NR2B Antagonist Research Articles

657. Effects of Imipramine, Scopolamine, Ketamine, NR2B and P2X7 Antagonists in a Chronic Model of Stress (CMS)-Induced Anhedonia

657. Effects of Imipramine, Scopolamine, Ketamine, NR2B and P2X7 Antagonists in a Chronic Model of Stress (CMS)-Induced Anhedonia

656. Different Effects of NR2B Antagonists on Behavior Paradigms in Mice

656. Different Effects of NR2B Antagonists on Behavior Paradigms in Mice

Soluble Epoxide Hydrolase Inhibitor and 14,15-Epoxyeicosatrienoic Acid-Facilitated Long-Term Potentiation through cAMP and CaMKII in the Hippocampus.

Epoxyeicosatrienoic acids (EETs) are derived from arachidonic acid and metabolized by soluble epoxide hydrolase (sEH). The role of EETs in synaptic function in the central nervous system is still largely unknown. We found that pharmacological inhibition of sEH to stabilize endogenous EETs and exogenous 14,15-EET significantly increased the field excitatory postsynaptic potential (fEPSP) response in the CA1 area of the hippocampus, while additionally enhancing high-frequency stimulation- (HFS-) induced long-term potentiation (LTP) and forskolin- (FSK-) induced LTP. sEH inhibitor (sEHI) N-[1-(oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy) phenyl)-urea (TPPU) and exogenous 14,15-EET increased HFS-LTP, which could be blocked by an N-methyl-D-aspartate (NMDA) receptor subunit NR2B antagonist. TPPU- or 14,15-EET-facilitated FSK-mediated LTP can be potentiated by an A1 adenosine receptor antagonist and a phosphodiesterase inhibitor, but is prevented by a cAMP-dependent protein kinase (PKA) inhibitor. sEHI and 14,15-EET upregulated the activation of extracellular signal-regulated kinases (ERKs) and Ca2+/calmodulin- (CaM-) dependent protein kinase II (CaMKII). Phosphorylation of synaptic receptors NR2B and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1 was increased by TPPU and 14,15-EET administration. These results indicated that EETs increased NMDAR- and FSK-mediated synaptic potentiation via the AC-cAMP-PKA signaling cascade and upregulated the ERKs and CaMKII, resulting in increased phosphorylation of NR2B and GluR1 in the hippocampus.

Effect of the ifenprodil administered into rostral anterior cingulate cortex on pain-related aversion in rats with bone cancer pain.

BackgroundTo assess the effect of rostral anterior cingulate cortex (rACC) administration with ifenprodil (NR2B receptor blocker) on bone cancer pain (BCP)-related aversion sentiment using the conditioned place avoidance experiments in rats.MethodsA total of 30 male Wistar rats without place preference were randomly assigned to three groups: control group (Group C, n = 10), BCP group (Group P, n = 10) and ifenprodil group (Group Ifen, n = 10). Three microliter MADB-106 cells were inoculated into right tibia bone marrow cavity in group P and Ifen, while the same dose of normal saline in group C as a control. Ifenprodil was administered into the rACC at the 14th day after inoculation in group Ifen and normal saline in group C and P. Mechanical stimulation pain thresholds of the rats’ right hind paws were measured using Von Frey stimulation method at 1 day before injection of the tumor cells and at 3, 7,10, 12 and 14 days after the injection. The pain-related aversion in rats with BCP was determined by the conditioned place avoidance (CPA) test at 14 days after injection of ifenprodil.ResultsThe mechanical stimulation pain thresholds substantially decreased in rats in groups P and Ifen from 10 days to 14 days after the incubation with the MADB-106 cells (P < 0.05). There were significant differences in pain thresholds in groups P and Ifen compared to group C at 10, 12 and 14 days after inoculation (P < 0.05). The percentage of residence time in chamber A was (30 ± 4%) in group P, which was lower than (52 ± 5%) in group C (P < 0.05). After ifenprodil treatment, the percentage time in chamber A increased to (42 ± 5%), which was higher than that in group P and still lower than that in group C (P < 0.05).ConclusionIfenprodil administered into rACC as a selective NR2B antagonist can effectively alleviate pain-related aversion sentiment in rats with BCP.

Involvement of protein phosphatases in the destabilization of methamphetamine-associated contextual memory.

Destabilization refers to a memory that becomes unstable when reactivated and is susceptible to disruption by amnestic agents. Here we delineated the cellular mechanism underlying the destabilization of drug memory. Mice were conditioned with methamphetamine (MeAM) for 3 d, and drug memory was assessed with a conditioned place preference (CPP) protocol. Anisomycin (ANI) was administered 60 min after the CPP retrieval to disrupt reconsolidation. We found that destabilization of MeAM CPP after the application of ANI was blocked by the N-methyl-d-aspartate receptor (NMDAR) antagonist MK-801 and the NR2B antagonist ifenprodil (IFN) but not by the NR2A antagonist NVP-AAM077 (NVP). In addition, decrease in the phosphorylation of GluR1 at Serine845 (p-GluR1-Ser845), decrease in spine density, and a reduction in the AMPAR/NMDAR ratio in the basolateral amygdala (BLA) were reversed after the MK-801 treatment. The effect of ANI on destabilization was prevented by the protein phosphatase 2B (calcineurin, CaN) inhibitors cyclosporine A (CsA) and FK-506 and the protein phosphatase 1 (PP1) inhibitors calyculin A (CA) and okadaic acid (OA). These results suggest that memory destabilization involves the activation of NR2B-containing NMDARs, which in turn allows the influx of Ca2+. Increased intracellular Ca2+ stimulates CaN, leading to the dephosphorylation and inactivation of inhibitor 1 and the activation of PP1. PP1 then dephosphorylates p-GluR1-Ser845 to elicit AMPA receptor (AMPAR) endocytosis and destabilization of the drug memory.

Unprecedented therapeutic potential with a combination of A2A/NR2B receptor antagonists as observed in the 6-OHDA lesioned rat model of Parkinson's disease

In Parkinson's disease, the long-term use of dopamine replacing agents is associated with the development of motor complications; therefore, there is a need for non-dopaminergic drugs. This study evaluated the potential therapeutic impact of six different NR2B and A2A receptor antagonists given either alone or in combination in unilateral 6-OHDA-lesioned rats without (monotherapy) or with (add-on therapy) the co-administration of L-Dopa: Sch-58261+ Merck 22; Sch-58261+Co-101244; Preladenant + Merck 22; Preladenant + Radiprodil; Tozadenant + Radiprodil; Istradefylline + Co-101244. Animals given monotherapy were assessed on distance traveled and rearing, whereas those given add-on therapy were assessed on contralateral rotations. Three-way mixed ANOVA were conducted to assess the main effect of each drug separately and to determine whether any interaction between two drugs was additive or synergistic. Additional post hoc analyses were conducted to compare the effect of the combination with the effect of the drugs alone. Motor activity improved significantly and was sustained for longer when the drugs were given in combination than when administered separately at the same dose. Similarly, when tested as add-on treatment to L-Dopa, the combinations resulted in higher levels of contralateral rotation in comparison to the single drugs. Of special interest, the activity observed with some combinations could not be described by a simplistic additive effect and involved more subtle synergistic pharmacological interactions. The combined administration of A2A/NR2B-receptor antagonists improved motor behaviour in 6-OHDA rats. Given the proven translatability of this model such a combination may be expected to be effective in improving motor symptoms in patients.

NMDA receptors promote neurogenesis in the neonatal rat subventricular zone following hypoxic‑ischemic injury.

Evidence suggests the involvement of N-methyl-D-aspartate receptors (NMDAR) in the regulation of neurogenesis. Functional properties of NMDAR are strongly influenced by the type of NR2 subunits in the receptor complex. NR2A- and NR2B-containing receptors are expressed in neonatal fore-brain regions, such as the subventricular zone (SVZ). The aim of the present study was to examine the effect of the protein expression of hypoxic-ischemic injury NMDAR subunits 2A and 2B in the SVZ of neonatal rats. Expression of these and other proteins of interest was performed using immunohistochemistry. The results showed that NR2A expression was decreased at 6 h after hypoxic-ischemic injury. By contrast, a significant increase in NR2B expression was observed at 24 h after hypoxic-ischemic injury, induced by the clamping of the right common carotid artery. The functional effect of NMDAR subunits on neurogenesis was also examined by quantifying Nestin and doublecortin (DCX), the microtubule-associated protein expressed only in immature neurons. In addition, the effects of selective non-competitive NMDAR antagonist MK-801 (0.5 mg/kg), NR2B antagonist Ro25-6981 (5 mg/kg), and NR2A antagonist NVP-AAM077 (5 mg/kg) administered 30 min prior to the hypoxic-ischemic injury were examined. The number of Nestin- and DCX-positive cells increased significantly 48 h after hypoxic-ischemic injury, which was reverted by the MK-801 and Ro25-6981 antagonists. Notably, NVP-AAM077 had no significant effect on the expression of Nestin and DCX. In conclusion, the results of the present study demonstrate that hypoxia-ischemia inhibited the expression of NR2A, but promoted the expression of NR2B. Furthermore, NMDAR promoted neurogenesis in the SVZ of neonatal brains.

Neuropathic Allodynia Involves Spinal Neurexin-1β-dependent Neuroligin-1/Postsynaptic Density-95/NR2B Cascade in Rats.

Neuroligin-1 (NL1) forms a complex with the presynaptic neurexin-1β (Nrx1b), regulating clustering of N-methyl-D-aspartate receptors with postsynaptic density-95 (PSD-95) to underlie learning-/memory-associated plasticity. Pain-related spinal neuroplasticity shares several common features with learning-/memory-associated plasticity. The authors thereby investigated the potential involvement of NL1-related mechanism in spinal nerve ligation (SNL)-associated allodynia. In 626 adult male Sprague-Dawley rats, the withdrawal threshold and NL1, PSD-95, phosphorylated NR2B (pNR2B) expressions, interactions, and locations in dorsal horn (L4 to L5) were compared between the sham operation and SNL groups. A recombinant Nrx1b Fc chimera (Nrx1b Fc, 10 μg, 10 μl, i.t., bolus), antisense small-interfering RNA targeting to NL1 (10 μg, 10 μl, i.t., daily for 4 days), or NR2B antagonist (Ro 25-6981; 1 μM, 10 μl, i.t., bolus) were administered to SNL animals to elucidate possible cascades involved. SNL-induced allodynia failed to affect NL1 or PSD-95 expression. However, pNR2B expression (mean ± SD from 13.1 ± 2.87 to 23.1 ± 2.52, n = 6) and coexpression of NL1-PSD-95, pNR2B-PSD-95, and NL1-total NR2B were enhanced by SNL (from 10.7 ± 2.27 to 22.2 ± 3.94, 11.5 ± 2.15 to 23.8 ± 3.32, and 8.9 ± 1.83 to 14.9 ± 2.27 at day 7, n = 6). Furthermore, neuron-localized pNR2B PSD-95-pNR2B double-labeled and NL1/PSD-95/pNR2B triple-labeled immunofluorescence in the ipsilateral dorsal horn was all prevented by Nrx1b Fc and NL1-targeted small-interfering RNA designed to block and prevent NL1 expression. Without affecting NL1-PSD-95 coupling, Ro 25-6981 decreased the SNL-induced PSD-95-pNR2B coprecipitation (from 18.7 ± 1.80 to 14.7 ± 2.36 at day 7, n = 6). SNL-induced allodynia, which is mediated by the spinal NL1/PSD-95/pNR2B cascade, can be prevented by blockade of transsynaptic Nrx1b-NL1 interactions.

Role of 2B subunits-containing NMDA receptors in arcuate nucleus in development of inflammatory pain in rats

Objective To investigate the role of 2B subunits-containing N-methyl-D-aspartate receptors (NR2B) in the arcuate nucleus in the development of inflammatory pain (IP) in rats. Methods One hundred and eight male Sprague-Dawley rats, aged 9 weeks, weighing 200-250 g, were randomly divided into 4 groups using a random number table: sham operation group (group S, n=47); group IP (n=47); dimethyl sulfoxide control group (group DMSO, n=7); selective NR2B antagonist Ro25-6981 group (group Ro25-6981, n=7). IP was induced by injecting complete Freund's adjuvant (CFA) 0.1 ml into the plantar surface of the left hindpaw.Ro25-6981 400 pmol was injected into the arcuate nucleus at 3 days after CFA injection.Seven rats in each group were selected for measurement of the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) at 1 day before CFA injection (T1) and at 2 days after CFA injection (T2), at 30 min before administration on 3rd day (T3), at 30 min after administration on 3rd day (T4), and on 5th day (T5). In S and IP groups, The rats were sacrificed at T1-3 and T5, and the arcuate nucleus of the hypothalamus was removed for determination of NR2B mRNA expression (by real-time reverse transcriptase polymerase chain reaction) and NR2B and phosphorylated NR2B (p-NR2B) expression (by Western blot). Conclusion Compared with group S, the MWT was significantly decreased, and the TWL was shortened at T2-5 in IP, DMSO and Ro25-6981 groups, and the expression of p-NR2B was up-regulated at each time point (P 0.05). Compared with group IP, the MWT was significantly increased, and the TWL was prolonged at T4 in group Ro25-6981 (P 0.05). Conclusion The activation of NR2B in the arcuate nucleus is involved in the development of IP in rats. Key words: Inflammation; Pain; Arcuate nucleus; Receptors, N-methyl-D-aspartate

Vagal afferent-dependent cholecystokinin modulation of visceral pain requires central amygdala NMDA-NR2B receptors in rats.

Cholecystokinin (CCK), a gut hormone that is released during feeding, exerts gastrointestinal effects in part through vagal pathway. It is reported to be a potential trigger for increased postprandial visceral sensitivity in healthy subjects and, especially in patients with irritable bowel syndrome. NR2B-containing N-methyl-d-aspartate (NMDA) receptors in the central amygdala (CeA) participate in pain modulation. Systemically administered CCK activates the CeA-innervating neurons. Here, we investigated whether CCK modulation of visceral sensitivity is mediated through CeA NMDA-NR2B receptors and whether this modulation involves vagal pathway. We first examined the visceromotor response (VMR) to colorectal distention (CRD) following i.p. injection of CCK octapeptide (CCK-8) in a rat model. Next, the NR2B antagonist ifenprodil and the NR2A antagonist NVP-AAM077 were microinjected into the CeA before systemic CCK injection. NR2B phosphorylation was detected by Western blot. To down-regulate NR2B gene expression, NR2B-specific small interfering RNA (siRNA) was delivered into CeA neurons by electroporation. In addition, the effects of functional deafferentation by perivagal application of capsaicin and pretreatment with the CCK1 receptor antagonist devazepide were investigated. CCK-8 increased VMR to CRD in a dose-dependent manner. This effect was blunted by intra-CeA administration of ifenprodil (but not NVP-AAM077) and was accompanied by phosphorylation of NR2B subunits in the CeA. CCK failed to increase VMR to CRD in NR2B siRNA-treated rats. Perivagal capsaicin application and pretreatment with devazepide prevented CCK-induced pronociception and CeA NR2B phosphorylation. The pronociception induced by systemic CCK, which is vagal afferent-dependent, requires activation of CeA NMDA-NR2B receptors.

Dissociable effects of NR2A and NR2B NMDA receptor antagonism on cognitive flexibility but not pattern separation.

N-methyl-D-aspartate (NMDA) receptors play crucial roles in learning and memory, but the role of each NMDA receptor subtype in a specific cognitive process is unclear. Non-selective blockers of NMDA receptor are used to model the cognitive impairment in schizophrenia and Alzheimer's disease. Counter-intuitively selective NR2A and 2B NMDA receptor antagonists are thought to have pro-cognitive properties. These seemingly contrasting findings might in part be the result of different compounds and behavioral measures used across studies. We compared the effect of NVP-AAM077 (NR2A antagonist), CP 101-606 (NR2B antagonist), and MK-801 (non-selective antagonist) in a series of touch screen tasks that can be used to measure spatial cognition and cognitive flexibility. NVP-AAM077, CP 101-606, and MK-801 were administered prior to testing, in adult male Lister-hooded rats trained in tasks of location discrimination, paired associate learning (PAL), and trial unique non-match to location (TUNL). Results showed that MK-801 impaired performance on all the tasks. In contrast, CP 101-606 only impaired reversal learning in location discrimination and had minimal effect on working memory in TUNL and caused a modest improvement in accuracy in PAL and acquisition of a spatial discrimination. NVP-AAM077 had little effect on performance across tasks, although these data allude to a potential enhancement of acquisition of a spatial location and impairments in spatial reversal learning in a separation-dependent manner. These data demonstrated that non-selective NMDA antagonism will disrupt numerous aspects of cognitive function. However, selective antagonism is capable of impairing or enhancing cognitive function in a task-dependent fashion.

Interleukin-6 reduces NMDAR-mediated cytosolic Ca2+ overload and neuronal death via JAK/CaN signaling

Cytosolic Ca2+ overload induced by N-methyl-d-aspartate (NMDA) is one of the major causes for neuronal cell death during cerebral ischemic insult and neurodegenerative disorders. Previously, we have reported that the cytokine interleukin-6 (IL-6) reduces NMDA-induced cytosolic Ca2+ overload by inhibiting both L-type voltage-gated calcium channel (L-VGCC) activity and intracellular Ca2+ store release in cultured cerebellar granule neurons (CGNs). Here we aimed to show that NMDA-gated receptor channels (i.e., NMDA receptors, NMDARs) are an inhibitory target of IL-6 via a mediation of calcineurin (CaN) signaling. As expected, IL-6 decreased NMDAR-mediated cytosolic Ca2+ overload and inward current in cultured CGNs. The NMDAR subunits, NR1, NR2A, NR2B and NR2C, were expressed in CGNs. Blocking either of NR2A, NR2B and NR2C with respective antagonist reduced NMDA-induced extracellular Ca2+ influx and neuronal death. Importantly, the reduced percentages in extracellular Ca2+ influx and neuronal death by either NR2B or NR2C antagonist were weaker in the presence of IL-6 than in the absence of IL-6, while the reduced percentage by NR2A antagonist was not significantly different between the presence and the absence of IL-6. AG490, an inhibitor of Janus kinase (JAK), abolished IL-6 protection against extracellular Ca2+ influx, mitochondrial membrane depolarization, neuronal death, and CaN activity impairment induced by NMDA. The CaN inhibitor FK506 reduced these IL-6 neuroprotective properties. Collectively, these results suggest that IL-6 exerts neuroprotection by inhibiting activities of the NMDAR subunits NR2B and NR2C (but not NR2A) via the intermediation of JAK/CaN signaling.

Effects of NMDA-Receptor Antagonist on the Expressions of Bcl-2 and Bax in the Subventricular Zone of Neonatal Rats with Hypoxia-Ischemia Brain Damage.

Neonatal hypoxia-ischemia brain damage is an important cause of death by affecting prognosis of neural diseases. It is difficult to find effective methods of prevention and treatment due to the complexity of its pathogenesis. N-methyl-D-aspartate (NMDA), as an excitotoxicity amino acids, has proven to play an important role in hypoxic-ischemic. However, the exact effects of the NMDA subunits, NR2A and NR2B, during hypoxic-ischemic have not been investigated in detail. Therefore, we sought to study whether the NMDA receptor antagonist could confer neuroprotective effects in a neonatal rat hypoxia-ischemia model. The effects of intraperitoneal injections of different drugs, namely MK-801 (0.5mg/kg), NVP-AAM077 (5mg/kg), and Ro25-6981 (5mg/kg), on the expressions of anti-apoptotic protein Bcl-2 and apoptosis protein Bax in the subventricular zone were analyzed by immunohistochemical staining to explore the roles of NMDA subunits (NR2A and NR2B) in hypoxic-ischemic. We found that the NR2B antagonist (Ro25-6981) could inhibit hypoxic-ischemic with the increasing Bcl-2 expression. NR2A antagonists (NVP-AAM077) can increase cerebral hypoxia-ischemia in neonatal rats, promoting the expression of apoptotic protein Bax.

Effect of NMDAR antagonists in the tetrabenazine test for antidepressants: comparison with the tail suspension test.

The N-methyl-d-aspartate receptor (NMDAR) antagonist ketamine, produces rapid and enduring antidepressant effect in patients with treatment-resistant depression. Similar dramatic effects have not been observed in clinical trials with other NMDAR antagonists indicating ketamine may possess unique pharmacological properties. Tetrabenazine induces ptosis (a drooping of the eyelids), and the reversal of this effect, attributed to a sympathomimetic action, has been used to detect first-generation antidepressants, as well as ketamine. Because the actions of other NMDAR antagonists have not been reported in this measure, we examined whether reversal of tetrabenazine-induced ptosis was unique to ketamine, or a class effect of NMDAR antagonists. The effects of ketamine and other NMDAR antagonists to reverse tetrabenazine-induced ptosis were examined and compared with their antidepressant-like effects in the tail suspension test (TST) in mice. All the NMDAR antagonists tested produced a partial reversal of tetrabenazine-induced ptosis and, as expected, reduced immobility in the TST. Ketamine, memantine, MK-801 and AZD6765 were all about half as potent in reversing tetrabenazine-induced ptosis compared to reducing immobility in the TST, while an NR2B antagonist (Ro 25-6981) and a glycine partial agonist (ACPC) were equipotent in both tests. The ability to reverse tetrabenazine-induced ptosis is a class effect of NMDAR antagonists. These findings are consistent with the hypothesis that the inability of memantine, AZD6765 (lanicemine) and MK-0657 to reproduce the rapid and robust antidepressant effects of ketamine in the clinic result from insufficient dosing rather than a difference in mechanism of action among these NMDAR antagonists.

Effect of acute NR2B antagonist treatment on long-term potentiation in the rat hippocampus

The long lasting antidepressant response seen following acute, i.v. ketamine administration in patients with treatment-resistant depression (TRD) is thought to result from enhanced synaptic plasticity in cortical and hippocampal circuits. Using extracellular field recordings in rat hippocampal slices, we show that a single dose of the non-selective NMDA receptor antagonist ketamine or CP-101,606, a selective antagonist of the NR2B subunit of the NMDA receptor, enhances hippocampal synaptic plasticity induced with high frequency stimulation (HFS) 24h after dosing – a time at which plasma concentrations of the drug are no longer detectable in the animal. These results indicate that acute inhibition of NMDA receptors containing the NR2B subunit can lead to long-lasting changes in hippocampal plasticity.

The NR2B antagonist, ifenprodil, corrects the l-DOPA-induced deficit of bilateral movement and reduces c-Fos expression in the subthalamic nucleus of hemiparkinsonian rats

The use of NR2B antagonists in Parkinsonism is still controversial. To examine their anti-parkinsonian effects, the NR2B antagonist, ifenprodil, and l-DOPA were administered together and separately in hemiparkinsonian rats (hemi-PD) that were subjected to a cylinder test. Recovery from hypoactivity was achieved by single administration of 3–7mg/kg of l-DOPA; however, improvement in the deficit of bilateral forelimb use was not observed. When administered alone, ifenprodil had no anti-parkinsonian effects; however, combined administration of ifenprodil and 7mg/kg of l-DOPA significantly reversed the deficit of bilateral forelimb use without adversely affecting the l-DOPA-induced improvement in motor activity. Next, in order to identify the brain area influenced by l-DOPA and ifenprodil, quantitative analysis of l-DOPA-induced c-Fos immunoreactivity was performed in various brain areas of hemi-PD following administration of l-DOPA with and without ifenprodil. Among brain areas with robust c-Fos expression within the motor loop circuit in dopamine-depleted hemispheres, co-administered ifenprodil markedly attenuated l-DOPA-induced c-Fos expression in only the subthalamic nucleus (STN), suggesting that the STN is the primary target for the anti-parkinsonian action of NR2B antagonists.

Involvement of NMDA receptor subtypes in cortical spreading depression in rats assessed by fMRI

Cortical spreading depression (CSD) is a phenomenon implicated in migraine with aura and associated with other neurological disorders (e.g. stroke, brain trauma). Current evidence points to the essential role of NMDA receptors in CSD mechanisms. However, the roles of multiple subunits of NMDA receptors expressed in neurons, glia and blood vessels in vivo, are little explored. Using BOLD fMRI of urethane anesthetized rats as an integrative CSD readout, we tested the involvement of different NMDA receptor subtypes in CSD induction and propagation. Rats were treated with a non-selective NMDA blocker (MK-801), NR2B antagonist (ifenprodil) or a NR2A selective antagonist (TCN-201). CSD was induced during fMRI scanning by application of KCl onto the cerebral cortex and fMRI data were collected by 9.4 T MRI. The non-specific NMDA antagonist MK-801 completely blocked CSD, which was not observed in the NR2A group where TCN-201 did not alter the CSD features. Unexpectedly, the NR2B specific antagonist ifenprodil largely promoted the initial negative phase of the BOLD CSD response, likely due to altered neurovascular coupling. Our data suggest key roles and differential involvement of NMDA receptor subtypes in CSD generation and propagation, highlighting an important role for the NR2B subtype.

NR2B antagonist CP-101,606 inhibits NR2B phosphorylation at tyrosine-1472 and its interactions with Fyn in levodopa-induced dyskinesia rat model

The augmented tyrosine phosphorylation of NR2B subunit of N-methyl-d-aspartate receptors (NMDAR) dependent on Fyn kinase has been associated with levodopa (l-dopa)-induced dyskinesia (LID). CP-101,606, one selective NR2B subunit antagonist, can improve dyskinesia. Yet, the accurate action mechanism is less well understood. In the present study, the evidences were investigated. Valid 6-hydroxydopamine-lesioned parkinsonian rats were treated with l-dopa intraperitoneally for 22 days to induce LID rat model. On day 23, rats received either CP-101,606 (0.5mg/kg) or vehicle with each l-dopa dose. On the day of 1, 8, 15, 22, and 23 during l-dopa treatment, we determined abnormal involuntary movements (AIMs) in rats. The levels of NR2B phosphorylation at tyrosine-1472 (pNR2B-Tyr1472) and interactions of NR2B with Fyn in LID rat model were detected by immunoblotting and immunoprecipitation. Results showed that CP-101,606 attenuated l-dopa-induced AIMs. In agreement with behavioral analysis, CP-101,606 reduced the augmented pNR2B-Tyr1472 and its interactions with Fyn triggered during the l-dopa administration in the lesioned striatum of parkinsonian rats. Moreover, CP-101,606 also decreased the level of Ca2+/calmodulin-dependent protein kinase II at threonine-286 hyperphosphorylation (pCaMKII-Thr286), which was the downstream signaling amplification molecule of NMDAR overactivation and closely associated with LID. However, the protein level of NR2B and Fyn had no difference under the above conditions. These data indicate that the inhibition of the interactions of NR2B with Fyn and NR2B tyrosine phosphorylation may contribute to the CP-101,606-induced downregulation of NMDAR function and provide benefit for the therapy of LID.

Unprecedented therapeutic potential with a combination of A2A/NR2B receptor antagonists as observed in the 6-OHDA lesioned rat model of Parkinson's disease.

In Parkinson's disease, the long-term use of dopamine replacing agents is associated with the development of motor complications; therefore, there is a need for non-dopaminergic drugs. This study evaluated the potential therapeutic impact of six different NR2B and A2A receptor antagonists given either alone or in combination in unilateral 6-OHDA-lesioned rats without (monotherapy) or with (add-on therapy) the co-administration of L-Dopa: Sch-58261+ Merck 22; Sch-58261+Co-101244; Preladenant + Merck 22; Preladenant + Radiprodil; Tozadenant + Radiprodil; Istradefylline + Co-101244. Animals given monotherapy were assessed on distance traveled and rearing, whereas those given add-on therapy were assessed on contralateral rotations. Three-way mixed ANOVA were conducted to assess the main effect of each drug separately and to determine whether any interaction between two drugs was additive or synergistic. Additional post hoc analyses were conducted to compare the effect of the combination with the effect of the drugs alone. Motor activity improved significantly and was sustained for longer when the drugs were given in combination than when administered separately at the same dose. Similarly, when tested as add-on treatment to L-Dopa, the combinations resulted in higher levels of contralateral rotation in comparison to the single drugs. Of special interest, the activity observed with some combinations could not be described by a simplistic additive effect and involved more subtle synergistic pharmacological interactions. The combined administration of A2A/NR2B-receptor antagonists improved motor behaviour in 6-OHDA rats. Given the proven translatability of this model such a combination may be expected to be effective in improving motor symptoms in patients.

Activation of Transient Receptor Potential Vanilloid 4 is Involved in Neuronal Injury in Middle Cerebral Artery Occlusion in Mice.

Transient receptor potential vanilloid 4 (TRPV4) is widely expressed in the central nervous system and can be activated by multiple stimuli during cerebral ischemia. Recently, we reported that intracerebroventricular (icv.) injection of HC-067047, a specific TRPV4 antagonist, reduced brain infarction following 60-min of middle cerebral artery occlusion (MCAO). This study was undertaken to investigate the molecular mechanisms underlying TRPV4-mediated neuronal injury in cerebral ischemia. We demonstrated that TRPV4 expression was upregulated in the ipsilateral hippocampus at 4 to 48h post-MCAO, peaking at 18h post-MCAO. Treatment with TRPV4 antagonists (HC-067047 and ruthenium red) dose-dependently reduced brain infarction at 24h post-MCAO. Phosphorylation of protein kinase B (p-Akt) was downregulated and that of extracellular signal-related kinase (p-ERK) was upregulated at 8 to 24h post-MCAO, which was markedly blocked by treatment with HC-067047. Icv. injection of GSK1016790A (a TRPV4 agonist), dose-dependently induced hippocampal neuronal death, accompanied by an increase in phosphorylation of the NR2B subunit of the N-methyl-D-aspartate receptor (NMDAR). In addition, the level of p-Akt was decreased and that of p-ERK was increased by GSK1016790A-injection, which was sensitive to an NR2B antagonist. The neuronal toxicity of GSK1016790A was blocked by treatment with an NR2B antagonist and a phosphatidylinositol-3-kinase (PI3K) agonist but not by administration of a MAPK/ERK kinase antagonist. We conclude that the activation of TRPV4 is upregulated and involved in neuronal injury during cerebral ischemia and that the neurotoxicity associated with TRPV4-activation is mediated through NR2B-NMDAR and the related downregulation of the Akt signaling pathway.