Neuropharmacology 42 (2002) 577–585 www.elsevier.com/locate/neuropharm Nimodipine prevents the effects of ethanol in tests of memory S.P. Brooks, G. Hennebry, G.P.R. McAlpin, G. Norman, H.J. Little ∗ Drug Dependence Unit, Psychology Department, Durham University, South Road, Durham DH1 3LE, UK Received 7 August 2001; received in revised form 21 November 2001; accepted 8 January 2002 Abstract The effects of acute administration of the dihydropyridine calcium channel antagonist, nimodipine, were studied on the actions of ethanol in the radial arm maze and the object recognition test. In the former test, the effects of the drugs were examined on the performance in finding the four baited arms, after previous training in this task. Ethanol, at 1 g/kg, increased both the number of re-entries into baited arms (counted as errors of working memory) and the total number of arm choices required to complete the task. Administration of nimodipine, 10 mg/kg, with the ethanol, completely prevented the deleterious effects on memory in this task, but had no effects on the performance when given in the absence of ethanol. In the object recognition task, ethanol, 1 g/kg, significantly decreased the differences in the time spent exploring novel and familiar objects. Nimodipine, 10 mg/kg, given with the ethanol, completely prevented this effect, but nimodipine alone had no effects. The lack of changes in total exploration times indicated that the effects of ethanol in these tests were not due to loss of motor co-ordination or of alertness. The results are discussed in the light of the known actions of the drugs on brain function.  2002 Elsevier Science Ltd. All rights reserved. Keywords: Ethanol; Nimodipine; Memory; Dihydropyridine; Radial arm maze; Object recognition 1. Introduction Dihydropyridine calcium channel antagonists are selective for the “L”-subtype of high-voltage activated calcium channel (Nowycky et al., 1985; Docherty and Brown, 1986). They have little effect on behaviour or neuronal activity in normal circumstances, although they easily enter the brain and have high affinity binding sites in the CNS. We have previously demonstrated neuronal actions of these drugs both in vitro and in vivo, after chronic alcohol consumption (Whittington et al., 1991; Bailey et al., 1998; Watson and Little, 1999). When administered acutely, dihydropyridine calcium channel antagonists prevented ethanol withdrawal hyperexcitability, both in vivo, and in hippocampal neurons in vitro (Littleton et al., 1990; Whittington and Little, 1991; Bailey et al., 1998). They also prevented the development of ethanol tolerance, when given chronically concurrent with the ethanol (Dolin and Little, 1989). Also prevented were some of the adaptive changes in neurons that are Corresponding author. Tel.: +44-191-374-7768; fax: +44-191374-7774. E-mail address: hilary.little@durham.ac.uk (H.J. Little). ∗ caused by chronic alcohol intake (Whittington et al., 1991; Dolin and Little, 1989). Dihydropyridine calcium channel antagonists have been shown to alleviate memory deficits due to a range of causes (Izquierdo, 1990; Schuurman, 1993). Nimodipine ameliorated age-related impairments in memory, measured by a trace conditioning response and by habituation to an open field (Deyo et al., 1989). Performance after hippocampal lesions was improved by nimodipine in several studies, including the radial arm maze (Nelson et al., 1992), the holeboard test (Weichman et al., 1994) and a delayed response latency (DRL) task (Finger et al., 1990). Memory impairments following cerebral hypoxia were found to be alleviated by nicardipine, felodipine and nifedipine (Zupan et al., 1993). Hoffmeister et al. (1992) showed that nimodipine, over a wide dose range (0.05 to 50 mg/kg) decreased the amnesic effects of electroshock, as measured by a passive avoidance paradigm. Impaired habituation to a novel environment caused by chronic corticosterone administration was counteracted by concurrent administration of nimodipine (Dachir et al., 1997). There are also reports that nimodipine may produce cognitive improvements in humans with Alzheimer’s disease (Fritze and Walden, 1995). 0028-3908/02/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 0 2 ) 0 0 0 0 6 - 0 578 S.P. Brooks et al. / Neuropharmacology 42 (2002) 577–585 Ethanol has long been known to cause deficits in memory and learning, affecting both spatial and nonspatial working memory (Melchior et al., 1993; Givens, 1995; Givens and McMahon, 1997) and decreasing contextual conditioning (Melia et al., 1996). Some selectivity has been reported, as it is said to disrupt the acquisition and performance of spatial reference memory tasks, but not tasks involving non-spatial reference memory (Matthews et al., 1995; White et al., 2000), and to change the use of information during a task from spatial to non-spatial (Matthews et al., 1999). The present study examines the effects of the dihydropyridine calcium channel antagonist, nimodipine, on the deficits produced by ethanol in tasks which involve different types of memory i.e. the radial arm maze and the object recognition test (Ennaceur and Delacour, 1988). The radial arm maze test involves the use of spatial information in learning the location of the baited arms and involves both working and reference memory. The object recognition test, in contrast, does not involve the use of spatial information but measures the familiarity of an object. 2. Methods Male Lister Hooded rats aged between 4 and 6 months old were housed in pairs under 12 hour on/off (08.00– 20.00) reversed phase lighting conditions. Throughout the experiment all animals had free access to water and were weighed daily. Those used in the radial maze test were maintained on a restricted food diet that stabilised their bodyweights at 80–85% of the normal weight for age. The animals used in the object recognition test had ad libitum access to food. 2.1. Drugs used Ethanol was 100%, diluted with distilled water to 20% v/v. Nimodipine was suspended in Tween 80 (0.5% in distilled water) and sonicated. The suspension was protected from light throughout the studies. All injections were made by the intraperitoneal route, with a volume of 1 ml/kg for nimodipine and Tween and 6.34 ml/kg for ethanol. The doses used in the radial arm maze test and in the object recognition were ethanol 1 g/kg and nimodipine 10 mg/kg. In all cases, control animals received equal volumes of the corresponding vehicle. N values were 15–16 per treatment group for the radial arm maze and 12 per treatment group for the object recognition experiment; different animals were used in the two tests. 2.2. Radial arm maze test The experiments were designed to test the effects of the drugs on both working memory and reference memory. The total number of arm choices needed to complete the task and the number of entries into unbaited arms were used as indices of reference memory, and the number of re-entries into the baited arms was used as an index of working memory. In addition, time taken to complete the task was also analysed, to demonstrate any detrimental drug-induced performance effects. A standard 8 arm radial maze was used, with arms of wooden construction with a metal and Perspex central octagonal hub. At the end of each arm was a sunken food container of approximately 6 cm diameter. Cues, including posters, were placed on the walls of the room and laboratory furniture and equipment around the maze. 2.3. Habituation Rats received 4 habituation sessions. In the first two of these sessions the rats were introduced to the maze in pairs and the sessions lasted 15 min. The subsequent sessions were on individual rats and lasted 10 min. In each of the sessions, rewards (Coco Pops) were scattered around the maze to encourage exploration. 2.4. Training The rats received once daily training sessions for a minimum of 21 days. Training sessions were run 6 days a week between 09.00–14.00, during which food pellets were placed along the whole length of the four arms that were allocated as baited arms. Over the training period the food was gradually removed so that eventually the animals ran along the arms to receive a single food pellet placed in the goal dish at the end of the allocated baited arm. The four arms allocated for baiting were not changed throughout the experiment for each rat, but differed between rats. At the start of each session the rat was initially placed in the central hub of the apparatus facing away from the experimenter. Each session lasted a maximum of 5 min, or was ended when the animal had taken the food from the four baited arms. After this initial training phase the animals were placed in the maze, with the appropriately baited arms, once daily for 6 days per week until the drug testing. They were deemed ready for the test phase if they could produce 3 sessions of steady performance. This was defined as finding all four baited arms within 5 min, with a minimum of 3/4 correct arms in the first four arm choices. If the animals met this criterion they were subsequently tested with the drugs on the following day. Throughout the sessions the time the animals took to retrieve the four baits was recorded, as was the number of arm choices necessary to complete the task. In S.P. Brooks et al. / Neuropharmacology 42 (2002) 577–585 addition, the sequential order of the arms that the animal visited in completing the task was noted. 2.5. Drug test On the test day the animals were injected with one of four drug combinations: Tween+saline, nimodipine+ saline, Tween+ethanol, or nimodipine+ethanol. Nimodipine or its vehicle were administered 30 min prior to testing and ethanol or saline were given 5 min prior to testing. Nimodipine has long lasting effects in rodents, with brain levels still in the micromolar range 2 h after intraperitoneal injection (Dolin and Little, 1989), while ethanol reaches maximal blood and brain concentrations between 5 and 30 min after intraperitoneal injection (Nurmi et al., 1994; Givens et al., 2000). After injection the animals were placed back into their home cages until tested. At the start of the test they were placed facing away from the experimenter into the central hub of the maze and permitted to search the maze to find the bait on the four baited arms. For the test session the animals were permitted a maximum of 10 min to complete the task. The time the animals took to retrieve the four baits, the number of arm choices necessary to complete the task, and the sequential order of the arms that the animal visited were recorded. 2.6. Statistical analysis The results were expressed as the differences between the four measures in the drug test and the mean of the corresponding data collected on the last three training sessions for each animal. One factor analysis of variance was carried out for the results for all four drug treatments, followed by post-hoc Tukey’s test. 579 mate pairs, the subsequent sessions lasted 5 min and rats were placed in the maze individually. During each habituation session one novel object was placed in the centre of the open field. 2.9. Testing Rats were placed individually in the open field which contained 2 identical copies of an object positioned equidistant from the sides of the maze, and were allowed to explore freely until they had spent 30 s exploring the objects. If they failed to explore for 30 s the rats were removed after 5 min and the actual time spent exploring was noted. Immediately after they were removed from the open field the rats were injected with one of four drug combinations: Tween + saline, nimodipine 10 mg/kg + saline, ethanol 1 g/kg + saline or nimodipine 10 mg/kg + ethanol 1 g/kg. At 15 min after the above exploration had ended, the rats were placed back in the open field, and allowed to explore for 3 min. The maze now contained a third copy of the object explored at the pre-drug exposure session and a novel object. Time spent exploring both the familiar and the novel object was recorded for each of the 3 min. This test procedure was repeated 1 h after the 15 min test, four hours after the one hour test and twenty-four hours after the four hour test. Test sessions were therefore at 15 min, 75 min, 5 h 15 min and 29 h 15 min after the pre-drug exposure session. For each test session a different novel object and a new copy of the familiar object were used. Effects of position were controlled for by alternating the position of the novel and familiar objects between animals and between test repetitions. The intrinsic interest of the objects was controlled for by alternating between rats which object was the designated familiar object. 2.7. Object recognition 2.10. Data analysis This test is designed to measure the discrimination between novel and familiar objects (Ennaceur and Delacour, 1988) and to test the effects of the drugs on the recall of a previously encountered object. The drugs were therefore given after the first encounter with the familiar object and prior to the test of recognition. The tests were carried out in an open field made out of aluminium, with base dimensions 85 cm2 and walls 48 cm high. The objects were placed into the open field, equidistant from the sides of the maze. Objects used included bottles, dishes, jars and candlesticks. The difference in exploration in seconds at a given time-point was calculated as the difference between the time spent exploring the novel object and the time spent exploring the familiar object. Secondly, discrimination between the objects as a proportion of total exploration was calculated as the difference between the time spent exploring the novel and the familiar objects, as a proportion of the total object exploration time. A one factor analysis of variance was carried out on each of these measures for each time point, followed by post-hoc Tukey’s test. 2.8. Habituation Prior to the habituation sessions, the animals were handled daily for several days to accustom them to the experience. They then received 3 habituation sessions. The first of these lasted 10 min and took place in cage- 3. Results 3.1. Radial arm maze The analysis of variance showed significant differences between treatment groups for re-entries into baited 580 S.P. Brooks et al. / Neuropharmacology 42 (2002) 577–585 arms (P ⬍ 0.001; F(3,57)=7.904, Fig. 1a), entries into unbaited arms (P = 0.05; F(3,57) = 2.73, Fig. 1b), and the total number of arm choices (P ⬍ 0.001; F(3,57) = 6.57, Fig. 2a). The time taken to visit the four baited Fig. 1. Effects of ethanol and nimodipine on behaviour in the radial arm maze. All values are mean ± s.e.m., for test scores minus the mean score of the previous 3 days. Negative scores are improvements, positive scores show increases in errors. The open columns indicate control values (CON); black columns: treatment with ethanol 1g/kg (ETH); shaded columns: nimodipine 10 mg/kg (NIM); and hatched columns ethanol 1g/kg plus nimodipine 10 mg/kg (ETH+NIM). (a) Reentries into baited arms. (b) Entries into unbaited arms. The results for ethanol alone were significantly different for the re-entries into baited arms, compared with those for vehicle alone (P ⬍ 0.001) and those for ethanol plus nimodipine (P ⬍ 0.01). Fig. 2. Effects of ethanol and nimodipine on behaviour in the radial arm maze. All values are mean ± s.e.m., for test scores minus the mean score of the previous 3 days. The open columns indicate control values (CON); black columns: treatment with ethanol 1g/kg (ETH); shaded columns: nimodipine 10 mg/kg (NIM); and hatched columns ethanol 1g/kg plus nimodipine 10 mg/kg (ETH+NIM). (a) Total numbers of arm choices required to complete the task. The number of total arm choices that the animals given ethanol 1g/kg required to complete the task was significantly greater than for vehicle-treated rats (P ⬍ 0.05) and for the corresponding measurement for the animals given ethanol plus nimodipine (P ⬍ 0.01). (b) Time taken to visit the four baited arms. There were no significant differences between any of the treatments in the times taken to visit the four baited arms. S.P. Brooks et al. / Neuropharmacology 42 (2002) 577–585 581 arms did not show a significant effect of treatment group (P ⬎ 0.05; F(3,57) = 1.79, Fig. 2b). The post-hoc analysis revealed that animals given ethanol at 1 g/kg had a significantly (P ⬍ 0.01) greater number of re-entries into baited arms, compared with those made by animals that received saline plus tween vehicle (Fig. 1a). The animals that received nimodipine, 10 mg/kg, in addition to 1 g/kg of ethanol, however, did not make significantly more of these errors than the vehicle controls and the results for this group were not significantly different from the control values (P ⬎ 0.05) and were significantly different from the values from animal given ethanol alone (P ⬍ 0.01). Those animals receiving nimodipine alone did not show any significant difference from the vehicle controls (P ⬎ 0.05). The post-hoc analysis for the number of entries into unbaited arms (Fig. 1b) detected a significant difference between the vehicle plus ethanol and the ethanol plus nimodipine treatment groups (P ⬍ 0.05) but no other differences were found. The total number of arm choices that the ethanol-treated animals required to complete the task was significantly greater than for the vehicle-treated rats (P ⬍ 0.05, Fig. 2a) and significantly different from the results for the rats that received ethanol plus nimodipine (P ⬍ 0.01). Treatment with nimodipine alone did not differ significantly from vehicle plus tween in any of the measures. There were no significant differences for any of the treatments in the mean times taken to visit the four baited arms. 3.2. Object recognition test The results from the object recognition experiment are illustrated in Figs. 3 and 4. The analysis of variance showed significant effects of treatment group on the difference between the time spent exploring each object at 15 min delay (P ⬍ 0.01, F(3,44) = 9.5) and the time spent exploring the novel object as a proportion of the total exploration time (P ⬍ 0.01, F (3, 44) = 15). Posthoc analysis revealed that this was due to the fact that the ethanol plus vehicle group showed a significantly smaller difference between the time spent exploring the familiar and novel objects than the other three treatment groups, whether this was expressed as the difference between the times (P ⬍ 0.001, Fig. 3a) or the difference as the proportion of the total exploration time (P ⬍ 0.001, Fig. 3b). When nimodipine was given with the ethanol, however, the post-hoc analysis showed that there was no difference from control values on either measure (P ⬎ 0.05 in both cases) but significant differences on both measures from the results for the rats receiving ethanol alone (P ⬍ 0.01 in both cases). Administration of nimodipine alone did not alter the difference between the time spent exploring the novel and familiar objects (P ⬎ 0.05). The analysis of variance showed that there were no Fig. 3. Effects of ethanol and nimodipine in the object recognition test at the 15 min time interval. All values are mean ± s.e.m. The open columns indicate control values (CON); black columns: treatment with ethanol 1g/kg (ETH); shaded columns: nimodipine 10 mg/kg (NIM); and hatched columns ethanol 1g/kg plus nimodipine 10 mg/kg (ETH+NIM). (a) Differences between time spent exploring the novel and familiar objects. (b) Differences between time spent in exploration of novel and familiar objects expressed a proportion of the total exploration time. The ethanol treatment significantly decreased the difference in time spent exploring the novel and familiar objects, whether this was expressed as actual time difference or as the proportion of total exploration time (P ⬍ 0.001 in both cases, for comparisons with vehicle control results, P ⬍ 0.01 for comparisons with effects of ethanol plus nimodipine) differences in the total time that the animals spent exploring both the objects (P ⬎ 0.05, F(3, 44) = 1.05), (Fig. 4a). At the other time intervals (Fig. 4b) there were no significant differences in any of the measures in this test (P ⬎ 0.05). 582 S.P. Brooks et al. / Neuropharmacology 42 (2002) 577–585 Fig. 4. Effects of ethanol and nimodipine in the object recognition test. All values are mean ± s.e.m. The open columns indicate control values (CON); black columns: treatment with ethanol 1g/kg (ETH); shaded columns: nimodipine 10 mg/kg (NIM); and hatched columns ethanol 1g/kg plus nimodipine 10 mg/kg (ETH+NIM). (a) Total exploration time for both objects at the 15 min time interval. (b) Time course of the differences between time spent in exploration of novel and familiar objects expressed a proportion of the total exploration time. There were no significant differences between any of the treatments for the total exploration times, or for any of the measures at the 75 min, 5 h and 29 h time intervals. 4. Discussion Actions of ethanol were seen on both reference and working memory in the radial arm maze, deficits in reference memory being reflected in the number of errors in correct arm entries and deficits in working memory by the re-entries into the baited arms after food had already been retrieved from these arms. In addition, ethanol clearly decreased recognition of previously encountered objects in the object recognition test, which, in contrast to the radial arm maze, involves nonspatial memory. All these effects of ethanol were prevented by the administration of nimodipine, no selectivity being seen with regard to the type of memory utilised in the tests. The effects of ethanol were not due to actions on motor co-ordination or alertness, since the time taken to visit all four baited arms in the radial arm maze test and the total exploration time in the object recognition test were not significantly altered. In addition, our previous work showed that the dihydropyridines nimodipine and nitrendipine acutely potentiated the ataxic and hypnotic effects of ethanol (Dolin and Little, 1986; Dolin and Little, 1989; Smith and Little, 2000), an interaction that contrasts with the antagonism by nimodipine of the amnesic effects of ethanol. When given alone, nimodipine did not alter behaviour in either test. This was not due to a ceiling effect, as there was scope for measurement of improvements in learning in both tests. The majority of studies have examined alleviation of memory deficits by dihydropyridine calcium channel antagonists, but Deyo et al. (1989) and McMonangle-Strucko and Fenelli (1993), studying rats in the Morris water maze, Vetulani et al. (1997) studying acquisition of an avoidance response, and Kane and Robinson (1999) using the Barnes circular platform task, all showed beneficial effects of nimodipine on learning in the absence of any specific deficits. The demonstration of such an effect is likely to depend on the test and dosing regime used. There is good evidence indicating that the behavioural effects of dihydropyridine calcium antagonists are due to neuronal actions, rather than to increased cerebral blood flow. The distribution of high affinity dihydropyridine binding sites in the CNS is consistent with a neuronal rather than a vascular location. Parallel electrophysiological studies have demonstrated effects of nimodipine on hippocampal neurons that may be related to the improvements in learning and memory, and these did not correlate with changes in cerebral blood flow (Disterhof et al., 1993). Administration of nifedipine directly into the hippocampus had a beneficial effect on avoidance learning (Quevedo et al., 1998). Although alcohol can constrict cerebral blood vessels, this is seen at higher concentrations than are likely to have occurred in the brain after administration of the low dose of ethanol used in the present study (Werber et al., 1997). In addition, it is considered unlikely that the memory deficits caused by alcohol are due to changes in cerebral blood flow (Givens et al., 2000; White et al., 2000). It is therefore unlikely that the antagonism of the effects of ethanol seen in the present study could be explained by changes in cerebral blood flow. One possibility is that nimodipine might alter the brain concentration of ethanol. However, Corso et al. (1998) showed that a dose of 600 mg/kg nimodipine, given concurrently with ethanol, did not affect the blood alcohol levels. In addition, we have previously shown that nitrendipine, a dihydropyridine with very similar properties and chemical structure to nimodipine, did not affect brain ethanol concentrations even at doses up to 50 S.P. Brooks et al. / Neuropharmacology 42 (2002) 577–585 mg/kg (Dolin and Little, 1989). The potentiation of ataxic and sedative properties of ethanol by dihydropyridines, described above, also argues against this explanation. Nimodipine decreased operant self-administration of ethanol (Smith et al., 1999) and contextnonspecific tolerance to ethanol, but not context-specific tolerance (Smith and Little, 2000). The latter effects were interpreted as due to a combination of decreased tolerance development (Dolin and Little, 1989) plus a suggested decrease in the amnesic actions of ethanol; the latter effect has now been demonstrated. It is particularly interesting that nimodipine prevented ethanol-induced amnesic effects on an object memory task; most research on dihydropyridines has concentrated either on non-episodic learning such as eye-blink conditioning (Disterhoft et al., 1988) or learning that is dependent on the septo-hippocampal system, in particular spatial tasks such as the Morris maze (Bannon et al., 1993) and the effects of ethanol on memory have been reported to be primarily on the use of spatial information (see Introduction). There is considerable evidence implicating the hippocampus, particularly area CA1, in spatial reference memory. The hippocampus does play a role in object memory, but this is mainly apparent when relatively complex tasks are used, such as the object-in-scene paradigm (Gaffan and Harrison, 1989). Such tasks, by placing the object in a complex context, are capable of engaging episodic memory and are therefore vulnerable to hippocampal damage. The object recognition task is dependent on the integrity of the rhinal cortices, specifically, the perirhinal cortex, as simple object memory is impaired by lesions to this area (Ennaceur et al., 1996), with a more restricted contribution from the entorhinal cortex (Meunier et al., 1993; Leonard et al., 1995). The effects of ethanol have been attributed to actions of this drug on the hippocampus (White and Best, 2000), and there is evidence of the importance of other areas, particularly the medial septum (Givens et al., 2000). As ethanol impaired memory for familiar objects, and this effect was prevented by the L-channel antagonist, any potential mechanism for this interaction cannot be limited to the hippocampus and its subcortical connections, but must also be capable of including the perirhinal cortex. Repeated high dose alcohol treatment caused degeneration in the perirhinal cortex, but this was increased, rather than prevented, by a high dose (600 mg/kg/day) nimodipine, although the latter prevented concurrent hippocampal damage (Corso et al., 1998). The effects of alcohol in the present study were tested primarily on recall of previously learned information. At 1 g/kg, ethanol had clear effects in these tests. The measurements at the later test times in the object recognition test were made in order to determine whether or not the subsequent recall of the familiarity of the object was affected either by alcohol or by nimodipine. Further 583 studies are under way to investigate the interactions between alcohol and dihydropyridines on acquisition of information. The mechanism(s) of the anti-amnesic actions of nimodipine and other dihydropyridine calcium channel antagonists is not yet understood. Ethanol does have some direct action on dihydropyridine-sensitive calcium channels, but this is a blocking effect at higher concentrations than would occur in the brain after the doses used in the current study (Little, 1999). However, other causes of cognitive deficits in which dihydropyridines had beneficial effects, such as administration of glucocorticoids (Dachir et al., 1997) and brain trauma (Westernbroek et al., 1998), have been associated with increased calcium flux through voltage-activated channels. In old age, increased calcium-dependent hyperpolarisation has been suggested to be involved in the deficits in memory and learning (Campbell et al., 1996), and this phenomenon is decreased by dihydropyridine calcium channel antagonists (Disterhof et al., 1993; Norris et al., 1998). Memory formation involves synaptic plasticity and long term potentiation (LTP) is thought to play an important role. Dihydropyridine calcium channel antagonists prevent some forms of LTP, for example the calcium-dependent potentiation produced in hippocampal CA1 neurons by very high frequency stimulation (Grover and Teyler, 1990), the LTP produced by tetraethylammonium in the CA1 field of the hippocampus (Ramakers et al., 2000) and LTP in the amygdala due to stimulation of the thalamus (Weisskopf et al., 1999). The recently described subtype of L-channel, the Lp channel, is thought to be particularly involved in NMDA-independent LTP (Schjott and Plumber, 2000) but the dihydropyridines do not distinguish between the L-channel subtypes. However, in all the above cases, dihydropyridine calcium channel antagonists decreased LTP. If this pattern of action were extrapolated to learning and memory, dihydropyridine calcium channel antagonists would be expected to produce deficits in learning and memory. The mechanism(s) of the effects of ethanol on memory are not yet fully understood. It has been suggested to involve selective effects of ethanol on hippocampal/septal pathways (Ryabinin, 1998; Givens et al., 2000). At the cellular level, ethanol reduces both NMDA receptor- and non-NMDA receptor-mediated LTP (Sinclair and Lo, 1986). The NMDA subtype of glutamate receptors may also be involved, as NMDA antagonists have deleterious effects on learning and memory and NMDA receptor-activated channels are blocked by ethanol at concentrations that would be found in the brain during its behavioural effects (Lovinger et al., 1989; Little, 1999). Chronic administration of nimodipine was not found to have effects on LTP in the hippocampal dentate gyrus 584 S.P. Brooks et al. / Neuropharmacology 42 (2002) 577–585 that paralleled improvement in acquisition of maze learning (Kane and Robinson, 1999). Evidence has been presented, however, that blockade of voltage-activated calcium channels can decrease long term depression (Calabresi et al., 1994; Norris et al., 1998). Such an effect may explain the behavioural results but more information is needed about the effects of dihydropyridines on the balance of neuronal responses between depression and excitation. Learning in behavioural paradigms is associated with increased expression of immediate early genes (IEGs). This occurs within minutes of the acquisition, so the effects of ethanol studied in the present project could have involved interference with this process. Although a large number of factors affect IEG expression, correlations have been demonstrated between the extent of learning and IEG expression in specific brain areas (Ryabinin, 1998). Dihydropyridine-sensitive calcium channels are intimately involved in gene expression (Murphy et al., 1991; Bading et al., 1993; Graeff et al., 1999): entry of calcium through L-channels causing activation of gene expression, a change in a similar direction to that found during memory formation. A dihydropyridine calcium channel antagonist might therefore be predicted to have amnesic actions via this effect, rather than the anti-amnesic actions demonstrated in the present study. The effects of ethanol on expression of IEGs, such as c-fos, are dependent on the brain area and the dose of ethanol. At amnesic ethanol doses (0.75 to 2 g/kg), c-fos expression was suppressed in the hippocampus but increased in several other brain areas (Ryabinin, 1998). In conclusion, the results show clear effects of nimodipine in alleviating the deleterious actions of ethanol in tests of memory. This action, together with the effects of dihydropyridines shown previously in preventing the alcohol withdrawal syndrome and some of the adaptive neuronal changes caused by chronic alcohol consumption (Whittington et al., 1991), suggest that this type of drug may have considerable potential in the treatment of alcohol dependence and the consequences of excessive alcohol consumption. Acknowledgements The authors thank the Wellcome Trust for financial support for this project. References Bading, H., Ginty, D.G., Greenburg, M.E., 1993. 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