Shin-ichi Ogawa

Motor discoordination and increased susceptibility to cerebellar injury in GLAST mutant mice

To study the function of GLAST, a glutamate transporter highly expressed in the cerebellar Bergmann astrocytes, the mouse GLAST gene was inactivated. GLAST‐deficient mice developed normally and could manage simple coordinated tasks, such as staying on a stationary or a slowly rotating rod, but failed more challenging task such as staying on a quickly rotating rod. Electrophysiological examination revealed that Purkinje cells in the mutant mice remained to be multiply innervated by climbing fibres even at the adult stage. We also found that oedema volumes in the mutant mice increased significantly after cerebellar injury. These results indicate that GLAST plays active roles both in the cerebellar climbing fibre synapse formation and in preventing excitotoxic cerebellar damage after acute brain injury.

NE-100, a novel sigma receptor ligand: In vivo tests

It has been suggested that sigma receptor antagonists may be useful as antipsychotic drugs. N, N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100) is a novel compound with high affinity for the sigma receptor (IC50 = 4.16 nM), but low affinity (IC50 > 10,000 nM) for D1, D2, 5-HT1A, 5-HT2 and phencyclidine (PCP) receptors. The head-weaving behavior induced by either (+)SKF10047 or PCP was dose-dependently antagonized by NE-100 with oral ED50 at 0.27 and 0.12 mg/kg, respectively. NE-100 did not affect dopamine agonists-induced stereotyped behavior and/or hyperactivity. NE-100 failed to induce catalepsy in rats. These findings indicate that NE-100 may have antipsychotic activity without the liability of motor side effects typical of neuroleptics.

Neuropharmacological profile of peripheral benzodiazepine receptor agonists, DAA1097 and DAA1106.

Receptor binding and behavioral profiles of N-(4-chloro-2-phenoxyphenyl)-N-(2-isopropoxybenzyl)acetamide (DAA1097) and N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide (DAA1106), novel, selective agonists for the peripheral benzodiazepine receptor (PBR) were examined. DAA1097 and DAA1106 inhibited [3H]PK 11195 binding to crude mitochondrial preparations of rat whole brain, with IC50 values of 0.92 and 0.28 nM. Likewise, DAA1097 and DAA1106 inhibited [3H]Ro 5-4864 binding to the same mitochondrial preparation, with IC50 values of 0.64 and 0.21 nM. In contrast, DAA1097 and DAA1106 did not inhibit [3H]-flunitrazepam, the central benzodiazepine receptor (CBR) ligand, binding to membranes of rat whole brain (IC50>10,000nM). Oral administration of DAA1097 and DAA1106 had anxiolytic effects in the mouse light/dark exploration test and in the rat elevated plus- maze test. Oral administration of DAA1106, diazepam and buspirone but not DAA1097 significantly increased sleeping time in hexobarbital-induced anesthesia in mice. The order of potency of potentiation of hexobarbital anesthesia was diazepam> buspirone> DAA1106> DAA1097. Oral administration of DAA1097 and DAA1106 but not diazepam and buspirone did not affect spontaneous locomotor activity in mice. These findings indicate that DAA1097 and DAA1106 are PBR selective ligands with potent anxiolytic-like properties, in laboratory animals.

Involvement of the melanocortin MC4 receptor in stress-related behavior in rodents

The melanocortin subtype 4 (MC4) receptor has been postulated to be involved in stress and stress-related behavior. We made use of melanocortin MC4 receptor agonists and antagonist to investigate the relationship between the melanocortin MC4 receptor and stress related disorders. The nonspecific melanocortin receptor agonist alpha-melanocyte stimulating hormone (alpha-MSH) and the melanocortin MC4 receptor agonist, Ac-[Nle4,Asp5,D-Phe7,Lys10]alpha-MSH-(4-10)-NH2 (MT II) dose-dependently and significantly reduced the number of licking periods in the rat Vogel conflict test, suggesting that stimulation of the melanocortin MC4 receptor causes anxiogenic-like activity in rats. We synthesized a peptidemimetic melanocortin MC4 receptor selective antagonist, Ac-D-2Nal-Arg-2Nal-NH2 (MCL0020), which has high affinity for the melanocortin MC4 receptor with IC50 values of 11.63 +/- 1.48 nM, in contrast, the affinities for melanocortin MC1 and MC3 receptors were negligible. In addition, MCL0020 significantly attenuated the cAMP formation induced by alpha-MSH in COS-1 cells expressing the melanocortin MC4 receptor without affecting basal cAMP contents. Thus, we considered MCL0020 to be a selective melanocrotin MC4 receptor antagonist among melanocortin receptors. Restraint stress significantly reduced food intake in rats, and i.c.v. administration of MCL0020 dose-dependently and significantly attenuated restraint stress-induced anorexia without affecting food intake. Swim stress induced reduction in the time spent in the light area in the mouse light/dark exploration test, and MCL0020 significantly prevented it. Taken together our findings suggest that the melanocortin MC4 receptor might be related to stress-induced changes in behavior, and blockade of the melanocortin MC4 receptor may prevent stress-induced disorders such as anxiety.

Effect of NE-100, a novel sigma receptor ligand, on phencyclidine-induced delayed cognitive dysfunction in rats

Phencyclidine (PCP), in a dose of 15 mg/kg, produced delayed cognitive dysfunction (at 24 h) in rats subjected to water maze tasks. At 24 h after PCP administration, ataxia, hyperlocomotion and stereotyped behavior were not induced. NE-100, N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-enthylamine monohydrochloride, a selective and potent sigma receptor ligand, was administered orally 10 min after PCP administration or 15 min before the first trial (24 h after PCP administration). In both cases, NE-100 dose-dependently attenuated the delayed cognitive dysfunction induced by PCP. As these findings show that ingestion of PCP led to delayed cognitive dysfunction similar to the cognitive signs of psychosis seen in humans, NE-100 is being further studied for possible treatment of subjects with schizophrenia.

The atypical antipsychotic profile of NRA0045, a novel dopamine D4 and 5-hydroxytryptamine2A receptor antagonist, in rats

The atypical antipsychotic profile of (R)‐(+)‐2‐amino‐4‐(4‐fluorophenyl)‐5‐[1‐[4‐(4‐fluorophenyl)‐4‐oxobutyl] pyrrolidin‐3‐yl] thiazole (NRA0045), a potent dopamine D4 and 5‐hydroxytryptamine (5‐HT)2A receptor antagonist, was examined in rats. Spontaneous locomotor activity was decreased dose‐dependently with i.p. administration of clozapine (ED50 3.7u2003mgu2003kg−1), haloperidol (ED50 0.1u2003mgu2003kg−1) and chlorpromazine (ED50 0.9u2003mgu2003kg−1), whereas inhibition of this type of behaviour induced by i.p. administration of NRA0045, at doses up to 10u2003mgu2003kg−1, did not exceed 50%. Locomotor hyperactivity induced by methamphetamine (MAP, 2u2003mgu2003kg−1, i.p.) in rats (a model of antipsychotic activity) was dose‐dependently antagonized by NRA0045 (ED50 0.4u2003mgu2003kg−1, i.p., and 0.3u2003mgu2003kg−1, p.o., respectively), clozapine (ED50 0.3u2003mgu2003kg−1, i.p. and 0.8u2003mgu2003kg−1, p.o., respectively), haloperidol (ED50 0.02u2003mgu2003kg−1, i.p. and 0.1u2003mgu2003kg−1, p.o., respectively), chlorpromazine (ED50 0.3u2003mgu2003kg−1, i.p. and 3.3u2003mgu2003kg−1, p.o., respectively). In contrast, the MAP (3u2003mgu2003kg−1, i.v.)‐induced stereotyped behaviour in rats (a model of extrapyramidal symptoms) was not affected by NRA0045 or clozapine, at the highest dose given (30u2003mgu2003kg−1, i.p.). Haloperidol (ED50 0.3u2003mgu2003kg−1, i.p.) and chlorpromazine (ED50 4.8u2003mgu2003kg−1, i.p.) strongly blocked the MAP‐induced stereotyped behaviour. NRA0045 and clozapine selectively blocked behaviour associated with activation of the mesolimbic/mesocortical dopamine neurones rather than nigrostriatal dopamine neurones. Extracellular single‐unit recording studies demonstrated that MAP (1u2003mgu2003kg−1, i.v.) decreased the firing rate in the substantia nigra (A9) and ventral tegmental area (A10) dopamine neurones in anaesthetized rats. NRA0045 completely reversed the inhibitory effects of MAP on A10 dopamine neurones (ED50 0.1u2003mgu2003kg−1, i.v.), whereas the inhibitory effects of MAP on A9 dopamine neurones were not affected by NRA0045, in doses up to 1u2003mgu2003kg−1 (i.v.). Clozapine completely reversed the inhibitory effects of MAP on A10 dopamine neurones (ED50 1.9u2003mgu2003kg−1, i.v.) and on A9 dopamine neurones (ED50 2.5u2003mgu2003kg−1, i.v.). Haloperidol completely reversed the inhibitory effects of MAP on A10 (ED50 0.03u2003mgu2003kg−1, i.v.) and on A9 dopamine neurones (0.02u2003mgu2003kg−1, i.v.). NRA0045, like clozapine, was more potent in reversing the effects of MAP on A10 than A9 dopamine neurones. Prepulse inhibition (PPI) is impaired markedly in humans with schizophrenia. The disruption of PPI in rats by apomorphine (0.5u2003mgu2003kg−1, s.c.) was reversed significantly by NRA0045 (3u2003mgu2003kg−1, i.p.), clozapine (3u2003mgu2003kg−1, i.p.) and haloperidol (0.3u2003mgu2003kg−1, i.p.). Phencyclidine (PCP) elicits predominantly psychotic symptoms in normal humans and in schizophrenics. NRA0045 (0.03–0.3u2003mgu2003kg−1, i.p.) and clozapine (0.1–1u2003mgu2003kg−1, i.p.) significantly and dose‐dependently shortened the PCP(1.25u2003mgu2003kg−1, i.p.)‐induced prolonged swimming latency in rats in a water maze task, whereas haloperidol (0.01–0.1u2003mgu2003kg−1, i.p.) did not significantly alter swimming latency. These findings suggest that NRA0045 may have unique antipsychotic activities without the liability of motor side effects typical of classical antipsychotics.

MCL0042: a nonpeptidic MC4 receptor antagonist and serotonin reuptake inhibitor with anxiolytic- and antidepressant-like activity.

In the present study, we examined the anxiolytic and antidepressant effects of MCL0042, a novel compound showing activity in both MC4 receptor antagonism and serotonin transporter inhibition. MCL0042 showed relatively high affinity for the MC4 receptor and serotonin reuptake site, as determined by receptor binding assays. MCL0042 attenuated [Nle(4),d-Phe(7)]alpha-MSH-increased cAMP formation in MC4 receptor expressing cells, and it inhibited [(3)H]serotonin uptake by rat brain synaptosomes; thus, MCL0042 is an MC4 receptor antagonist and serotonin transporter inhibitor. Subcutaneous administration of MCL0042 significantly increased the number of licks in a Vogel punished drinking test in rats, and it also significantly attenuated swim stress-induced reduction in time spent in open arms in an elevated plus-maze task in rats, showing the anxiolytic-like potential of MCL0042. Moreover, repeated administration of MCL0042 for 14 days attenuated olfactory bulbectomy-induced locomotor hyperactivity in rats, indicating antidepressant-like potential. These data show that MCL0042 has unique properties of both the MC4 receptor antagonist and serotonin transporter inhibitor, and produces anxiolytic and antidepressant activity in rats. Moreover, blockade of both the MC4 receptor and serotonin reuptake sites might represent a useful approach in the treatment of anxiety and depression.

Effect of NE-100, a novel σ receptor ligand, on phencyclidine-induced cognitive dysfunction

N,N-Dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100) is a selective and potent σ receptor ligand. We investigated the effects of NE-100 on phencyclidine (PCP)-induced cognitive dysfunction in rats in a water maze task. NE-100 significantly shortened the PCP-induced prolonged swimming latency as did 1-(cyclopropylmethyl)-4-[2′(4″-fluorophenyl)-2′-oxoethyl]- piperidine monohydrobromide (Dup 734), 4-[2′-(4″-cyanophenyl)-2′-oxoethyl]-1-(cyclopropyl-methyl)piperidine (XJ 448), α-(4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazine butanol (BMY 14802) and rimcazole, all of which are σ receptor ligands and possibly antagonists. Ritanserin, a 5-HT2 receptor antagonist, also showed a tendency to shorten swimming latencies. Latencies of haloperidol-, cis-N-(1-benzyl-2-methyl-pyrrolidin-3-yl)-2-mrthoxy-5-chloro-4- methyl-aminobenzamide (YM-09151-2)- and sulpiride-, dopamine D2 receptor antagonists, treated groups did not differ from that seen in the PCP-treated group. Thus, PCP-induced cognitive dysfunciton may be improved by σ receptor ligands.

Regulation of NMDA-induced [3H] dopamine release from rat hippocampal slices through sigma-1 binding sites

To examine the interaction between ionotropic glutamate receptors and sigma binding sites, we made use of [3H]dopamine release from rat hippocampal slices. Agonists for ionotropic glutamate receptors such as N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate evoked release of [3H]dopamine from rat hippocampal slices, in a dose-dependent manner. (+)-Pentazocine, a prototype sigma1 agonist, attenuated the NMDA-induced [3H]dopamine release dose-dependently and significantly as did non-competitive NMDA antagonists such as 5-methyl-10,11-dihydro-5H-dibenzo(a,b)cyclohepten-5,10-imine maleate (MK-801) and phencyclidine. In contrast, (+)-pentazocine had no effect on AMPA- or on kainate-induced [3H]dopamine release. Sigma-1 receptor antagonists including N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl] ethylamine monohydrochloride (NE-100), 1(cyclopropylmethyl)-4-(2-(4-fluorophenyl)-2-oxoethylpiperidine (DuP734) and 1-(cyclopropylmethyl)-4-(2,4-cianophenyl)-2-oxoethyl)-pip eridine hydrobromide (XJ448) prevented significantly the inhibitory effect of (+)-pentazocine on NMDA-induced [3H]dopamine release, without affecting the release of [3H]dopamine evoked by NMDA. The inhibitory effect of (+)-pentazocine on [3H]dopamine release was preserved even in the presence of tetrodotoxin. These results suggest that sigma1 binding sites selectively interact with the NMDA receptor channel complex among ionotropic glutamate receptors, and that sigma1 binding sites may be involved in modulating the release of dopamine in the rat hippocampus by interacting with the NMDA receptor on dopaminergic nerve terminal.

A rat model of phencyclidine psychosis

Phencyclidine (PCP)-induced behavior in rats was investigated in water maze and diving behavior tasks. The swimming and diving latencies of PCP-treated groups placed in a water maze apparatus were gradually shortened, and prolonged, respectively, while rats in a control group performed well. In all rats, stereotyped behavior and hyperlocomotion were absent. We propose that this animal model induced by lower doses of PCP may be useful for further studies to research schizophrenia.