Selegiline potentiates the effects of EGb 761 in response to ischemic brain injury.

We evaluated whether combined treatment with selegiline, a selective MAO-B inhibitor, and EGb 761, a standard extract of Ginkgo biloba, has synergistic effects against ischemic reperfusion injury (IRI) in gerbils. Interestingly, we observed that pretreatment with EGb 761 significantly attenuated selegiline-induced hyperactivity. This finding paralleled striatal fos-related antigen immunoreactivity (FRA-IR) in mice. Four minutes of bilateral carotid artery occlusion caused substantial cell loss in the CA1 of the hippocampus 5 days post-ischemic insult. Pretreatment with EGb 761, with or without selegiline, significantly attenuated this neuronal loss. Combined treatment with EGb 761 plus selegiline was more efficacious in preventing this loss. Synaptosomal formations of protein carbonyl, lipid peroxidation (malondialdehyde (MDA) + 4-hydroxyalkenal (4-HDA)), and reactive oxygen species (ROS) in the hippocampus remained elevated 5 days post-ischemic insult. The antioxidant effects appeared to be most significant in the group treated with EGb 761 plus selegiline. This combined treatment produced more significant attenuation of IRI-induced alterations in intramitochondrial calcium accumulation, the mitochondrial transmembrane potential, and mitochondrial Mn-superoxide dismutase-like immunoreactivity (Mn-SOD-IR) than either treatment alone. Our results suggest that co-administration of EGb 761 and selegiline produces significant neuroprotective effects via suppression of oxidative stress and mitochondrial dysfunction without affecting neurological function.

Hyperfunction of dopaminergic and serotonergic neuronal systems in mice lacking the NMDA receptor epsilon1 subunit.

NMDA receptors, an ionotropic subtype of glutamate receptors (GluRs) forming high Ca(2+)-permeable cation channels, are composed by assembly of the GluRzeta subunit (NR1) with any one of four GluRepsilon subunits (GluRepsilon1-4; NR2A-D). In the present study, we investigated neuronal functions in mice lacking the GluRepsilon1 subunit. GluRepsilon1 mutant mice exhibited a malfunction of NMDA receptors, as evidenced by alterations of [(3)H]MK-801 binding as well as (45)Ca(2+) uptake through the NMDA receptors. A postmortem brain analysis revealed that both dopamine and serotonin metabolism were increased in the frontal cortex and striatum of GluRepsilon1 mutant mice. The NMDA-stimulated [(3)H]dopamine release from the striatum was increased, whereas [(3)H]GABA release was markedly diminished in GluRepsilon1 mutant mice. When (+)bicuculline, a GABA(A) receptor antagonist, was added to the superfusion buffer, NMDA-stimulated [(3)H]dopamine release was significantly increased in wild-type, but not in the mutant mice. GluRepsilon1 mutant mice exhibited an increased spontaneous locomotor activity in a novel environment and an impairment of latent learning in a water-finding task. Hyperlocomotion in GluRepsilon1 mutant mice was attenuated by treatment with haloperidol and risperidone, both of which are clinically used antipsychotic drugs, at doses that had no effect in wild-type mice. These findings provide evidence that NMDA receptors are involved in the regulation of behavior through the modulation of dopaminergic and serotonergic neuronal systems. In addition, our findings suggest that GluRepsilon1 mutant mice are useful as an animal model of psychosis that is associated with NMDA receptor malfunction and hyperfunction of dopaminergic and serotonergic neuronal systems.

Involvement of cyclic AMP systems in morphine physical dependence in mice: prevention of development of morphine dependence by rolipram, a phosphodiesterase 4 inhibitor.

In this study, we examined whether morphine dependence was inhibited by rolipram, a cyclic AMP selective phosphodiesterase inhibitor in mice, since a role for the cyclic AMP systems in the development of morphine dependence has been reported. Mice, which received morphine (10 mg kg(-1) s.c.) twice a day for 5 days showed withdrawal syndromes such as jumping, rearing and forepaw tremor following naloxone challenge (5 mg kg(-1) i.p.) on the 6th day. Such mice exhibited a significant elevation of cyclic AMP levels in the thalamus compared to control mice. However, co-administration of rolipram (1 mg kg(-1) i.p.) with morphine for 5 days significantly attenuated the severity of the withdrawal syndrome and the increase in the cyclic AMP levels after the administration of naloxone. In naïve mice, acute morphine treatment (10 mg kg(-1) s.c.) decreased cyclic AMP levels in the thalamus and cerebral cortex 10 min later. The decrease of cyclic AMP levels induced by acute morphine treatment was blocked by co-administration of rolipram (1 mg kg(-1) i.p.). However, acute rolipram did not affect the naloxone-precipitated morphine withdrawal syndrome. These results suggest that the elevation of the cyclic AMP levels is involved in the development of morphine withdrawal syndrome and that blockade of the morphine-induced reduction of cyclic AMP levels by chronic rolipram inhibits the development of dependence and the behavioural and biochemical changes induced by naloxone. Furthermore, rolipram may be a useful drug for attenuating the development of morphine dependence.

Involvement of brain-derived neurotrophic factor in spatial memory formation and maintenance in a radial arm maze test in rats.

Brain-derived neurotrophic factor (BDNF) regulates both short-term synaptic functions and activity-dependent synaptic plasticity such as long-term potentiation. In the present study, we investigated the role of BDNF in the spatial reference and working memory in a radial arm maze test. The radial arm maze training resulted in a significant increase in the BDNF mRNA expression in the hippocampus, although the expression in the frontal cortex did not change. When spatial learning was inhibited by treatment with 7-nitroindazole, an inhibitor of brain nitric oxide synthase, the increase in the hippocampal BDNF mRNA did not occur. To clarify the causal relation between BDNF mRNA expression and spatial memory formation, we examined the effects of antisense BDNF treatment on spatial learning and memory. A continuous intracerebroventricular infusion of antisense BDNF oligonucleotide resulted in an impairment of spatial learning, although the sense oligonucleotide had no effect. Treatment with antisense, but not sense, BDNF oligonucleotide was associated with a significant reduction of BDNF mRNA and protein levels in the hippocampus. Furthermore, treatment with antisense BDNF oligonucleotide in rats, which had previously acquired spatial memory by an extensive training, impaired both reference and working memory. There were no differences in locomotor activity, food consumption, and body weight between the antisense and sense oligonucleotide-treated rats. These results suggest that BDNF plays an important role not only in the formation, but also in the retention and/or recall, of spatial memory.

Modest neuropsychological deficits caused by reduced noradrenaline metabolism in mice heterozygous for a mutated tyrosine hydroxylase gene.

Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme for the biosynthesis of catecholamines that are considered to be involved in a variety of neuropsychiatric functions. Here, we report behavioral and neuropsychological deficits in mice carrying a single mutated allele of the TH gene in which TH activity in tissues is reduced to approximately 40% of the wild-type activity. In the mice heterozygous for the TH mutation, noradrenaline accumulation in brain regions was moderately decreased to 73-80% of the wild-type value. Measurement of extracellular noradrenaline level in the frontal cortex by the microdialysis technique showed a reduction in high K(+)-evoked noradrenaline release in the mutants. The mutant mice displayed impairment in the water-finding task associated with latent learning performance. They also exhibited mild impairment in long-term memory formation in three distinct forms of associative learning, including active avoidance, cued fear conditioning, and conditioned taste aversion. These deficits were restored by the drug-induced stimulation of noradrenergic activity. In contrast, the spatial learning and hippocampal long-term potentiation were normal in the mutants. These results provide genetic evidence that the central noradrenaline system plays an important role in memory formation, particularly in the long-term memory of conditioned learning.

[Development of sigma-receptor ligands and clinical trials].

There are at least two classes of sigma-receptors, termed sigma 1 and sigma 2. Recently, the sigma 1-receptor has been completely sequenced in different species. The amino acid sequences of the different purified proteins are highly homologous, but it shares no homology to know mammalian proteins. These results suggest that the sigma 1 receptor is a distinct entity from any often known receptors. sigma-Receptors are involved in many physiological functions. Therefore, sigma-ligands show many different pharmacological effects such as glucose utilization, neuroprotective, antipsychotic, antidepressive, anxiolytic, nootropic, antiepileptic, antiabuse, antitussive, antidiarrhea, anti-inflammatory, tear protein releasing stimulant and central anti-micturition reflex actions. These results suggest that sigma-receptors are very promising targets for the development of new drugs that have new mechanisms of action.

Improvement by nefiracetam of beta-amyloid-(1-42)-induced learning and memory impairments in rats.

1. We have previously demonstrated that continuous i.c.v. infusion of amyloid beta-peptide (A beta), the major constituent of senile plaques in the brains of patients with Alzheimer's disease, results in learning and memory deficits in rats. 2. In the present study, we investigated the effects of nefiracetam [N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl) acetamide, DM-9384] on A beta-(1-42)-induced learning and memory deficits in rats. 3. In the A beta-(1-42)-infused rats, spontaneous alternation behaviour in a Y-maze task, spatial reference and working memory in a water maze task, and retention of passive avoidance learning were significantly impaired as compared with A beta-(40-1)-infused control rats. 4. Nefiracetam, at a dose range of 1-10 mg kg(-1), improved learning and memory deficits in the A beta-(1-42)-infused rats when it was administered p.o. 1 h before the behavioural tests. 5. Nefiracetam at a dose of 3 mg kg(-1) p.o. increased the activity of choline acetyltransferase in the hippocampus of A beta-(1-42)-infused rats. 6. Nefiracetam increased dopamine turnover in the cerebral cortex and striatum of A beta-(1-42)-infused rats, but failed to affect the noradrenaline, serotonin and 5-hydroxyindoleacetic acid content. 7. These results suggest that nefiracetam may be useful for the treatment of patients with Alzheimer's disease.

NC-1900, an active fragment analog of arginine vasopressin, improves learning and memory deficits induced by beta-amyloid protein in rats.

We have reported that the continuous infusion of beta-amyloid protein-(1-40) into the rat cerebral ventricle produces learning and memory deficits accompanied by dysfunction in the cholinergic and dopaminergic systems. L-Pyroglutamyl-L-asparaginyl-L-seryl-L-prolyl-L-arginylglycinamide (NC-1900), an active fragment analog of arginine vasopressin in the rat brain, is a stable peptide with a five-fold longer half-life than that of arginine vasopressin-(4-9). In the present study, we examined the effects of NC-1900 on learning and memory deficits in beta-amyloid protein-(1-40)-infused rats. The rats were injected subcutaneously with NC-1900 (0.1 and 1 ng kg(-1)) once a day throughout the period of behavioral examination. In the beta-amyloid protein-infused rats, learning and memory in water maze and passive avoidance tasks were impaired compared with these in the control rats. NC-1900 prevented the learning and memory deficits in beta-amyloid protein-infused rats. Moreover, NC-1900 tended to increase the choline acetyltransferase activity in the frontal cortex of the beta-amyloid protein-infused rats. These results suggested that NC-1900 could be useful for the treatment of patients with Alzheimer's disease.

Continuous infusion of beta-amyloid protein into the rat cerebral ventricle induces learning impairment and neuronal and morphological degeneration.

To investigate the toxicity of beta-amyloid protein, a component of the senile plaques in Alzheimer's disease, it was infused into the cerebral ventricle of rats for 14 days by a mini-osmotic pump. Performances in the water maze and passive avoidance tasks in beta-amyloid protein-treated rats were impaired. Choline acetyltransferase activity significantly decreased in the hippocampus both immediately and 2 weeks after the cessation of the infusion. However, the learning impairment was recoverable 2 weeks after cessation of the infusion. Both immediately and 2 weeks after the cessation of the infusion, glial fibrillary acidic protein immunoreactivity increased. Furthermore, beta-amyloid protein altered the staining in the nuclei of hippocampal cells for only 2 weeks after the cessation. These results suggest that beta-amyloid protein produces some damage in the central nervous system in vivo.

Effects of dietary vegetable oils on behavior and drug responses in mice.

Previously, we noted significant differences in the behavioral patterns of mice fed safflower oil with a very low alpha-linolenate/linoleate ratio and perilla oil with a high alpha-linolenate/linoleate ratio from mothers to offsprings. In this report, we compared the behavior and drug responses in mice fed diets containing six different vegetable oils-corn, rapeseed, soybean, safflower, perilla and a mixture of perilla and safflower oils- for a relatively short period: 8 months after weaning. Soybean oil is a component of most conventional diets and was used as a control. The alpha-linolenate/linoleate ratios of the oils appeared to affect the locomotor activities in a wheel cage: the activity decreased in the order of safflower, the mixture (1:1) and the perilla oil groups. However, the rapeseed oil group exhibited much higher locomotor activity than that expected from the alpha-linolenate/linoleate ratio. Additionally, the rapeseed oil group exhibited unusual behavior patterns, including higher ambulation and rearing activities, faster acquisition of the water maze task and slower habituation behavior as compared with the control group. Susceptibility to pentobarbital anesthesia tended to be higher in the rapeseed oil group. The differences in the alpha-linolenate/linoleate ratios of these oils alone do not account for the observed differences in the behavioral patterns among the six dietary groups. Although we cannot exclude the possibility that the observed behavioral anomaly is due to the unique fatty acid composition of rapeseed oil, we speculate that a factor(s) other than fatty acids in rapeseed oil affected nervous system functions.

Dysfunction of cholinergic and dopaminergic neuronal systems in beta-amyloid protein--infused rats.

Accumulations of beta-amyloid protein are characteristic and diagnostic features of the brain of Alzheimer's disease patients; however, the physiological role of this protein in CNS is unknown. We have previously reported that continuous infusion of beta-amyloid protein into rat cerebral ventricle impairs learning ability and decreases choline acetyltransferase activity, a marker enzyme of cholinergic neuron. In this study, the effects of beta-amyloid protein infusion on the release of neurotransmitters in cholinergic and dopaminergic neuronal systems were investigated by using an in vivo brain microdialysis method. Nicotine-stimulated release of acetylcholine and dopamine in these animals was significantly lower than that in vehicle-infused rats. Further, dopamine release induced by high-K stimulation was decreased in beta-amyloid protein-infused rats compared with vehicle-infused rats. These results suggest that the release of the two transmitters, acetylcholine and dopamine, was decreased by beta-amyloid protein and that learning deficits observed in the beta-amyloid protein-infused rats are partly due to the impairment of neurotransmitter release. Furthermore, continuous infusion of beta-amyloid protein may be a useful method to produce the animal model of Alzheimer's disease.

Involvement of nitric oxide in phencyclidine-induced hyperlocomotion in mice.

The present study was undertaken to investigate the involvement of nitric oxide (NO) in the behaviors induced by 1-(1-phenylcyclohexyl) piperidine (phencyclidine; PCP) in mice, using N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase. PCP (1, 3, and 10 mg/kg s.c.) dose dependently induced hyperlocomotion and stereotyped behaviors, including sniffing, head movement, and ataxia, in mice. PCP also caused a marked deficit of motor coordination in mice, the effect being exerted in a dose-dependent manner. Although pretreatment with L-NAME (50 mg/kg i.p.) slightly enhanced the ataxia induced by PCP (3 mg/kg), it failed to modify other stereotyped behaviors and the lack of motor coordination induced by PCP (2 mg/kg). The hyperlocomotion induced by PCP (3 mg/kg) was significantly enhanced by L-NAME (5 and 50 mg/kg) and 7-nitro indazole (25 mg/kg), but not by D-NAME (50 mg/kg), a less active enantiomer of L-NAME. However, the behavioral changes induced by PCP, at the high dose, 10 mg/kg, were not enhanced by L-NAME and D-NAME. The enhancing effects of L-NAME on the PCP (3 mg/kg)-induced hyperlocomotion were significantly prevented by L-arginine (1 g/kg i.p.). However, D-arginine (1 g/kg i.p.) and L-lysine (1 g/kg i.p.) had no effect in this regard. These results suggested the involvement of central NO production in the mediation of PCP-induced behaviors, hyperlocomotion in particular, in mice.

Effects of traditional Chinese medicines on pharmacokinetics of levofloxacin.

The effects of single coadministrations of one of three traditional Chinese medicines, Hotyu-ekki-to, Rikkunshi-to, and Juzen-taiho-to, on the pharmacokinetics of levofloxacin (LVFX) were investigated with eight healthy volunteers in an open, random crossover fashion. Subjects each received a single oral dose of LVFX (200 mg) alone and then with a single coadministration of each Chinese medicine. There were no significant differences in any pharmacokinetic parameters of LVFX between the groups. Also, no significant changes in the urinary recovery (> 80%) and renal clearance of LVFX were observed. These results indicate that the Chinese medicines tested have no significant effect on the rate and extent of bioavailability or renal excretion of LVFX.

Memory impairment and neuronal dysfunction induced by beta-amyloid protein in rats.

Alzheimer's disease (AD) is characterized by the presence of senile plaques. The core of the plaque consists of beta-amyloid protein. In AD patients, learning and memory are impaired with a concomitant loss of the cholinergic marker enzyme, choline acetyltransferase (ChAT). However, direct evidence that beta-amyloid protein is related to the impairment of learning and memory has not been demonstrated. In this study, we investigated whether memory impairment and neuronal dysfunction were produced after 2 weeks continuous infusion of beta-amyloid protein (3, 30 and 300 pmol/day) into the cerebral ventricles in adult rats. To investigate the ability of learning and memory in beta-amyloid protein-treated rats, water maze and passive avoidance tasks were carried out. The performance of both tasks in beta-amyloid protein-treated rats was impaired. ChAT activity in the frontal cortex (3 and 30 pmol/day) and hippocampus (300 pmol/day) significantly decreased. These results suggest that beta-amyloid protein is related to the impairment of learning and memory, and neurodegeneration, and that beta-amyloid protein-treated rats could be used as an animal model for AD.

[Experimental techniques for developing new drugs acting on dementia (6)--Carbon monoxide-induced amnesia model in experimental animals].

Cell death, neuronal dysfunction and deterioration of memory function can be produced after carbon monoxide exposure in mice as in human. These deficiencies are developed in a delayed manner (delayed amnesia). The neurotoxicity of excitatory amino acids may be involved in this model, since dizocilpine (MK-801) fully protects against carbon monoxide-induced cell death, learning impairment and delayed amnesia. In the present paper, we described the method of carbon monoxide exposure and the characteristic of behavioral and biochemical changes after carbon monoxide exposure. These data indicate that carbon monoxide can provide an amnesic model for the investigation of memory deterioration and the development of new anti-amnesic drugs.

Effects of Kamikihito, a traditional Chinese medicine, on neurotransmitter receptor binding in the aged rat brain determined by in vitro autoradiography (2): changes in GABAA and benzodiazepine receptor binding.

We investigated the effects of the long-term administration of Kamikihito (KKT) on the specific binding of [3H]muscimol and [3H]flunitrazepam in the brains of young and aged rats using in vitro quantitative autoradiography. Specific [3H]muscimol binding in aged rats was decreased in all brain regions examined compared with that in young rats, whereas [3H]flunitrazepam binding did not change in any of the brain regions. Scatchard analysis revealed that the maximal number of [3H]muscimol binding sites in the cortex and thalamus was significantly decreased in aged rats compared with young rats, while its affinity remained unchanged. Long-term administration of KKT in young rats had no effect on either [3H]muscimol or [3H]flunitrazepam binding. In contrast, the same treatment in aged rats produced a significant increase in [3H]flunitrazepam binding to the cortex, caudate/putamen and accumbens, and it tended to decrease the [3H]muscimol binding. These results suggest that the selective reduction of specific [3H]muscimol binding in the brain may be responsible, at least in part, for anxiety-related behavior in aged rats. Furthermore, it appears that the significant increase in specific [3H]flunitrazepam binding produced in the brains of aged rats by the long-term administration of KKT may be responsible for the anxiolytic effects of this agent.

Pharmacokinetic characteristics of 5-fluorouracil and mitomycin C in intraperitoneal chemotherapy.

Eight patients with malignancies confined to the peritoneal space participated in this study. Five hundred milligrams 5-fluorouracil or 10 mg mitomycin C was diluted in 1 L saline. The mixed solution was injected intraperitoneally through the semi-permanent peritoneal catheter. Blood and peritoneal fluid were collected after injection. 5-Fluorouracil concentrations in the peritoneal fluid were 1000 times those in serum, while mitomycin C concentrations were 100 times those in serum. Areas under the concentration vs time curve (AUC) were calculated by the trapezoidal method with extrapolation to infinity. The ratio of peritoneal fluid AUC to serum AUC was about 1400 for 5-fluorouracil and 80 for mitomycin C. Patterns for the absorption and elimination from systemic circulation were similar for both compounds. Drug concentrations in the peritoneal fluid and serum were analysed according to the compartment model. The half-life in the peritoneal fluid (t1/2p) and the rate constant from the peritoneal fluid to the systemic circulation (ka) were nearly equal for both 5-fluorouracil and mitomycin C (t1/2p, 1.0 h for 5-fluorouracil and 1.3 h for mitomycin C; ka 0.71 h-1 for 5-fluorouracil and 0.68 h-1 for mitomycin C), although the apparent volume of distribution (Vds/F) and clearance in the peritoneal cavity (CLp) for mitomycin C (78 L m-2 and 1.8 L h-1 m-2) were about twice the values for 5-fluorouracil (149 L m-2 and 0.8 L h-1 m-2).

Effects of kamikihito, a traditional Chinese medicine, on neurotransmitter receptor binding in the aged rat brain determined by in vitro autoradiography: changes in dopamine D1 and serotonin 5-HT2A receptor binding.

Using in vitro autoradiography, we investigated the effects of Kamikihito (KKT), a traditional Chinese medicine, on specific [3H]SCH23390 binding to dopamine D1 receptors and [3H]ketanserine binding to serotonin 5-HT2A receptors in the rat brain. Specific binding of both compounds was affected by aging. Long-term administration of KKT resulted in decreases in [3H]SCH23390 binding to the cortex and hippocampus in aged rats, and in decreases in [3H]ketanserine binding to the caudate/putamen in young rats. These results suggest that the changes in dopamine Di and serotonin 5-HT2A receptor binding may be involved in the central effects of KKT.

Effects of kamikihito, a traditional Chinese medicine, on neurotransmitter receptor binding in the aged rat brain determined by in vitro autoradiography (1): Changes in the [3H]QNB binding.

Using in vitro autoradiography, we investigated the effects of Kamikihito (KKT), a traditional Chinese medicine, on the specific binding of [3H]quinuclidinyl benzilate (QNB) and [3H]N-(1-[2-thienyl]cyclohexyl)-3,4-piperidine (TCP) in the rat brain. The Bmax but not the Kd values for [3H]QNB binding to the caudate/putamen and accumbens in aged rats were lower than those in young rats. The [3H]TCP binding was also decreased in aged rats compared with that in young rats. Long-term administration of KKT modulated the [3H]QNB binding in young but not aged rats.