Pharmacological Reports 66 (2014) 931–936
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
CP55,940 attenuates spatial memory retrieval in mice Izabela Bialuk *,1, Katarzyna Dobosz 1, Bartosz Potrzebowski, Maria Małgorzata Winnicka Department of General and Experimental Pathology, Medical University of Białystok, Białystok, Poland
A R T I C L E I N F O
Article history: Received 20 May 2014 Accepted 4 June 2014 Available online 19 June 2014 Keywords: CP55,940 Spatial memory retrieval Morris water maze Hole-board test Mice
A B S T R A C T
Background: Cannabinoids constitute a varied group of lipophilic substances able to infiltrate the blood– brain barrier and influence neuronal processes. Clinical observations supported by experimental data have revealed that these compounds exert a deleterious effect on cognitive processes. The present study was carried out to determine the influence of a single systemic administration of CP55,940, a potent synthetic agonist of cannabinoid receptors, on spatial memory retrieval assessed in a Morris water maze. Methods: C57BL/6J male mice were submitted to three consecutive days of training to find a hidden platform in the water maze. CP55,940 was given intraperitoneally once, at doses of 0.025, 0.125 or 0.25 mg/kg on the fourth day, 30 min before testing memory retrieval, and in separate groups before testing psychomotor activity and anxiety level in a hole-board test. Results: CP55,940 only at the highest dose of 0.25 mg/kg significantly altered all parameters used to assess spatial memory. It increased the latency in the first crossing of the former platform location (target area), decreased the number of target area crossings and shortened the time spent in the target quadrant. Moreover, CP55,940 at doses of 0.25 and 0.125 mg/kg attenuated motor and exploratory activity in hole-board test. Conclusion: Since the attenuated psychomotor activity after a dose of 0.125 mg/kg did not interfere with memory retrieval, we assume that the impairment of spatial memory observed after the highest dose of CP55,940 (0.25 mg/kg) was exerted by its influence on cognitive processes, however, the impact on locomotion could not be excluded. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Marijuana has been used for medical and narcotic purposes for thousands of years, and remains the most common illicit drug [1]. Its constituents responsible for psychoactive effects are known as cannabinoids, highly lipophilic compounds, able to infiltrate the blood–brain barrier [2,3]. Intensive research on cannabinoids has led to the discovery of a tonically active endocannabinoid system, composed of cannabinoid receptors, their endogenous ligands and enzymes involved in their metabolism [2]. High density of cannabinoid receptors in the brain suggests an important role of endocannabinoids in the physiological activity of the central
Abbreviations: CP55,940, ( )-cis-3-[2-Hydroxy-4-(1,1-dimethy-lheptyl)phenyl]trans-4-(3-hydroxypropyl)cyclohexanol; CB1 receptor, cannabinoid receptor type 1; delta-9 THC, delta9-tetrahydrocannabinol; SR141716A, [N-(piperidin-1-yl)-5(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride]. * Corresponding author. E-mail address: [email protected] (I. Bialuk). 1 These authors have contributed equally to this work.
nervous system (CNS). Currently, the diverse group of cannabinoids, consisting of a broad range of endogenous, exogenous and synthetic compounds, creates potential drugs for the treatment of variety diseases [3,4], and therefore better understanding of cannabinoid properties is of critical value. The increasing evidence supports a negative effect of cannabinoid receptor type 1 (CB1) agonists on learning and memory processes [5–8]. Among cognitive processes, spatial memory is of significant importance in adaptation to the environment, both for human and animals. Two types of behavioural tests are commonly used as animal models for its evaluation: radial maze [9] and Morris water maze [10]. There is support for the notion that different CB1 receptor agonists impair spatial memory in rats. Lichtman et al. [11] showed that systemic administration of delta-9 THC, the main active constituent of marijuana, and two potent synthetic cannabinoids: WIN-55,212-2 and CP55,940 increased the number of errors and completion time delay in the radial maze. Moreover, they revealed that also intrahippocampal administration of CP55,940 caused a dose-dependent impairment of maze performance. Egashira et al. [12] demonstrated that bilateral microinjections of delta-9 THC
http://dx.doi.org/10.1016/j.pharep.2014.06.002 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
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into dorsal hippocampus, ventral hippocampus or dorsomedial thalamic nucleus impaired spatial memory in the eight-arm radial maze in rats. Systemic pre-treatment with SR141716A, a selective CB1 receptor antagonist, prevented delta-9 THC-induced memory deficits in the radial-arm maze, providing strong evidence that delta 9-THC impairs working memory through direct action at cannabinoid receptors. The involvement of CB1 receptors in this effect was supported by intrahippocampal administration of rimonabant (SR141716A) that reversed delta-9 THC- and CP55,940-evoked memory deficit in the radial maze [13,14]. The results of experiments performed in the Morris water maze are less consistent and predominantly demonstrate the effects of delta-9 THC. Varvel et al. [15] reported a selective impairment of working memory in mice by delta-9 THC mediated by CB1 receptors. Da and Takahashi [16] confirmed the impairment of mice performance in the working memory task by delta 9-THC, which was prevented by CB1 receptor antagonist – SR141716A. Moreover, they reported that delta 9-THC impaired acquisition of spatial learning, but did not affect consolidation and retrieval of the previously learned task. Contrary to previous data, Niyuhire et al. [17] demonstrated that delta 9-THC and exposure to marijuana smoke impaired memory retrieval in mice through a CB1 receptor mechanism. There are no current reports on acute CP55,940 effects on mice tested in the Morris water maze, and the problem whether the activation of CB1 receptors impairs memory retrieval remains unclear. Therefore, the present study was performed in an attempt to evaluate whether CP55,940, a potent synthetic agonist of cannabinoid receptors, affects spatial memory retrieval in C57BL/6J mice in the Morris water maze. Since cannabinoid receptors agonists are known to alter animals’ locomotor activity [18,19] and anxiety level [20–22], the influence of CP55,940 on ambulation and anxiety was assessed in separate groups of mice in a hole-board test. Materials and methods All experiments were approved by the Local Animal Ethics Committee in Bialystok, Poland and were performed in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Subjects Subjects were experimentally naı¨ve 4-month-old male mice – C57BL/6J, weighing approximately 30 g. The experiments were performed after 7 days of acclimatization to the laboratory conditions. The mice were maintained in a temperature-controlled environment (22 1 8C) with a 12 h light–dark cycles beginning at 7 a.m. and were housed in polycarbonate cages (27 cm 21 cm 14 cm), cm), five animals per cage, with water and commercial food available ad libitum. The experiments were carried out between 9.00 am and 1.00 pm in the air-conditioned, sound-isolated room with regulated light intensity. Drugs CP55,940 {( )-cis-3-[2-Hydroxy-4-(1,1-dimethy-lheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol} (Tocris) was dissolved in the 19% solution of cyclodextrin (SIGMA). Solutions were prepared before each behavioural test and protected from light. CP55,940 was given intraperitoneally randomly, at a single dose of 0.025, 0.125 or 0.25 mg/kg in a volume of 10 ml/kg. In order to assess the influence of CP55,940 on spatial memory retrieval the tested compound was administered on the fourth day, 30 min before testing animals in the Morris water maze. To evaluate the influence of CP55,940 on the locomotor and exploratory activity separate groups of animals received the same doses of the
Table 1 Starting points in the Morris water maze on the consecutive days of the experiment. Experiment day
Trial 1
Trial 2
Trial 3
Trial 4
1 2 3 4 Testing day
N SE NW NE
E N SE
SE NW E
NW E N
compound, 30 min before testing in a hole-board test. The control groups of animals were given a vehicle solution only at indicated time points. Behavioural tests Morris water maze The maze consisted of a circular, galvanized and painted in white steel pool (120 cm in diameter, 30 cm height), filled with water maintained at 23 1 8C [10,23,24]. The pool was divided into four equal quadrants: NE, NW, SE, SW. Four visual cues (A4-size sheets of white laminated paper with black geometric symbols) were attached on the internal marginates (N, W, E, S) of the pool and constituted navigation points. An escape platform (9 cm in diameter, 21 cm height) at a fixed location was made of transparent Plexiglas and was immersed 1 cm under the surface of water in the centre of the SW quadrant (target quadrant). The maze was lighted with the intensity of 45 lx. For three consecutive days mice were assigned to training sessions (one session a day) consisting of four trials separated by 10 min interval. Each trial started from one of the semi-random start positions (Table 1). If an animal did not find a submerged platform within 60 s, it was placed on the platform for the time of 15 s. On the fourth day the platform was removed from the pool. Thirty minutes after injection of CP55,940 or vehicle solution the animal was placed in the water maze for 60 s. Latency in the first crossing of the former platform location (target area), the number of crossings of the target area, and the time spent in the target quadrant were measured. If a mouse did not find the platform’s place within 60 s, it was given a latency score of 60 s. Hole-board test The experiments were performed according to the modified method described by Boissier and Simon [25]. The apparatus was a wooden box with a square floor of 55 cm 55 cm divided into 25 equal parts and surrounded by a 42 cm high wall. Four holes in the floor (2.5 cm in diameter) were designed as objects of possible interest of the animals. The apparatus was placed on the floor and lighted with the intensity of 30 lx. Thirty minutes after injection of CP55,940 or vehicle solution, the animal was placed in the centre of the hole-board box and its behaviour was observed for 5 min. Locomotor activity (ambulation) was measured as the number of squares crossed with all four limbs. Exploratory activity was measured as the number of rearings (rises of animal on its rear limbs, either with forelegs leaning against the wall or away from the wall) and the number of head-dips (dips of head into the hole). Moreover, during assessment of ambulation, external and internal locomotor activities of the mice were recorded. The number of crossed squares adjacent to the apparatus wall was used to measure peripheral activity, whereas the remaining crossed squares were considered as central activity. The percentage of central ambulation was used as an indication of anxiety level. Statistics The data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test using GraphPad Prism 5. Fratios, degrees of freedom and p-values are reported for significant differences at the level of p < 0.05.
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Table 2 Locomotor activity of mice evaluated in hole-board test, 30 min after CP 55,940 administration.
Results Morris water maze
CP55,940
CP55,940 given ip once, 30 min before the testing trial, significantly attenuated spatial memory retrieval. ANOVA of three groups of mice injected with increasing doses of CP55,940 and a control group showed significant differences in all parameters used for the evaluation of memory retrieval: latency in the first crossing of the former platform location (target area) (F3,36 = 8.12; p = 0.0003), the number of crossings of the target area (F3,36 = 6.57; p = 0.002) and the time spent in the target quadrant (F3,36 = 3.22; p = 0.033). Post hoc comparisons with Dunnett’s test revealed a significant increase in the latency to reach the former platform location in the group of mice given the highest dose of CP55,940 (0.25 mg/kg) (p < 0.0005) (Fig. 1B). Moreover, a significant decrease in the number of target crossings (p < 0.005) (Fig. 1C) and a significant reduction in the time spent in the target quadrant (p < 0.05) (Fig. 1D) were observed in comparison to the control group only after the highest dose of the compound tested. Hole-board test The ANOVA analysis of total, peripheral and central locomotor activity measured by the number of crossed squares showed significant differences between study groups (F3,30 = 16.43,
40 30 20 10 0 1
3
2
training day
C Crossings over target area
4
3
2
**
1
0 Control
0.025
mg/kg
0.125
0.25
Peripheral
Central
110.222 (9.416) 117.889 (18.421) 54.667* (13.733) 15.143*** (7.446)
44.111 (6.750) 28.556 (4.939) 8.889*** (3.791) 3.857*** (1.204)
27.8 23.9 19.9 22.1
(0.031) (0.050) (0.104) (0.086)
p < 0.0001; F3,30 = 12.03, p < 0.0001 and F3,30 = 14.08, p < 0.0001 for total, peripheral and central locomotor activity, respectively). The post hoc comparison made with the Dunnett’s test revealed significant attenuation of total and central ambulation in groups treated with CP55,940 at doses of 0.125 mg/kg and 0.25 mg/kg (p < 0.0005), as well as of peripheral ambulation in mice treated with CP55,940 at doses of 0.125 mg/kg (p < 0.05) and 0.25 mg/kg (p < 0.0005) as compared to the control group (Table 2 and Fig. 2A). Calculation of the percentage of central ambulation showed a
Latency to reach platform's palce (s)
50
% Of central ambulation
Data are presented as means (SEM) of the values obtained from 7 to 9 animals. Percentage of squares crossed in the central area of the apparatus (% of central ambulation = 100 central ambulation/central + peripheral ambulation). * Significant differences: p < 0.05 vs. control group (ANOVA followed by Dunnett’s test). *** Significant differences: p < 0.0005 vs. control group (ANOVA followed by Dunnett’s test).
Time spent in the target quadrant (sec)
Latency to reach platform (s)
A
Control 0.025 mg/kg 0.125 mg/kg 0.25 mg/kg
Locomotor activity
B 80
***
70 60 50 40 30 20 10 0 Control
0.025
0.125
0.25
mg/kg
D 25 20 15
* 10 5 0 Control
0.025
0.125
0.25
mg/kg
Fig. 1. Effect of a single ip injection of CP55,940 on spatial memory retrieval, administered 30 min before testing the animals in a Morris water maze. Learning was performed for three consecutive days of training to find a hidden platform (A) – data are presented as means of the values obtained from 40 animals. Twenty-four hours later, during a 60s observation in a platform-free water maze the following parameters of spatial memory were measured: (A) latency in the first crossing of the former platform location (target area), (B) the number of crossings of the target area, (C) the time spent in the target quadrant. Data are presented as means SEM of the values obtained from 10 animals. Statistical differences: *p < 0.05, **p < 0.001, ***p < 0.0005 vs. control group (ANOVA followed by Dunnett’s test).
I. Bialuk et al. / Pharmacological Reports 66 (2014) 931–936
C
B
200
150
***
100
***
50
0 Control
0.025
0.125
0.25
30
**
20
10
***
***
0.125
0.25
0 Control
0.025
mg/kg
mg/kg
Number of head dips / 5 min
A Number of rearings / 5 min
Number of squares crossed / 5 min
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8
6
4
*** ***
2
0 Control
0.025
0.125
0.25
mg/kg
Fig. 2. Effect of a single ip injection of CP55,940 on psychomotor activity, administered 30 min before testing the animals in a hole-board test. During a 5-min observation the following parameters were counted: locomotor activity measured by the number of squares crossed = sum of central and peripheral ambulation (A), exploratory activity measured by the number of rearings (B), as well as the number of head dips (C). Data are presented as means SEM of the values obtained from 7 to 9 animals. Statistical differences: **p < 0.01, ***p < 0.001 vs. control group (ANOVA followed by Dunnett’s test).
tendency to be dose-dependently decreased, although there were no statistical differences between the control group and groups of animals injected with CP55,940 (Table 2). The exploratory activity measured by the number of rearings and head-dips varied between the groups (Fig. 2B and C). The ANOVA of the three groups injected with the cannabinoid agonist and the control group yielded F3,30 = 56.39, p < 0.0001 and F3,30 = 15.62, p < 0.0001 for rearings and head-dips, respectively. The post hoc comparison between these groups made with the Dunnett’s test revealed a significant dose-dependent decrease in the number of rearings (0.025 mg/kg – p < 0.005; 0.125 and 0.25 mg/kg – p < 0.0005) in comparison to the control group (Fig. 2B) and a decrease in the number of head-dips, which reached a significant level at doses of 0.125 and 0.25 mg/kg (p < 0.0005) (Fig. 2C). Discussion In the present study we showed that CP55,940, the potent synthetic agonist of cannabinoid receptors, attenuates retrieval of spatial memory in mice, evaluated in the Morris water maze. CP55,940, given intraperitoneally once, 30 min before testing trial, at the highest dose (0.25 mg/kg), but not at the lower doses (0.025 and 0.125 mg/kg), significantly changed all parameters used for spatial memory evaluation. It increased the latency in the first crossing of the former platform location (target area), diminished the number of crossings over the target area, and reduced the time spent in the target quadrant. The results of the present study are in agreement with those reported by Lichtman et al. [11], as well as by Wise et al. [14] in rats, and recently in mice by Avdesh et al. [26] in the radial arm test. Lichtman et al. [11] reported that systemic and intrahippocampal administration of CP55,940 impaired maze performance in a dose-dependent manner. In their study, working memory impairment and significantly delayed test time duration were observed after systemic injection of CP55,940 at doses of 0.15; 0.18 or 0.25 mg/kg, but not at the lower dose of 0.125 mg/kg, whereas in our study spatial memory impairment caused by CP55,940 was noted only after a dose of 0.25 mg/kg, and significant attenuation of locomotor activity took place in mice injected with 0.125 and 0.25 mg/kg. However, we used a Morris water maze to assess spatial memory and a hole-board test to evaluate locomotor activity. Moreover, our study was conducted on mice. Furthermore, the effect of intrahippocampal CP55,940 infusion, evaluated by Lichtman et al. [11] was apparently specific to cognition as except for memory impairment no other pharmacological effects such as antinociception, hypothermia or catalepsy were detected. Also infusions of other cannabinoids into the hippocampus
e.g. delta-9 THC, as compared to other brain regions, impaired memory performance in the radial arm maze task [12]. Similarly, administration of WIN55,212-2 only to the dorsal but not to the ventral hippocampus, nucleus accumbens, ventral tegmental area or medial prefrontal cortex, selectively impaired memory retrieval in the radial arm maze without affecting locomotor activity [12]. Moreover, post-training intrahippocampal administration of WIN55,212-2 disrupted long-term spatial memory in a rat reference task in the water maze [27]. The above observations are consistent with the findings reported by Wise et al. [14], demonstrating that microinjections of CB1 receptor antagonist, rimonabant (SR141716A), into the rat hippocampus blocked spatial memory deficits caused by CP55,940 and delta-9 THC in the radial arm maze task. The observation that intrahippocampal administration of the CB1 antagonist prevented memory impairment caused by systemically applied CP55,940 and delta-9 THC provided strong evidence that the memory altering effects of these agonists are mediated by the hippocampal CB1 receptors. Moreover, the effect of rimonabant was behaviourally selective since it did not alter the non-mnemonic effect of cannabinoids. It was also regionally selective, as its administration to sites just dorsal or ventral to the borders of the hippocampus did not antagonize the memory disruptive effect of systemically injected delta-9 THC. In the study performed on mice in the radial arm maze by Avdesh et al. [26], two strains of mice, C57BL/6J and C3H/HEJ were used, and only one, selected on the basis of previous research, a dose of 0.1 mg/kg CP55,940 was tested. While CP55,940 at this dose impaired both working and reference memory in C57BL/6J mice, it did not affect working memory in C3H/HEJ mice, probably because of the strain sensitivity to the compound tested. Contrary to earlier research performed by Wise et al. [14], CP55,940 impairment of working memory was not prevented by SR141716A, which according to the authors may indicate that in mice other than CB1 receptors mediate this effect. The results of experiments performed in the Morris water maze on mice are less consistent and present mainly effects of the naturally occurring cannabinoid delta-9 THC. In experiments conducted on C57BL/6 mice Varvel et al. [15] showed that delta-9 THC disrupted performance of the working memory task at a dose of 3 mg/kg, which was much lower than the dose required to disrupt performance of the reference memory task (100 mg/kg) or elicit hypomotility, antinociception, hypothermia and catalepsy. In another study performed on C57BL/6 mice, delta-9 THC impaired acquisition of spatial learning at doses of 3 and 10 mg/kg and only at the higher dose deteriorated the retrieval of previously learned information exclusively in the hidden platform task, thus indicating that delta-9 THC selectively impairs memory retrieval independently of its effects on sensorimotor performance,
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motivation and initial acquisition [28]. The effects reported in both studies were prevented by 3 mg/kg of CB1 receptor antagonist SR141716A, demonstrating a CB1 receptor mechanism of action. In a study performed by Da and Takahashi [16], delta-9 THC at a dose of 8 mg/kg, but not in the lower doses (4 and 6 mg/kg), impaired spatial learning acquisition and working memory task performance in male albino mice, similarly to the above mentioned studies. These effects were also prevented by SR141716A administration. However, delta-9 THC did not have any influence on consolidation and retrieval processes. In our present study performed on C57BL/6J mice, much more potent than delta-9 THC, the CB1 receptor agonist, CP55,940 caused impairment of spatial memory retrieval only at the highest dose (0.25 mg/kg), being higher than those eliciting motor inhibition in the hole-board test. Lack of delta-9 THC effect on consolidation and retrieval process in albino mice reported by Da and Takahashi [16] could be explained by lower sensitivity of this strain to cannabinoids. These data seem to indicate the possibility that learning can be more easily disrupted than retrieval process. However, the likelihood that acquisition and retrieval are qualitatively different processes with regard to neurochemical events could not be excluded. Moreover, memory enhancement in cannabinoid CB1 receptor knock-out mice, confirming an important role of the endocannabinoid system in the processes of memory storage and retrieval [29], is in accordance with the finding of memory improving effect caused by SR141716A [30,31] and other CB1 receptor antagonists/inverse agonists: AM251 [32] and CE [33]. Since motor performance is essential for memory expression, and cannabinoids may attenuate psychomotor activity of animals, the influence of CP55,940 on locomotor and exploratory activity, in the time related to animal testing in the Morris water maze, was examined in a hole-board test, to clarify whether motor activity may affect the parameters used for memory evaluation. CP55,940 at higher doses of 0.125 and 0.25 mg/kg significantly attenuated locomotor activity measured by the number of crossed squares and decreased exploratory activity determined by the number of rearings and head-dips in comparison to the control group. CP55,940 at the lowest dose (0.025 mg/kg) did not alter the number of crossed squares and head-dips, but significantly attenuated the number of rearings. Since CP55,940 at a dose of 0.125 mg/kg did not alter any parameter used for memory evaluation in the Morris water maze, but decreased locomotor and exploratory activity, it may indicate that the memory disrupting effect observed in the present study, caused by the highest dose of CP55,940 (0.25 mg/kg), was independent of its influence on psychomotor activity. Our data are in agreement with experiments reporting a dissociation of CB1 receptor agonists’ effects on memory and locomotor activity. In our earlier study performed on rats [6], attenuation of locomotor activity occurred at a dose of 0.25 mg/kg of CP55,940, while impairment of recognition memory at a lower dose of 0.025 mg/kg. Also Lichtman et al. [11] reported that delta-9 THC and CP55,940 were approximately twice as potent in eliciting maze failure than locomotor suppression. Similarly, Nakamura et al. [34] using a low dose of delta-9 THC (1.25 mg/kg) described a significant increase in errors in an 8-arm radial maze with no apparent effects on locomotor activity, whereas Wegener et al. [35] showed that WIN55,212-2 at a dose of 1.2 mg/kg impaired retrieval of spatial memory in rats and had no effect on locomotion in an open field. Although in rats, spatial memory impairment was found to occur after lower doses of CB1 receptor agonists than motor inhibition, in the only study performed on mice, similarly to our present study, significant attenuation of their spontaneous activity was caused by a lower dose of CP55,940 as compared to the impairment of reference memory in the Morris water maze [15].
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The dissociation of cannabinoid effects on locomotion and cognition could be explained by the activity of the endogenous cannabinoid system regulating locomotion, located in the cerebellum and in the basal ganglia, supported by high density of CB1 receptors in these structures [36–38]. Dose-dependent motor incoordination reported following direct intracerebellar microinjection of CP55,940 or HU-210 [38] supports the existence of this system. Moreover, significant prevention of motor disturbances caused by local application of SR141716A or a CB1 receptor antisense provides strong evidence that a crucial role in this system is played by CB1 cannabinoid receptors. This hypothesis is in accordance with the observation that microinjection of CP55,940 into the hippocampus, an area with high density of CB1 receptors, did not impair motor coordination, but exclusively disrupted cognitive processes [39]. There is support for the notion that cannabinoids also alter the anxiety level, which can interfere with the memory expression in behavioural tests. In experiments performed in the elevated plus maze and in light–dark crossing tests [20–22,40,41], low doses of CB1 receptor agonists have been shown to induce anxiolytic-like, while high doses anxiogenic-like effects. This bidirectional effect of cannabinoids on anxiety has been recently explained by the differential sensitivity and basal activity of the CB1 receptors present either on glutamatergic or GABA-ergic terminals and by cannabinoid regulation of the cross-talk between these two neurotransmitter systems in a study performed by Rey et al. [21]. In their research, CP55,940 at a low (1 mg/kg) and high (50 mg/kg) dose evoked anxiolytic and anxiogenic effects, respectively, in wild-type C57BL/6N mice assessed in the elevated plus maze and in hole-board tests. The application of these two doses of CP 55,940 to GABA-CB1-KO and GLU-CB1-KO C57BL/6N mice has shown that CB1 receptor activation in forebrain GABA-ergic neurons is required to induce an anxiogenic-like effect, while CB1 receptors in cortical glutamatergic neurons mediate anxiolytic-like effect. In the wild-type mice, CP55, 940 at a dose of 50 mg/kg significantly decreased the percentage of open arms entries in plus-maze test and the percentage of central ambulation in holeboard test. In our present study a similar, although insignificant, approximately 10% decrease in central ambulation in a hole-board test was observed after 0.125 and 0.25 mg/kg, but not after the lowest dose (0.025 mg/kg) of CP55,940, which may indicate that the two higher doses exerted an anxiogenic-like effect. To sum up, the present study indicated that CP55,940, a potent synthetic cannabinoid receptor agonist, administered systemically once at the highest dose (0.25 mg/kg) impaired retrieval of spatial memory in C57BL/6J male mice in the Morris water maze, and at doses of 0.125 and 0.25 mg/kg attenuated locomotor and exploratory activity as well as increased anxiety. Given that the attenuation of locomotor activity and anxiogenic-like effect exerted by a dose of 0.125 mg/kg did not have any influence on spatial memory retrieval, it may suggest that the impairment of spatial memory observed after the highest dose of CP55,940 (0.25 mg/kg) was exerted by its influence on cognitive processes, however, the impact on locomotion could not be entirely excluded. This data support observations that popular habit of a Marijuana smoking may be dangerous because it can affect motor coordination and cognitive functions. Conflict of interest The authors declare that there is no conflict of interest. Funding This research was supported by the statutory funds from the Medical University of Białystok: UMB grant No. 3-24630
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Acknowledgment The authors wish to thank Krystyna Filon´czuk, M.Sc. for outstanding technical assistance. References [1] Zuardi AW. History of cannabis as a medicine: a review. Rev Bras Psiquiatr 2006;28:153–7. [2] Di MV. Biosynthesis and inactivation of endocannabinoids: relevance to their proposed role as neuromodulators. Life Sci 1999;65:645–55. [3] Hill A, Williams C, Whalley B, Stephens G. Phytocannabinoids as novel therapeutic agents in CNS disorders. Pharmacol Ther 2012;133:79–97. [4] Grotenhermen F. Pharmacokinetics and pharmacodynamics of cannabinoids. Clin Pharmacokinet 2003;42:327–60. [5] Castellano C, Rossi-Arnaud C, Cestari V, Costanzi M. Cannabinoids and memory: animal studies. Curr Drug Targets CNS Neurol Disord 2003;2: 389–402. [6] Kosiorek P, Hryniewicz A, Bialuk I, Zawadzka A, Winnicka MM. Cannabinoids alter recognition memory in rats. Pol J Pharmacol 2003;55:903–10. [7] Suenaga T, Kaku M, Ichitani Y. Effects of intrahippocampal cannabinoid receptor agonist and antagonist on radial maze and T-maze delayed alternation performance in rats. Pharmacol Biochem Behav 2008;91:91–6. [8] Thompson RF, Kim JJ. Memory systems in the brain and localization of a memory. Proc Natl Acad Sci U S A 1996;93:13438–44. [9] Olton DS, Samuelson RJ. Remembrance of places passed – spatial memory in rats. J Exp Psychol Anim Behav Process 1976;2:97–116. [10] Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 1984;11:47–60. [11] Lichtman AH, Dimen KR, Martin BR. Systemic or intrahippocampal cannabinoid administration impairs spatial memory in rats. Psychopharmacology (Berl) 1995;119:282–90. [12] Egashira N, Mishima K, Iwasaki K, Fujiwara M. Intracerebral microinjections of delta 9-tetrahydrocannabinol: search for the impairment of spatial memory in the eight-arm radial maze in rats. Brain Res 2002;952:239–45. [13] Lichtman AH, Martin BR. Delta 9-tetrahydrocannabinol impairs spatial memory through a cannabinoid receptor mechanism. Psychopharmacology (Berl) 1996;126:125–31. [14] Wise LE, Thorpe AJ, Lichtman AH. Hippocampal CB(1) receptors mediate the memory impairing effects of Delta(9)-tetrahydrocannabinol. Neuropsychopharmacology 2009;34:2072–80. [15] Varvel SA, Hamm RJ, Martin BR, Lichtman AH. Differential effects of delta 9THC on spatial reference and working memory in mice. Psychopharmacology (Berl) 2001;157:142–50. [16] Da S, Takahashi RN. SR 141716A prevents delta 9-tetrahydrocannabinolinduced spatial learning deficit in a Morris-type water maze in mice. Prog Neuropsychopharmacol Biol Psychiatry 2002;26:321–5. [17] Niyuhire F, Varvel SA, Martin BR, Lichtman AH. Exposure to marijuana smoke impairs memory retrieval in mice. J Pharmacol Exp Ther 2007;322: 1067–75. [18] Jarbe TU, DiPatrizio NV, Li C, Makriyannis A. Effects of AM1346, a high-affinity CB1 receptor selective anandamide analog, on open-field behavior in rats. Behav Pharmacol 2007;18:673–80. [19] Romero EM, Fernandez B, Sagredo O, Gomez N, Uriguen L, Guaza C, et al. Antinociceptive, behavioural and neuroendocrine effects of CP 55,940 in young rats. Brain Res Dev Brain Res 2002;136:85–92.
[20] Genn RF, Tucci S, Marco EM, Viveros MP, File SE. Unconditioned and conditioned anxiogenic effects of the cannabinoid receptor agonist CP 55,940 in the social interaction test. Pharmacol Biochem Behav 2004;77:567–73. [21] Rey AA, Purrio M, Viveros MP, Lutz B. Biphasic effects of cannabinoids in anxiety responses: CB1 and GABA(B) receptors in the balance of GABAergic and glutamatergic neurotransmission. Neuropsychopharmacology 2012;37:2624–34. [22] Rutkowska M, Jamontt J, Gliniak H. Effects of cannabinoids on the anxiety-like response in mice. Pharmacol Rep 2006;58:200–6. [23] Varvel SA, Lichtman AH. Evaluation of CB1 receptor knockout mice in the Morris water maze. J Pharmacol Exp Ther 2002;301:915–24. [24] Vorhees CV, Williams MT. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 2006;1:848–58. [25] Boissier JR, Simon P. The exploration reaction in the mouse. Preliminary note. Therapie 1962;17:1225–32. [26] Avdesh A, Hoe Y, Martins RN, Martin-Iverson MT. Pharmacological effects of cannabinoids on the reference and working memory functions in mice. Psychopharmacology (Berl) 2013;225:483–94. [27] Yim TT, Hong NS, Ejaredar M, McKenna JE, McDonald RJ. Post-training CB1 cannabinoid receptor agonist activation disrupts long-term consolidation of spatial memories in the hippocampus. Neuroscience 2008;151:929–36. [28] Niyuhire F, Varvel SA, Thorpe AJ, Stokes RJ, Wiley JL, Lichtman AH. The disruptive effects of the CB1 receptor antagonist rimonabant on extinction learning in mice are task-specific. Psychopharmacology (Berl) 2007;191:223–31. [29] Reibaud M, Obinu MC, Ledent C, Parmentier M, Bohme GA, Imperato A. Enhancement of memory in cannabinoid CB1 receptor knock-out mice. Eur J Pharmacol 1999;379:R1–2. [30] Lichtman AH. SR 141716A enhances spatial memory as assessed in a radialarm maze task in rats. Eur J Pharmacol 2000;404:175–9. [31] Wolff MC, Leander JD. SR 141716A, a cannabinoid CB1 receptor antagonist, improves memory in a delayed radial maze task. Eur J Pharmacol 2003;477:213–7. [32] Bialuk I, Winnicka MM. AM251, cannabinoids receptors ligand, improves recognition memory in rats. Pharmacol Rep 2011;63:670–9. [33] Wise LE, Iredale PA, Lichtman AH. The cannabinoid CB(1) receptor antagonist CE prolongs spatial memory duration in a rat delayed radial arm maze memory task. Eur J Pharmacol 2008;590:246–9. [34] Nakamura EM, da Silva EA, Concilio GV, Wilkinson DA, Masur J. Reversible effects of acute and long-term administration of delta-9-tetrahydrocannabinol (THC) on memory in the rat. Drug Alcohol Depend 1991;28:167–75. [35] Wegener N, Kuhnert S, Thuns A, Roese R, Koch M. Effects of acute systemic and intra-cerebral stimulation of cannabinoid receptors on sensorimotor gating, locomotion and spatial memory in rats. Psychopharmacology (Berl) 2008;198: 375–85. [36] Herkenham M, Lynn AB, Johnson MR, Melvin LS, De Costa BR, Rice KC. Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. J Neurosci 1991;11:563–83. [37] Mailleux P, Vanderhaeghen JJ. Distribution of neuronal cannabinoid receptor in the adult rat brain: a comparative receptor binding radioautography and in situ hybridization histochemistry. Neuroscience 1992;48:655–68. [38] Matsuda LA, Bonner TI, Lolait SJ. Localization of cannabinoid receptor mRNA in rat brain. J Comp Neurol 1993;327:535–50. [39] DeSanty KP, Dar MS. Cannabinoid-induced motor incoordination through the cerebellar CB(1) receptor in mice. Pharmacol Biochem Behav 2001;69:251–9. [40] Marco EM, Perez-Alvarez L, Borcel E, Rubio M, Guaza C, Ambrosio E, et al. Involvement of 5-HT1A receptors in behavioural effects of the cannabinoid receptor agonist CP 55,940 in male rats. Behav Pharmacol 2004;15:21–7. [41] Berrendero F, Maldonado R. Involvement of the opioid system in the anxiolytic-like effects induced by Delta(9)-tetrahydrocannabinol. Psychopharmacology (Berl) 2002;163:111–7.
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
CP55,940 attenuates spatial memory retrieval in mice Izabela Bialuk *,1, Katarzyna Dobosz 1, Bartosz Potrzebowski, Maria Małgorzata Winnicka Department of General and Experimental Pathology, Medical University of Białystok, Białystok, Poland
A R T I C L E I N F O
Article history: Received 20 May 2014 Accepted 4 June 2014 Available online 19 June 2014 Keywords: CP55,940 Spatial memory retrieval Morris water maze Hole-board test Mice
A B S T R A C T
Background: Cannabinoids constitute a varied group of lipophilic substances able to infiltrate the blood– brain barrier and influence neuronal processes. Clinical observations supported by experimental data have revealed that these compounds exert a deleterious effect on cognitive processes. The present study was carried out to determine the influence of a single systemic administration of CP55,940, a potent synthetic agonist of cannabinoid receptors, on spatial memory retrieval assessed in a Morris water maze. Methods: C57BL/6J male mice were submitted to three consecutive days of training to find a hidden platform in the water maze. CP55,940 was given intraperitoneally once, at doses of 0.025, 0.125 or 0.25 mg/kg on the fourth day, 30 min before testing memory retrieval, and in separate groups before testing psychomotor activity and anxiety level in a hole-board test. Results: CP55,940 only at the highest dose of 0.25 mg/kg significantly altered all parameters used to assess spatial memory. It increased the latency in the first crossing of the former platform location (target area), decreased the number of target area crossings and shortened the time spent in the target quadrant. Moreover, CP55,940 at doses of 0.25 and 0.125 mg/kg attenuated motor and exploratory activity in hole-board test. Conclusion: Since the attenuated psychomotor activity after a dose of 0.125 mg/kg did not interfere with memory retrieval, we assume that the impairment of spatial memory observed after the highest dose of CP55,940 (0.25 mg/kg) was exerted by its influence on cognitive processes, however, the impact on locomotion could not be excluded. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Marijuana has been used for medical and narcotic purposes for thousands of years, and remains the most common illicit drug [1]. Its constituents responsible for psychoactive effects are known as cannabinoids, highly lipophilic compounds, able to infiltrate the blood–brain barrier [2,3]. Intensive research on cannabinoids has led to the discovery of a tonically active endocannabinoid system, composed of cannabinoid receptors, their endogenous ligands and enzymes involved in their metabolism [2]. High density of cannabinoid receptors in the brain suggests an important role of endocannabinoids in the physiological activity of the central
Abbreviations: CP55,940, ( )-cis-3-[2-Hydroxy-4-(1,1-dimethy-lheptyl)phenyl]trans-4-(3-hydroxypropyl)cyclohexanol; CB1 receptor, cannabinoid receptor type 1; delta-9 THC, delta9-tetrahydrocannabinol; SR141716A, [N-(piperidin-1-yl)-5(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride]. * Corresponding author. E-mail address: [email protected] (I. Bialuk). 1 These authors have contributed equally to this work.
nervous system (CNS). Currently, the diverse group of cannabinoids, consisting of a broad range of endogenous, exogenous and synthetic compounds, creates potential drugs for the treatment of variety diseases [3,4], and therefore better understanding of cannabinoid properties is of critical value. The increasing evidence supports a negative effect of cannabinoid receptor type 1 (CB1) agonists on learning and memory processes [5–8]. Among cognitive processes, spatial memory is of significant importance in adaptation to the environment, both for human and animals. Two types of behavioural tests are commonly used as animal models for its evaluation: radial maze [9] and Morris water maze [10]. There is support for the notion that different CB1 receptor agonists impair spatial memory in rats. Lichtman et al. [11] showed that systemic administration of delta-9 THC, the main active constituent of marijuana, and two potent synthetic cannabinoids: WIN-55,212-2 and CP55,940 increased the number of errors and completion time delay in the radial maze. Moreover, they revealed that also intrahippocampal administration of CP55,940 caused a dose-dependent impairment of maze performance. Egashira et al. [12] demonstrated that bilateral microinjections of delta-9 THC
http://dx.doi.org/10.1016/j.pharep.2014.06.002 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
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into dorsal hippocampus, ventral hippocampus or dorsomedial thalamic nucleus impaired spatial memory in the eight-arm radial maze in rats. Systemic pre-treatment with SR141716A, a selective CB1 receptor antagonist, prevented delta-9 THC-induced memory deficits in the radial-arm maze, providing strong evidence that delta 9-THC impairs working memory through direct action at cannabinoid receptors. The involvement of CB1 receptors in this effect was supported by intrahippocampal administration of rimonabant (SR141716A) that reversed delta-9 THC- and CP55,940-evoked memory deficit in the radial maze [13,14]. The results of experiments performed in the Morris water maze are less consistent and predominantly demonstrate the effects of delta-9 THC. Varvel et al. [15] reported a selective impairment of working memory in mice by delta-9 THC mediated by CB1 receptors. Da and Takahashi [16] confirmed the impairment of mice performance in the working memory task by delta 9-THC, which was prevented by CB1 receptor antagonist – SR141716A. Moreover, they reported that delta 9-THC impaired acquisition of spatial learning, but did not affect consolidation and retrieval of the previously learned task. Contrary to previous data, Niyuhire et al. [17] demonstrated that delta 9-THC and exposure to marijuana smoke impaired memory retrieval in mice through a CB1 receptor mechanism. There are no current reports on acute CP55,940 effects on mice tested in the Morris water maze, and the problem whether the activation of CB1 receptors impairs memory retrieval remains unclear. Therefore, the present study was performed in an attempt to evaluate whether CP55,940, a potent synthetic agonist of cannabinoid receptors, affects spatial memory retrieval in C57BL/6J mice in the Morris water maze. Since cannabinoid receptors agonists are known to alter animals’ locomotor activity [18,19] and anxiety level [20–22], the influence of CP55,940 on ambulation and anxiety was assessed in separate groups of mice in a hole-board test. Materials and methods All experiments were approved by the Local Animal Ethics Committee in Bialystok, Poland and were performed in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Subjects Subjects were experimentally naı¨ve 4-month-old male mice – C57BL/6J, weighing approximately 30 g. The experiments were performed after 7 days of acclimatization to the laboratory conditions. The mice were maintained in a temperature-controlled environment (22 1 8C) with a 12 h light–dark cycles beginning at 7 a.m. and were housed in polycarbonate cages (27 cm 21 cm 14 cm), cm), five animals per cage, with water and commercial food available ad libitum. The experiments were carried out between 9.00 am and 1.00 pm in the air-conditioned, sound-isolated room with regulated light intensity. Drugs CP55,940 {( )-cis-3-[2-Hydroxy-4-(1,1-dimethy-lheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol} (Tocris) was dissolved in the 19% solution of cyclodextrin (SIGMA). Solutions were prepared before each behavioural test and protected from light. CP55,940 was given intraperitoneally randomly, at a single dose of 0.025, 0.125 or 0.25 mg/kg in a volume of 10 ml/kg. In order to assess the influence of CP55,940 on spatial memory retrieval the tested compound was administered on the fourth day, 30 min before testing animals in the Morris water maze. To evaluate the influence of CP55,940 on the locomotor and exploratory activity separate groups of animals received the same doses of the
Table 1 Starting points in the Morris water maze on the consecutive days of the experiment. Experiment day
Trial 1
Trial 2
Trial 3
Trial 4
1 2 3 4 Testing day
N SE NW NE
E N SE
SE NW E
NW E N
compound, 30 min before testing in a hole-board test. The control groups of animals were given a vehicle solution only at indicated time points. Behavioural tests Morris water maze The maze consisted of a circular, galvanized and painted in white steel pool (120 cm in diameter, 30 cm height), filled with water maintained at 23 1 8C [10,23,24]. The pool was divided into four equal quadrants: NE, NW, SE, SW. Four visual cues (A4-size sheets of white laminated paper with black geometric symbols) were attached on the internal marginates (N, W, E, S) of the pool and constituted navigation points. An escape platform (9 cm in diameter, 21 cm height) at a fixed location was made of transparent Plexiglas and was immersed 1 cm under the surface of water in the centre of the SW quadrant (target quadrant). The maze was lighted with the intensity of 45 lx. For three consecutive days mice were assigned to training sessions (one session a day) consisting of four trials separated by 10 min interval. Each trial started from one of the semi-random start positions (Table 1). If an animal did not find a submerged platform within 60 s, it was placed on the platform for the time of 15 s. On the fourth day the platform was removed from the pool. Thirty minutes after injection of CP55,940 or vehicle solution the animal was placed in the water maze for 60 s. Latency in the first crossing of the former platform location (target area), the number of crossings of the target area, and the time spent in the target quadrant were measured. If a mouse did not find the platform’s place within 60 s, it was given a latency score of 60 s. Hole-board test The experiments were performed according to the modified method described by Boissier and Simon [25]. The apparatus was a wooden box with a square floor of 55 cm 55 cm divided into 25 equal parts and surrounded by a 42 cm high wall. Four holes in the floor (2.5 cm in diameter) were designed as objects of possible interest of the animals. The apparatus was placed on the floor and lighted with the intensity of 30 lx. Thirty minutes after injection of CP55,940 or vehicle solution, the animal was placed in the centre of the hole-board box and its behaviour was observed for 5 min. Locomotor activity (ambulation) was measured as the number of squares crossed with all four limbs. Exploratory activity was measured as the number of rearings (rises of animal on its rear limbs, either with forelegs leaning against the wall or away from the wall) and the number of head-dips (dips of head into the hole). Moreover, during assessment of ambulation, external and internal locomotor activities of the mice were recorded. The number of crossed squares adjacent to the apparatus wall was used to measure peripheral activity, whereas the remaining crossed squares were considered as central activity. The percentage of central ambulation was used as an indication of anxiety level. Statistics The data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test using GraphPad Prism 5. Fratios, degrees of freedom and p-values are reported for significant differences at the level of p < 0.05.
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Table 2 Locomotor activity of mice evaluated in hole-board test, 30 min after CP 55,940 administration.
Results Morris water maze
CP55,940
CP55,940 given ip once, 30 min before the testing trial, significantly attenuated spatial memory retrieval. ANOVA of three groups of mice injected with increasing doses of CP55,940 and a control group showed significant differences in all parameters used for the evaluation of memory retrieval: latency in the first crossing of the former platform location (target area) (F3,36 = 8.12; p = 0.0003), the number of crossings of the target area (F3,36 = 6.57; p = 0.002) and the time spent in the target quadrant (F3,36 = 3.22; p = 0.033). Post hoc comparisons with Dunnett’s test revealed a significant increase in the latency to reach the former platform location in the group of mice given the highest dose of CP55,940 (0.25 mg/kg) (p < 0.0005) (Fig. 1B). Moreover, a significant decrease in the number of target crossings (p < 0.005) (Fig. 1C) and a significant reduction in the time spent in the target quadrant (p < 0.05) (Fig. 1D) were observed in comparison to the control group only after the highest dose of the compound tested. Hole-board test The ANOVA analysis of total, peripheral and central locomotor activity measured by the number of crossed squares showed significant differences between study groups (F3,30 = 16.43,
40 30 20 10 0 1
3
2
training day
C Crossings over target area
4
3
2
**
1
0 Control
0.025
mg/kg
0.125
0.25
Peripheral
Central
110.222 (9.416) 117.889 (18.421) 54.667* (13.733) 15.143*** (7.446)
44.111 (6.750) 28.556 (4.939) 8.889*** (3.791) 3.857*** (1.204)
27.8 23.9 19.9 22.1
(0.031) (0.050) (0.104) (0.086)
p < 0.0001; F3,30 = 12.03, p < 0.0001 and F3,30 = 14.08, p < 0.0001 for total, peripheral and central locomotor activity, respectively). The post hoc comparison made with the Dunnett’s test revealed significant attenuation of total and central ambulation in groups treated with CP55,940 at doses of 0.125 mg/kg and 0.25 mg/kg (p < 0.0005), as well as of peripheral ambulation in mice treated with CP55,940 at doses of 0.125 mg/kg (p < 0.05) and 0.25 mg/kg (p < 0.0005) as compared to the control group (Table 2 and Fig. 2A). Calculation of the percentage of central ambulation showed a
Latency to reach platform's palce (s)
50
% Of central ambulation
Data are presented as means (SEM) of the values obtained from 7 to 9 animals. Percentage of squares crossed in the central area of the apparatus (% of central ambulation = 100 central ambulation/central + peripheral ambulation). * Significant differences: p < 0.05 vs. control group (ANOVA followed by Dunnett’s test). *** Significant differences: p < 0.0005 vs. control group (ANOVA followed by Dunnett’s test).
Time spent in the target quadrant (sec)
Latency to reach platform (s)
A
Control 0.025 mg/kg 0.125 mg/kg 0.25 mg/kg
Locomotor activity
B 80
***
70 60 50 40 30 20 10 0 Control
0.025
0.125
0.25
mg/kg
D 25 20 15
* 10 5 0 Control
0.025
0.125
0.25
mg/kg
Fig. 1. Effect of a single ip injection of CP55,940 on spatial memory retrieval, administered 30 min before testing the animals in a Morris water maze. Learning was performed for three consecutive days of training to find a hidden platform (A) – data are presented as means of the values obtained from 40 animals. Twenty-four hours later, during a 60s observation in a platform-free water maze the following parameters of spatial memory were measured: (A) latency in the first crossing of the former platform location (target area), (B) the number of crossings of the target area, (C) the time spent in the target quadrant. Data are presented as means SEM of the values obtained from 10 animals. Statistical differences: *p < 0.05, **p < 0.001, ***p < 0.0005 vs. control group (ANOVA followed by Dunnett’s test).
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C
B
200
150
***
100
***
50
0 Control
0.025
0.125
0.25
30
**
20
10
***
***
0.125
0.25
0 Control
0.025
mg/kg
mg/kg
Number of head dips / 5 min
A Number of rearings / 5 min
Number of squares crossed / 5 min
934
8
6
4
*** ***
2
0 Control
0.025
0.125
0.25
mg/kg
Fig. 2. Effect of a single ip injection of CP55,940 on psychomotor activity, administered 30 min before testing the animals in a hole-board test. During a 5-min observation the following parameters were counted: locomotor activity measured by the number of squares crossed = sum of central and peripheral ambulation (A), exploratory activity measured by the number of rearings (B), as well as the number of head dips (C). Data are presented as means SEM of the values obtained from 7 to 9 animals. Statistical differences: **p < 0.01, ***p < 0.001 vs. control group (ANOVA followed by Dunnett’s test).
tendency to be dose-dependently decreased, although there were no statistical differences between the control group and groups of animals injected with CP55,940 (Table 2). The exploratory activity measured by the number of rearings and head-dips varied between the groups (Fig. 2B and C). The ANOVA of the three groups injected with the cannabinoid agonist and the control group yielded F3,30 = 56.39, p < 0.0001 and F3,30 = 15.62, p < 0.0001 for rearings and head-dips, respectively. The post hoc comparison between these groups made with the Dunnett’s test revealed a significant dose-dependent decrease in the number of rearings (0.025 mg/kg – p < 0.005; 0.125 and 0.25 mg/kg – p < 0.0005) in comparison to the control group (Fig. 2B) and a decrease in the number of head-dips, which reached a significant level at doses of 0.125 and 0.25 mg/kg (p < 0.0005) (Fig. 2C). Discussion In the present study we showed that CP55,940, the potent synthetic agonist of cannabinoid receptors, attenuates retrieval of spatial memory in mice, evaluated in the Morris water maze. CP55,940, given intraperitoneally once, 30 min before testing trial, at the highest dose (0.25 mg/kg), but not at the lower doses (0.025 and 0.125 mg/kg), significantly changed all parameters used for spatial memory evaluation. It increased the latency in the first crossing of the former platform location (target area), diminished the number of crossings over the target area, and reduced the time spent in the target quadrant. The results of the present study are in agreement with those reported by Lichtman et al. [11], as well as by Wise et al. [14] in rats, and recently in mice by Avdesh et al. [26] in the radial arm test. Lichtman et al. [11] reported that systemic and intrahippocampal administration of CP55,940 impaired maze performance in a dose-dependent manner. In their study, working memory impairment and significantly delayed test time duration were observed after systemic injection of CP55,940 at doses of 0.15; 0.18 or 0.25 mg/kg, but not at the lower dose of 0.125 mg/kg, whereas in our study spatial memory impairment caused by CP55,940 was noted only after a dose of 0.25 mg/kg, and significant attenuation of locomotor activity took place in mice injected with 0.125 and 0.25 mg/kg. However, we used a Morris water maze to assess spatial memory and a hole-board test to evaluate locomotor activity. Moreover, our study was conducted on mice. Furthermore, the effect of intrahippocampal CP55,940 infusion, evaluated by Lichtman et al. [11] was apparently specific to cognition as except for memory impairment no other pharmacological effects such as antinociception, hypothermia or catalepsy were detected. Also infusions of other cannabinoids into the hippocampus
e.g. delta-9 THC, as compared to other brain regions, impaired memory performance in the radial arm maze task [12]. Similarly, administration of WIN55,212-2 only to the dorsal but not to the ventral hippocampus, nucleus accumbens, ventral tegmental area or medial prefrontal cortex, selectively impaired memory retrieval in the radial arm maze without affecting locomotor activity [12]. Moreover, post-training intrahippocampal administration of WIN55,212-2 disrupted long-term spatial memory in a rat reference task in the water maze [27]. The above observations are consistent with the findings reported by Wise et al. [14], demonstrating that microinjections of CB1 receptor antagonist, rimonabant (SR141716A), into the rat hippocampus blocked spatial memory deficits caused by CP55,940 and delta-9 THC in the radial arm maze task. The observation that intrahippocampal administration of the CB1 antagonist prevented memory impairment caused by systemically applied CP55,940 and delta-9 THC provided strong evidence that the memory altering effects of these agonists are mediated by the hippocampal CB1 receptors. Moreover, the effect of rimonabant was behaviourally selective since it did not alter the non-mnemonic effect of cannabinoids. It was also regionally selective, as its administration to sites just dorsal or ventral to the borders of the hippocampus did not antagonize the memory disruptive effect of systemically injected delta-9 THC. In the study performed on mice in the radial arm maze by Avdesh et al. [26], two strains of mice, C57BL/6J and C3H/HEJ were used, and only one, selected on the basis of previous research, a dose of 0.1 mg/kg CP55,940 was tested. While CP55,940 at this dose impaired both working and reference memory in C57BL/6J mice, it did not affect working memory in C3H/HEJ mice, probably because of the strain sensitivity to the compound tested. Contrary to earlier research performed by Wise et al. [14], CP55,940 impairment of working memory was not prevented by SR141716A, which according to the authors may indicate that in mice other than CB1 receptors mediate this effect. The results of experiments performed in the Morris water maze on mice are less consistent and present mainly effects of the naturally occurring cannabinoid delta-9 THC. In experiments conducted on C57BL/6 mice Varvel et al. [15] showed that delta-9 THC disrupted performance of the working memory task at a dose of 3 mg/kg, which was much lower than the dose required to disrupt performance of the reference memory task (100 mg/kg) or elicit hypomotility, antinociception, hypothermia and catalepsy. In another study performed on C57BL/6 mice, delta-9 THC impaired acquisition of spatial learning at doses of 3 and 10 mg/kg and only at the higher dose deteriorated the retrieval of previously learned information exclusively in the hidden platform task, thus indicating that delta-9 THC selectively impairs memory retrieval independently of its effects on sensorimotor performance,
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motivation and initial acquisition [28]. The effects reported in both studies were prevented by 3 mg/kg of CB1 receptor antagonist SR141716A, demonstrating a CB1 receptor mechanism of action. In a study performed by Da and Takahashi [16], delta-9 THC at a dose of 8 mg/kg, but not in the lower doses (4 and 6 mg/kg), impaired spatial learning acquisition and working memory task performance in male albino mice, similarly to the above mentioned studies. These effects were also prevented by SR141716A administration. However, delta-9 THC did not have any influence on consolidation and retrieval processes. In our present study performed on C57BL/6J mice, much more potent than delta-9 THC, the CB1 receptor agonist, CP55,940 caused impairment of spatial memory retrieval only at the highest dose (0.25 mg/kg), being higher than those eliciting motor inhibition in the hole-board test. Lack of delta-9 THC effect on consolidation and retrieval process in albino mice reported by Da and Takahashi [16] could be explained by lower sensitivity of this strain to cannabinoids. These data seem to indicate the possibility that learning can be more easily disrupted than retrieval process. However, the likelihood that acquisition and retrieval are qualitatively different processes with regard to neurochemical events could not be excluded. Moreover, memory enhancement in cannabinoid CB1 receptor knock-out mice, confirming an important role of the endocannabinoid system in the processes of memory storage and retrieval [29], is in accordance with the finding of memory improving effect caused by SR141716A [30,31] and other CB1 receptor antagonists/inverse agonists: AM251 [32] and CE [33]. Since motor performance is essential for memory expression, and cannabinoids may attenuate psychomotor activity of animals, the influence of CP55,940 on locomotor and exploratory activity, in the time related to animal testing in the Morris water maze, was examined in a hole-board test, to clarify whether motor activity may affect the parameters used for memory evaluation. CP55,940 at higher doses of 0.125 and 0.25 mg/kg significantly attenuated locomotor activity measured by the number of crossed squares and decreased exploratory activity determined by the number of rearings and head-dips in comparison to the control group. CP55,940 at the lowest dose (0.025 mg/kg) did not alter the number of crossed squares and head-dips, but significantly attenuated the number of rearings. Since CP55,940 at a dose of 0.125 mg/kg did not alter any parameter used for memory evaluation in the Morris water maze, but decreased locomotor and exploratory activity, it may indicate that the memory disrupting effect observed in the present study, caused by the highest dose of CP55,940 (0.25 mg/kg), was independent of its influence on psychomotor activity. Our data are in agreement with experiments reporting a dissociation of CB1 receptor agonists’ effects on memory and locomotor activity. In our earlier study performed on rats [6], attenuation of locomotor activity occurred at a dose of 0.25 mg/kg of CP55,940, while impairment of recognition memory at a lower dose of 0.025 mg/kg. Also Lichtman et al. [11] reported that delta-9 THC and CP55,940 were approximately twice as potent in eliciting maze failure than locomotor suppression. Similarly, Nakamura et al. [34] using a low dose of delta-9 THC (1.25 mg/kg) described a significant increase in errors in an 8-arm radial maze with no apparent effects on locomotor activity, whereas Wegener et al. [35] showed that WIN55,212-2 at a dose of 1.2 mg/kg impaired retrieval of spatial memory in rats and had no effect on locomotion in an open field. Although in rats, spatial memory impairment was found to occur after lower doses of CB1 receptor agonists than motor inhibition, in the only study performed on mice, similarly to our present study, significant attenuation of their spontaneous activity was caused by a lower dose of CP55,940 as compared to the impairment of reference memory in the Morris water maze [15].
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The dissociation of cannabinoid effects on locomotion and cognition could be explained by the activity of the endogenous cannabinoid system regulating locomotion, located in the cerebellum and in the basal ganglia, supported by high density of CB1 receptors in these structures [36–38]. Dose-dependent motor incoordination reported following direct intracerebellar microinjection of CP55,940 or HU-210 [38] supports the existence of this system. Moreover, significant prevention of motor disturbances caused by local application of SR141716A or a CB1 receptor antisense provides strong evidence that a crucial role in this system is played by CB1 cannabinoid receptors. This hypothesis is in accordance with the observation that microinjection of CP55,940 into the hippocampus, an area with high density of CB1 receptors, did not impair motor coordination, but exclusively disrupted cognitive processes [39]. There is support for the notion that cannabinoids also alter the anxiety level, which can interfere with the memory expression in behavioural tests. In experiments performed in the elevated plus maze and in light–dark crossing tests [20–22,40,41], low doses of CB1 receptor agonists have been shown to induce anxiolytic-like, while high doses anxiogenic-like effects. This bidirectional effect of cannabinoids on anxiety has been recently explained by the differential sensitivity and basal activity of the CB1 receptors present either on glutamatergic or GABA-ergic terminals and by cannabinoid regulation of the cross-talk between these two neurotransmitter systems in a study performed by Rey et al. [21]. In their research, CP55,940 at a low (1 mg/kg) and high (50 mg/kg) dose evoked anxiolytic and anxiogenic effects, respectively, in wild-type C57BL/6N mice assessed in the elevated plus maze and in hole-board tests. The application of these two doses of CP 55,940 to GABA-CB1-KO and GLU-CB1-KO C57BL/6N mice has shown that CB1 receptor activation in forebrain GABA-ergic neurons is required to induce an anxiogenic-like effect, while CB1 receptors in cortical glutamatergic neurons mediate anxiolytic-like effect. In the wild-type mice, CP55, 940 at a dose of 50 mg/kg significantly decreased the percentage of open arms entries in plus-maze test and the percentage of central ambulation in holeboard test. In our present study a similar, although insignificant, approximately 10% decrease in central ambulation in a hole-board test was observed after 0.125 and 0.25 mg/kg, but not after the lowest dose (0.025 mg/kg) of CP55,940, which may indicate that the two higher doses exerted an anxiogenic-like effect. To sum up, the present study indicated that CP55,940, a potent synthetic cannabinoid receptor agonist, administered systemically once at the highest dose (0.25 mg/kg) impaired retrieval of spatial memory in C57BL/6J male mice in the Morris water maze, and at doses of 0.125 and 0.25 mg/kg attenuated locomotor and exploratory activity as well as increased anxiety. Given that the attenuation of locomotor activity and anxiogenic-like effect exerted by a dose of 0.125 mg/kg did not have any influence on spatial memory retrieval, it may suggest that the impairment of spatial memory observed after the highest dose of CP55,940 (0.25 mg/kg) was exerted by its influence on cognitive processes, however, the impact on locomotion could not be entirely excluded. This data support observations that popular habit of a Marijuana smoking may be dangerous because it can affect motor coordination and cognitive functions. Conflict of interest The authors declare that there is no conflict of interest. Funding This research was supported by the statutory funds from the Medical University of Białystok: UMB grant No. 3-24630
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Acknowledgment The authors wish to thank Krystyna Filon´czuk, M.Sc. for outstanding technical assistance. References [1] Zuardi AW. History of cannabis as a medicine: a review. Rev Bras Psiquiatr 2006;28:153–7. [2] Di MV. Biosynthesis and inactivation of endocannabinoids: relevance to their proposed role as neuromodulators. Life Sci 1999;65:645–55. [3] Hill A, Williams C, Whalley B, Stephens G. Phytocannabinoids as novel therapeutic agents in CNS disorders. Pharmacol Ther 2012;133:79–97. [4] Grotenhermen F. Pharmacokinetics and pharmacodynamics of cannabinoids. Clin Pharmacokinet 2003;42:327–60. [5] Castellano C, Rossi-Arnaud C, Cestari V, Costanzi M. Cannabinoids and memory: animal studies. Curr Drug Targets CNS Neurol Disord 2003;2: 389–402. [6] Kosiorek P, Hryniewicz A, Bialuk I, Zawadzka A, Winnicka MM. Cannabinoids alter recognition memory in rats. Pol J Pharmacol 2003;55:903–10. [7] Suenaga T, Kaku M, Ichitani Y. Effects of intrahippocampal cannabinoid receptor agonist and antagonist on radial maze and T-maze delayed alternation performance in rats. Pharmacol Biochem Behav 2008;91:91–6. [8] Thompson RF, Kim JJ. Memory systems in the brain and localization of a memory. Proc Natl Acad Sci U S A 1996;93:13438–44. [9] Olton DS, Samuelson RJ. Remembrance of places passed – spatial memory in rats. J Exp Psychol Anim Behav Process 1976;2:97–116. [10] Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 1984;11:47–60. [11] Lichtman AH, Dimen KR, Martin BR. Systemic or intrahippocampal cannabinoid administration impairs spatial memory in rats. Psychopharmacology (Berl) 1995;119:282–90. [12] Egashira N, Mishima K, Iwasaki K, Fujiwara M. Intracerebral microinjections of delta 9-tetrahydrocannabinol: search for the impairment of spatial memory in the eight-arm radial maze in rats. Brain Res 2002;952:239–45. [13] Lichtman AH, Martin BR. Delta 9-tetrahydrocannabinol impairs spatial memory through a cannabinoid receptor mechanism. Psychopharmacology (Berl) 1996;126:125–31. [14] Wise LE, Thorpe AJ, Lichtman AH. Hippocampal CB(1) receptors mediate the memory impairing effects of Delta(9)-tetrahydrocannabinol. Neuropsychopharmacology 2009;34:2072–80. [15] Varvel SA, Hamm RJ, Martin BR, Lichtman AH. Differential effects of delta 9THC on spatial reference and working memory in mice. Psychopharmacology (Berl) 2001;157:142–50. [16] Da S, Takahashi RN. SR 141716A prevents delta 9-tetrahydrocannabinolinduced spatial learning deficit in a Morris-type water maze in mice. Prog Neuropsychopharmacol Biol Psychiatry 2002;26:321–5. [17] Niyuhire F, Varvel SA, Martin BR, Lichtman AH. Exposure to marijuana smoke impairs memory retrieval in mice. J Pharmacol Exp Ther 2007;322: 1067–75. [18] Jarbe TU, DiPatrizio NV, Li C, Makriyannis A. Effects of AM1346, a high-affinity CB1 receptor selective anandamide analog, on open-field behavior in rats. Behav Pharmacol 2007;18:673–80. [19] Romero EM, Fernandez B, Sagredo O, Gomez N, Uriguen L, Guaza C, et al. Antinociceptive, behavioural and neuroendocrine effects of CP 55,940 in young rats. Brain Res Dev Brain Res 2002;136:85–92.
[20] Genn RF, Tucci S, Marco EM, Viveros MP, File SE. Unconditioned and conditioned anxiogenic effects of the cannabinoid receptor agonist CP 55,940 in the social interaction test. Pharmacol Biochem Behav 2004;77:567–73. [21] Rey AA, Purrio M, Viveros MP, Lutz B. Biphasic effects of cannabinoids in anxiety responses: CB1 and GABA(B) receptors in the balance of GABAergic and glutamatergic neurotransmission. Neuropsychopharmacology 2012;37:2624–34. [22] Rutkowska M, Jamontt J, Gliniak H. Effects of cannabinoids on the anxiety-like response in mice. Pharmacol Rep 2006;58:200–6. [23] Varvel SA, Lichtman AH. Evaluation of CB1 receptor knockout mice in the Morris water maze. J Pharmacol Exp Ther 2002;301:915–24. [24] Vorhees CV, Williams MT. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 2006;1:848–58. [25] Boissier JR, Simon P. The exploration reaction in the mouse. Preliminary note. Therapie 1962;17:1225–32. [26] Avdesh A, Hoe Y, Martins RN, Martin-Iverson MT. Pharmacological effects of cannabinoids on the reference and working memory functions in mice. Psychopharmacology (Berl) 2013;225:483–94. [27] Yim TT, Hong NS, Ejaredar M, McKenna JE, McDonald RJ. Post-training CB1 cannabinoid receptor agonist activation disrupts long-term consolidation of spatial memories in the hippocampus. Neuroscience 2008;151:929–36. [28] Niyuhire F, Varvel SA, Thorpe AJ, Stokes RJ, Wiley JL, Lichtman AH. The disruptive effects of the CB1 receptor antagonist rimonabant on extinction learning in mice are task-specific. Psychopharmacology (Berl) 2007;191:223–31. [29] Reibaud M, Obinu MC, Ledent C, Parmentier M, Bohme GA, Imperato A. Enhancement of memory in cannabinoid CB1 receptor knock-out mice. Eur J Pharmacol 1999;379:R1–2. [30] Lichtman AH. SR 141716A enhances spatial memory as assessed in a radialarm maze task in rats. Eur J Pharmacol 2000;404:175–9. [31] Wolff MC, Leander JD. SR 141716A, a cannabinoid CB1 receptor antagonist, improves memory in a delayed radial maze task. Eur J Pharmacol 2003;477:213–7. [32] Bialuk I, Winnicka MM. AM251, cannabinoids receptors ligand, improves recognition memory in rats. Pharmacol Rep 2011;63:670–9. [33] Wise LE, Iredale PA, Lichtman AH. The cannabinoid CB(1) receptor antagonist CE prolongs spatial memory duration in a rat delayed radial arm maze memory task. Eur J Pharmacol 2008;590:246–9. [34] Nakamura EM, da Silva EA, Concilio GV, Wilkinson DA, Masur J. Reversible effects of acute and long-term administration of delta-9-tetrahydrocannabinol (THC) on memory in the rat. Drug Alcohol Depend 1991;28:167–75. [35] Wegener N, Kuhnert S, Thuns A, Roese R, Koch M. Effects of acute systemic and intra-cerebral stimulation of cannabinoid receptors on sensorimotor gating, locomotion and spatial memory in rats. Psychopharmacology (Berl) 2008;198: 375–85. [36] Herkenham M, Lynn AB, Johnson MR, Melvin LS, De Costa BR, Rice KC. Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. J Neurosci 1991;11:563–83. [37] Mailleux P, Vanderhaeghen JJ. Distribution of neuronal cannabinoid receptor in the adult rat brain: a comparative receptor binding radioautography and in situ hybridization histochemistry. Neuroscience 1992;48:655–68. [38] Matsuda LA, Bonner TI, Lolait SJ. Localization of cannabinoid receptor mRNA in rat brain. J Comp Neurol 1993;327:535–50. [39] DeSanty KP, Dar MS. Cannabinoid-induced motor incoordination through the cerebellar CB(1) receptor in mice. Pharmacol Biochem Behav 2001;69:251–9. [40] Marco EM, Perez-Alvarez L, Borcel E, Rubio M, Guaza C, Ambrosio E, et al. Involvement of 5-HT1A receptors in behavioural effects of the cannabinoid receptor agonist CP 55,940 in male rats. Behav Pharmacol 2004;15:21–7. [41] Berrendero F, Maldonado R. Involvement of the opioid system in the anxiolytic-like effects induced by Delta(9)-tetrahydrocannabinol. Psychopharmacology (Berl) 2002;163:111–7.
