Tsutomu Kameyama

Utility of an elevated plus-maze for the evaluation of memory in mice: effects of nootropics, scopolamine and electroconvulsive shock

An elevated plus-maze consisting of two open and two enclosed arms was employed for an evaluation of memory in mice. Mice in the plus-maze escaped from the open arm to the enclosed arm because mice apparently dislike open and high spaces. The time it took for the mice to move from the open arm to the enclosed arm (transfer latency) was recorded. The transfer latency after the 2nd day was significantly shorter than that on the 1st day when it was recorded at a rate of one trial a day for 5 days. The transfer latency on the 2nd day was significantly prolonged in the mice administered electroconvulsive shock (300 V, 1 s) or scopolamine (20 µg, ICV) immediately after the first trial compared to the transfer latency in the control group. The prolongation of transfer latency in the mice administered an electroconvulsive shock was reversed by pretreatment with aniracetam (20 mg/kg, PO), but not tacrine and physostigmine. The prolongation of transfer latency in the mice administered scopolamine was reversed by pretreatment with aniracetam (10 and 20 mg/kg, PO) tacrine (1 and 3 mg/kg, PO), or physostigmine (0.025–0.2 mg/kg, IP). These results suggest that transfer latency may be one of the parameters of learning and memory.

Protective effects of idebenone and α-tocopherol on β-amyloid-(1–42)-induced learning and memory deficits in rats: implication of oxidative stress in β-amyloid-induced neurotoxicity in vivo

Amyloid β‐peptide (Aβ), the major constituent of the senile plaques in the brains of patients with Alzheimers disease, is cytotoxic to neurons and has a central role in the pathogenesis of the disease. Previous studies have suggested that oxidative stress is involved in the mechanisms of Aβ‐induced neurotoxicity in vitro. In the present study, we examined whether oxidative stress contributes to learning and memory deficits caused by continuous intracerebroventricular infusion of Aβ‐(1–42). In the Aβ‐(1–42)‐infused rats, spontaneous alternation behaviour in a Y‐maze and spatial memory in a water maze task were significantly impaired, as compared with Aβ‐(40–1)‐infused control rats. The retention of passive avoidance learning was also significantly impaired by treatment with Aβ‐(1–42). Potent antioxidants idebenone and α‐tocopherol prevented the behavioural deficits in Y‐maze and water maze, but not passive avoidance, tasks in Aβ‐(1–42)‐infused rats when they were repeatedly administered by mouth once a day from 3 days before the start of Aβ infusion to the end of behavioural experiments. Lipid peroxide levels in the hippocampus and cerebral cortex of Aβ‐(1–42)‐infused rats did not differ from those in control animals, and neither idebenone nor α‐tocopherol affected the lipid peroxide levels. These results suggest that treatment with antioxidants such as idebenone and α‐tocopherol prevents learning and memory deficits caused by Aβ.

Behavioral evidence for a modulating role of σ ligands in memory processes. I. Attenuation of dizocilpine (MK-801)-induced amnesia

The potentiating effect of low doses of sigma ligands on the N-methyl-D-aspartate (NMDA)-induced excitation of pyramidal CA3 dorsal hippocampal neurons has recently been reported. In the present study, we investigated behavioral effects relevant to these findings in the experimental amnesia induced by the non-competitive NMDA antagonist, dizocilpine (MK-801), in mice. At doses below 1 mg/kg s.c., the sigma ligands, 1,3-di-(2-tolyl)guanidine (DTG), (+)-SKF 10,047, and (+)-pentazocine, but not their (-)-isomers, significantly decreased MK-801 (100 microgram/kg s.c.)-induced impairment of spontaneous alternation performances in 8-min sessions of a Y-maze exploration, an index of spatial working memory, without affecting the concomitant hyperlocomotion. The effect of DTG (100 micrograms/kg s.c.) was completely antagonized by the simultaneous administration of BMY 14802 (10 mg/kg i.p.) and NE-100 (1 mg/kg i.p.), two putative sigma antagonists, which had no effect by themselves. In long-term memory tests (step-down and step-through types of passive avoidance, elevated plus-maze), DTG exhibited a significant attenuation of MK-801-induced amnesia, at doses of 10 and 100 micrograms/kg s.c. In all tests of short- and long-term memory, the effects exhibited by the sigma ligands tested had a bell-shaped curve; no effect was seen at 1 mg/kg. DTG did not affect the impairment of alternation induced by CPP (5 mg/kg i.p.): the modulation may selectively target the blockade of NMDA receptor-associated ion channels. Moreover, DTG (1-1000 micrograms/kg) did not affect the impairment induced by scopolamine (1 mg/kg i.p.) or diazepam (4 mg/kg i.p.), but significantly prevented the impairment induced by mecamylamine (10 mg/kg i.p.). These results suggest that the potentiating effect of sigma ligands on NMDA receptor-mediated glutamatergic neurotransmission, already demonstrated electrophysiologically, may have some relevance to learning and memory processes in the hippocampus. A similar modulation may also affect cholinergic nicotinic systems.

Utility of an elevated plus-maze for dissociation of amnesic and behavioral effects of drugs in mice

Learning and memory were previously evaluated by using the elevated plus-maze test in mice. We investigated whether this method could be used for the evaluation of amnesic properties of drugs, including those which alter behavior on the first (training) trial. Six drugs of different types, scopolamine, MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate), diazepam, butylscopolamine, methamphetamine and haloperidol were administered before training. The transfer latency of vehicle-treated mice on retention testing was significantly shorter than that on training. The transfer latencies in mice given scopolamine (1 and 3 mg/kg s.c.), butylscopolamine (6 mg/kg s.c.), methamphetamine (2 and 4 mg/kg i.p.), or haloperidol (0.4 mg/kg i.p.) were significantly prolonged on training compared with those of the corresponding vehicle groups. However, significant prolongation of the transfer latency in the retention test, compared to the vehicle groups, was observed only in mice given scopolamine (3 mg/kg s.c.), MK-801 (0.1 and 0.15 mg/kg i.v.), diazepam (4 mg/kg i.p.), or methamphetamine (4 mg/kg i.p.). These results suggested that the prolongation of the transfer latency on retention testing in the plus-maze method might be used as an indicator for impairment of learning and memory induced by the drugs which have amnesic properties, and is not related to the change in transfer latency on training.

Nitric oxide synthase inhibitors impair reference memory formation in a radial arm maze task in rats.

We investigated the role of nitric oxide (NO) in learning and memory formation in a radial maze test, by using the NO synthase (NOS) inhibitors, NG-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI), and an NO precursor, L-arginine. Rats were trained on a working and reference memory task in an eight arm radial maze for 30 days, wherein the same four arms were baited for each daily training trial. Daily administration of the specific neuronal NOS inhibitor 7-NI, as well as the non-selective NOS inhibitor L-NAME, impaired the acquisition of the task. Analysis of memory categories affected by NOS inhibitors revealed that reference memory formation was impaired by the treatment with L-NAME and 7-NI. L-NAME also impaired working memory although 7-NI had no effect. L-Arginine significantly increased the choice accuracy, by reducing the reference memory errors, of the radial arm maze task during the last ten days although it had no effect during the first 20 trials. These results suggest that NO plays an important role in spatial reference memory formation.

Role of nitric oxide and cyclic GMP in the dizocilpine-induced impairment of spontaneous alternation behavior in mice.

The activation of N-methyl-D-aspartate receptors induces the synthesis of nitric oxide, which activates soluble guanylate cyclase and leads to the formation of cyclic GMP in the brain. The inhibition of nitric oxide production, as well as the blockade of N-methyl-D-aspartate receptors, has been reported to prevent the induction of hippocampal long-term potentiation and learning and memory formation in vivo, although the effects of inhibitors of nitric oxide synthase are still controversial. We investigated the putative role of nitric oxide and cyclic GMP in dizocilpine-induced memory impairment in mice. The nitric oxide synthase inhibitors, NG-nitro-L-arginine methyl ester and 7-nitro indazole, as well as dizocilpine, a non-competitive N-methyl-D-aspartate receptor antagonist, dose-dependently impaired spatial working memory in mice, assessed by their spontaneous alternation behavior in a Y-maze. The inhibitory effects of both NG-nitro-L-arginine methyl ester and dizocilpine on their behavior were completely reversed by 8-bromo-cyclic GMP. Cyclic GMP levels in the cerebellum were reduced by treatment with dizocilpine. NG-Nitro-L-arginine methyl ester and 7-nitro indazole reduced cyclic GMP levels in the cerebral cortex/hippocampus and cerebellum, and the suppressive effect of NG-nitro-L-arginine methyl ester on cyclic GMP levels in the cerebral cortex/hippocampus was reversed by co-treatment with L-arginine. Cyclic AMP levels in the brain were not affected by treatment with either dizocilpine, NG-nitro-L-arginine methyl ester, or 7-nitro indazole. Neither NG-nitro-L-arginine methyl ester nor L-arginine had any effect on monoamine and acetylcholine metabolism in the brain. These results suggest that the reduction in nitric oxide/cyclic GMP production in the brain may be responsible for dizocilpine-induced impairment of spontaneous alternation behavior in a Y-maze.

Step-down-type passive avoidance- and escape-learning method. Suitability for experimental amnesia models.

A method for evaluating passive avoidance- and escape-learning responses simultaneously has been developed for the study of learning and memory in mice. Prolongation of the step-down latency and shortening of the escape latency in the retention test depended on the strength of the voltage of the electric shocks delivered during the training test. Therefore, the step-down latency and escape latency may be good parameters of learning and memory performance. By cycloheximide treatment immediately after training, the step-down latency and escape latency were shortened and prolonged, respectively, in a dose-related manner, and the relationship between the step-down latency and escape latency was significant. Treatment with cycloheximide within 30 min after training caused significant amnesia, but not after more than 60 min. Furthermore, amnesic action of cycloheximide developed 24 hr after the treatment, but not within 4 hr. On the other hand, the step-down latency and/or the escape latency in the training test were changed by pretreatment with diethyldithiocarbamate and scopolamine. Therefore, the amnesic action of these drugs administered before the training should be investigated in detail. The present method, simultaneously estimating passive avoidance- and escape-learning responses, may be useful for the development of experimental amnesia models.

Improvement by nefiracetam of β‐amyloid‐(1‐42)‐induced learning and memory impairments in rats

We have previously demonstrated that continuous i.c.v. infusion of amyloid β‐peptide (Aβ), the major constituent of senile plaques in the brains of patients with Alzheimers disease, results in learning and memory deficits in rats. In the present study, we investigated the effects of nefiracetam [N‐(2,6‐dimethylphenyl)‐2‐(2‐oxo‐1‐pyrrolidinyl) acetamide, DM‐9384] on Aβ‐(1–42)‐induced learning and memory deficits in rats. In the Aβ‐(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 Aβ‐(40–1)‐infused control rats. Nefiracetam, at a dose range of 1–10 mg kg−1, improved learning and memory deficits in the Aβ‐(1–42)‐infused rats when it was administered p.o. 1 h before the behavioural tests. Nefiracetam at a dose of 3 mg kg−1 p.o. increased the activity of choline acetyltransferase in the hippocampus of Aβ‐(1–42)‐infused rats. Nefiracetam increased dopamine turnover in the cerebral cortex and striatum of Aβ‐(1–42)‐infused rats, but failed to affect the noradrenaline, serotonin and 5‐hydroxyindoleacetic acid content. These results suggest that nefiracetam may be useful for the treatment of patients with Alzheimers disease.

Development of tolerance and supersensitivity to phencyclidine in rats after repeated administration of phencyclidine

In rats treated with phencyclidine (PCP) repeatedly (PCP 10 mg/kg per day for 14 days), the back-pedalling, head-weaving and turning induced by PCP were attenuated (tolerance), while PCP-induced sniffing, rearing and ambulation were potentiated (supersensitivity). The behavior induced by the direct and indirect serotonin (5-HT) agonists, 5-methoxy-N,N-dimethyltryptamine and p-chloroamphetamine, was attenuated, while the sniffing, rearing or licking induced by the direct and indirect dopamine (DA) agonists, apomorphine and methamphetamine, were potentiated in the chronic PCP-treated rats. The DA and 5-HT contents in the nucleus accumbens and the ratio of HVA to DA in the striatum increased following the repeated PCP administration. Pentobarbital-induced sleep time did not change in the chronic PCP-treated rats as compared with the control rats. In addition, there was no significant difference between the disappearance rate of PCP in the brain of the rats treated with PCP repeatedly and the rate in the control rats. These results suggest that functional changes in the dopaminergic and serotonergic neuronal systems develop on repeated administration of PCP but that such changes do not develop in the hepatic drug-metabolizing system. In addition, tolerance develops in the serotonergic neuronal system while supersensitivity develops in the dopaminergic neuronal system. Biochemical findings suggest that increased mesolimbic dopaminergic neuronal function plays an important role in the development of the supersensitivity.

Serotonergic involvement in phencyclidine-induced behaviors

Administration of 5-10 mg/kg of phencyclidine (PCP) caused stereotyped behaviors including sniffing, backpedalling, head weaving and turning in rats. The PCP-induced stereotyped behaviors (backpedalling, head weaving and turning) were attenuated by serotonin (5-HT) depleters [reserpine, p-chlorophenylalanine, p-chloroamphetamine (PCA)] and 5-HT receptor antagonist (cyproheptadine). PCP-induced head weaving and turning were potentiated by 5-HT precursor (tryptophan) and 5-HT releaser (PCA). PCP-induced head weaving were potentiated also by monoamine oxidase inhibitor (pargyline) and 5-HT reuptake inhibitor (imipramine). PCP 5-10 mg/kg significantly increased the content of 5-HT in the thalamus/hypothalamus at 30 and 60 min after the injection, except PCP 5 mg/kg at 60 min. PCP 7.5 and 10 mg/kg increased the rate of increment of 5-HT by pargyline in the thalamus/hypothalamus at 30 and 60 min after the injection, respectively. PCP 10 mg/kg significantly increased the contents of 5-HIAA in the striatum and thalamus/hypothalamus at 30 min, but decreased that of 5-HIAA in all discrete brain areas except the stratium at 60 min after the injection. PCP also significantly prevented the depletion of 5-HT by PCA in all discrete brain areas except the stratium at 60 min after the injection. From these results, PCP-induced stereotyped behaviors are related to an increased serotonergic neuronal activity due to 5-HT releasing action and/or inhibitory action of 5-HT uptake-by this drug.