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Abstract
The cannabinoid receptor system plays an integral role in learning and memory. Moreover, the cannabinoid CB1 receptor antagonist rimonabant has been found to improve performance in a variety of animal memory models. The present study tested whether a novel and potent cannabinoid CB1 receptor antagonist, CE, would prolong the duration of spatial memory. Rats were trained in a two-phase radial arm maze procedure, consisting of acquisition and retrieval tests, which were separated by an 18 h delay. CE was administered 30 min before the acquisition phase, immediately after the acquisition phase, or 30 min before the retrieval test to assess its effects on acquisition and retrieval processes. CE administered before and immediately after the acquisition phase significantly decreased the number of errors committed during the retrieval test. On the other hand, CE administered 30 min before the retrieval test had no effect on the number of errors committed. These findings demonstrate that CE improves memory by acting on consolidation, rather than retrieval, processes and further suggest that the endocannabinoid system has an important role in modulating memory duration.
Introduction
The centrally occurring endocannabinoid system consists of the G-protein coupled cannabinoid CB1 receptor (Matsuda et al., 1990) and endogenous ligands, anandamide (Devane et al., 1992) and 2-arachidonyl glycerol (2-AG; Mechoulam et al., 1995; Sugiura et al., 1995). The high concentration of cannabinoid CB1 receptors (Herkenham et al., 1991) and the presence of anandamide and 2-AG in the hippocampus and other forebrain regions associated with memory function (Di Marzo et al., 2000; Felder et al., 1996) are consistent with the notion that the endocannabinoid system modulates cognitive processes. Administration of the cannabinoid CB1 receptor antagonist rimonabant (SR 141716; Rinaldi-Carmona et al., 1994) has been reported to enhance performance in several memory models (Deadwyler et al., 2007; Lichtman, 2000; Robinson et al., 2008; Shiflett et al., 2004; Takahashi et al., 2005; Terranova et al., 1996; Wolff and Leander, 2003). However, the efficacy of other cannabinoid CB1 receptor antagonists in memory models has yet to be explored.
CE (1-[7-(2-Chlorophenyl)-8-(4-chlorophenyl)-2-methylpyrazolo[1,5-a]-[1,3,5] triazin-4-yl]-3-ethylaminoazetidine-3-carboxylic acid amide benzenesulfonate) is described as a highly selective full antagonist for the cannabinoid CB1 receptor that is well absorbed and readily crosses the blood brain barrier (Cao et al., 2007). This compound was recently reported to block the effects of the cannabinoid receptor agonist CP 55,940 in behavioral measures well-known to be produced by cannabinoid agonists including hypomotility, antinociception, hypothermia, and catalepsy, as well as to block CP-55,940-stimulated GTPγ[35S] binding (Cao et al., 2007). Additionally, CE dose-dependently significantly increased the ability of suboptimal doses of L-DOPA to reduce motor disability in severely Parkinsonian monkeys with a duration of action of approximately two h (Cao et a., 2007).
Findings from our laboratory have shown that rimonabant prolongs spatial memory in a rat radial arm maze delay paradigm that employs a two-phase procedure in which acquisition and test phases are separated by an 18 h delay to increase the task difficulty (Lichtman, 2000; Wise et al., 2007). Thus, the main goal of the present study was to determine whether administration of CE would enhance memory in this model. The dose-response relationship of CE administered 30 min before the acquisition phase was evaluated. In addition, CE was given immediately after the acquisition phase or 30 min before retrieval testing to infer whether this compound influences attentional/acquisition, consolidation, or retrieval processes.
Methods
2.1. Subjects
All experiments were performed in nine Sprague Dawley (Harlan, IN) male rats aged 4-12 months that were individually housed in a temperature-controlled (20—22°C) environment with a 12-h light/dark cycle. Subjects were maintained on a food-restricted diet in order to sustain body weights of 290—320 g, approximately 85% of their free-feeding weight. Water was available ad libitum. All animal protocols were approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee and were in concordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).
2.2. Drugs
CE (0.03, 0.1, 0.3, 1.0 and 3.0 mg/kg; Pfizer) was dissolved in a 1:1 mixture of absolute ethanol and alkamuls-620 (Rhone-Poulenc, Princeton, NJ) and diluted with saline to a final ratio of 1
18 (ethanol/alkamuls/saline). Injections were given i.p. in a volume of 1 ml/kg.
2.3. Radial arm maze procedure
All rats received approximately five weeks of repeated acquisition radial arm maze training, as previously described (Lichtman, 2000; Wise et al., 2007). Each session incorporated acquisition and retrieval test phases. During the acquisition phase, one of the arms was randomly selected and a Plexiglas barricade blocked its entryway. The remaining seven arms were baited with 45 mg rodent formula, dustless precision food pellets (Bioserve, Frenchtown, NJ) before the subject's placement in the maze. After the subject entered the seven available arms and consumed each of the available food pellets, it was removed from the maze and returned to its home cage. During the test phase, all arms were available; however, only the previously blocked arm was baited with a food pellet. An error was defined as a re-entry into an arm from which a food pellet had been previously consumed. The number of entries and the duration of time required for each subject to enter the baited arm(s) and consume available food pellets were recorded for each phase. An 18 h delay between the acquisition and test phases was used, which we previously have shown to reduce choice accuracy to chance performance during the test phase (Wise et al., 2007).
An initial pilot study tested the effects of 0 and 3 mg/kg CE (n= 4/group) administered 30 min before the acquisition phase. After treatment with 3 mg/kg CE, two of the four rats tested did not enter the arms, thus precluding memory assessment in the test phase. The two rats that did complete the acquisition phase required more time to complete this phase of the task than typically is observed (38 and 24 s per arm as opposed to 10 s/arm typically observed). Accordingly, the effects of 0.03, 0.1, 0.3, and 1.0 mg/kg of CE administered 30 min before the acquisition phase were assessed (n = 9 rats per dose). Next, either an effective dose of CE (0.1 mg/kg; n = 9) or vehicle (n= 9) was administered immediately after the acquisition phase and errors in the test phase were assessed. Finally, the effect of administering an effective dose of CE (0.1 mg/kg; n = 8) or vehicle (n= 8) 30 min before the test phase was determined. Treatments in each experiment were counter-balanced to control for any order effects. Subjects were given a maximum of two tests/week, with at least 48 h between test sessions.
2.4. Data Analyses
A within subject analysis of variance (ANOVA) was used to analyze the dose effect curve of CE. Dunnett's post hoc test was used to analyze differences between vehicle and each drug condition. T-tests were used to analyze the effect of administering CE or vehicle after the acquisition phase or before the retention phase, as these experiments included two groups. Differences were considered significant at the P < 0.05 level.
Results
Choice accuracy was virtually perfect during the acquisition phase in all experiments, as subjects entered each baited arm, ate all available pellets, and rarely made any errors of re-entry. While CE administered 30 min before the acquisition phase had no effect on acquisition performance (P = 0.26; Fig. 1A), it significantly reduced the number of errors committed during the retrieval test, F (4, 32) = 7.32, P < 0.01 (Fig. 1B). The 0.1 (P < 0.01), 0.3 (P < 0.05), and 1.0 (P < 0.01) mg/kg doses of CE reduced the number of errors compared to the vehicle condition. In addition, CE had no effect on rate of arm entry during the acquisition phase (P = 0.15; Fig. 1C) or the retrieval test (P = 0.29; Fig. 1D).
Next, the effects of an effective dose of CE (0.1 mg/kg) or vehicle administered immediately after the acquisition phase or 30 min before the test phase were assessed. An effective dose of CE (0.1 mg/kg) reduced errors in the retrieval test when given immediately after acquisition (P < 0.05; Fig. 2A, left panel), but failed to affect performance when given 30 min before the retrieval test (P = 0.44; Fig. 2A, right panel). The rate of arm entry during the retrieval test in rats treated with an effective dose of CE (0.1 mg/kg) given either immediately after acquisition (P = 0.54; Fig. 2B, left panel) or 30 min before the retrieval test (P = 0.46; Fig. 2B, right panel) did not differ from the vehicle treatment.
Discussion
In the present study we make the observation that the novel cannabinoid CB1 receptor antagonist CE significantly enhances memory as assessed in rat delayed radial arm maze task when administered 30 min before or immediately after the acquisition phase. However, an effective dose of CE failed to affect memory performance when administered 30 min before the retrieval phase. These results suggest that CE improves choice accuracy by its actions on consolidation processes rather than on retrieval processes. Additionally, doses of CE that improved memory duration did not affect either the rate of entry into the arms or consumption of the food pellets. Thus, it is unlikely that the facilitated memory performance effects observed in these studies are due to altered locomotor activity or increased salience of the food reward. CE blocks the effects of the cannabinoid CB1 agonist CP 55,940 on locomotor activity, antinociception, hypothermia, and catalepsy, as well as CP-55,940-stimulated GTPγ[35S] binding (Cao et al., 2007) indicating that it is a behaviorally active antagonist for the cannabinoid CB1 receptor. In the present study, as well as in that of Cao et al. (2007), 0.03—1.0 mg/kg of CE did not affect motor activity. However, 3.0 mg/kg of CE disrupted performance in the radial arm maze task.
We have previously found that rimonabant enhances performance in the delayed radial arm maze paradigm used in the present study when given before acquisition, but not when administered immediately after the acquisition phase or prior to the retrieval phase (Lichtman, 2000; Wise et al., 2007). Using a different version of the radial arm task in which four arms were blocked, Wolff and Leander (2003) found that 1 mg/kg rimonabant, suspended in a different vehicle solution (i.e., 15% cyclodextrin and tween 80) than the vehicle solution used in our laboratory (Lichtman, 2000; Wise et al., 2007), given immediately after the acquisition phase, but not before the test phase, enhanced performance during the retrieval test. Rimonabant also has been demonstrated to enhance performance in a rodent social recognition memory task, as well as attenuate deficits in aged mice and rats in this same task, when administered 5 min after acquisition (Terranova et al., 1996). Avoidance behavior in an elevated T-maze task in mice was also enhanced when rimonabant was administered before or immediately after the acquisition phase, but not before the test phase (Takahashi et al., 2005). The observations that both CE and rimonabant can improve memory when given immediately after acquisition, but not when given before the retrieval test, suggest that these drugs are acting upon consolidation processes.
While the results of the aforementioned studies demonstrate that disruption of cannabinoid CB1 receptor signaling can enhance memory duration, rimonabant failed to improve memory in a variety of operant tasks (Brodkin and Moerschbaecher, 1997; Hampson and Deadwyler, 2000; Mallet and Beninger, 1998; Mansbach et al., 1996). However, in a more recent study, rimonabant significantly increased the strength of firing pattern ensembles of CA1 and CA3 hippocampal neurons, which was strongly associated with improved performance on a delayed non-match to sample task, in trials with delay intervals greater than 10 s (Deadwyler et al., 2007). These findings indicate that cannabinoid CB1 receptor antagonists can enhance memory duration in spatial memory, social recognition, and operant tasks.
On the other hand, manipulations of the endogenous cannabinoid system produce differential effects on extinction learning in which learned behavior is actively suppressed when the reinforcer is withheld. Specifically, CB1 (−/−) mice as well as rimonabant-treated mice display impaired extinction learning in aversive conditioning tasks, but show normal extinction learning in operant tasks that employ palatable food reward (Lutz, 2007). Accordingly, the apparent paradoxical effects of cannabinoid receptor antagonists in the memory duration and extinction studies may result from the differential roles that the endogenous cannabinoid system plays in mnemonic processes and in response to fear or stressful events.
In conclusion, we report that CE, a novel cannabinoid CB1 receptor antagonist structurally distinct from rimonabant, prolongs spatial memory in a delayed radial arm maze task at doses that did not affect motor behavior. CE prolonged memory when administered 30 min before or immediately after the acquisition phase, but did not improve performance when administered 30 min before the retrieval phase. These findings suggest that CE acts on acquisition/consolidation processes, rather than retrieval process.
Source, Graphs and Figures: The cannabinoid CB1 receptor antagonist CE prolongs spatial memory duration in a rat delayed radial arm maze memory task
The cannabinoid receptor system plays an integral role in learning and memory. Moreover, the cannabinoid CB1 receptor antagonist rimonabant has been found to improve performance in a variety of animal memory models. The present study tested whether a novel and potent cannabinoid CB1 receptor antagonist, CE, would prolong the duration of spatial memory. Rats were trained in a two-phase radial arm maze procedure, consisting of acquisition and retrieval tests, which were separated by an 18 h delay. CE was administered 30 min before the acquisition phase, immediately after the acquisition phase, or 30 min before the retrieval test to assess its effects on acquisition and retrieval processes. CE administered before and immediately after the acquisition phase significantly decreased the number of errors committed during the retrieval test. On the other hand, CE administered 30 min before the retrieval test had no effect on the number of errors committed. These findings demonstrate that CE improves memory by acting on consolidation, rather than retrieval, processes and further suggest that the endocannabinoid system has an important role in modulating memory duration.
Introduction
The centrally occurring endocannabinoid system consists of the G-protein coupled cannabinoid CB1 receptor (Matsuda et al., 1990) and endogenous ligands, anandamide (Devane et al., 1992) and 2-arachidonyl glycerol (2-AG; Mechoulam et al., 1995; Sugiura et al., 1995). The high concentration of cannabinoid CB1 receptors (Herkenham et al., 1991) and the presence of anandamide and 2-AG in the hippocampus and other forebrain regions associated with memory function (Di Marzo et al., 2000; Felder et al., 1996) are consistent with the notion that the endocannabinoid system modulates cognitive processes. Administration of the cannabinoid CB1 receptor antagonist rimonabant (SR 141716; Rinaldi-Carmona et al., 1994) has been reported to enhance performance in several memory models (Deadwyler et al., 2007; Lichtman, 2000; Robinson et al., 2008; Shiflett et al., 2004; Takahashi et al., 2005; Terranova et al., 1996; Wolff and Leander, 2003). However, the efficacy of other cannabinoid CB1 receptor antagonists in memory models has yet to be explored.
CE (1-[7-(2-Chlorophenyl)-8-(4-chlorophenyl)-2-methylpyrazolo[1,5-a]-[1,3,5] triazin-4-yl]-3-ethylaminoazetidine-3-carboxylic acid amide benzenesulfonate) is described as a highly selective full antagonist for the cannabinoid CB1 receptor that is well absorbed and readily crosses the blood brain barrier (Cao et al., 2007). This compound was recently reported to block the effects of the cannabinoid receptor agonist CP 55,940 in behavioral measures well-known to be produced by cannabinoid agonists including hypomotility, antinociception, hypothermia, and catalepsy, as well as to block CP-55,940-stimulated GTPγ[35S] binding (Cao et al., 2007). Additionally, CE dose-dependently significantly increased the ability of suboptimal doses of L-DOPA to reduce motor disability in severely Parkinsonian monkeys with a duration of action of approximately two h (Cao et a., 2007).
Findings from our laboratory have shown that rimonabant prolongs spatial memory in a rat radial arm maze delay paradigm that employs a two-phase procedure in which acquisition and test phases are separated by an 18 h delay to increase the task difficulty (Lichtman, 2000; Wise et al., 2007). Thus, the main goal of the present study was to determine whether administration of CE would enhance memory in this model. The dose-response relationship of CE administered 30 min before the acquisition phase was evaluated. In addition, CE was given immediately after the acquisition phase or 30 min before retrieval testing to infer whether this compound influences attentional/acquisition, consolidation, or retrieval processes.
Methods
2.1. Subjects
All experiments were performed in nine Sprague Dawley (Harlan, IN) male rats aged 4-12 months that were individually housed in a temperature-controlled (20—22°C) environment with a 12-h light/dark cycle. Subjects were maintained on a food-restricted diet in order to sustain body weights of 290—320 g, approximately 85% of their free-feeding weight. Water was available ad libitum. All animal protocols were approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee and were in concordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).
2.2. Drugs
CE (0.03, 0.1, 0.3, 1.0 and 3.0 mg/kg; Pfizer) was dissolved in a 1:1 mixture of absolute ethanol and alkamuls-620 (Rhone-Poulenc, Princeton, NJ) and diluted with saline to a final ratio of 1
18 (ethanol/alkamuls/saline). Injections were given i.p. in a volume of 1 ml/kg.2.3. Radial arm maze procedure
All rats received approximately five weeks of repeated acquisition radial arm maze training, as previously described (Lichtman, 2000; Wise et al., 2007). Each session incorporated acquisition and retrieval test phases. During the acquisition phase, one of the arms was randomly selected and a Plexiglas barricade blocked its entryway. The remaining seven arms were baited with 45 mg rodent formula, dustless precision food pellets (Bioserve, Frenchtown, NJ) before the subject's placement in the maze. After the subject entered the seven available arms and consumed each of the available food pellets, it was removed from the maze and returned to its home cage. During the test phase, all arms were available; however, only the previously blocked arm was baited with a food pellet. An error was defined as a re-entry into an arm from which a food pellet had been previously consumed. The number of entries and the duration of time required for each subject to enter the baited arm(s) and consume available food pellets were recorded for each phase. An 18 h delay between the acquisition and test phases was used, which we previously have shown to reduce choice accuracy to chance performance during the test phase (Wise et al., 2007).
An initial pilot study tested the effects of 0 and 3 mg/kg CE (n= 4/group) administered 30 min before the acquisition phase. After treatment with 3 mg/kg CE, two of the four rats tested did not enter the arms, thus precluding memory assessment in the test phase. The two rats that did complete the acquisition phase required more time to complete this phase of the task than typically is observed (38 and 24 s per arm as opposed to 10 s/arm typically observed). Accordingly, the effects of 0.03, 0.1, 0.3, and 1.0 mg/kg of CE administered 30 min before the acquisition phase were assessed (n = 9 rats per dose). Next, either an effective dose of CE (0.1 mg/kg; n = 9) or vehicle (n= 9) was administered immediately after the acquisition phase and errors in the test phase were assessed. Finally, the effect of administering an effective dose of CE (0.1 mg/kg; n = 8) or vehicle (n= 8) 30 min before the test phase was determined. Treatments in each experiment were counter-balanced to control for any order effects. Subjects were given a maximum of two tests/week, with at least 48 h between test sessions.
2.4. Data Analyses
A within subject analysis of variance (ANOVA) was used to analyze the dose effect curve of CE. Dunnett's post hoc test was used to analyze differences between vehicle and each drug condition. T-tests were used to analyze the effect of administering CE or vehicle after the acquisition phase or before the retention phase, as these experiments included two groups. Differences were considered significant at the P < 0.05 level.
Results
Choice accuracy was virtually perfect during the acquisition phase in all experiments, as subjects entered each baited arm, ate all available pellets, and rarely made any errors of re-entry. While CE administered 30 min before the acquisition phase had no effect on acquisition performance (P = 0.26; Fig. 1A), it significantly reduced the number of errors committed during the retrieval test, F (4, 32) = 7.32, P < 0.01 (Fig. 1B). The 0.1 (P < 0.01), 0.3 (P < 0.05), and 1.0 (P < 0.01) mg/kg doses of CE reduced the number of errors compared to the vehicle condition. In addition, CE had no effect on rate of arm entry during the acquisition phase (P = 0.15; Fig. 1C) or the retrieval test (P = 0.29; Fig. 1D).
Next, the effects of an effective dose of CE (0.1 mg/kg) or vehicle administered immediately after the acquisition phase or 30 min before the test phase were assessed. An effective dose of CE (0.1 mg/kg) reduced errors in the retrieval test when given immediately after acquisition (P < 0.05; Fig. 2A, left panel), but failed to affect performance when given 30 min before the retrieval test (P = 0.44; Fig. 2A, right panel). The rate of arm entry during the retrieval test in rats treated with an effective dose of CE (0.1 mg/kg) given either immediately after acquisition (P = 0.54; Fig. 2B, left panel) or 30 min before the retrieval test (P = 0.46; Fig. 2B, right panel) did not differ from the vehicle treatment.
Discussion
In the present study we make the observation that the novel cannabinoid CB1 receptor antagonist CE significantly enhances memory as assessed in rat delayed radial arm maze task when administered 30 min before or immediately after the acquisition phase. However, an effective dose of CE failed to affect memory performance when administered 30 min before the retrieval phase. These results suggest that CE improves choice accuracy by its actions on consolidation processes rather than on retrieval processes. Additionally, doses of CE that improved memory duration did not affect either the rate of entry into the arms or consumption of the food pellets. Thus, it is unlikely that the facilitated memory performance effects observed in these studies are due to altered locomotor activity or increased salience of the food reward. CE blocks the effects of the cannabinoid CB1 agonist CP 55,940 on locomotor activity, antinociception, hypothermia, and catalepsy, as well as CP-55,940-stimulated GTPγ[35S] binding (Cao et al., 2007) indicating that it is a behaviorally active antagonist for the cannabinoid CB1 receptor. In the present study, as well as in that of Cao et al. (2007), 0.03—1.0 mg/kg of CE did not affect motor activity. However, 3.0 mg/kg of CE disrupted performance in the radial arm maze task.
We have previously found that rimonabant enhances performance in the delayed radial arm maze paradigm used in the present study when given before acquisition, but not when administered immediately after the acquisition phase or prior to the retrieval phase (Lichtman, 2000; Wise et al., 2007). Using a different version of the radial arm task in which four arms were blocked, Wolff and Leander (2003) found that 1 mg/kg rimonabant, suspended in a different vehicle solution (i.e., 15% cyclodextrin and tween 80) than the vehicle solution used in our laboratory (Lichtman, 2000; Wise et al., 2007), given immediately after the acquisition phase, but not before the test phase, enhanced performance during the retrieval test. Rimonabant also has been demonstrated to enhance performance in a rodent social recognition memory task, as well as attenuate deficits in aged mice and rats in this same task, when administered 5 min after acquisition (Terranova et al., 1996). Avoidance behavior in an elevated T-maze task in mice was also enhanced when rimonabant was administered before or immediately after the acquisition phase, but not before the test phase (Takahashi et al., 2005). The observations that both CE and rimonabant can improve memory when given immediately after acquisition, but not when given before the retrieval test, suggest that these drugs are acting upon consolidation processes.
While the results of the aforementioned studies demonstrate that disruption of cannabinoid CB1 receptor signaling can enhance memory duration, rimonabant failed to improve memory in a variety of operant tasks (Brodkin and Moerschbaecher, 1997; Hampson and Deadwyler, 2000; Mallet and Beninger, 1998; Mansbach et al., 1996). However, in a more recent study, rimonabant significantly increased the strength of firing pattern ensembles of CA1 and CA3 hippocampal neurons, which was strongly associated with improved performance on a delayed non-match to sample task, in trials with delay intervals greater than 10 s (Deadwyler et al., 2007). These findings indicate that cannabinoid CB1 receptor antagonists can enhance memory duration in spatial memory, social recognition, and operant tasks.
On the other hand, manipulations of the endogenous cannabinoid system produce differential effects on extinction learning in which learned behavior is actively suppressed when the reinforcer is withheld. Specifically, CB1 (−/−) mice as well as rimonabant-treated mice display impaired extinction learning in aversive conditioning tasks, but show normal extinction learning in operant tasks that employ palatable food reward (Lutz, 2007). Accordingly, the apparent paradoxical effects of cannabinoid receptor antagonists in the memory duration and extinction studies may result from the differential roles that the endogenous cannabinoid system plays in mnemonic processes and in response to fear or stressful events.
In conclusion, we report that CE, a novel cannabinoid CB1 receptor antagonist structurally distinct from rimonabant, prolongs spatial memory in a delayed radial arm maze task at doses that did not affect motor behavior. CE prolonged memory when administered 30 min before or immediately after the acquisition phase, but did not improve performance when administered 30 min before the retrieval phase. These findings suggest that CE acts on acquisition/consolidation processes, rather than retrieval process.
Source, Graphs and Figures: The cannabinoid CB1 receptor antagonist CE prolongs spatial memory duration in a rat delayed radial arm maze memory task
