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. 2017 Dec 11;24(2):144–153. doi: 10.1111/cns.12784

CIQ, a positive allosteric modulator of GluN2C/D‐containing N‐methyl‐d‐aspartate receptors, rescues striatal synaptic plasticity deficit in a mouse model of Parkinson's disease

Mona Nouhi 1, Xiaoqun Zhang 1,2, Ning Yao 1, Karima Chergui 1,✉
PMCID: PMC6490094  PMID: 29230960

Summary

Aims

To investigate if CIQ, a positive allosteric modulator of N‐methyl‐d‐aspartate receptors (NMDARs) containing GluN2C/D subunits, rescues the loss of long‐term potentiation (LTP) and forelimb‐use asymmetry in a mouse model of Parkinson's disease (PD).

Methods

We have used electrophysiology in brain slices and the cylinder test to examine the effect of CIQ on glutamatergic synaptic transmission, synaptic plasticity, and forelimb‐use in the unilateral 6‐hydroxydopamine‐lesion mouse model of PD.

Results

CIQ, applied in the perfusion solution, reversibly reduced glutamatergic synaptic transmission in the dopamine‐depleted striatum and had no effect in the dopamine‐intact striatum. LTP, a dopamine‐ and NMDAR‐dependent form of synaptic plasticity, was induced in the dopamine‐intact striatum but was lost in the dopamine‐depleted striatum. This impaired LTP was restored in the presence of CIQ applied in the perfusion solution. This treatment, however, prevented LTP induction in control slices. In brain slices from mice which received single and chronic intraperitoneal injections of CIQ, LTP was restored in the dopamine‐depleted striatum and unaffected in the dopamine‐intact striatum. Forelimb‐use asymmetry, a test which assesses deficits in paw usage in the unilateral lesion model of PD, was reversed by systemic chronic treatment with CIQ.

Conclusion

A positive allosteric modulator of GluN2C/D‐containing NMDARs rescues LTP and forelimb‐use asymmetry in a mouse model of PD. This study proposes GluN2D as a potential candidate for therapeutic intervention in PD.

Keywords: allosteric modulator, long‐term potentiation, N‐methyl‐d‐aspartate receptor, Parkinson's disease, striatum

1. INTRODUCTION

Parkinson's disease (PD) is characterized by a dramatic reduction in the content of dopamine in the striatum and a progressive degeneration of dopaminergic neurons in the substantia nigra.1 As a consequence of dopamine loss, glutamatergic neurotransmission is altered.2 Amantadine, a noncompetitive antagonist at the N‐methyl‐d‐aspartate (NMDA) type of glutamate receptor (NMDAR), is used in the treatment of particular motor symptoms as well as dyskinesia induced by levodopa treatment. Because the beneficial actions of amantadine are associated with unwanted side effects, subunit‐specific NMDAR antagonists were suggested as alternative strategies for therapeutic intervention in PD.3 NMDARs are heteromeric assemblies of at least one GluN1 subunit and other GluN2 (A‐D) and GluN3 (A, B) subunits. Functional and pharmacological properties of NMDARs are determined by the GluN2 subunits they contain.4, 5 Although GluN2B‐containing NMDAR antagonists reverse motor symptoms in animal models of PD, these compounds failed to provide clear benefit in PD patients.6, 7 Targeting the other GluN2 subunits that compose NMDARs in the basal ganglia might suggest alternative therapeutic strategies for treatment of PD.

In the striatum of adult humans and rodents, the expression of GluN2A is moderate, that of GluN2B is high, GluN2C is scarce, and GluN2D is expressed in some classes of interneurons. Medium spiny projection neurons (MSNs), which represent 95% of the total striatal population, express high levels of GluN2B and moderate levels of GluN2A.8 Large aspiny cholinergic interneurons in the striatum express moderate levels of GluN2A, GluN2B, and GluN2D.8, 9, 10 In the striatum of animal models of PD, the expression of GluN2A is unaffected but that of GluN2B is decreased.8, 11, 12, 13, 14 GluN2D is reduced in striatal cholinergic interneurons, and a switch between GluN2B and GluN2D occurs in projection neurons.15, 16, 17 These studies suggest that neuronal adaptations that occur in the dopamine‐depleted striatum involve a change in the subunit composition of NMDARs and an altered role of these receptors in NMDAR‐dependent forms of synaptic plasticity.

Long‐term changes in glutamatergic synaptic strength are potential candidates for cellular mechanisms of learning and memory.18 In the striatum, long‐term potentiation (LTP) and long‐term depression (LTD) are believed to underlie motor learning. NMDARs, in particular those containing GluN2A,19 are involved in LTP, but not LTD, induction in the striatum.20, 21, 22 Several studies have shown that LTP and LTD are lost in the striatum of animal models of PD and that aberrant synaptic plasticity occurs in models of L‐DOPA‐induced dyskinesia.2, 20, 23, 24, 25 Loss of LTP likely results from reduced content of dopamine in the striatum, but the possibility exists that an overall reduced function of NMDARs in the dopamine‐depleted striatum contributes to the inability to induce LTP, despite an upregulation of GluN2D in MSNs. Recent studies suggest a potential benefit in potentiating GluN2D‐containing NMDARs in experimental Parkinsonism. Thus, using the positive allosteric modulator of GluN2C/D‐containing NMDARs, CIQ,26 we found that dopamine release from residual axon terminals was enhanced in the partially dopamine‐depleted striatum.27 These observations suggest a potential way to rescue neurochemical deficits in early, presymptomatic stages of PD, when a proportion of dopamine neurons are still functioning, by modulating the activity of GluN2D‐containing NMDARs. Whether potentiation of GluN2D‐containing NMDARs with CIQ rescues other dopamine‐dependent deficits remains to be examined.

The aims of our study were to examine if CIQ rescues the loss of LTP in the striatum and deficits in paw usage (forelimb‐use asymmetry), in the unilateral 6‐hydroxydopamine (6‐OHDA)‐lesioned mouse model of PD. Our results demonstrate improvement of both neurophysiological and behavioral deficits by positive allosteric modulation of GluN2C/2D‐containing NMDARs with CIQ.

2. METHODS

2.1. Animals

Experiments were approved by our local ethical committee (Stockholms norra djurförsöksetiska nämnd) and were performed as described previously.15, 28 All efforts were made to minimize animal suffering. We used male C57Bl/6 mice aged 4‐9 weeks (Janvier Labs, Le Genest‐Saint‐Isle, France). Mice were maintained on a 12:12 hours light/dark cycle and had free access to food and water. Control mice did not undergo surgery prior to the experiments. Another group of mice underwent unilateral stereotaxic injection of the toxin 6‐OHDA in the substantia nigra pars compacta to produce degeneration of dopaminergic neurons and dopamine depletion of the striatum. 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 minutes, with 3 μg of 6‐OHDA in 0.01% ascorbate 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.29 Mice were allowed to recover from the surgery for 1‐3 weeks before they were used for electrophysiological and behavioral experiments. The severity of the lesion induced by 6‐OHDA was analyzed by Western blotting and immunodetection of the protein amounts of tyrosine hydroxylase (TH), the rate‐limiting enzyme in the synthesis of dopamine as described previously.15, 16, 27, 30 For the mice used in the experiments presented in Figures 1 and 2, the levels of TH in the lesioned striatum were reduced to 29.7 ± 6.5% of the levels in the intact striatum (n = 29 mice). TH levels in the striatum of mice that received i.p. injections of vehicle or CIQ (Figures 3, 4, 5) were 35.7 ± 6.6% (n = 21 mice) and 41.6 ± 7.6% (n = 25 mice) of intact striatum, respectively. For i.p. administrations, a single injection of CIQ ((3‐chlorophenyl)(6,7‐dimethoxy‐1‐((4‐methoxyphenoxy)methyl)‐3,4‐dihydroisoquinolin‐2(1H)‐yl)methanone, 20) racemate, 10 or 20 mg/kg) or vehicle was administered 45‐60 minutes before the mice were sacrificed for electrophysiological experiments or 90 minutes before behavioral testing. These time points were chosen on the basis of the pharmacokinetic of CIQ and its effect on mouse behavior described earlier.31 Indeed, selectivity assays and pharmacokinetic studies performed in C57Bl/6 mice have demonstrated that brain concentrations of CIQ peaked 30 minutes after a single i.p. injection of CIQ (20 mg/kg) and remained elevated for at least 3 hours following the injection.31 The half‐life of CIQ was 81 minutes after i.p. injection at 20 mg/kg. 31 For chronic treatments, CIQ or vehicle was administered everyday for seven consecutive days, and, on the seventh day, they were sacrificed for brain slice electrophysiological experiments 45‐60 minutes after the seventh injection.

Figure 1.

Figure 1

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The GluN2C/D potentiator CIQ depresses glutamatergic synaptic transmission in the dopamine‐depleted striatum through a presynaptic mechanism. (A) Time course of the effect of CIQ (20 μmol/L), applied at the time indicated by the black horizontal bar, on the amplitude of the field excitatory postsynaptic potentials/population spikes (fEPSP/PSs) in the intact striatum (open circles) and dopamine‐depleted striatum (filled circles). (B) Average effect of CIQ applied alone on the amplitude of the fEPSP/PS in the intact striatum (n = 8) and dopamine‐depleted striatum (n = 11) and in the presence of a GluN2D‐containing NMDAR antagonist UBP141 (n = 9) in the dopamine‐depleted striatum. **P < 0.01 compared with baseline (Student's t test); #P < 0.05 (ANOVA). (C) Time course of the effect of CIQ on the paired‐pulse ratio in the intact striatum (n = 8) and dopamine‐depleted striatum (n = 9). (D) Representative records of fEPSP/PSs, obtained from an intact slice and a lesioned slice during a paired‐pulse stimulation protocol, at the time points indicated in (A) and (C), ie, before (1) and during (2) bath application of CIQ

Figure 2.

Figure 2

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CIQ applied in the perfusion solution rescues long‐term potentiation (LTP) in the dopamine‐depleted striatum. (A) High‐frequency stimulation (HFS), applied at the time indicated by the arrow, induces LTP of the field excitatory postsynaptic potentials/population spikes (fEPSP/PSs) in the intact striatum (open circles) but not in the 6‐OHDA‐lesioned striatum (filled circles). Representative fEPSP/PS traces recorded before (1) and after (2) HFS are illustrated on the right of the graph. (B) LTP is induced in the dopamine‐depleted striatum when CIQ (20 μmol/L) is applied before and during HFS. (C) Magnitude of LTP in the intact striatum (white bar, n = 10) and in the dopamine‐depleted striatum (black bars) in control slices (n = 10) and in the presence of CIQ applied during the entire recording session (CIQ, n = 10) or only before and during HFS [CIQ during HFS, n = 8, as shown in (B)]. *P < 0.05, ***P < 0.001 compared with baseline (Student's t test); #P < 0.05 (ANOVA)

Figure 3.

Figure 3

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CIQ applied in the perfusion solution impairs long‐term potentiation (LTP) in the striatum of control mice. In control mice, high‐frequency stimulation induces LTP of the field excitatory postsynaptic potentials/population spikes in the striatum (A, n = 11) but not in the presence of CIQ applied in the perfusion solution at the time indicated by the horizontal bar (B, n = 8)

Figure 4.

Figure 4

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A single intraperitoneal injection of CIQ rescues long‐term potentiation (LTP) in the dopamine‐depleted striatum. (A) High‐frequency stimulation (HFS), applied at the time indicated by the arrows, induces LTP of the field excitatory postsynaptic potentials/population spikes in the striatum of control mice that received a single i.p. injection of vehicle (left) or CIQ (10 mg/kg, right). (B) HFS fails to induce LTP in the 6‐OHDA‐lesioned striatum of mice that received a single injection of vehicle (left). LTP is partially and fully rescued in the 6‐OHDA‐lesioned striatum of mice that received a single injection of CIQ at 10 mg/kg (middle) and 20 mg/kg (right). (C) Magnitude of LTP in the striatum of control mice (white bars) treated with vehicle (n = 10) or CIQ 10 mg/kg (n = 10), and in the dopamine‐depleted striatum (black bars) of mice treated with vehicle (n = 8), CIQ 10 mg/kg (n = 11), and CIQ 20 mg/kg (n = 6). *P < 0.05, **P < 0.01 compared with baseline (Student's t test); #P < 0.05, ##P < 0.01 (ANOVA)

Figure 5.

Figure 5

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Chronic administration of CIQ rescues long‐term potentiation (LTP) in the dopamine‐depleted striatum. (A) High‐frequency stimulation (HFS), applied at the time indicated by the arrows, induces LTP of the field excitatory postsynaptic potentials/population spikes in the intact striatum of lesioned mice that received chronic i.p. injections (daily for 7 days) of vehicle (left) or CIQ (10 mg/kg, right). (B) HFS fails to induce LTP in the 6‐OHDA‐lesioned striatum of mice that received chronic injections of vehicle (left). LTP is rescued in the 6‐OHDA‐lesioned striatum of mice that received chronic injections of CIQ (10 mg/kg, right). (C) Magnitude of LTP in the intact striatum (white bars) and in the dopamine‐depleted striatum (black bars) of mice treated with vehicle (n = 14 and 9) or CIQ (10 mg/kg, n = 11 and 7). **P < 0.01 compared with baseline (Student's t test); #P < 0.05, ##P < 0.01 (ANOVA)

2.2. Electrophysiology in brain slices

Mice underwent cervical dislocation followed by decapitation. Their brains were rapidly removed, and coronal brain slices (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 hour, at 32°C in oxygenated (95% O2 + 5% CO2) artificial cerebrospinal fluid (aCSF) containing (in mmol/L): 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 and were continuously perfused with oxygenated aCSF at 28°C. Data were acquired and analyzed with the pClamp 9 or pClamp 10 software (Axon Instruments, Foster City CA, USA). Data are expressed as percent of the baseline response measured for each slice during the 5‐10 minutes preceding the start of perfusion with CIQ or high‐frequency stimulation (HFS). Extracellular field potentials were recorded using a glass micropipette filled with aCSF positioned on the slice surface in the dorsolateral part of the striatum. These synaptic responses were evoked by stimulation pulses applied every 15 seconds to the brain slice through a concentric bipolar stimulating electrode (FHC, Bowdoinham, ME) placed near the recording electrode on the surface of the slice. 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 postsynaptic potentials/population spikes (fEPSP/PSs) evoked by increasing stimulation intensities. We previously showed that these fEPSP/PSs were mediated by glutamate acting on AMPA receptors.28 Paired‐pulse stimulations consisted of two stimulation pulses separated by a 20‐ms interval. HFS was used to induce LTP of the fEPSP/PS and consisted of 100‐Hz trains of 1‐second duration repeated 4 times with a 10‐second inter‐train 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.

2.3. Cylinder test

We examined the effect of acute and chronic i.p. injections of CIQ on forelimb‐use in the cylinder test. Mice were lesioned as described above and received an i.p. injection of apomorphine (1 mg/kg) 1 week later to confirm the efficacy of the 6‐OHDA stereotaxic injection and to select the mice that were presumably sufficiently lesioned (ie, mice that did not demonstrate rotations were not included in the behavioral tests) as described previously.32 One week later, mice were injected with either vehicle or CIQ and, 90 minutes after the first and the seventh injections, were placed in a transparent glass cylinder (13 cm diameter, 24 cm height) to examine forelimb‐use. We counted the number of times the mice touched the wall of the cylinder with their left forepaw (contralateral to the lesion) and right forepaw (ipsilateral to the lesion) during 5 minutes to evaluate forelimb‐use asymmetry.

2.4. Statistical analyses

Data are expressed as mean ± SEM with n indicating the number of slices or mice tested. Statistical significance of the results was assessed using the Student's t test for paired observations, the one‐way ANOVA followed by Dunnett's multiple comparison test, or the two‐way ANOVA followed by Bonferroni multiple comparison test. Significant levels were set at P < 0.05.

2.5. Materials

Chemicals and drugs were purchased from Sigma‐Aldrich (Stockholm, Sweden), Tocris Bioscience (Bristol, UK), and Abcam Biochemicals (Cambridge, UK). For brain slice electrophysiology, CIQ and UBP141 were prepared in stock solutions, aliquoted, and stored at −20°C. CIQ was dissolved in DMSO. On the day of the experiment, single aliquots were thawed, and 20 μmol/L (CIQ) or 6 μmol/L (UBP141) solutions were made in aCSF and applied in the perfusion solution. For i.p. injections, CIQ was first dissolved in 10% (v/v) dimethylacetamide, 40% (v/v) PEG‐400, and thereafter in 50% (v/v) of a 5% glucose (w/v) solution. Vehicle contained all chemicals except CIQ.

3. RESULTS

3.1. Effect of CIQ on glutamatergic synaptic transmission

We first examined if CIQ altered glutamatergic synaptic transmission in the striatum by measuring fEPSP/PSs in mouse brain slices, as described previously.15, 28, 33 When a stable fEPSP/PS amplitude was obtained, we applied CIQ (20 μmol/L) in the perfusion solution. In the intact striatum, CIQ did not significantly modify the amplitude of the fEPSP/PS (95.9 ± 2.8% of baseline, n = 8 slices from 5 mice, P = 0.0686. Figure 1A,B,D). However, in the dopamine‐depleted striatum, CIQ reversibly depressed the amplitude of the fEPSP/PS (68.9 ± 7.7% of baseline, n = 11 slices from 8 mice, P = 0.0031. Figure 1A,B,D). This depressant action was mediated by GluN2C/2D‐containing NMDARs because CIQ failed to reduce the fEPSP/PS amplitude in the presence of the GluN2C/2D antagonist UPB141 (6 μmol/L, 96.3 ± 7.2% of baseline, n = 9 slices from 6 mice, P = 0.5303. Figure 1B). We sought to determine if the inhibition induced by CIQ in the dopamine‐depleted striatum was mediated through a presynaptic mechanism. We applied a paired‐pulse stimulation protocol, a commonly used experimental design to investigate the ability of pharmacological compounds to modulate the release of glutamate. Both paired‐pulse facilitation and paired‐pulse depression have been observed in several brain regions and are determined by the release probability which can vary between different synapses34 and within the striatum.35, 36 In our experimental conditions, we found a paired‐pulse synaptic depression, ie, the amplitude of the second fEPSP/PS is smaller than the amplitude of the first fEPSP/PS (Figure 1C,D). We found that CIQ did not affect the ratio between the second and the first fEPSP/PS in the intact striatum but increased this ratio in the dopamine‐depleted striatum (Figure 1C,D). This change in the paired‐pulse ratio indicates that CIQ modulates the release of glutamate at synapses onto striatal neurons. Thus, CIQ applied in the perfusion solution reversibly depresses glutamatergic synaptic transmission in the dopamine‐depleted striatum, but not in the intact striatum, through presynaptic inhibition of glutamate release.

3.2. Effect of CIQ on LTP induction

We then examined the effect of CIQ on a form of NMDAR‐dependent synaptic plasticity, LTP, in the dorsolateral part of the striatum. After a stable fEPSP/PS amplitude was obtained, we applied a HFS consisting of 100‐Hz trains of 1‐second duration repeated 4 times with a 10‐second inter‐train interval. In the intact striatum, HFS induced a lasting increase in the amplitude of the fEPSP/PS (147.5 ± 14.2% baseline, n = 10 slices from 10 mice, P = 0.0161 Figure 2A,C). This LTP was significantly reduced in the lesioned striatum (109.5 ± 4.0% baseline, n = 10 slices from 9 mice, P = 0.0541, Figure 2A,C). We previously found that NMDARs are functional in the dopamine‐depleted striatum although their overall activity is reduced.16 We reasoned that HFS failed to trigger LTP because of an insufficient activation of NMDARs and that potentiating their activation might promote LTP induction. We first examined if CIQ applied in the perfusion solution during the entire recording session affected the ability of HFS to induce LTP in the dopamine‐depleted striatum. We found that LTP was induced in the presence of CIQ (20 μmol/L, 134.5 ± 5.8% baseline, n = 10 slices from 5 mice, P = 0.0002, Figure 2C). We then tested the effect of CIQ applied only before and during HFS, and we found that LTP was again rescued (147.9 ± 13.7% baseline, n = 8 slices from 7 mice, P = 0.0122, Figure 2B,C), demonstrating a requirement for potentiation of NMDARs primarily during HFS. These results demonstrate that a GluN2C/D potentiator is able to rescue LTP in the dopamine‐depleted striatum and that potentiation of NMDARs during the induction phase is required for this LTP.

We have also investigated the effect of CIQ in slices from control mice. LTP was induced in control slices (142.7 ± 11.3% baseline, P = 0.0046, n = 11 slices from 8 mice) but was impaired in slices perfused with CIQ before and during HFS (100.9 ± 4.8% baseline, P = 0.5039, n = 8 slices from 4 mice, Figure 3).

CIQ crosses the blood‐brain‐barrier and was shown to modify specific behaviors and learning when administered systemically. In particular, CIQ facilitates fear acquisition and increases fear extinction when injected either at a single dose of 10 or 20 mg/kg or with chronic administrations at 10 mg/kg. 31, 37 We first examined if a single i.p. injection of CIQ could restore LTP in the striatum of 6‐OHDA‐lesioned mice. In control mice, one injection of CIQ at 10 mg/kg did not affect the magnitude of LTP as compared to vehicle‐treated mice (vehicle: 161.8 ± 14.8% baseline, n = 10 slices from 7 mice, P = 0.0021; CIQ: 136.7 ± 13.7% baseline, n = 10 slices from 6 mice, P = 0.0348, Figure 4A,C). LTP was impaired in the dopamine‐depleted striatum of vehicle‐treated 6‐OHDA‐lesioned mice (98.6 ± 5.0% baseline, n = 8 slices from 6 mice, P = 0.6693, Figure 4B,C) but was partially rescued by a single injection of CIQ 10 mg/kg (125.5 ± 8.9% baseline, n = 11 slices from 8 mice, P = 0.0240, Figure 4B,C). We then tested if a higher dose of CIQ could fully restore impaired LTP, and we found that a single injection of CIQ at 20 mg/kg resulted in a fully recovered LTP following HFS (139.3 ± 13.00% baseline, n = 6 slices from 5 mice, P = 0.0332, Figure 4B,C). We found that the stimulus/response curves of the fEPSP/PS were similar in CIQ‐ and vehicle‐treated mice (not shown), which demonstrates that the effect of CIQ on glutamatergic synaptic transmission seen with bath application is transient.

To avoid the use of a high dose of CIQ in the following behavioral experiments, we examined whether chronic treatment with the lower dose of CIQ (10 mg/kg) produced a complete rescue of LTP. Mice received daily injections of either vehicle or CIQ for 7 days (7 injections in total). In the intact striatum, CIQ did not significantly alter the ability of HFS to induce LTP as compared to vehicle‐treated mice (vehicle: 153.0 ± 12.6% baseline, n = 14 slices from 9 mice, P = 0.0009; CIQ: 172.2 ± 14.4% baseline, n = 11 slices from 8 mice, P = 0.0005, Figure 5A,C). LTP was lost in the dopamine‐depleted striatum of vehicle‐treated mice (91.9 ± 6.9% baseline, n = 9 slices from 6 mice, P = 0.7649, Figure 5B,C) and was fully induced in CIQ‐treated mice (152.6 ± 10.8% baseline, n = 7 slices from 6 mice, P = 0.0021, Figure 5B,C).

Thus, CIQ applied in the perfusion solution or administered systemically does not affect LTP in the intact striatum but rescues impaired LTP in the dopamine‐depleted striatum.

3.3. Effect of CIQ on forelimb‐use asymmetry

We have used the cylinder test to examine if acute and chronic i.p. injections of CIQ (10 mg/kg) could rescue forelimb‐use asymmetry, a motor impairment induced by unilateral 6‐OHDA lesion, as shown by other groups in earlier studies.38, 39 After the first (acute) and the seventh (chronic) injections of vehicle or CIQ, mice were placed in a glass cylinder, and we counted the number of times they touched the wall of the cylinder with their left forepaw (contralateral to the lesion) and right forepaw (ipsilateral to the lesion). The total number of touches was similar in vehicle‐ and CIQ‐treated mice (Day 1 vehicle: 42.2 ± 5.3; Day 1 CIQ: 46.6 ± 5.4; Day 7 vehicle: 41.2 ± 4.4; Day 7 CIQ: 29.8 ± 5.4; n = 11 mice in each group, two‐way ANOVA followed by Bonferroni multiple comparisons test, P = 0.4662). However, vehicle‐treated mice demonstrated forelimb‐use asymmetry, ie, they used the forepaw contralateral to the lesion less frequently than the forepaw ipsilateral to the lesion (contralateral touches: 42.7 ± 2.9 and 41.0 ± 1.7% of total touches for acute and chronic vehicle, respectively, n = 11 mice, Figure 6). These values are within the range of those published earlier for mice that received a 6‐OHDA injection in the substantia nigra.38 Mice treated with CIQ used the left and right forepaws equally (contralateral touches: 47.9 ± 1.0 and 48.7 ± 1.0% of total touches for acute and chronic CIQ, respectively, n = 11 mice, Figure 6), and there was a significant difference between mice chronically treated with vehicle and mice chronically treated with CIQ (Two‐way ANOVA analysis followed by Bonferroni multiple comparisons test, P = 0.0051, Figure 6). These observations demonstrate that CIQ reverses forelimb‐use asymmetry in 6‐OHDA‐lesioned mice.

Figure 6.

Figure 6

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Effect of CIQ on forelimb‐use asymmetry in the cylinder test. Mice received a single (Day 1) and chronic (Day 7) i.p. injections of either vehicle (open bars, n = 11 mice) or CIQ (10 mg/kg, black bars, n = 11 mice). Data represent the number of contralateral touches as a percentage of the total touches. **P < 0.01 (ANOVA)

4. DISCUSSION

Our study demonstrates that a positive allosteric modulator of GluN2C/D‐containing NMDARs rescues impaired LTP, a potential mechanism for cellular mechanism of motor learning, in the striatum of a mouse model of PD. Our results also show that CIQ reverses a behavioral impairment, ie, forelimb‐use asymmetry, which is produced by unilateral striatal dopamine loss. Modulating the function of GluN2D‐containing NMDARs might be a novel approach to reduce deficits in experimental Parkinsonism.

CIQ is a positive allosteric modulator of GluN2C/D‐containing NMDARs, ie, it potentiates the action of glutamate, or of an agonist, on the NMDAR. In this study, glutamate released upon electrical stimulation is the agonist that activates NMDARs. Because GluN2C is absent from the striatum,10 the effects of CIQ observed in our slice experiments are likely mediated by NMDARs that contain GluN2D in the striatum. CIQ was shown to potentiate NMDA responses in neurons which express GluN2D such as neurons in the subthalamic nucleus, cholinergic interneurons in the striatum, and medium spiny neurons in the dopamine‐depleted striatum. CIQ does not potentiate NMDA responses in neurons which express mostly GluN2A and GluN2B, such as medium spiny neurons in the intact striatum and hippocampal pyramidal neurons.26, 27, 30, 40 In this study, we found that CIQ depresses glutamatergic synaptic transmission in the dopamine‐depleted striatum but not in the intact striatum. This finding is in agreement with the observed upregulation of GluN2D in the lesioned striatum.16, 30 Indeed, GluN2D is exclusively present in interneurons in the striatum of adult rodents but is upregulated and forms functional NMDARs in MSNs in the dopamine‐depleted striatum. We previously found that NMDA applied in the perfusion solution depresses glutamatergic synaptic transmission in the striatum through a presynaptic mechanism.15, 28, 33 Here, the inhibitory effect of CIQ also involves a presynaptic mechanism. Thus, it is possible that potentiation of GluN2D‐containing NMDARs by CIQ leads to the release of a retrograde signaling substance, such as adenosine, that acts on axon terminals to inhibit glutamate release, as shown previously for NMDA.28

Because the presynaptic inhibitory action of CIQ is transient, we could examine the effect of this compound on the induction of LTP. Our results show that HFS induces LTP of the fEPSP/PS in the striatum, which likely corresponds to an increased glutamatergic synaptic transmission and firing in projection neurons, as described in previous studies.20, 41 In accordance with previous findings,42 we found that LTP is lost in the dopamine‐depleted striatum. In this study, we demonstrate that this impaired LTP is rescued by CIQ applied in the perfusion solution or administered systemically. It is unlikely that this rescued LTP by CIQ is due to residual dopamine released upon HFS and potentiated by CIQ, as shown previously in the partially lesioned striatum,27 because LTP induction is highly sensitive to a reduction in striatal dopamine content and is impaired even in partially lesioned striatum.13 The contribution of cholinergic interneurons is unlikely because, in the lesioned striatum, GluN2D is reduced in these interneurons, and CIQ does not increase their firing or NMDA‐evoked currents.16, 30 However, GluN2D contributes to functional NMDARs in projection neurons in the dopamine‐depleted striatum, but not in the dopamine‐intact striatum, as a result of a switch between GluN2B and GluN2D.16 Because the conductance and calcium permeability of NMDARs made of GluN2D are lower than those made of GluN2A and GluN2B,4, 5 it is likely that the overall functions of NMDARs are reduced in the dopamine‐depleted striatum. An insufficient calcium entry through NMDARs during HFS might underlie impairment of LTP in the lesioned striatum. Therefore, a likely mechanism that might contribute to the rescue of LTP in the dopamine‐depleted striatum by CIQ involves potentiation of GluN2D‐containing NMDARs in MSNs during the induction phase. This is possible if CIQ is still present in the brain slices. A previous study showed that CIQ, administered i.p. at a dose which was higher (20 mg/kg) than the one used in our study, remains in the brain for at least 3 hours after its injection.31 It is probable that CIQ is washed out during incubation and perfusion of the slices with aCSF. In line with this possibility, we found that a single i.p. injection of CIQ does not affect LTP induction but that CIQ applied in the perfusion solution prevents LTP induction in control slices. This latter observation is likely due to the transient depressant action of CIQ on dopamine release evoked upon HFS trains that we identified in a previous study.27 It is probable that the amounts of dopamine released during HFS are reduced by CIQ applied in the perfusion solution, which prevents LTP induction. The inability of acute treatment with CIQ to affect LTP, although there was a trend for reduced LTP as compared with vehicle‐treated mice (see Figure 4), is possibly due to the short duration of the initial CIQ‐induced decrease in dopamine release or to the washout of this compound. Interestingly, chronic administration of CIQ was more effective in restoring LTP than a single injection. This finding suggests that chronic, and to a lesser extent acute, administration of CIQ induces neuronal adaptations in the dopamine‐depleted striatum that allow for LTP to be induced. These adaptations, as well as the precise mechanisms that underlie the rescue of LTP by CIQ, remain to be investigated.

In this study, we demonstrate that chronic systemic administration of CIQ rescues a form of synaptic plasticity which is impaired in models of PD. Future studies will determine whether CIQ also rescues other forms of synaptic plasticity described in the striatum, such as LTP and LTD of AMPA receptor‐mediated synaptic responses measured in projection neurons and in interneurons with whole‐cell patch clamp recordings. Interestingly, systemic administration of CIQ also rescues a behavior which is specific for unilateral lesion of the dopaminergic pathway, ie, forelimb‐use asymmetry. Potentiation of NMDARs by CIQ in striatal projection neurons, which constitute 95% of the total striatal population, could contribute to the behavioral benefits obtained with CIQ. Indeed, the contribution of GluN2D to NMDARs is increased in these neurons in the dopamine‐depleted striatum,16 and we now demonstrate that LTP in the striatum is rescued by CIQ. However, GluN2D is expressed in other basal ganglia regions, ie, the subthalamic nucleus, substantia nigra, and globus pallidus.43, 44, 45 The possible involvement of the subthalamic nucleus in the behavioral effect of CIQ is unlikely although CIQ was shown to potentiate NMDA responses in control mice.43 Indeed, NMDAR antagonists infused in this brain region reverse locomotor deficits in the hemiparkinsonian rat.46 It is unlikely that CIQ improves behavioral deficits by potentiating GluN2C‐containing NMDARs in the cerebellum. Indeed, two studies have demonstrated that locomotor activity and motor coordination measured in control mice were not affected by systemic administration of CIQ, even at the highest dose tested (20 mg/kg),31, 37 suggesting a lack of involvement of GluN2C‐containing NMDARs in the cerebellum in the behavioral effects of CIQ. The brain regions and neuronal populations that contribute to CIQ‐induced behavioral improvement remain to be identified. Future studies aimed to determine if the functions of GluN2C/2D‐containing NMDARs are altered in the basal ganglia in experimental Parkinsonism and if potentiation of these receptors by CIQ contributes to behavioral benefits are warranted.

GluN2A and GluN2B are highly expressed in the striatum. Interestingly, an article by Li et al19 found that GluN2A, but not GluN2B, is involved in the induction of LTP in the dorsolateral striatum. These findings suggest that GluN2A could be targeted for the rescue of neurophysiological and behavioral impairments in models of PD. Future studies might reveal the ability of positive allosteric modulators of GluN2A‐ and GluN2B‐containing NMDARs to rescue impairments in experimental Parkinsonism.

5. CONCLUSIONS

We recently demonstrated that CIQ increases dopamine release from residual terminals in the partially dopamine‐depleted striatum.27 In this study, the use of CIQ allowed us to confirm dysfunctions of GluN2D‐containing NMDARs in experimental Parkinsonism and to identify a novel way to pharmacologically rescue the loss of LTP and a behavioral impairment induced by unilateral dopamine depletion of the striatum. In light of our previously published observations,15, 16, 27, 30 our results strengthen our suggestion that GluN2D is a potential target for the development of antiparkinsonian compounds.

CONFLICT OF INTEREST

The authors report no conflict of interest.

ACKNOWLEDGMENTS

This study was supported by the Swedish Research Council (grant 2014‐3254), Parkinsonfonden, Stiftelse Lars Hiertas Minne, Karolinska Institute funding for PhD student, Stiftelsen för ålderssjukdomar at Karolinska Institute and Karolinska Institute research fund.

Nouhi M, Zhang X, Yao N, Chergui K. CIQ, a positive allosteric modulator of GluN2C/D‐containing N‐methyl‐d‐aspartate receptors, rescues striatal synaptic plasticity deficit in a mouse model of Parkinson's disease. CNS Neurosci Ther. 2018;24:144–153. 10.1111/cns.12784

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