B-containing NMDARs in a subpopulation of MSNs, and to some extent in cholinergic interneurons. We found that this loss correlates with a decreased protein levels of GluN2B, in accordance with previous studies (Dunah et al., 2000; Paille et al., 2010). Future experiments will determine whether the two populations of MSNs, which display different RIs and respond differently to Ro 25-6981, belong to the direct and indirect pathway or whether these differences are due to
variable levels of dopamine depletion. At the synapse, however, GluN2B is replaced by GluN2D in MSNs, allowing maintenance of NMDAR-mediated synaptic transmission. Our findings have potential clinical implications. Indeed, antagonists acting on GluN2B-containing NMDARs have been investigated as possible therapeutic agents for treatment of PD with reduced propensity to elicit side effects as compared to broad spectrum NMDAR antagonists (Gogas, 2006; Loftis and Janowsky, 2003). Unfortunately, clinical trials with GluN2B-selective antagonists failed to provide clear benefit in PD patients (Addy et al., 2009). Taken together with our observations, it is probable that the locus of action of GluN2B-antagonists, in the behavioral improvement in animal models of PD, is not in MSNs. Alternatively, the beneficial effects of GluN2B antagonists could be related to their ability to potentiate, rather than inhibit, NMDAR-mediated responses in a subpopulation of MSNs.
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4.2. GluN2D-containing NMDARs
References
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5. Conclusions Our study provides evidence for cell-type specific alterations in the functions and subunit composition of NMDARs in the striatum of the 6-OHDA lesioned mouse model of PD. Thus, one of the consequences of dopamine depletion includes a switch, in various amounts, between GluN2B and GluN2D in MSNs NMDARs and a reduced function of GluN2D-containing NMDARs in cholinergic interneurons. Our results propose GluN2D as a potential novel target for the development of antiparkinsonian compounds. Further studies will determine whether manipulations of GluN2Dcontaining NMDARs could be of therapeutic benefit in the management of symptoms of PD.
Acknowledgments This study was supported by the Swedish Research Council (grants 2008e2636 and 2011e2770), the Loo and Hans Ostermans foundation for geriatric research, Parkinsonfonden, Stiftelse Lars €r medicinsk forskning, Hiertas Minne, Tore Nilsons Stiftelse fo €kares€ Svenska La allskapet and the Karolinska Institute. X.Z. was a recipient of a postdoctoral fellowship from the Swedish Society for Medical Research (SSMF).
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