1 Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, 2 School of Medicine, Tehran University of Medical Sciences, Tehran and 3 Department of Pharmacology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
* Author to whom correspondence should be addressed at: Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145784, Tehran, Iran. Tel.: +98 21 611 2801; Fax: +98 21 640 2569; E-mail: zarinmr{at}ams.ac.ir
(Received 19 February 2004; first review notified 2 April 2004; in revised form 6 May 2004; accepted 21 June 2004)
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ABSTRACT |
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INTRODUCTION |
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Ethanol has been shown to affect many processes involved with central nervous system functions including memory. However, when administered as a single dose, the effects of ethanol on memory depend on several factors e.g. the animal, the dose and duration of ethanol administration and the performed test. Taking together the above variables, ethanol has been reported to impair (Holloway, 1972; Bammer and Chesher, 1982
; Castellano and Pavone, 1988
; Sasaki et al., 1995
) or to enhance memory (Mikolajczak et al., 2001
; Prediger and Takahashi, 2003
). Several hypotheses have been proposed to explain the acute effects of ethanol on memory in the laboratory animals. Henn et al. (1998)
have suggested that the effect of ethanol on memory may be due to an involvement of the GABAergic system. Moreover, there is evidence suggesting that the reinforcing effects of ethanol on the memory are mediated by the endogenous opioid system (Kalant, 1977
; Prediger and Takahashi, 2003
) or the cholinergic system (Stancampiano et al., 2004
).
Our preliminary results show that ethanol administration on the pretest day enhanced the memory recall in the step-down avoidance task. To explore the possible involvement of the GABAergic, endogenous opioidergic and cholinergic systems on the observed effects of ethanol, the animals were pretreated with bicuculline (GABAA antagonist), naloxone (opioid antagonist), atropine (muscarinic cholinergic antagonist) and mecamylamine (nicotinic cholinergic antagonist), respectively. The locomotor activity of the animals was studied as well.
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MATERIALS AND METHODS |
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Apparatus
The passive avoidance apparatus consisted of a wooden box (30 x 30 x 40 cm high) with a steel-rod floor (29 parallel rods, 0.3 cm in diameter set 1 cm apart). A wooden platform (4 x 4 x 4 cm) was set in the center of the grid floor. Intermittent electric shocks (1 Hz, 0.5 s and 50 V DC) were delivered to the grid floor by an insulated stimulator. Locomotor activity was measured with an activity meter, Animex, type S (LKB Farrad). The locomotor activity meter had a Plexiglas box (40 x 25 x 15 cm high).
Passive avoidance testing
Each mouse was gently placed on the wooden platform. When the mouse stepped down from the platform and placed its paws on the grid floor, intermittent electric shocks (1 Hz, 0.5 s and 50 V DC) were delivered for 15 s (Hiramatsu and Kameyama, 1995). This training procedure was carried out between 10:00 a.m. and 3:00 p.m.
Memory testing was performed 24 h later, in which each mouse was placed on the platform again and the step-down latency measured, in the absence of electric foot shocks, with a step-watch as passive avoidance behaviour. An upper cut-off of 300 s was set. The retention test was also carried out between 10:00 a.m. and 3:00 p.m. Saline, morphine and ethanol were injected 30 min before training or testing. This interval was set at 15 min for bicuculline, atropine and mecamylamine, and 5 min for naloxone.
Locomotion study
For locomotor activity study, each animal was placed in a plastic cage for 30 min to acclimatize to the environment before testing. Immediately after drug injection, animals were returned to the cage for measuring the locomotion. Counts were made for a period of 30 min.
Drug treatment
Ten animals were used to examine the effect of each dose of the tested drug. In experiments in which the animals received two or three saline or vehicle injections, the doses were adjusted in a way for each animal to receive a volume of at most 10 ml/kg.
In experiment 1, 10 animals received saline before training and testing. Other animals received 5 mg/kg morphine as pre-training treatment. These animals were treated 24 h later as pre-test treatment with saline, three different doses of morphine (0.5, 1 and 5 mg/kg), four different doses of ethanol (0.125, 0.25, 1 and 2 g/kg) or a combination of morphine (0.5 mg/kg) + ethanol (0.125 g/kg). We have also studied the effect of the above mentioned doses of morphine or ethanol on pre-training saline-treated mice.
In experiment 2, animals received morphine (5 mg/kg) as pre-training treatment. These animals received as pre-test treatment three different doses of bicuculline (0.5, 1 and 2 mg/kg), three different doses of naloxone (0.06, 0.25 and 1 mg/kg), two different doses of atropine (0.625 and 1.25 mg/kg) or three different doses of mecamylamine (0.5, 1 and 2 mg/kg). The same animals received ethanol at a dose of 0.25 g/kg or saline. In Fig. 2, data of the effects of pre-test treatment with saline, morphine (5 mg/kg) and ethanol (0.25 g/kg) from Fig. 1, are illustrated for comparison of the effects of bicuculline, naloxone, atropine and mecamylamine on the improvement of memory by ethanol.
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Data analysis
The retention latencies were expressed as the median and interquartile range. Because of the large individual variations, the data were analyzed by using KruskalWallis non-parametric one-way analysis of variance (ANOVA) followed by 2-tailed MannWhitney's U-test followed by Bonferoni's correction for the paired comparisons. For locomotor activity the counts were expressed as the mean ± standard errors. The data were analyzed by analysis of variance (ANOVA) followed by Tukey post hoc. In all statistical evaluations P < 0.05 was considered as the criterion for statistical significance.
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RESULTS |
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The injection of morphine at a dose of 5 mg/kg (s.c.) on the training day impaired memory retention. When the drug (0.5, 1 and 5 mg/kg) was injected 30 min before testing, the impaired memory was reversed: P = 0.052, 0.0036 and 0.004 for 0.5, 1 and 5 mg/kg morphine respectively, compared with the saline injected group (Morphine State-Dependency). The best effect was observed with 5 mg/kg of morphine.
Ethanol produced no change in training-day saline-treated animals (data not shown), but reversed the memory impairing effect of morphine (P = 0.015, 0.008, 0.0003 and 0.009 for 0.125, 0.25, 1 and 2 g/kg ethanol respectively). The co-administration of 0.125 g/kg ethanol with 0.5 mg/kg morphine did not increase the memory recall compared with that of the same dose of morphine (P = 0.531).
Bicuculline, naloxone, atropine and mecamylamine did not change the memory retention in saline or morphine treated animals (data not shown).
The administration of bicuculline after ethanol (0.25 g/kg) prevented the memory recall by ethanol (P = 0.238, 0.042 and 0.392 for 0.5, 1 and 2 mg/kg bicuculline, respectively). The effect of bicuculline was significant only at 1 mg/kg.
Naloxone prevented the ethanol-induced memory retrieval (P = 0.103, 0.012 and 0.024 for 0.06, 0.25 and 1 mg/kg, respectively). The effect of naloxone was significant at the doses of 0.25 and 1 mg/kg.
When atropine was injected after ethanol (0.25 g/kg), it prevented significantly the memory recall by ethanol (P = 0.038 and 0.036 for 0.625 and 1.25 mg/kg, respectively).
When mecamylamine was injected after ethanol (0.25 g/kg) it prevented the memory recall by ethanol (P = 0.024, 0.02 and 0.144 for 0.5, 1 and 2 mg/kg, respectively). The effect of mecamylamine was significant at the doses of 0.5 and 1 mg/kg.
Effect of the drugs on the locomotor activity of mice
Ethanol with the doses used in the present experiment showed no effect on locomotor activity either by itself or in combination with morphine.
The same results were obtained with different doses of bicuculline. When the drug was used at a dose of 0.5 mg/kg in combination with ethanol (0.25 g/kg), it significantly increased the locomotor activity (P = 0.032).
Naloxone, atropine and mecamylamine, at the doses used in the present experiment showed no effect on the locomotor activity either alone or in combination with a fixed dose of morphine (0.5 mg/kg) or ethanol (0.25 g/kg).
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DISCUSSION |
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Acute ethanol administration has been reported to have state-dependent effects on conditioned avoidance responding (Crow, 1966; Overton, 1966
). Moreover, the independent effects of ethanol on memory in man and laboratory animals have also been studied in detail (Fadda and Rossetti, 1998
; Ryabinin, 1998
).
There were two findings in the present report. First, the acute administration of different doses of ethanol (0.125, 0.25, 1 and 2 g/kg) improved morphine-induced memory impairment in a step-down passive avoidance task when they replaced morphine on the test day. Second, drugs which antagonized GABAergic, endogenous opioidergic or cholinergic systems (but not all doses of each antagonist) prevented the improving action of ethanol on memory (see Results).
Reports on the effects of ethanol administration on memory processes in laboratory animals are contradictory. Ethanol administration has been widely reported to impair memory in man and laboratory animals (Acheson et al., 2001). Passive avoidance procedures have also shown a decrease in memory retention following the administration of ethanol (Holloway, 1972
; Bammer and Chesher, 1982
; Castellano and Pavone, 1988
; Sasaki et al., 1995
). Contrary to the above findings Mikolajczak et al. (2001)
and Prediger and Takahashi (2003)
have demonstrated that under some experimental conditions ethanol facilitates short-term memory in laboratory animals. Recently Stancampiano et al. (2004)
have demonstrated that ethanol could have a biphasic action on the cholinergic system to cause deficit or to exert facilitator effects on memory. The same authors suggested that increased cholinergic transmission may contribute to the improving effects of ethanol on memory.
In the present study bicuculline, a GABAA receptor antagonist, prevented the improving effect of ethanol on memory recall. Several behavioural effects of ethanol are enhanced by GABAA receptor agonist and attenuated by antagonists or inverse agonists (Lister et al., 1991). Despite the plethora of data linking ethanol action to GABAA receptors, in vivo studies examining a direct interaction between these two are inconclusive (Morrow et al., 2001). Although initially the effects of ethanol on memory were considered to be hippocampus-independent, recent studies have shown that the hippocampus is involved in passive avoidance learning (for review, see Ryabinin, 1998
).
There is considerable evidence that the reinforcing effects of ethanol on memory are mediated by the endogenous opioid system and that some of the behavioural and pharmacological effects of ethanol are similar to those produced by opioids (Kalant, 1977; Prediger and Takahashi, 2003
). The fact that opioid antagonist reversed the effects of ethanol on the CNS also suggested an involvement of endogenous opioid peptides in the mechanism of action of this substance (Gianoulakis, 1993
). Taken together, it is unlikely that ethanol has a dramatic effect on opioid receptor binding. Our results showed that naloxone prevented the improving effect of ethanol on memory recall. According to Prunell et al. (1987)
both the CNS stimulant and depressant effects of ethanol in rats have been antagonized by naloxone. However, other investigators suggested that the effects of naloxone are probably due to a non-specific analeptic action rather than blockade of opioid receptors (Saddler et al., 1985
).
The activity of the cholinergic septo-hippocampal pathway plays a major role in the memory processes and is a possible target for the acute effect of ethanol (Cocco et al., 2002). Impairment of the hippocampal cholinergic system has only so far been observed after long term ethanol administration (Henn et al., 1998
). Higher ethanol doses decrease cortical cholinergic functions and lower doses stimulate hippocampal ACh release (Henn et al., 1998
). This is in agreement with the results of the present experiment in which both atropine and mecamylamine prevented the improving effect of ethanol on memory recall.
Apart from the involvement of GABAergic, endogenous opioidergic and cholinergic systems in the mechanism of action of ethanol on memory improvement, Davis and Walsh (1970) have suggested that ethanol metabolism in the body may result in the formation of morphine-like alkaloids (tetrahydropapaveroline) which may replace the effect of morphine on the test day to improve memory recall.
Moreover, Hoffman et al. (1990) have reviewed the part played by NMDA receptors on certain acute behavioural effects of ethanol and have concluded that these receptors may be involved in the effects of ethanol on memory. Nakagawa and Iwasaki (1996)
have investigated the part played by NMDA receptors in state-dependent learning (SDL) induced by ethanol in rats. They have concluded that NMDA receptor complex may not be involved in ethanol SDL. Napiorkowska-Pawlak et al. (2000)
have shown that dizacilpine (MK-801), a non competitive NMDA receptor antagonist, failed to change the amnesiac effect of ethanol in a passive avoidance task in mice. The above data do not support the hypothesis concerning the involvement of NMDA receptors in the effects of ethanol on memory.
The doses of ethanol used in the present study did not show any sedative effect, which was assessed by measuring locomotor activity. In fact, with the exception of one group of animals which were treated with bicuculline (0.5 mg/kg) + ethanol (0.25 g/kg), there were no significant changes in the locomotor activity of other groups of animals compared to the proper controls. The above results suggest that the locomotor activity and memory recall of the step-down passive avoidance task are not inter-related. This hypothesis is in agreement with the results reported by other investigators. Sanberg and Fibiger (1979) have demonstrated that oral administration of taurine resulted in the impairment in retention of a step-down passive avoidance task in rats without changes on spontaneous locomotor activity. McNamara et al. (1995)
have reported that treatment with (+/)3,4-methylenedioxymethamphetamine (MDMA) increased locomotor activity without a significant change in step-down passive avoidance behaviour in rats. Vianna et al. (2000)
have studied the involvement of protein kinase C isoforms on memory retrieval and found it unrelated to locomotor activity or anxiety level of rats. Barros et al. (2002)
have studied the effects of bupropion and sertraline on memory retrieval and found it unrelated to locomotor activity as well. Experiments with ethanol also failed to show a direct relationship between locomotor activity and memory in laboratory animals (McMillen et al., 1998
; Prediger and Takahashi, 2003
).
In conclusion, single injection of different doses of ethanol, when they replaced morphine on the test day, improved the impaired memory in step-down passive avoidance task. In the present study, blockade of GABAergic, endogenous opioidergic and cholinergic systems prevented this effect of ethanol, which is suggestive of the involvement of the above systems in the effects of ethanol on memory.
A review of the literature suggests other possibilities such as modulation of the release of intermediate neurotransmitters by ethanol. Studies concerning the effect of ethanol as pre-training treatment on memory, the use of different timing between ethanol administration and the test and the effects of chronic ethanol administration seem to be of interest.
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