Substitution for PCP, disruption of prepulse inhibition and hyperactivity induced by N-methyl-D-aspartate receptor antagonists: preferential involvement of the NR2B rather than NR2A subunit

The non-competitive NMDA receptor antagonist phencyclidine (PCP) is known to produce a discriminative stimulus in rats. The first aim of the present study was to investigate which NMDA receptor subtype(s) is involved in this effect of PCP. Rats were trained to discriminate PCP (2 mg/kg; i.p.) from saline in a two lever operant task. The NMDA channel blocker, (+)MK-801 (0.1 mg/kg; i.p.) and the competitive NMDA receptor antagonist SDZ 220-581 (3 mg/kg; i.p.) produced 76% of PCP-lever selection (ED50=0.045 and 2 mg/kg, respectively), whereas their respective inactive enantiomers (-)MK-801 (0.025-0.1 mg/kg) and SDZ 221-653 (2-5 mg/kg) induced less than 30% of PCP-appropriate responding. Another competitive NMDA antagonist, SDZ EAB-515 (30 mg/kg; i.p.), induced 63% of PCP-lever responding (ED50=23.48 mg/kg). The selective antagonist of NMDA receptors containing the NR1A/NR2B-subunits Ro 25-6981 (20 mg/kg; i.p.) resulted in a complete substitution (more than 80% of PCP-lever selection) for PCP (ED50=8.59 mg/kg). In contrast, the NR1A/NR2A NMDA receptor-preferring antagonist NVP-AAM077 (2-10 mg/kg; i.p.) failed to produce PCP-like discriminative stimuli. At high doses SDZ 220-581 (ED50=2.44), NVP-AAM077 (ED50=8.33) and SDZ EAB-515 (ED50=25.81) decreased the performance of the rats in this operant task. The ability of these NMDA receptor antagonists to disrupt the prepulse inhibition (PPI) of the startle response and to alter locomotor activity was also studied. PCP (0.5-2 mg/kg; s.c.), SDZ 220-581 (0.5-5 mg/kg; s.c.), SDZ EAB-515 (1-30 mg/kg; i.p.) and Ro 25-6981 (5-20 mg/kg; i.p.) disrupted PPI and at high doses produced hyperlocomotion. In contrast, NVP-AAM077 (5-20 mg/kg; i.p.) did not disrupt PPI and reduced locomotor activity. In conclusion, it appears that the NMDA receptor containing the NR2B, rather than the NR2A subunit, may play a major role in the PCP-like discriminative stimulus. In addition, sensory motor gating disturbances associated with NMDA antagonists do not seem to result from a blockade of NR1/NR2A-containing NMDA receptors.     

Brain but not spinal NR2B receptor is responsible for the anti-allodynic effect of an NR2B subunit-selective antagonist CP-101,606 in a rat chronic constriction injury model

In order to examine the site of action of an NR2B subtype-selective NMDA antagonist CP-101,606, we investigated its analgesic effect in a rat model of neuropathic pain at various routes of administration. Mechanical allodynia was induced by chronic constriction injury (CCI) of the sciatic nerve in male Sprague-Dawley rats. Subcutaneous treatment of the animals with CP-101,606 at 10 mg/kg significantly inhibited CCI-induced mechanical allodynia. Intracerebroventricular injection of CP-101,606 at 10, 30 and 100 nmol also inhibited the mechanical allodynia in a dose-dependent manner, the statistically significant effect being achieved at the highest dose tested (100 nmol) without producing any behavioral abnormalities. However, intrathecal injection of CP-101,606 at a dose of 300 nmol failed to inhibit CCI-induced allodynia. A receptor binding assay using rat forebrain and spinal cord membrane preparations demonstrated that [3H]CP-101,606 bound to the brain NR2B receptor with a greater extent compared to the spinal cord one. These findings suggest that the anti-allodynia effect of CP-101,606 is ascribable to blockade of NR2B receptors at the brain, but not at the spinal cord. In contrast, intrathecal injection of a non-selective NMDA antagonist, memantine, significantly inhibited CCI-induced mechanical allodynia at a dose of 300 nmol, indicating the difference in the site of action between the non-selective NMDA antagonist and the NR2B-specific NMDA antagonist.     

Evidence for improved performance in cognitive tasks following selective NR2B NMDA receptor antagonist pre-treatment in the rat

Rationale We previously reported that the NR2B subunit-selective N-methyl-d-aspartate (NMDA) antagonist Ro 63-1908 produced a marked deficit in response control in the five-choice serial reaction time task (5-CSRTT).Objectives The present studies were designed to investigate this further by studying the NR2B NMDA antagonists, ifenprodil, traxoprodil (CP101,606), Ro 25-6981 as well as Ro 63-1908 in this test.Methods Following training in the 5-CSRTT, separate groups of rats were either tested under (1) standard test conditions [5 s inter-trial interval (ITI), 0.5 s stimulus duration, 100 trials], (2) high (3 s ITI) and low (10 s ITI) event rate of stimulus presentation and (3) a 250-trial protocol in aged 2-year-old rats. In a final study, the effects of traxoprodil were investigated in an operant delayed match to position (DMTP) task, a test of working memory, and compared to dizocilpine and Ro 63-1908.Results Similar to Ro 63-1908, both traxoprodil (1–10 mg/kg) and Ro 25-6981 (3–30 mg/kg) increased premature responding but also increased response speed with no error trade-off. Conversely, ifenprodil (1–10 mg/kg) slowed response speed and increased omissions with no effect on premature responding. Tested under a variable ITI, Ro 63-1908 (1 mg/kg) increased premature responding at all ITIs, but this change was proportional to controls. At short ITI (3 s), Ro 63-1908 reliably improved performance both in terms of response speed and accuracy (percent correct). In a 250-trial protocol in aged rats, both Ro 63-1908 (0.1–0.3 mg/kg) and, particularly, traxoprodil (1–3 mg/kg) improved performance—increasing response speed and increasing the number of rewards earned during test. Finally, traxoprodil (1–10 mg/kg) improved accuracy and increased response speed in the DMTP task.Conclusions The present studies support the view that selective NR2B NMDA antagonists promote impulsive-type responding in the 5-CSRTT; however, under certain test conditions, drugs of this class—notably traxoprodil—may also improve task performance.     

The NMDA receptor NR2B subtype selective antagonist Ro 25-6981 aggravates paroxysmal dyskinesia in the dt(sz) mutant

Previously, enhanced levels of spermine which stimulates N-methyl-D-aspartate (NMDA) receptors, particularly those containing the NR2B subunit, were found in brains of dt(sz) mutant hamsters, a model of paroxysmal dyskinesia in which dystonic episodes occur in response to stress. Therefore, the effects of the NR2B selective NMDA receptor antagonist Ro 25-6981 ([R-(R,S)]-alpha-(4-hydroxyphenyl)-beta-methyl-4-phenyl-methyl)-1-piperidine-propanol] on severity of dystonia were investigated in the dt(sz) hamster. Ro 25-6981 failed to exert antidystonic effects, but even caused a moderate aggravation at higher doses (10.0, 12.5 mg/kg). This result indicates that overstimulation of receptors that include the NR2B subunit by polyamines is not involved in the dystonic syndrome. NR2B-selective NMDA receptor antagonists seem not to provide a novel approach in the treatment of hereditary paroxysmal dyskinesias.     

NR2B containing NMDA receptor dependent windup of single spinal neurons

Windup, the frequency dependent build-up of spinal neuronal responses, is implicated in the development of central sensitization of nociceptive pathways. N-methyl-D-aspartate (NMDA) receptors have been shown to be involved in these processes but the role of various receptor subtypes at the spinal level is not fully understood. In our experiments, we compared the inhibitory effect of MK-801 (a nonselective NMDA receptor antagonist, 0.01-3 mg/kg i.v.) and CI-1041 (an NR2B subunit specific NMDA receptor antagonist, 0.3-10 mg/kg i.v.) on the formation of dorsal horn neuronal windup in spinalized rats, in vivo. Both types of antagonist blocked windup considerably at doses not affecting the normal synaptic transmission. These results are in agreement with the well-documented effectivity of NR2B subtype selective NMDA receptor antagonists in chronic pain models and give the first direct evidence that spinal mechanisms are involved in this effect.     

In vivo neurochemical effects of the NR2B selective NMDA receptor antagonist CR 3394 in 6-hydroxydopamine lesioned rats

Microdialysis in intact and denervated striatum of unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats was used to investigate whether CR 3394, N-[2-(3,5-dimethyl-1-adamantyl)ethyl]acetamidine, an adamantane derivative with preferential selectivity for the NR2B subunit of the NMDA receptor, has dopamine releasing properties in vivo. We also investigated whether this NMDA antagonist can potentiate the effects of L-Dopa on extracellular dopamine in these animals. After systemic injection, there was no significant effect of CR 3394 on extracellular dopamine, at all doses studied (1, 5 and 20 mg/kg i.p.), in either intact or in denervated striatum. On the other hand, striatal perfusion with 100 microM of the compound elicited release of dopamine in intact, but not in denervated striatum. In denervated striatum of the 6-OHDA-lesioned rats, CR 3394 (5 mg/kg) significantly enhanced the dopamine release induced by L-Dopa administration (25 mg/kg i.p.) in combination with benserazide (10 mg/kg i.p.). In particular, the onset of action of L-Dopa was potentiated. However, when combined with a subthreshold dose of L-Dopa (5 mg/kg), the effects of CR 3394 were lost. We conclude that CR 3394, like other NR2B receptor antagonists, has dopamine releasing properties in vivo. It enhances the effects of suprathreshold doses of L-Dopa in the denervated striatum, but not of low doses of L-Dopa. Therefore, future studies are necessary to establish the potential of selective NR2B receptor antagonists as L-Dopa-sparing agents.     

NMDA NR2B subtype-selective receptor antagonists fail to antagonize electrically-precipitated seizures and elicit popping in mice

NR2B-subtype-selective antagonists differ from MK-801, a nonselective NMDA receptor antagonist. MK-801 antagonizes electrical seizures at doses as low as 0.1 to 0.18 mg/kg and elicits popping at doses as low as 0.5 mg/kg, whereas ifenprodil and Ro 8-4304 were unable to do so at the doses tested. Ro 25-6981, however, was able to antagonize electrically-precipitated tonic hindlimb extension at 100 mg/kg, but was not able to elicit popping behavior at this dose.     

Pharmacological characterization of the chronic constriction injury model of neuropathic pain

The chronic constriction injury model is a rat model of neuropathic pain based on a unilateral loose ligation of the sciatic nerve. The aim of the present study was to test its sensitivity to various clinically validated and experimental drugs. Mechanical allodynia and thermal hyperalgesia developed within one week post-surgery and were reliably present for at least 7 weeks. Mechanical allodynia was strongly attenuated by morphine (minimal effective dose in brackets: 8 mg/kg, p.o.) and the cannabinoids Delta9-tetrahydrocannabinol (3 mg/kg, p.o.) and (-)-cis-3-[2-hydroxy-4(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP 55,940; 0.05 mg/kg, i.p.), and weakly/moderately attenuated by the anticonvulsants gabapentin (50 mg/kg, i.p.) and carbamazepine (32 mg/kg, i.p.), the muscle relaxant baclofen (3 mg/kg, i.p.), and the adenosine kinase inhibitor 4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido[2,3-d]pyrimidine (ABT-702; 30 mg/kg, i.p.). Thermal hyperalgesia was strongly attenuated by morphine (16 mg/kg, p.o.), Delta9-tetrahydrocannabinol (6 mg/kg, p.o.), CP 55,940 (0.025 mg/kg, i.p.), carbamazepine (32 mg/kg, i.p.) and the antidepressant amitriptyline (32 mg/kg, p.o.), and weakly/moderately attenuated by gabapentin (50 mg/kg, i.p.), the anti-inflammatory cyclooxygenase-2 inhibitor rofecoxib (30 mg/kg, i.p.) and the adenosine A1 receptor positive allosteric modulator 2-amino-4,5,6,7-tetrahydrobenzo(b)thiophen-3-yl 4-chlorophenylmethanone (T62; 30 mg/kg, i.p.). Both symptoms were hardly or not affected by the nonselective N-methyl-d-aspartate receptor antagonists ketamine and dizocilpine, and the N-methyl-d-aspartate receptor NR2B-selective antagonists ifenprodil and R-(R*,S*)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenyl-methyl)-1-piperidine propranol (Ro 25-6981). The finding that mechanical allodynia and/or thermal hyperalgesia are attenuated by various established compounds further supports the validity of the chronic constriction injury model for the study of neuropathic pain and its use for the identification of novel treatments.     

Role of the NMDA receptor subunit in the expression of the discriminative stimulus effect induced by ketamine.

Ketamine, which is a non-competitive NMDA receptor antagonist, has been used as a dissociative anesthetic agent. However, chronic use of ketamine produces psychotomimetic effects, such as nightmares, hallucination and delusion. Therefore, the present study was designed to ascertain the role of the NMDA receptor and sigma receptor in the discriminative stimulus effect induced by ketamine. Fischer 344 rats were trained to discriminate between ketamine (5 mg/kg, i.p.) and saline under a fixed-ratio 10 food-reinforced procedure. Non-competitive antagonists for both NR2A- and NR2B-containing NMDA receptors, such as phencyclidine (0.1--1 mg/kg, i.p.) and dizocilpine (3--30 microg/kg, i.p.), and the NR2A-containing NMDA receptor-preferred antagonist dextromethorphan (3--56 mg/kg, i.p.) fully substituted for the ketamine cue in a dose-dependent manner. By contrast, the NR2B-containing NMDA receptor antagonist ifenprodil (5--20 mg/kg, i.p.) exhibited no generalization. Additionally, the competitive NMDA antagonist 3-[(+/-)-2-carboxypiperazine-4-yl] propyl-1-phosphonic acid ((+/-)-CPP; 0.3--5.6 mg/kg, i.p.) and a sigma receptor ligand DTG (0.3--3 mg/kg, s.c.) displayed no generalization to the ketamine cue. These results suggest that NR1/NR2A subunit containing NMDA antagonism may be critical for the production of the ketamine cue.     

Role of the NMDA receptor NR2B subunit in the discriminative stimulus effects of ketamine

The noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, ketamine, is a dissociative anesthetic with antihyperalgesic properties. However, its clinical use is compromised by psychotomimetic side-effects. As ketamine and other noncompetitive NMDA antagonists, such as phencyclidine and dizocilpine, are not selective for the NR2A-2D subunits of the NMDA receptor, it is unclear which of these subunits is responsible for the psychotomimetic side-effects. This study investigated the role of the NR2B subunit in the ketamine drug discrimination model, a possible correlate for such side-effects. In a first experiment aimed at assessing general potency and time dependency, ketamine, dizocilpine, phencyclidine and the NR2B-selective antagonists ifenprodil and Ro 25-6981, dose-dependently suppressed fixed ratio 10 food-reinforced responding in rats, with peak efficacy obtained around 15-40 min. In rats trained to discriminate ketamine from vehicle in a two-lever fixed ratio 10 food-reinforced procedure, ketamine, dizocilpine, phencyclidine and Ro 25-6981 induced complete generalization (>80%); whereas ifenprodil induced partial generalization (33%). These findings suggest that the NR2B subunit is involved in the discriminative stimulus effects of noncompetitive NMDA antagonists, and that selective NR2B antagonists may also induce psychotomimetic side-effects.     

NR2B subunit-specific NMDA antagonist Ro25-6981 inhibits the expression of conditioned fear: a comparison with the NMDA antagonist MK-801 and fluoxetine

N-methyl-D-asparate (NMDA)-mediated glutamatergic neurotransmission is strongly involved in the development of trauma-induced behavioral dysfunctions, and indirect evidence suggests that NR2B subunit-expressing NMDA receptors are primarily involved in this process. Earlier studies showed that NR2B blockers inhibit the acquisition of conditioned fear, a frequently used model of post-traumatic stress disorder, but their effects on the expression of conditioned fear was poorly studied. We investigated here the effects of the selective serotonin reuptake blocker, fluoxetine, the NMDA blocker, MK-801, and the NR2B subunit blocker, Ro25-6981 on the expression of conditioned fear. Rats received 10 foot shocks administered over 5 min and were tested 24 h later in the shocking context. Treatments were administered 1 h before testing. Shocks dramatically increased freezing and reduced exploration. MK-801 and Ro25-6981 significantly ameliorated both changes. The effects of fluoxetine were less pronounced. In the open field, MK-801 increased locomotion, ataxia, and stereotypy (effects typical of NMDA blockade). Neither fluoxetine nor Ro25-6981 affected locomotion in the open field. Thus, the NR2B-specific NMDA blockade preserved the beneficial effects of general NMDA antagonists on the expression of conditioned fear but did not produce the locomotor side-effects typical of the latter. These findings warrant further studies on the effects of NR2B antagonists in models of post-traumatic stress disorder.     

Studies on the subtype selectivity of CP-101,606: evidence for two classes of NR2B-selective NMDA receptor antagonists

The subtype-selectivity of racemic [(3)H]CP-101,606, a novel high-affinity NMDA receptor radioligand was determined using defined recombinant NMDA receptor subunits expressed in HEK 293 cells. [(3)H]CP-101,606 binds to adult rodent forebrain and NR1/NR2B receptors expressed in HEK 293 cells with K(D)=4.2 nM and 6.0 nM, respectively. In contrast, no high affinity specific binding was detected to NR1, NR2A, NR2B subunits expressed alone or NR1/NR2A receptors. HEK 293 cells were transfected with NR1, NR2A and NR2B receptor subunits and complexes comprising all three subunits were isolated by anti-NR2A immunoaffinity chromatography. Based on immunoblotting with subunit-selective antibodies, the immunopurified material contained all three NMDA receptor subunit polypeptides. However, in contrast to parallel studies in which high affinity [(3)H]Ro-25,6981 binding activity was observed, no high affinity [(3)H]CP-101,606 binding sites were detected to the immunopurified material. This study provides further evidence for two distinct classes of NR2B-directed NMDA receptor antagonists, one which binds with high affinity irrespective whether another NR2 subunit type is present (Ro-25,6981) and a second class which is affected significantly by the presence of another NR2 subunit type within the receptor complex, exemplified by CP-101,606.     

Pharmacological and genetic evidence indicates that combined inhibition of NR2A and NR2B subunit containing NMDA receptors is required to disrupt prepulse inhibition

Rationale Glutamate signalling through the N-methyl-d-aspartate (NMDA) receptor is of critical importance for normal central nervous system (CNS) function, as indicated by the marked behavioural disturbances produced by non-subtype selective NMDA antagonists such as dizocilpine (MK-801).Objective The present studies were designed to investigate the involvement of the two major NMDA receptor subunits in the central nervous system, i.e. NR2A and NR2B, on sensorimotor gating in mice.Methods These experiments utilised the non-subtype-selective NMDA antagonist dizocilpine, a line of NR2A-KO mice and the selective NR2B antagonist Ro 63–1908, in the study of pre-pulse inhibition of the startle response (PPI).Results The non-selective NMDA receptor antagonist dizocilpine (0.1–1 mg/kg, IP) robustly disrupted PPI in wild-type mice. Conversely, selective genetic or pharmacological inhibition of either the NMDA NR2A or NR2B receptor subunit containing receptors, respectively, had no effect on PPI. Thus, NR2A KO mice showed normal PPI compared with wild-type littermate controls, and administration of Ro 63-1908 (1–10 mg/kg IP) to wild-type mice did not affect PPI. However, selective inhibition of NR2A and NR2B by administration of Ro 63–1908 to NR2A KO mice significantly disrupted PPI.Conclusions These data imply that concomitant inhibition of both NR2A and NR2B subunit-containing NMDA receptors is necessary to disrupt PPI, suggesting that inhibition of NR2A and NR2B-containing NMDA receptors is required to elicit behaviours suggestive of psychomimetic effects in man.     

Implication of Src family kinase-dependent phosphorylation of NR2B subunit-containing NMDA receptor in the rewarding effect of morphine.

The present study was undertaken to further clarify the role of tyrosine phosphorylation of NR2B subunit-containing N-methyl-D-aspartate (NMDA) receptor in the development of the morphine-induced rewarding effect in mice. The morphine (5 mg/kg, sc)-induced rewarding effect was completely inhibited by pretreatment with a selective NR2B subunit-containing NMDA receptor antagonist ifenprodil (20 mg/kg, i.p.). The protein level of phospho-Tyr-1472, but not phospho-Ser-1303, NR2B subunit was significantly increased in the mouse limbic forebrain containing the nucleus accumbens (N.Acc.) of mice that had shown the morphine-induced rewarding effect. In addition, the level of phospho-Tyr-416 Src family kinase was also increased in the limbic forebrain of mice that had shown the morphine-induced rewarding effect. These findings suggest that Tyr-1472 phosphorylation of NR2B subunit-containing NMDA receptor associated with activation of Src family kinase in the limbic forebrain may be involved in the morphine-induced rewarding effect.     

Activation of NR2B-containing NMDA receptors is not required for NMDA receptor-dependent long-term depression

The triggering of both NMDA receptor-dependent long-term potentiation (LTP) and long-term depression (LTD) in the CA1 region of the hippocampus requires a rise in postsynaptic calcium. A prominent hypothesis has been that the detailed properties of this postsynaptic calcium signal dictate whether LTP or LTD is generated by a given pattern of synaptic activity. Recently, however, evidence has been presented that the subunit composition of the NMDA receptor (NMDAR) determines whether a synapse undergoes LTP or LTD with NR2A-containing NMDARs triggering LTP and NR2B-containing NMDARs triggering LTD. In the present study, the role of NR2B-containing synaptic NMDARs in the induction of LTD in CA1 pyramidal cells has been studied using the selective NR2B antagonists, ifenprodil and Ro25-6981. While both antagonists reduced NMDAR-mediated synaptic currents, neither prevented induction of LTD. These results demonstrate that activation of NR2B-containing NMDARs is not an absolute requirement for the induction of LTD in the hippocampus.     

Effects of N-methyl-d-aspartate receptor antagonism on neuroleptic-induced orofacial dyskinesias

Rationale Tardive dyskinesia is a syndrome of abnormal, involuntary movements, which occurs as a complication of long-term neuroleptic therapy. The pathophysiology of this potentially irreversible syndrome is still an enigma.Objective The objective of the present study was to elucidate the role of N-methyl-d-aspartate (NMDA) receptor involvement in neuroleptic-induced orofacial dyskinesia in rats.Methods Animals chronically treated with haloperidol for a period of 40 weeks exhibited significantly more vacuous chewing movements (VCMs), as compared to vehicle-treated controls. In a series of acute experiments, rats received: amantadine (10, 20, and 40 mg/kg i.p.), a low-affinity, uncompetitive NMDA-receptor antagonist (open channel blocker); dextrorphan (5, 10, and 20 mg/kg i.p.), an NMDA receptor channel antagonist; ifenprodil (2.5, 5, and 10 mg/kg i.p.), a noncompetitive allosteric NMDA receptor antagonist acting at the polyamine site; and Ro 25-6981 (2.5, 5, and 10 mg/kg i.p.), a potent and selective blocker of NMDA receptors which contain the NR2B subunit.Results All the drugs tested, except dextrorphan, reduced VCMs and tongue protrusions with varying efficacies and side effects profiles. Ro 25-6981 was found significantly more potent than amantadine and ifenprodil in reducing VCMs and tongue protrusions at all doses tested, and at the higher dose, it completely eliminated orofacial dyskinesia (p<0.05).Conclusions These results suggest that NMDA receptors may play a significant role in the pathophysiology of tardive dyskinesia. Furthermore, antagonists showing selectivity for NMDA receptors containing the NR2B subunit may be particularly efficacious as novel therapeutic agents for the treatment of tardive dyskinesia and deserve further testing.     

CP-101,606, an NR2B subunit selective NMDA receptor antagonist, inhibits NMDA and injury induced c-fos expression and cortical spreading depression in rodents

(1S, 2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP-101,606) is a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptors containing the NR2B subunit. This compound was used to investigate the role of NR2B containing receptors in three responses to NMDA receptor activation in vivo. In mouse, CP-101,606 completely inhibited increases in fos-like immunoreactivity in dentate gyrus caused by a subconvulsant intraperitoneal dose of NMDA. In rat, the compound completely blocked cortical c-fos mRNA induction following focal injury in parietal cortex and the initiation and propagation of electrically induced cortical spreading depression. Inhibition of these responses by CP-101,606 indicates that c-fos induction and cortical spreading depression are dependent on activation of NMDA receptors containing the NR2B subunit. Since NMDA receptor dependent c-fos induction and cortical spreading depression may contribute to neuron loss after focal CNS injury, inhibition of these responses by CP-101,606 may contribute to the neuroprotective efficacy of the compound.     

Evaluation of the NR2B-selective NMDA receptor antagonist Ro 63-1908 on rodent behaviour: evidence for an involvement of NR2B NMDA receptors in response inhibition

We have characterised the effects of the recently described NMDA NR2B subtype selective antagonist, Ro 63-1908, on spontaneous behaviour and in tasks sensitive to non-selective NMDA antagonists. In both rats and wild type mice, Ro 63-1908 (1-30mg/kg sc) produced a mild increase in motor activity of lesser magnitude than that elicited by dizocilpine. No signs of overt PCP-like stereotypy were seen in either species at equivalent doses. PPI was also unaffected. However, in mice lacking the NR2A subunit, Ro 63-1908 (3-30mg/kg) produced a profound hyperactivity of similar magnitude to dizocilpine but few other 'PCP-like' behaviours. In rats, Ro 63-1908 (1-10mg/kg) did not affect Morris water maze or delayed matching performance. In a 5-choice serial reaction time task, requiring rats to respond to a visual stimulus presented after a fixed time interval, Ro 63-1908 (0.3-3mg/kg) produced a dramatic increase in premature responses - accuracy was relatively unaffected. Finally in a DRL24 task, Ro 63-1908 (0.3-3mg/kg) reduced inter-response time, increased response rate, and consequently reduced efficiency. We conclude that the improved profile of Ro 63-1908 compared to NMDA channel blockers is due to both its selectivity for the NR2B vs. NR2A subunit containing receptors and its activity-dependent mechanism of action. However, in the 5-CSRT and DRL24 tasks, Ro 63-1908 produced behaviours suggestive of impaired response inhibition, implicating a critical role of NMDA NR2B transmission in this process.     

Differential roles of NR2A and NR2B subtypes in NMDA receptor-dependent protein synthesis in dendrites

Protein synthesis in dendrites is critical for long-term synaptic plasticity. Previous studies have identified an essential role of NMDA receptors in control of activity-dependent dendritic protein synthesis, but the contribution of NR2A- and NR2B-containing NMDA receptors, the two predominant subtypes of NMDA receptors in the forebrain, has not been determined. Using a pharmacological approach, we investigated the role of NR2A and NR2B subtypes in the regulation of NMDA-induced dendritic translation of a GFP reporter mRNA controlled by CaMKII untranslated regions (UTRs). We found that ifenprodil and Ro25-6981, two specific inhibitors of NR2B-containing NMDA receptors, did not affect dendritic GFP synthesis induced by NMDA. In contrast, NVP-AAM077, an antagonist that preferentially blocks the NR2A subtype, completely abolished NMDA-induced GFP synthesis in dendrites. Our results together suggest that NR2A but not NR2B subtypes are indispensable for NMDA receptor-dependent dendritic protein synthesis.     

Preclinical evaluation of CHF3381 as a novel antiepileptic agent

CHF3381 [n-(2-indanyl)-glycinamide hydrochloride] has been selected on the basis of a screening program as the compound displaying the highest anticonvulsant activity in the maximal electroshock seizure (MES) test and the best therapeutic index with reference to the rotarod test in mice and rats. In this study, the antiepileptic activity and the behavioural toxicity of CHF3381 were characterised in multiple model systems. CHF3381 effectively prevented MES-induced convulsions when administered i.p. (ED50, 24 mg/kg and 7.5 mg/kg) or p.o. (ED50, 21 mg/kg and 21 mg/kg) in both mice and rats, respectively. The time course of oral anti-MES activity in the rat was related to the brain concentration profile of unchanged CHF3381. Interestingly, the brain drug levels were about 4-5 times higher than in plasma. CHF3381 was very effective in mice against picrotoxin-, and i.c.v. N-methyl-D-aspartate (NMDA)-induced hind limb tonic extension (ED50 Approximately/=10 mg/kg), but was a weaker antagonist of 4-amynopyridine- and bicuculline-induced tonic seizures (ED50 approximately/=100 mg/kg), and ineffective against pentylentetrazole- and picrotoxin-induced clonic seizures. CHF3381 antagonised the behavioural effects and lethality of i.p. administered NMDA (ED50 = 57 mg/kg p.o.), indicating that the compound may act as a functional NMDA antagonist. In keeping with this idea, CHF3381 weakly displaced [(3)H]-TCP from binding to NMDA receptor channels (Ki, 8.8 microM). In the rat amygdala kindling model, CHF3381 was more efficient against kindling development than against kindled seizures (minimally active dose = 80 vs. 120 mg/kg i.p). Furthermore, it significantly increased the seizure threshold in kindled rats at relatively low doses (40 mg/kg i.p.). In contrast with MK-801-induced hyperactivity, CHF3381 moderately reduced the spontaneous locomotor activity in mice at anticonvulsant doses. Toxic effects on motor performance (rotarod test) were found at high doses only (TD50 approximately/= 300 mg/kg p.o., congruent with 100 mg/kg i.p. in both mice and rats). Furthermore, CHF3381 did not impair passive avoidance and Morris water maze responding in the therapeutic range of doses. Finally, the development of tolerance after repeated doses was negligible. These data indicate that CHF3381 exerts anticonvulsant and antiepileptogenic effects in various seizure models and possesses good therapeutic window, with scarce propensity to cause neurological side-effects.