J Pharmacol Sci 101, 325 – 328 (2006) Journal of Pharmacological Sciences ©2006 The Japanese Pharmacological Society Full Paper Potentiation of Ethanol in Spatial Memory Deficits Induced by Some Benzodiazepines

Atsushi Takiguchi1, Takayoshi Masuoka1, Yasuko Yamamoto1, Azusa Mikami1, and Chiaki Kamei1,* 1Department of Medicinal Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8530, Japan

Received May 9, 2006; Accepted June 28, 2006

Abstract. Triazolam caused no significant increase in the total error at 0.05 and 0.1 mg/kg. However, at 0.2 mg/kg, it caused a significant increase in total error. Almost the same findings were observed with brotizolam and rilmazafone. That is, at 0.2 and 0.5 mg/kg of brotizolam, 0.5 and 1.0 mg/kg of rilmazafone caused no significant increase in the total error. However, brotizolam at 1.0 mg/kg and rilmazafone at 2.0 mg/kg caused a significant increase in total error. Triazolam (0.05 mg/kg) and ethanol (1.0 g/kg) showed no significant effect on the numbers of errors when used alone separately, but the simultaneous use of triazolam and ethanol caused a significant increase in total error. Almost the same findings were observed with the coadministration of brotizolam (0.2 mg/kg) or rilmazafone (0.5 mg/kg) with ethanol. These results clearly indicate that all the short-acting benzodiazepines used in the study showed potentiation by ethanol in spatial memory deficits in mice.

Keywords: triazolam, brotizolam, rilmazafone, ethanol, radial maze

Introduction in comparison with benzodiazepines alone, in human beings. For example, Seppala et al. (7) reported that the Benzodiazepines are widely used as a remedy for disturbance of short-term memory and learning induced insomnia. However, it is known that benzodiazepines by diazepam was potentiated by ethanol in humans. cause anterograde amnesia as a side effect (1, 2). In Moreover, Simpson and Rush (8) also found that ethanol addition, it has been reported that triazolodiazepines, exacerbated the memory deficit caused by temazepam in such as triazolam and brotizolam, caused retrograde an eight-digit number recall task and the repeated amnesia as well as anterograde amnesia in animals acquisition procedure in human beings. In the animal (3, 4). Noguchi et al. (5) reported that benzodiazepines, experiments, Kitaichi et al. (9) reported that ethanol such as triazolam and nitrazepam, impaired the spatial exacerbated the memory deficits by triazolam or chlor- memory learning, that is, acquisition on Morris water diazepoxide in the condition fear stress task in mice. maze in rats. On the other hand, White et al. (6) reported Shimizu et al. (10) also demonstrated that the combined that diazepam produced a spatial working memory use of triazolam with ethanol potently impaired the deficit on the retrieval process of six-arm maze perfor- spatial memory of the water maze task in rats. These mance in rats. These reports indicate that benzo- two reports dealt with the potentiation by ethanol in diazepines impaired both the acquisition process and the memory deficits by benzodiazepines in the acquisi- retrieval process of spatial memory performance. There- tion process. However, it is still unclear about the fore, particular attention has been directed at the clinical coadministration effect of benzodiazepines with ethanol use of benzodiazepines. on the retrieval process of spatial memory in animals. It is generally recognized that the combined use of In the present study, therefore, we examined the benzodiazepines and ethanol potentiates memory deficits, effects of some benzodiazepines on the retrieval process of spatial memory using an eight-arm radial maze in *Corresponding author. [email protected] mice, and we also investigated whether or not these Published online in J-STAGE: August 5, 2006 benzodiazepines-induced memory deficits are potentiated doi: 10.1254/jphs.FPJ06008X by ethanol.

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Materials and Methods as the total error. Running time per choice was defined as the total running time divided by the number of arm Animals choice. Male ddY mice, 6-week-old at the beginning of the experiment (body weight: 28 – 31 g; Nippon SLC, Data analysis Shizuoka) were used. All animals were maintained in Values shown are each a mean ± S.E.M. One-way an air-conditioned room with controlled temperature analysis of variance (ANOVA) with Dunnett’s test or (24 ± 2°C) and humidity (55 ± 15%). Body weight Mann-Whitney U test was used for statistical analysis of was kept at 80% – 90% of normal mice in the experi- the results of radial maze performance. ment. Water was provided ad libitum. All procedures involving animals were conducted in accordance with Results the Guidelines for Animal Experiments at Okayama University Advanced Science Research Center. Effects of benzodiazepines on radial maze performance Triazolam caused no significant increase in total Drugs error at doses of 0.05 and 0.1 mg/kg. However, at a dose The following drugs were used: triazolam (Halcion®; of 0.2 mg/kg, it caused a significant increase in total Pharmacia & Upjohn, London, UK), brotizolam error. Almost the same findings were observed with (Lendormin®; Nippon Boehringer Ingelheim, Hyogo), brotizolam and rilmazafone. That is, at smaller doses rilmazafone (Rhythmy®; Shionogi, Osaka), and ethanol (0.2 and 0.5 mg/kg of brotizolam and 0.5 and 1.0 mg/kg (99.5% (v/v); Sigma-Aldrich Japan, Tokyo). Triazolam, of rilmazafone), there was no significant increase in total brotizolam, and rilmazafone were suspended in 0.5% error. However, brotizolam at a dose of 1.0 mg/kg and (w/v) carboxymethyl cellulose solution. Ethanol was rilmazafone at a dose of 2.0 mg/kg caused a significant diluted with 0.5% carboxymethyl cellulose solution to a increase in total error (Fig. 1). concentration of 10% (w/v). Ethanol was administered orally 60 min before the test trial; and triazolam, Effect of ethanol on radial maze performance brotizolam, and rilmazafone were administered orally Ethanol at doses of 0.5 and 1.0 g/kg showed no 30 min before the test trial. significant effect on the total error (Fig. 2).

Experimental apparatus and procedure Effects of simultaneous use of benzodiazepines and The apparatus used was as described in a previous ethanol on radial maze performance report (11). To familiarize the mouse with the radial Triazolam at a dose of 0.05 mg/kg, brotizolam at a maze, they received 1 daily habituation session for 2 dose of 0.2 mg/kg, and rilmazafone at a dose of days prior to training. On the first day, food pellets 0.5 mg/kg had no effect on the total number of errors (20 mg; Bio-Serv, A Holton Industries, Frenchtown, NJ, when used alone (Fig. 1). Therefore, these doses were USA) were scattered over the entire maze surface, and used to estimate the potentiation effect of ethanol on the four or five mice were simultaneously placed on the benzodiazepine-induced memory deficits. The simulta- radial maze and allowed to take pellets freely. On the neous use of triazolam at a dose of 0.05 mg/kg and next day, a pellet was placed in the food cup in each of ethanol at a dose of 1.0 g/kg caused a significant the eight arms, and the mouse was allowed to explore increase in total error in comparison with triazolam at a freely until it had taken all the pellets. After adaptation, dose of 0.05 mg/kg alone or ethanol at a dose of all mice were trained with two trials per day. In each 1.0 g/kg alone. Almost the same findings were observed trial, only four arms were baited, and the sequence was with brotizolam, that is, at a dose of 0.2 mg/kg or not changed throughout the experiment. The mouse rilmazafone at a dose of 0.5 mg/kg with ethanol showed was placed on the center platform, which was closed off a significant potentiation of total error in comparison by a door. After 20 s, the door was opened and the mouse with brotizolam, rilmazafone, or ethanol alone (Fig. 3). was allowed to make an arm choice to obtain food pellets until all four pellets had been eaten or 10 min Effects of simultaneous use of benzodiazepines and had elapsed. Mice were trained until reaching a criterion ethanol on running time per choice of at most one error per trial for five successive trials. No significant changes were observed with running Test trials were carried out the next day after reaching time per choice used as an index of locomotor activity the criterion. After the drugs administration, test trials in mice after the simultaneous use of these benzo- were performed in the same manner as training trials. diazepines and ethanol compared with benzodiazepines The number of entries into the unbaited arms was scored alone or ethanol alone (Fig. 4). Potentiation of Ethanol and Benzodiazepines 327

Fig. 2. Effect of ethanol on radial maze performance in mice. Mice showing no or one error per trial for five successive trials were used in the test. Ethanol was administered orally 60 min before the test trial. Column and vertical bars represent means ± S.E.M. (n = 12 – 15).

Fig. 1. Effects of benzodiazepines on radial maze performance in mice. Mice showing no or one error per trial for five successive trials were used in the test. Benzodiazepines were administered orally Fig. 3. Effects of simultaneous use of benzodiazepines and ethanol 30 min before the test trial. Column and vertical bars represent on radial maze performance in mice. Ethanol was administered ± = < < means S.E.M. (n 15 – 19). *P 0.05, **P 0.01, as compared with orally 60 min before the test trial; and triazolam, brotizolam, and the control group. rilmazafone were administered orally 30 min before the test trials. Column and vertical bars represent means ± S.E.M. (n = 11 – 14). *P<0.05, as compared with the benzodiazepine-treated group. # Discussion P<0.05, as compared with the ethanol-treated group.

In the present study, it was confirmed that the oral administration of triazolam (0.2 mg/kg), brotizolam (1.0 mg/kg), and rilmazafone (2.0 mg/kg) caused an increase in total error when used orally. Saraf et al. (3) reported that the intraperitoneal injection of triazolam and brotizolam impaired the spatial learning (acquisi- tion) and memory (retrieval) in the Morris water maze in mice. Shimizu et al. (10) also demonstrated that the intracerebroventricular injection of triazolam and rilmazafone resulted in spatial learning deficits in the Morris water maze task in rats. From these findings, it was revealed that triazolam, brotizolam, and rilmazafone impaired not only acquisition process but also the Fig. 4. Effects of simultaneous use of benzodiazepines and ethanol retrieval process of spatial memory performance in on running time per choice. Ethanol was administered orally 60 min before the test trial; and triazolam, brotizolam, and rilmazafone were mice. It is well recognized that a decrease in locomotor administered orally 30 min before the test trials. Column and vertical activity and an occurrence of muscle relaxant activity bars represent means ± S.E.M. (n = 11 – 14). 328 A Takiguchi et al may exert some influence on spatial memory. Therefore, benzodiazepine-induced memory deficits not only in the we measured the running time per choice as an index acquisition process but also in the retrieval process of of the spontaneous activity of mice. As a result, three memory. Thus, it can be assumed that the potentiating benzodiazepines caused no significant changes in effect of benzodiazepines with ethanol is mainly due to running time per choice compared with benzodiazepines an increase in the brain levels of benzodiazepines. alone or ethanol alone. These results clearly confirmed that an increase in total error induced by three benzo- References diazepines have no correlation with motor disturbance. 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