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Effects of sub-anesthetic doses of ketamine on rats' spatial and non-spatial recognition memory
Neuroscience 154 (2008) 454 – 460
EFFECTS OF SUB-ANESTHETIC DOSES OF KETAMINE ON RATS’ SPATIAL AND NON-SPATIAL RECOGNITION MEMORY N. PITSIKAS,* A. BOULTADAKIS AND N. SAKELLARIDIS
It has been shown that MK-801 and PCP usually impair either non-spatial (de Lima et al., 2005; Grayson et al., 2007; Nilsson et al., 2007) or spatial (Robinson and Crawley, 1993; Long and Kessner, 1995; Verma and Moghaddam, 1996) recognition memory in the rodent. At the moment, there is poor experimental evidence concerning the role of ketamine on recognition memory. It has been reported that ketamine disrupted rats’ recognition memory (Verma and Moghaddam, 1996), although another study did not support this finding (Robinson and Crawley, 1993). In addition, it is not clear yet if ketamine affects different stages of memory formation (acquisition, storage or retrieval of information). Taking the above evidences into account the aim of our study was to clarify the exact role of ketamine on recognition memory using different training procedures. For this purpose, the object recognition and the object location task were selected. The former is a non-spatial working memory task, does not involve at all, the learning of a rule since it is based on the spontaneous exploratory behavior of rats toward objects (Ennaceur and Delacour, 1988). The same authors developed a novel version of this procedure named object location task, aiming to evaluate spatial working memory in rodents (Ennaceur et al., 1997). This task assesses the ability to discriminate the novelty of the object location, but not the object itself, since the behavioral test arena is already familiar to the animal (Ennaceur et al., 1997).
Department of Pharmacology, School of Medicine, University of Thessaly, 22 Papakiriazi str., 412-22 Larissa, Greece
Abstract—There are experimental evidences indicating that the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist ketamine impairs cognition and produces a series of schizophrenia-like symptoms in rodents (hyperactivity, stereotypies and ataxia). The present study was designed to investigate the effects of ketamine on rats’ non-spatial and spatial recognition memory. For this purpose the object recognition and the object location task were selected. Pre- or post-training systemic administration of ketamine (0.3, 1 and 3 mg/kg; i.p.) in a dose-dependent manner disrupted animals’ performance in both these recognition memory paradigms, suggesting that this compound affected pre- and posttraining memory components. The current results indicate that the non-competitive NMDA antagonist ketamine may modulate either spatial or non-spatial recognition memory. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: ketamine, object recognition, object location, working memory, rat.
Non-competitive antagonists of the N-methyl-D-aspartate (NMDA) receptor such as phencyclidine (PCP), MK-801 or the dissociative anesthetic ketamine are known for their strong psychotomimetic effects in humans (Javitt and Zukin, 1991; Krystal et al., 1994). Ketamine, at sub-anesthetic doses, produces wideranging effects on rodent behavior, including disruption of sensory motor gating (Mansbach and Geyer, 1991), memory deficits (Jones et al., 1990; Uchihashi et al., 1994; Verma and Moghaddam, 1996; Imre et al., 2006; Kos et al., 2006), hypermotility (Willetts et al., 1990; Carlsson, 1993), stereotypy and ataxia (Tricklebank et al., 1989). Therefore, these compounds are used routinely as clinical and animal models of schizophrenia (Geyer, 1998). Recognition memory stems from a series of neural processes by which a subject is aware that a stimulus has been previously experienced, recognition being the behavioral outcome of these processes. It requires that the perceived characteristics of events be discriminated, identified and compared (matched) against a memory of the characteristics of previously experienced events (Steckler et al., 1998).
EXPERIMENTAL PROCEDURES Animals Male, 3-month-old Wistar rats (Hellenic Pasteur Institute, Athens, Greece), weighing 250 –300 g were used in this study. The animals were housed in Makrolon cages (45 cm long⫻35 cm high⫻20 cm wide) three per cage, in a regulated environment (21⫾1 °C; 50 –55% relative humidity; 12-h light/dark cycle, lights on at 07:00 h) with free access to food and water. Experiments were conducted in the room where only these animals where housed, and took place between 09:00 and 13:00 h. Animals’ behavior was video-recorded. Behavioral observations and evaluations were performed by experimenters who were unaware of the pharmacological treatment. Since both object recognition and object location are visuallydependent paradigms, a pilot study was carried out in order to examine whether or not ketamine affected rats’ visual abilities. The procedure described by Markowska et al., 1989, was used. This method, in brief, consists of lowering rats toward the edge of a table. If visual abilities of animals are not reduced they should always extend their forepaws well before they touch it. In this short investigation, male, 3-month-old rats were divided in four experimental groups (four rats per group) as follows: vehicle (NaCl 0.9%); ketamine 0.3 mg/kg; ketamine 1 mg/kg and ketamine
*Corresponding author. Tel: ⫹30-2410-565268; fax: ⫹30-2410-565236. E-mail address: [email protected] (N. Pitsikas). Abbreviations: ANOVA, analysis of variance; D, discrimination index; F, familiar object; FL, familiar location; ITI, intertribal interval; N, new object; NL, new location; NMDA, N-methyl-D-aspartate; n.s., not significant; PCP, phencyclidine; T1, sample trial; T2, choice trial.
0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.04.001
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N. Pitsikas et al. / Neuroscience 154 (2008) 454 – 460 3 mg/kg. Vehicle or ketamine was delivered 20 min before testing animals’ visual acuity. None of the treated rats presented evident signs of reduced visual acuity. Our findings are in agreement with a prior report in which it was observed that ketamine did not affect rats’ visual abilities up to 20 mg/kg (Lalonde and Joyal, 1991). All animal treatments were approved by the local ethical committee and were conducted in conformity with the international guidelines in compliance with national and international laws and policies (EEC Council Directive 86/609, JL 358, 1, December, 12, 1987; NIH Guide for Care and Use of Laboratory Animals, NIH publication no. 65-23, 1985). Every effort was made to minimize the number of animals used and their suffering.
Object recognition test The test apparatus consisted of a dark open box made of Plexiglas (80 cm long⫻50 cm high⫻60 cm wide) which was illuminated by a 60-W lamp suspended 60 cm above the box. In the different parts of the apparatus the light intensity was equal. The objects to be discriminated (in triplicate) made of glass, plastic, or metal, were in three different shapes: cubes, pyramids and cylinders 7 cm high; they could not be displaced by rats. The object recognition test was performed as described elsewhere (Ennaceur and Delacour, 1988). In the week preceding testing, the animals were handled twice daily. On the day before testing, they were allowed to explore the apparatus for 2 min, while on the testing day, a session of two 2-min trials was given. During the “sample” trial (T1), two identical samples (objects) were placed in two opposite corners of the apparatus. A rat was placed in the middle of the apparatus and was left to explore these two identical objects. After T1, the rat was put back in its home cage and an intertrial interval (ITI) of 60 min was given, since at this delay condition recognition memory is still intact in the untreated rat (Bartolini et al., 1996). Subsequently, the “choice” trial (T2) was performed. During T2, a new object (N) replaced one of the samples presented in T1, thence, the rats were re-exposed to two objects: the familiar object (F) and the N. All combinations and locations of objects were used in a balanced manner to reduce potential biases due to preferences for particular locations or objects. To avoid the presence of olfactory trails, the apparatus and the objects after each trial were thoroughly cleaned. Exploration was defined as follows: directing the nose toward the object at a distance of no more than 2 cm and/or touching the object with the nose. Turning around or sitting on the object was not considered as exploratory behavior. The times spent by rats in exploring each object during T1 and T2 were recorded manually by using a stopwatch. From this measure a series of variables was then calculated: the total time spent in exploring the two identical objects in T1, and that spent in exploring the two different objects, F and N in T2. The discrimination between F and N during T2 was measured by comparing the time spent in exploring the F with that spent in exploring the N. As this time may be biased by differences in overall levels of exploration (Cavoy and Delacour, 1993) a discrimination index (D) was then calculated; D⫽N-F/N⫹F. D is the discrimination ratio and represents the difference in exploration time expressed as a proportion of the total time spent exploring the two objects in T2 (Cavoy and Delacour, 1993). In addition, motor activity of each animal expressed as total number of steps during each trial was also recorded.
Object location test The test apparatus was the same apparatus used in the object recognition test. The test arena was located in a large observation room with external cues (large and distinctive objects) surrounding the experimental box to help rats to resolve this spatial memory task. These cues were kept in a constant location throughout the period of testing. The objects were the same objects in the object recognition task.
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The object location test was performed as described elsewhere (Ennaceur et al., 1997; Pitsikas, 2007). This test consisted of a period of habituation, a T1 and a T2. During habituation, the animals were allowed to freely explore the apparatus without objects for 2 min, once a day for three consecutive days before testing. On the testing day, a session of two 2-min trials was given. During the T1 two identical samples (objects) were placed in two opposite corners of the apparatus 10 cm from the sidewall. A rat was placed in the middle of the apparatus and was left to explore these two identical objects. After T1, the rat was put back in its home cage and an ITI of 20 min was given, since at this delay condition recognition memory is still intact in the untreated rat (Pitsikas, 2007). Subsequently, T2 was conducted. The rat was re-introduced to the apparatus. During T2, one of the two similar objects was moved to a different location (new location, NL) while the other object remained in the same position (familiar location, FL) as in the T1. All locations of objects were used in a balanced manner to reduce potential biases due to preferences for particular locations. To avoid the presence of olfactory trails, the apparatus and the objects after each trial were thoroughly cleaned. Exploration was defined as follows: directing the nose toward the object at a distance of no more than 2 cm and/or touching the object with the nose. Turning around or sitting on the object was not considered as exploratory behavior. The times spent by rats in exploring each object during T1 and T2 were recorded manually by using a stopwatch. From this measure, a series of variables was then calculated: the total time spent in exploring the two identical objects in T1 and that spent in exploring the two objects in the two different locations in T2. The discrimination between FL and NL during T2 was measured by comparing the time spent in exploring the object in the FL with that spent in exploring the object in the NL. As this time may be biased by differences in overall levels of exploration (Cavoy and Delacour, 1993) the D was then calculated; D⫽NL-FL/ NL⫹FL. D represents the difference in exploration time expressed as a proportion of the total time spent exploring the two objects in T2 (Cavoy and Delacour, 1993). In addition, motor activity of each animal expressed as total number of steps during each trial was also recorded.
Drugs Ketamine hydrochloride was dissolved in saline (NaCl 0.9%) and administered i.p. Control animals received the vehicle (NaCl 0.9%).
Experimental protocol Experiment 1: effects of pre-training administration of different doses of ketamine on animals’ performance in the object recognition task. Rats were randomly divided into four experimental groups (10 rats per group) as follows: vehicle; ketamine 0.3 mg/ kg; ketamine 1 mg/kg and ketamine 3 mg/kg. To study the effects on acquisition, ketamine and vehicle were administered 20 min before T1. Experiment 2: effects of post-training administration of different doses of ketamine on animals’ performance in the object recognition task. Rats were randomly divided into four experimental groups (10 rats per group) as follows: vehicle; ketamine 0.3 mg/kg; ketamine 1 mg/kg and ketamine 3 mg/kg. To study the effects on post-mnemonic stages (storage and/or retrieval), ketamine and vehicle were administered immediately after T1. Experiment 3: effects of pre-training administration of different doses of ketamine on animals’ performance in the object location task. Rats were randomly divided into four experimental groups (10 rats per group) as follows: vehicle; ketamine 0.3 mg/kg; ketamine 1 mg/kg and ketamine 3 mg/kg. To study the effects on
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Table 1. Comparison of the exploration time of the two similar objects (F1 and F2) during T1 displayed by different groups of rats in the object recognition task Group
N
Total exploration time (s), mean⫾S.E.M. Acquisition experiment
Vehicle Ketamine (0.3 mg/kg) Ketamine (1 mg/kg) Ketamine (3 mg/kg)
10 10 10 10
Storage experiment
F1
F2
F1
F2
7.3⫾0.7 6.0⫾0.3 6.5⫾0.5 7.0⫾0.7
7.9⫾0.6 6.4⫾0.4 6.9⫾0.7 6.4⫾0.4
8.2⫾0.9 6.2⫾0.8 7.3⫾0.8 7.2⫾0.6
8.9⫾0.4 7.4⫾0.9 6.1⫾0.7 8.1⫾0.4
N⫽number of rats.
acquisition, ketamine and vehicle were administered 20 min before T1. Experiment 4: effects of post-training administration of different doses of ketamine on animals’ performance in the object location task. Rats were randomly divided into four experimental groups (10 rats per group) as follows: vehicle; ketamine 0.3 mg/ kg; ketamine 1 mg/kg and ketamine 3 mg/kg. To study the effects on post-mnemonic stages, ketamine and vehicle were administered immediately after T1.
Statistical analysis All data are expressed as mean⫾S.E.M. Preference of animals for objects or locations during T1 was analyzed by the Student’s t-test for each experimental group. In experiments 1 and 3, motor activity and total exploration times during T1 and T2, were evaluated by the two-way analysis of variance (ANOVA) with a split-plot design (between-within subjects). The between factor was ketamine (three levels) and the within factor were trials (two levels). Post hoc comparisons were made by the Tukey’s post hoc test. In experiments 2 and 4, motor activity and total exploration times during T2 were assessed by the one-way ANOVA followed by the Tukey’s post hoc test. The factor was ketamine (three levels). Discrimination index D data were calculated by the one-way ANOVA test. The factor was ketamine (three levels). Post hoc comparisons were made by the Tukey’s test.
RESULTS No difference within any group when the exploration time was compared accordingly to the nature of objects (cubes, pyramids or cylinders) and their locations (left or right), in the apparatus was observed during T1 either in the object recognition or in the object location paradigm (Tables 1 and 2, respectively).
Experiment 1: effects of pre-training administration of different doses of ketamine on animals’ performance in the object recognition task Overall analysis of motor activity results did not reveal a main effect either of ketamine [F(3,36)⫽2, P⫽0.12, not significant (n.s.)] or of trials [F(1,36)⫽2.7, P⫽0.1, n.s.] or a significant interaction between ketamine and trials [F(3,36)⫽0.95, P⫽0.4, n.s., Fig. 1A]. Similarly, analysis of total exploration times did not show a main effect either of ketamine [F(3,36)⫽1.9, P⫽0.16, n.s.] or of trials [F(1,36)⫽0.2, P⫽0.65, n.s.] or a significant interaction between ketamine and trials [F(3,36)⫽1.2, P⫽0.3, n.s., Fig. 1B]. D results showed that the vehicle-treated rats discriminated better the novel than the F during T2 with respect to their counterparts which received ketamine [F(3,36)⫽7.8, P⬍0.01; Tukey’s post hoc test, P⬍0.05 vs. ketamine 1 and 3 mg/kg groups, Fig. 1C]. Experiment 2: effects of post-training administration of different doses of ketamine on animals’ performance in the object recognition task Statistical analyses of the motor activity data did not evidence a main effect of treatment [F(3,36)⫽1.2, P⫽0.31, n.s., Fig. 2A]. Total exploration levels during T2 were not different among the various groups of animals [F(3,36)⫽0.8, P⫽0.5, n.s., Fig. 2B]. Data for index D (Fig. 2C) revealed a significant effect of treatment [F(3,36)⫽10.6, P⬍0.01]. Post hoc comparisons have shown that the ketamine 1 and ketamine 3 mg/kg–treated animals were unable to discriminate between the familiar and the novel object with respect to the vehicle-treated rats
Table 2. Comparison of the exploration time of the two similar objects (F1 and F2) during T1 displayed by different groups of rats in the object location task. Group
N
Total exploration time (s), mean⫾S.E.M. Acquisition experiment
Vehicle Ketamine (0.3 mg/kg) Ketamine (1 mg/kg) Ketamine (3 mg/kg) N⫽number of rats.
10 10 10 10
Storage experiment
F1
F2
F1
F2
8.4⫾1.0 7.4⫾0.6 9.4⫾0.7 8.8⫾1.5
7.7⫾1.4 8.5⫾1.3 8.6⫾1.0 7.5⫾1.4
8.0⫾0.8 8.6⫾0.6 8.8⫾1.0 8.4⫾0.9
7.4⫾0.8 9.7⫾1.2 8.0⫾1.2 7.4⫾0.7
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P⫽0.94, n.s., Fig. 3A]. Similarly, analysis of total exploration times did not reveal a main effect either of ketamine [F(3,36)⫽0.9, P⫽0.43, n.s.] or of trials [F(1,36)⫽2.3, P⫽0.12, n.s.] or a significant interaction between ketamine and trials [F(3,36)⫽0.4, P⫽0.74, n.s., Fig. 3B]. D results showed that the vehicle-treated rats discriminated better the novel than the FL during T2 with respect to their counterparts which received ketamine; [F(3,36)⫽4.2, P⬍0.01; Tukey’s post hoc test, P⬍0.05 vs. ketamine 1 and 3 mg/kg groups, Fig. 3C]. Moreover, the ketamine 1 and
Fig. 1. Object recognition test. Acquisition experiment. Vehicle and ketamine were injected i.p., 20 min before starting T1. (A) Total motor activity displayed by different groups of rats. (B) Total exploration time displayed by different groups of rats. (C) Discrimination index D performance expressed by different groups of rats during T2. Results are expressed as mean⫾S.E.M. * P⬍0.05 vs. the vehicle group.
(P⬍0.05). In addition, the ketamine 3 mg/kg–treated rats’ performance was significantly lower as compared with ketamine 0.3 mg/kg–treated rodents (P⬍0.05). Experiment 3: effects of pre-training administration of different doses of ketamine on animals’ performance in the object location task Overall analysis of motor activity results did not show a main effect either of ketamine [F(3,36)⫽1, P⫽0.39, n.s.] or of trials [F(1,36)⫽0.96, P⫽0.33, n.s.] or a significant interaction between ketamine and trials [F(3,36)⫽0.14,
Fig. 2. Object recognition test. Storage experiment. Vehicle and ketamine were injected i.p., just after T1. (A) Total motor activity displayed by different groups of rats during T2. (B) Total exploration time displayed by different groups of rats during T2. (C) Discrimination index D performance expressed by different groups of rats during T2. Results are expressed as mean⫾S.E.M. * P⬍0.05 vs. the vehicle group; ⫹ P⬍0.05 vs. ketamine 0.3 mg/kg–treated rats.
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groups of animals [F(3,36)⫽1.5, P⫽0.8, n.s., Fig. 4B]. Data for index D (Fig. 4C) revealed a significant effect of treatment [F(3,36)⫽6.4, P⬍0.01]. Post hoc comparisons have shown that the ketamine 1 and ketamine 3 mg/kg– treated animals were unable to discriminate between the familiar and the novel location with respect to the vehicletreated rats (P⬍0.05). In addition, the ketamine 3 mg/kg– treated rats’ performance was significantly lower as compared with ketamine 0.3 mg/kg–treated rodents (P⬍0.05).
Fig. 3. Object location test. Acquisition experiment. Vehicle and ketamine were injected i.p., 20 min before starting T1. (A) Total motor activity displayed by different groups of rats. (B) Total exploration time displayed by different groups of rats. (C) Discrimination index D performance expressed by different groups of rats during T2. Results are expressed as mean⫾S.E.M. * P⬍0.05 vs. the vehicle group; ⫹ P⬍0.05 vs. ketamine 0.3 mg/kg–treated rats.
3 mg/kg–treated rats’ performance was significantly lower as compared with ketamine 0.3 mg/kg–treated rodents (P⬍0.05). Experiment 4: effects of post-training administration of different doses of ketamine on animals’ performance in the object location task Statistical analyses of the motor activity data did not show differences among the various experimental groups during T2 [F(3,36)⫽0.92, P⫽0.4, n.s., Fig. 4A]. Total exploration levels during T2 were not different among the various
Fig. 4. Object location test. Storage experiment. Vehicle and ketamine were injected i.p., just after T1. (A) Total motor activity displayed by different groups of rats during T2. (B) Total exploration time displayed by different groups of rats during T2. (C) Discrimination index D performance expressed by different groups of rats during T2. Results are expressed as mean⫾S.E.M. * P⬍0.05 vs. the vehicle group; ⫹ P⬍0.05 vs. ketamine 0.3 mg/kg–treated rats.
N. Pitsikas et al. / Neuroscience 154 (2008) 454 – 460
DISCUSSION Our results indicate that ketamine dose-dependently impaired both non-spatial and spatial recognition memory paradigms. At the dose of 0.3 mg/kg, it seemed to affect animals’ performance, though this effect did not reach statistical significance. However, when given at 1 and 3 mg/kg, ketamine, consistently impaired rats’ performance in the object recognition and object location procedures. In the present study, treatment was applied either before T1, or just after T1, and a certain ITI was given (60 min in the object recognition task and 20 min in the object location test). It is therefore impossible to dissect on which specific memory component ketamine was acting. This paper is providing evidence that, at least, pre-training administration of ketamine affected acquisition, while its posttraining administration affected storage and/or retrieval. Ketamine was applied systematically, thus, it cannot be excluded that non-specific factors (attentional, sensory motor) might have influenced animals’ performance. This possibility, however, could be ruled out since this compound did not affect rats’ visual acuity and did not influence rodents’ motor activity or exploration levels either in the object recognition procedure or in the object location task. It is also unlikely that the effects of ketamine on memory were due to residual presence of the drug during testing since this compound has a very short half-life (10 –15 min) (Krystal et al., 1994). Results of ours related to the effects of pre-training administration of ketamine are in line with previous reports in which pre-training administration of ketamine disrupted rodents’ performance in various memory paradigms (Jones et al., 1990; Uchihashi et al., 1994; Imre et al., 2006; Kos et al., 2006). The involvement of ketamine in post-training memory processes (storage and/or retrieval) has yet to be clarified. Reportedly, ketamine (2.5, 5 and 10 mg/kg) did not affect the retention latency when administered immediately after training or prior to retention of a passive avoidance task suggesting that in this behavioral paradigm it did not impair posttraining memory components in the mouse (Uchihashi et al., 1994). Conversely, ketamine disrupted post-training memory components in a spatial delayed alternation paradigm (Verma and Moghaddam, 1996) and in the autoshaping task (Liy-Salmeron and Meneses, 2007) performed in the rat. The present results are consistent with the latter studies and support a role of ketamine also in post-training memory processes. The above controversial findings on ketamine role might depend on differences in the experimental settings; e.g. the type of animal model (mouse vs. rat in our study), the dose regimen, and the behavioral pattern investigated (passive avoidance, a negatively reinforced paradigm utilized in the study by Uchihashi et al., vs. the object recognition and object location tasks—not rewarded tasks— used in our studies). To our knowledge, few studies aiming to investigate the role of non-competitive NMDA antagonists on object recognition memory have been carried out. Pre- or post-training administration either of MK-801 or PCP decreased retention abilities either in the male (Pitsikas et al., 2006; Pichat et al.,
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2007) or in the female rat (de Lima et al., 2005; Grayson et al., 2007). A recent investigation however, did not support these findings. It has been reported that MK-801 disrupted the encoding but not the post-training mnemonic stages (storage and retrieval) in the object recognition task in the male mouse (Nilsson et al., 2007). The species utilized (NMRI mouse vs. rat used in the other studies) and the low discrimination index displayed by the control mice may underlie this discrepancy (Nilsson et al., 2007). In this context, the possibility that the psychotomimetic properties of MK-801 might result in an amplification of the perceived salience of the novel object, and/or anxiolytic mechanisms could result in neophilic effects may to be considered (Nilsson et al., 2007). There is scant evidence whether or not object location is sensitive to pharmacological manipulation (Pitsikas, 2007). It is the first time to our knowledge that the effects of a noncompetitive NMDA receptor antagonist were evaluated in this spatial working memory paradigm. Object recognition and object location measure nonspatial and spatial recognition memory respectively. As compared with other cognitive tests, these paradigms do not involve a reward or a punishment and thus the behavioral outcome is not influenced by reinforcement/response interactions (Dere et al., 2007). The tasks are quite similar to procedures used in humans and should therefore have a significant level of predictive validity (Ennaceur and Delacour, 1988; Ennaceur et al., 1997). The mechanism by which ketamine produces its adverse behavioral effects, at least partly, have been attributed to the blockade of NMDA receptors located on GABA interneurons, which in turn leads to disinhibition of neural activity in limbic structures (Moghaddam et al., 1997). This disinhibitory action elicits an increase in terms of neuronal activity and excessive glutamate and dopamine release in the prefrontal cortex and limbic regions (Moghaddam et al., 1997; Lorrain et al., 2003; Razoux et al., 2007). In this context, controversial results have been reported concerning the dopamine D2 receptor contribution to the psychotominetic effects of the NMDA receptor antagonists including ketamine (Seeman et al., 2005; Jordan et al., 2006). Object recognition and object location were demonstrated sensitive to ketamine treatment. Both these procedures might represent valid tools for assessing the action of potential antipsychotic compounds on non-spatial and spatial working memory. Interestingly, ketamine impaired animals’ performance in these tasks at very low doses (1 and 3 mg/kg) as compared with those usually utilized in order to disrupt working memory in rodents (10 –30 mg/kg) (Jones et al., 1990; Uchihashi et al., 1994; Verma and Moghaddam, 1996; Imre et al., 2006; Kos et al., 2006). Although low doses of ketamine affected performance in object recognition and object location, certainly there is experimental evidence that anesthetic doses did not alter memory consolidation (LiySalmeron and Meneses, 2007; Meneses, 2007).
CONCLUSION In summary, studies herein presented indicate that systemic administration of sub-anesthetic doses of ketamine
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dose-dependently impaired non-spatial and spatial recognition memory in the rat. In addition, this non-competitive NMDA receptor antagonist seems to modulate pre- and post-training recognition memory components.
REFERENCES Bartolini L, Casamenti F, Pepeu G (1996) Aniracetam restores object recognition impaired by age, scopolamine and nucleus basalis lesions. Pharmacol Biochem Behav 53:277–283. Carlsson ML (1993) Are the disparate pharmacological profiles of competitive and un-competitive NMDA antagonists due to different baseline activities of distinct glutamatergic pathways? J Neural Transm 94:1–10. Cavoy A, Delacour J (1993) Spatial but not object recognition is impaired by aging in rats. Physiol Behav 53:527–530. de Lima MNM, Laranja DC, Bromberg E, Roesler R, Schroder N (2005) Pre- or post-training administration of the NMDA receptor blocker MK-801 impairs object recognition memory in rats. Behav Brain Res 156:139 –143. Dere E, Huston JP, De Souza MA (2007) The pharmacology, neuroanatomy and neurogenetics of one-trial object recognition in rodents. Neurosci Biobehav Rev 31:673–704. Ennaceur A, Delacour J (1988) A new one-trial test for neurobiological studies of memory in rats. 1. Behavioral data. Behav Brain Res 31:47–59. Ennaceur A, Neave N, Aggleton JP (1997) Spontaneous object recognition and object location memory in rats: the effects of lesions in the cingulated cortices, the medialprefrontal cortex, the cingulum bundle and the fornix. Exp Brain Res 113:509 –519. Geyer MA (1998) Behavioural studies of hallucinogenic drugs in animals: implications for schizophrenia research. Pharmacopsychiatry 31:73–79. Grayson B, Idris NF, Neill JC (2007) Atypical antipsychotics attenuate a sub-chronic PCP-induced cognitive deficits in the novel object recognition task in the rat. Behav Brain Res 184:31–38. Imre G, Fokkema DS, Den Boer JA, Ter Horst GJ (2006) Dose-response characteristics of ketamine effect on locomotion, cognitive function and central neuronal activity. Brain Res Bull 69:338–345. Javitt DC, Zukin RS (1991) Recent advances in the phencyclidine model of schizophrenia. Am J Psychiatry 148:1301–1308. Jones KW, Bauerle LM, DeNoble VJ (1990) Differential effects of and phencyclidine receptor ligands on learning. Eur J Pharmacol 179:97–102. Jordan S, Chen R, Fernalld R, Johnson J, Regardie K, Kambayashi J, Tadori Y, Kitagawa H, Kikuchi T (2006) In vitro biochemical evidence that the psychotomimetic phencyclidine, ketamine and dizocilpine (MK-801) are inactive at cloned human and rat dopamine D2 receptors. Eur J Pharmacol 540:53–56. Kos T, Popik P, Pietraszek M, Schafer D, Danysz W, Dravolina O, Blokhina E, Galankin T, Bespalov AY (2006) Effect of 5-HT3 receptor antagonist MDL72222 on behaviours induced by ketamine in rats and mice. Eur Neuropsychopharmacol 16:297–310. Krystal JH, Karper LP, Seibyl JP, Freeman GK, Delaney R, Bremner JD, Heninger GR, Bowers MB, Charney DS (1994) Subanesthetic effects of the noncompetitive NMDA antagonist, ketamine, in humans. Psychotomimetic, perceptual, cognitive, and neuroendocrine responses. Arch Gen Psychiatry 51:199 –214. Lalonde R, Joyal CC (1991) Effects of ketamine and 1-glutamic acid diethyl ester on concept learning in rats. Pharmacol Biochem Behav 39:829 – 833. Liy-Salmeron G, Meneses A (2007) Role of 5HT1-7 receptors in shortand long-term memory for an autoshaping task: Intrahippocampal manipulations. Brain Res 1147:140 –147.
Long JM, Kessner RP (1995) Phencyclidine impairs temporal order memory for spatial location in rats. Pharmacol Biochem Behav 52:645– 648. Lorrain DS, Baccei CS, Bristow LJ, Anderson JJ, Varney MA (2003) Effects of ketamine and N-methyl-D-aspartate on glutamate and dopamine release in the rat prefrontal cortex: modulation by a group II selective metabotropic glutamate receptor agonist LY379268. Neuroscience 117:697–706. Mansbach RS, Geyer MA (1991) Parametric determinants in prestimulus modifications of acoustic startle: interaction with ketamine. Psychopharmacology 105:162–168. Markowska AL, Stone WS, Ingram DK, Reynolds J, Gold PE, Conti LH, Pontecorvo MJ, Wenk GL, Olton DS (1989) Individual differences in aging: behavioural and neurobiological correlates. Neurobiol Aging 10:31– 43. Meneses A (2007) Do serotonin1–7 receptors modulate short and long-term memory? Neurobiol Learn Mem 87:561–572. Moghaddam B, Adams B, Verma A, Daly D (1997) Activation of glutamatergic neurotransmission by ketamine: a novel step in the pathway from NMDA receptor blockade to dopaminergic and cognitive disruptions associated with the prefrontal cortex. J Neurosci 17:2921–2927. Nilsson M, Hansson S, Carlsson A, Carlsson ML (2007) Differential effects of the N-methyl-D- aspartate receptor antagonist MK-801 on different stages of object recognition memory in mice. Neuroscience 149:123–130. Pichat P, Bergis OE, Terranova JP, Urani A, Duarte C, Santucci V, Guedet C, Voltz C, Steinberg R, Stemmelin J, Oury-Donat F, Avenet P, Griebel G, Scatton B (2007) SSR180711, a novel selective alpha7 nicotinic receptor partial agonist: (II) efficacy in experimental models predictive of activity against cognitive symptoms of schizophrenia. Neuropsychopharmacology 32:17–34. Pitsikas N, Zisopoulou S, Sakellaridis N (2006) Nitric oxide donor molsidomine attenuates psychotomimetic effects of the NMDA receptor antagonist MK-801. J Neurosci Res 84:299 –305. Pitsikas N (2007) Effects of scopolamine and L-NAME on rats’ performance in the object location test. Behav Brain Res 179:294 –298. Razoux F, Garcia R, Lena I (2007) Ketamine at a dose that disrupts motor behavior and latent inhibition, enhances prefrontal cortex synaptic efficacy and glutatmate release in the nucleus accumbens. Neuropsychopharmacology 32:719 –727. Robinson JK, Crawley JN (1993) Intraventricular galanin impairs delayed non-matching to sample performance in rats. Behav Neurosci 107:458 – 467. Seeman P, Ko F, Tallerico T (2005) Dopamine receptor contribution to the action of PCP, LSD and ketamine psychotomimetics. Mol Psychiatry 10:877– 883. Steckler T, Drinkenburg WHIM, Sahgal A, Aggleton JP (1998) Recognition memory in rats. I. Concepts and classification. Prog Neurobiol 54:289 –311. Tricklebank MD, Singh L, Oles RJ, Preston C, Iversen SD (1989) The behavioural effects of MK-801: a comparison with antagonists acting non-competitively and competitively at the NMDA receptor. Eur J Pharmacol 167:127–135. Uchihashi Y, Kuribara H, Isa Y, Morita T, Sato T (1994) The disruptive effects of ketamine on passive avoidance learning in mice: involvement of dopaminergic mechanism. Psychopharmacology 116: 40 – 44. Verma A, Moghaddam B (1996) NMDA receptor antagonists impair prefrontal cortex function as assessed via spatial delayed alternation performance in rats: modulation by dopamine. J Neurosci 16:373–379. Willetts J, Balster RL, Leander JD (1990) The behavioural pharmacology of NMDA receptor antagonists. Trends Pharmacol Sci 11:423– 428.
(Accepted 1 April 2008) (Available online 7 April 2008)
EFFECTS OF SUB-ANESTHETIC DOSES OF KETAMINE ON RATS’ SPATIAL AND NON-SPATIAL RECOGNITION MEMORY N. PITSIKAS,* A. BOULTADAKIS AND N. SAKELLARIDIS
It has been shown that MK-801 and PCP usually impair either non-spatial (de Lima et al., 2005; Grayson et al., 2007; Nilsson et al., 2007) or spatial (Robinson and Crawley, 1993; Long and Kessner, 1995; Verma and Moghaddam, 1996) recognition memory in the rodent. At the moment, there is poor experimental evidence concerning the role of ketamine on recognition memory. It has been reported that ketamine disrupted rats’ recognition memory (Verma and Moghaddam, 1996), although another study did not support this finding (Robinson and Crawley, 1993). In addition, it is not clear yet if ketamine affects different stages of memory formation (acquisition, storage or retrieval of information). Taking the above evidences into account the aim of our study was to clarify the exact role of ketamine on recognition memory using different training procedures. For this purpose, the object recognition and the object location task were selected. The former is a non-spatial working memory task, does not involve at all, the learning of a rule since it is based on the spontaneous exploratory behavior of rats toward objects (Ennaceur and Delacour, 1988). The same authors developed a novel version of this procedure named object location task, aiming to evaluate spatial working memory in rodents (Ennaceur et al., 1997). This task assesses the ability to discriminate the novelty of the object location, but not the object itself, since the behavioral test arena is already familiar to the animal (Ennaceur et al., 1997).
Department of Pharmacology, School of Medicine, University of Thessaly, 22 Papakiriazi str., 412-22 Larissa, Greece
Abstract—There are experimental evidences indicating that the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist ketamine impairs cognition and produces a series of schizophrenia-like symptoms in rodents (hyperactivity, stereotypies and ataxia). The present study was designed to investigate the effects of ketamine on rats’ non-spatial and spatial recognition memory. For this purpose the object recognition and the object location task were selected. Pre- or post-training systemic administration of ketamine (0.3, 1 and 3 mg/kg; i.p.) in a dose-dependent manner disrupted animals’ performance in both these recognition memory paradigms, suggesting that this compound affected pre- and posttraining memory components. The current results indicate that the non-competitive NMDA antagonist ketamine may modulate either spatial or non-spatial recognition memory. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: ketamine, object recognition, object location, working memory, rat.
Non-competitive antagonists of the N-methyl-D-aspartate (NMDA) receptor such as phencyclidine (PCP), MK-801 or the dissociative anesthetic ketamine are known for their strong psychotomimetic effects in humans (Javitt and Zukin, 1991; Krystal et al., 1994). Ketamine, at sub-anesthetic doses, produces wideranging effects on rodent behavior, including disruption of sensory motor gating (Mansbach and Geyer, 1991), memory deficits (Jones et al., 1990; Uchihashi et al., 1994; Verma and Moghaddam, 1996; Imre et al., 2006; Kos et al., 2006), hypermotility (Willetts et al., 1990; Carlsson, 1993), stereotypy and ataxia (Tricklebank et al., 1989). Therefore, these compounds are used routinely as clinical and animal models of schizophrenia (Geyer, 1998). Recognition memory stems from a series of neural processes by which a subject is aware that a stimulus has been previously experienced, recognition being the behavioral outcome of these processes. It requires that the perceived characteristics of events be discriminated, identified and compared (matched) against a memory of the characteristics of previously experienced events (Steckler et al., 1998).
EXPERIMENTAL PROCEDURES Animals Male, 3-month-old Wistar rats (Hellenic Pasteur Institute, Athens, Greece), weighing 250 –300 g were used in this study. The animals were housed in Makrolon cages (45 cm long⫻35 cm high⫻20 cm wide) three per cage, in a regulated environment (21⫾1 °C; 50 –55% relative humidity; 12-h light/dark cycle, lights on at 07:00 h) with free access to food and water. Experiments were conducted in the room where only these animals where housed, and took place between 09:00 and 13:00 h. Animals’ behavior was video-recorded. Behavioral observations and evaluations were performed by experimenters who were unaware of the pharmacological treatment. Since both object recognition and object location are visuallydependent paradigms, a pilot study was carried out in order to examine whether or not ketamine affected rats’ visual abilities. The procedure described by Markowska et al., 1989, was used. This method, in brief, consists of lowering rats toward the edge of a table. If visual abilities of animals are not reduced they should always extend their forepaws well before they touch it. In this short investigation, male, 3-month-old rats were divided in four experimental groups (four rats per group) as follows: vehicle (NaCl 0.9%); ketamine 0.3 mg/kg; ketamine 1 mg/kg and ketamine
*Corresponding author. Tel: ⫹30-2410-565268; fax: ⫹30-2410-565236. E-mail address: [email protected] (N. Pitsikas). Abbreviations: ANOVA, analysis of variance; D, discrimination index; F, familiar object; FL, familiar location; ITI, intertribal interval; N, new object; NL, new location; NMDA, N-methyl-D-aspartate; n.s., not significant; PCP, phencyclidine; T1, sample trial; T2, choice trial.
0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.04.001
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N. Pitsikas et al. / Neuroscience 154 (2008) 454 – 460 3 mg/kg. Vehicle or ketamine was delivered 20 min before testing animals’ visual acuity. None of the treated rats presented evident signs of reduced visual acuity. Our findings are in agreement with a prior report in which it was observed that ketamine did not affect rats’ visual abilities up to 20 mg/kg (Lalonde and Joyal, 1991). All animal treatments were approved by the local ethical committee and were conducted in conformity with the international guidelines in compliance with national and international laws and policies (EEC Council Directive 86/609, JL 358, 1, December, 12, 1987; NIH Guide for Care and Use of Laboratory Animals, NIH publication no. 65-23, 1985). Every effort was made to minimize the number of animals used and their suffering.
Object recognition test The test apparatus consisted of a dark open box made of Plexiglas (80 cm long⫻50 cm high⫻60 cm wide) which was illuminated by a 60-W lamp suspended 60 cm above the box. In the different parts of the apparatus the light intensity was equal. The objects to be discriminated (in triplicate) made of glass, plastic, or metal, were in three different shapes: cubes, pyramids and cylinders 7 cm high; they could not be displaced by rats. The object recognition test was performed as described elsewhere (Ennaceur and Delacour, 1988). In the week preceding testing, the animals were handled twice daily. On the day before testing, they were allowed to explore the apparatus for 2 min, while on the testing day, a session of two 2-min trials was given. During the “sample” trial (T1), two identical samples (objects) were placed in two opposite corners of the apparatus. A rat was placed in the middle of the apparatus and was left to explore these two identical objects. After T1, the rat was put back in its home cage and an intertrial interval (ITI) of 60 min was given, since at this delay condition recognition memory is still intact in the untreated rat (Bartolini et al., 1996). Subsequently, the “choice” trial (T2) was performed. During T2, a new object (N) replaced one of the samples presented in T1, thence, the rats were re-exposed to two objects: the familiar object (F) and the N. All combinations and locations of objects were used in a balanced manner to reduce potential biases due to preferences for particular locations or objects. To avoid the presence of olfactory trails, the apparatus and the objects after each trial were thoroughly cleaned. Exploration was defined as follows: directing the nose toward the object at a distance of no more than 2 cm and/or touching the object with the nose. Turning around or sitting on the object was not considered as exploratory behavior. The times spent by rats in exploring each object during T1 and T2 were recorded manually by using a stopwatch. From this measure a series of variables was then calculated: the total time spent in exploring the two identical objects in T1, and that spent in exploring the two different objects, F and N in T2. The discrimination between F and N during T2 was measured by comparing the time spent in exploring the F with that spent in exploring the N. As this time may be biased by differences in overall levels of exploration (Cavoy and Delacour, 1993) a discrimination index (D) was then calculated; D⫽N-F/N⫹F. D is the discrimination ratio and represents the difference in exploration time expressed as a proportion of the total time spent exploring the two objects in T2 (Cavoy and Delacour, 1993). In addition, motor activity of each animal expressed as total number of steps during each trial was also recorded.
Object location test The test apparatus was the same apparatus used in the object recognition test. The test arena was located in a large observation room with external cues (large and distinctive objects) surrounding the experimental box to help rats to resolve this spatial memory task. These cues were kept in a constant location throughout the period of testing. The objects were the same objects in the object recognition task.
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The object location test was performed as described elsewhere (Ennaceur et al., 1997; Pitsikas, 2007). This test consisted of a period of habituation, a T1 and a T2. During habituation, the animals were allowed to freely explore the apparatus without objects for 2 min, once a day for three consecutive days before testing. On the testing day, a session of two 2-min trials was given. During the T1 two identical samples (objects) were placed in two opposite corners of the apparatus 10 cm from the sidewall. A rat was placed in the middle of the apparatus and was left to explore these two identical objects. After T1, the rat was put back in its home cage and an ITI of 20 min was given, since at this delay condition recognition memory is still intact in the untreated rat (Pitsikas, 2007). Subsequently, T2 was conducted. The rat was re-introduced to the apparatus. During T2, one of the two similar objects was moved to a different location (new location, NL) while the other object remained in the same position (familiar location, FL) as in the T1. All locations of objects were used in a balanced manner to reduce potential biases due to preferences for particular locations. To avoid the presence of olfactory trails, the apparatus and the objects after each trial were thoroughly cleaned. Exploration was defined as follows: directing the nose toward the object at a distance of no more than 2 cm and/or touching the object with the nose. Turning around or sitting on the object was not considered as exploratory behavior. The times spent by rats in exploring each object during T1 and T2 were recorded manually by using a stopwatch. From this measure, a series of variables was then calculated: the total time spent in exploring the two identical objects in T1 and that spent in exploring the two objects in the two different locations in T2. The discrimination between FL and NL during T2 was measured by comparing the time spent in exploring the object in the FL with that spent in exploring the object in the NL. As this time may be biased by differences in overall levels of exploration (Cavoy and Delacour, 1993) the D was then calculated; D⫽NL-FL/ NL⫹FL. D represents the difference in exploration time expressed as a proportion of the total time spent exploring the two objects in T2 (Cavoy and Delacour, 1993). In addition, motor activity of each animal expressed as total number of steps during each trial was also recorded.
Drugs Ketamine hydrochloride was dissolved in saline (NaCl 0.9%) and administered i.p. Control animals received the vehicle (NaCl 0.9%).
Experimental protocol Experiment 1: effects of pre-training administration of different doses of ketamine on animals’ performance in the object recognition task. Rats were randomly divided into four experimental groups (10 rats per group) as follows: vehicle; ketamine 0.3 mg/ kg; ketamine 1 mg/kg and ketamine 3 mg/kg. To study the effects on acquisition, ketamine and vehicle were administered 20 min before T1. Experiment 2: effects of post-training administration of different doses of ketamine on animals’ performance in the object recognition task. Rats were randomly divided into four experimental groups (10 rats per group) as follows: vehicle; ketamine 0.3 mg/kg; ketamine 1 mg/kg and ketamine 3 mg/kg. To study the effects on post-mnemonic stages (storage and/or retrieval), ketamine and vehicle were administered immediately after T1. Experiment 3: effects of pre-training administration of different doses of ketamine on animals’ performance in the object location task. Rats were randomly divided into four experimental groups (10 rats per group) as follows: vehicle; ketamine 0.3 mg/kg; ketamine 1 mg/kg and ketamine 3 mg/kg. To study the effects on
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Table 1. Comparison of the exploration time of the two similar objects (F1 and F2) during T1 displayed by different groups of rats in the object recognition task Group
N
Total exploration time (s), mean⫾S.E.M. Acquisition experiment
Vehicle Ketamine (0.3 mg/kg) Ketamine (1 mg/kg) Ketamine (3 mg/kg)
10 10 10 10
Storage experiment
F1
F2
F1
F2
7.3⫾0.7 6.0⫾0.3 6.5⫾0.5 7.0⫾0.7
7.9⫾0.6 6.4⫾0.4 6.9⫾0.7 6.4⫾0.4
8.2⫾0.9 6.2⫾0.8 7.3⫾0.8 7.2⫾0.6
8.9⫾0.4 7.4⫾0.9 6.1⫾0.7 8.1⫾0.4
N⫽number of rats.
acquisition, ketamine and vehicle were administered 20 min before T1. Experiment 4: effects of post-training administration of different doses of ketamine on animals’ performance in the object location task. Rats were randomly divided into four experimental groups (10 rats per group) as follows: vehicle; ketamine 0.3 mg/ kg; ketamine 1 mg/kg and ketamine 3 mg/kg. To study the effects on post-mnemonic stages, ketamine and vehicle were administered immediately after T1.
Statistical analysis All data are expressed as mean⫾S.E.M. Preference of animals for objects or locations during T1 was analyzed by the Student’s t-test for each experimental group. In experiments 1 and 3, motor activity and total exploration times during T1 and T2, were evaluated by the two-way analysis of variance (ANOVA) with a split-plot design (between-within subjects). The between factor was ketamine (three levels) and the within factor were trials (two levels). Post hoc comparisons were made by the Tukey’s post hoc test. In experiments 2 and 4, motor activity and total exploration times during T2 were assessed by the one-way ANOVA followed by the Tukey’s post hoc test. The factor was ketamine (three levels). Discrimination index D data were calculated by the one-way ANOVA test. The factor was ketamine (three levels). Post hoc comparisons were made by the Tukey’s test.
RESULTS No difference within any group when the exploration time was compared accordingly to the nature of objects (cubes, pyramids or cylinders) and their locations (left or right), in the apparatus was observed during T1 either in the object recognition or in the object location paradigm (Tables 1 and 2, respectively).
Experiment 1: effects of pre-training administration of different doses of ketamine on animals’ performance in the object recognition task Overall analysis of motor activity results did not reveal a main effect either of ketamine [F(3,36)⫽2, P⫽0.12, not significant (n.s.)] or of trials [F(1,36)⫽2.7, P⫽0.1, n.s.] or a significant interaction between ketamine and trials [F(3,36)⫽0.95, P⫽0.4, n.s., Fig. 1A]. Similarly, analysis of total exploration times did not show a main effect either of ketamine [F(3,36)⫽1.9, P⫽0.16, n.s.] or of trials [F(1,36)⫽0.2, P⫽0.65, n.s.] or a significant interaction between ketamine and trials [F(3,36)⫽1.2, P⫽0.3, n.s., Fig. 1B]. D results showed that the vehicle-treated rats discriminated better the novel than the F during T2 with respect to their counterparts which received ketamine [F(3,36)⫽7.8, P⬍0.01; Tukey’s post hoc test, P⬍0.05 vs. ketamine 1 and 3 mg/kg groups, Fig. 1C]. Experiment 2: effects of post-training administration of different doses of ketamine on animals’ performance in the object recognition task Statistical analyses of the motor activity data did not evidence a main effect of treatment [F(3,36)⫽1.2, P⫽0.31, n.s., Fig. 2A]. Total exploration levels during T2 were not different among the various groups of animals [F(3,36)⫽0.8, P⫽0.5, n.s., Fig. 2B]. Data for index D (Fig. 2C) revealed a significant effect of treatment [F(3,36)⫽10.6, P⬍0.01]. Post hoc comparisons have shown that the ketamine 1 and ketamine 3 mg/kg–treated animals were unable to discriminate between the familiar and the novel object with respect to the vehicle-treated rats
Table 2. Comparison of the exploration time of the two similar objects (F1 and F2) during T1 displayed by different groups of rats in the object location task. Group
N
Total exploration time (s), mean⫾S.E.M. Acquisition experiment
Vehicle Ketamine (0.3 mg/kg) Ketamine (1 mg/kg) Ketamine (3 mg/kg) N⫽number of rats.
10 10 10 10
Storage experiment
F1
F2
F1
F2
8.4⫾1.0 7.4⫾0.6 9.4⫾0.7 8.8⫾1.5
7.7⫾1.4 8.5⫾1.3 8.6⫾1.0 7.5⫾1.4
8.0⫾0.8 8.6⫾0.6 8.8⫾1.0 8.4⫾0.9
7.4⫾0.8 9.7⫾1.2 8.0⫾1.2 7.4⫾0.7
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P⫽0.94, n.s., Fig. 3A]. Similarly, analysis of total exploration times did not reveal a main effect either of ketamine [F(3,36)⫽0.9, P⫽0.43, n.s.] or of trials [F(1,36)⫽2.3, P⫽0.12, n.s.] or a significant interaction between ketamine and trials [F(3,36)⫽0.4, P⫽0.74, n.s., Fig. 3B]. D results showed that the vehicle-treated rats discriminated better the novel than the FL during T2 with respect to their counterparts which received ketamine; [F(3,36)⫽4.2, P⬍0.01; Tukey’s post hoc test, P⬍0.05 vs. ketamine 1 and 3 mg/kg groups, Fig. 3C]. Moreover, the ketamine 1 and
Fig. 1. Object recognition test. Acquisition experiment. Vehicle and ketamine were injected i.p., 20 min before starting T1. (A) Total motor activity displayed by different groups of rats. (B) Total exploration time displayed by different groups of rats. (C) Discrimination index D performance expressed by different groups of rats during T2. Results are expressed as mean⫾S.E.M. * P⬍0.05 vs. the vehicle group.
(P⬍0.05). In addition, the ketamine 3 mg/kg–treated rats’ performance was significantly lower as compared with ketamine 0.3 mg/kg–treated rodents (P⬍0.05). Experiment 3: effects of pre-training administration of different doses of ketamine on animals’ performance in the object location task Overall analysis of motor activity results did not show a main effect either of ketamine [F(3,36)⫽1, P⫽0.39, n.s.] or of trials [F(1,36)⫽0.96, P⫽0.33, n.s.] or a significant interaction between ketamine and trials [F(3,36)⫽0.14,
Fig. 2. Object recognition test. Storage experiment. Vehicle and ketamine were injected i.p., just after T1. (A) Total motor activity displayed by different groups of rats during T2. (B) Total exploration time displayed by different groups of rats during T2. (C) Discrimination index D performance expressed by different groups of rats during T2. Results are expressed as mean⫾S.E.M. * P⬍0.05 vs. the vehicle group; ⫹ P⬍0.05 vs. ketamine 0.3 mg/kg–treated rats.
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groups of animals [F(3,36)⫽1.5, P⫽0.8, n.s., Fig. 4B]. Data for index D (Fig. 4C) revealed a significant effect of treatment [F(3,36)⫽6.4, P⬍0.01]. Post hoc comparisons have shown that the ketamine 1 and ketamine 3 mg/kg– treated animals were unable to discriminate between the familiar and the novel location with respect to the vehicletreated rats (P⬍0.05). In addition, the ketamine 3 mg/kg– treated rats’ performance was significantly lower as compared with ketamine 0.3 mg/kg–treated rodents (P⬍0.05).
Fig. 3. Object location test. Acquisition experiment. Vehicle and ketamine were injected i.p., 20 min before starting T1. (A) Total motor activity displayed by different groups of rats. (B) Total exploration time displayed by different groups of rats. (C) Discrimination index D performance expressed by different groups of rats during T2. Results are expressed as mean⫾S.E.M. * P⬍0.05 vs. the vehicle group; ⫹ P⬍0.05 vs. ketamine 0.3 mg/kg–treated rats.
3 mg/kg–treated rats’ performance was significantly lower as compared with ketamine 0.3 mg/kg–treated rodents (P⬍0.05). Experiment 4: effects of post-training administration of different doses of ketamine on animals’ performance in the object location task Statistical analyses of the motor activity data did not show differences among the various experimental groups during T2 [F(3,36)⫽0.92, P⫽0.4, n.s., Fig. 4A]. Total exploration levels during T2 were not different among the various
Fig. 4. Object location test. Storage experiment. Vehicle and ketamine were injected i.p., just after T1. (A) Total motor activity displayed by different groups of rats during T2. (B) Total exploration time displayed by different groups of rats during T2. (C) Discrimination index D performance expressed by different groups of rats during T2. Results are expressed as mean⫾S.E.M. * P⬍0.05 vs. the vehicle group; ⫹ P⬍0.05 vs. ketamine 0.3 mg/kg–treated rats.
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DISCUSSION Our results indicate that ketamine dose-dependently impaired both non-spatial and spatial recognition memory paradigms. At the dose of 0.3 mg/kg, it seemed to affect animals’ performance, though this effect did not reach statistical significance. However, when given at 1 and 3 mg/kg, ketamine, consistently impaired rats’ performance in the object recognition and object location procedures. In the present study, treatment was applied either before T1, or just after T1, and a certain ITI was given (60 min in the object recognition task and 20 min in the object location test). It is therefore impossible to dissect on which specific memory component ketamine was acting. This paper is providing evidence that, at least, pre-training administration of ketamine affected acquisition, while its posttraining administration affected storage and/or retrieval. Ketamine was applied systematically, thus, it cannot be excluded that non-specific factors (attentional, sensory motor) might have influenced animals’ performance. This possibility, however, could be ruled out since this compound did not affect rats’ visual acuity and did not influence rodents’ motor activity or exploration levels either in the object recognition procedure or in the object location task. It is also unlikely that the effects of ketamine on memory were due to residual presence of the drug during testing since this compound has a very short half-life (10 –15 min) (Krystal et al., 1994). Results of ours related to the effects of pre-training administration of ketamine are in line with previous reports in which pre-training administration of ketamine disrupted rodents’ performance in various memory paradigms (Jones et al., 1990; Uchihashi et al., 1994; Imre et al., 2006; Kos et al., 2006). The involvement of ketamine in post-training memory processes (storage and/or retrieval) has yet to be clarified. Reportedly, ketamine (2.5, 5 and 10 mg/kg) did not affect the retention latency when administered immediately after training or prior to retention of a passive avoidance task suggesting that in this behavioral paradigm it did not impair posttraining memory components in the mouse (Uchihashi et al., 1994). Conversely, ketamine disrupted post-training memory components in a spatial delayed alternation paradigm (Verma and Moghaddam, 1996) and in the autoshaping task (Liy-Salmeron and Meneses, 2007) performed in the rat. The present results are consistent with the latter studies and support a role of ketamine also in post-training memory processes. The above controversial findings on ketamine role might depend on differences in the experimental settings; e.g. the type of animal model (mouse vs. rat in our study), the dose regimen, and the behavioral pattern investigated (passive avoidance, a negatively reinforced paradigm utilized in the study by Uchihashi et al., vs. the object recognition and object location tasks—not rewarded tasks— used in our studies). To our knowledge, few studies aiming to investigate the role of non-competitive NMDA antagonists on object recognition memory have been carried out. Pre- or post-training administration either of MK-801 or PCP decreased retention abilities either in the male (Pitsikas et al., 2006; Pichat et al.,
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2007) or in the female rat (de Lima et al., 2005; Grayson et al., 2007). A recent investigation however, did not support these findings. It has been reported that MK-801 disrupted the encoding but not the post-training mnemonic stages (storage and retrieval) in the object recognition task in the male mouse (Nilsson et al., 2007). The species utilized (NMRI mouse vs. rat used in the other studies) and the low discrimination index displayed by the control mice may underlie this discrepancy (Nilsson et al., 2007). In this context, the possibility that the psychotomimetic properties of MK-801 might result in an amplification of the perceived salience of the novel object, and/or anxiolytic mechanisms could result in neophilic effects may to be considered (Nilsson et al., 2007). There is scant evidence whether or not object location is sensitive to pharmacological manipulation (Pitsikas, 2007). It is the first time to our knowledge that the effects of a noncompetitive NMDA receptor antagonist were evaluated in this spatial working memory paradigm. Object recognition and object location measure nonspatial and spatial recognition memory respectively. As compared with other cognitive tests, these paradigms do not involve a reward or a punishment and thus the behavioral outcome is not influenced by reinforcement/response interactions (Dere et al., 2007). The tasks are quite similar to procedures used in humans and should therefore have a significant level of predictive validity (Ennaceur and Delacour, 1988; Ennaceur et al., 1997). The mechanism by which ketamine produces its adverse behavioral effects, at least partly, have been attributed to the blockade of NMDA receptors located on GABA interneurons, which in turn leads to disinhibition of neural activity in limbic structures (Moghaddam et al., 1997). This disinhibitory action elicits an increase in terms of neuronal activity and excessive glutamate and dopamine release in the prefrontal cortex and limbic regions (Moghaddam et al., 1997; Lorrain et al., 2003; Razoux et al., 2007). In this context, controversial results have been reported concerning the dopamine D2 receptor contribution to the psychotominetic effects of the NMDA receptor antagonists including ketamine (Seeman et al., 2005; Jordan et al., 2006). Object recognition and object location were demonstrated sensitive to ketamine treatment. Both these procedures might represent valid tools for assessing the action of potential antipsychotic compounds on non-spatial and spatial working memory. Interestingly, ketamine impaired animals’ performance in these tasks at very low doses (1 and 3 mg/kg) as compared with those usually utilized in order to disrupt working memory in rodents (10 –30 mg/kg) (Jones et al., 1990; Uchihashi et al., 1994; Verma and Moghaddam, 1996; Imre et al., 2006; Kos et al., 2006). Although low doses of ketamine affected performance in object recognition and object location, certainly there is experimental evidence that anesthetic doses did not alter memory consolidation (LiySalmeron and Meneses, 2007; Meneses, 2007).
CONCLUSION In summary, studies herein presented indicate that systemic administration of sub-anesthetic doses of ketamine
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dose-dependently impaired non-spatial and spatial recognition memory in the rat. In addition, this non-competitive NMDA receptor antagonist seems to modulate pre- and post-training recognition memory components.
REFERENCES Bartolini L, Casamenti F, Pepeu G (1996) Aniracetam restores object recognition impaired by age, scopolamine and nucleus basalis lesions. Pharmacol Biochem Behav 53:277–283. Carlsson ML (1993) Are the disparate pharmacological profiles of competitive and un-competitive NMDA antagonists due to different baseline activities of distinct glutamatergic pathways? J Neural Transm 94:1–10. Cavoy A, Delacour J (1993) Spatial but not object recognition is impaired by aging in rats. Physiol Behav 53:527–530. de Lima MNM, Laranja DC, Bromberg E, Roesler R, Schroder N (2005) Pre- or post-training administration of the NMDA receptor blocker MK-801 impairs object recognition memory in rats. Behav Brain Res 156:139 –143. Dere E, Huston JP, De Souza MA (2007) The pharmacology, neuroanatomy and neurogenetics of one-trial object recognition in rodents. Neurosci Biobehav Rev 31:673–704. Ennaceur A, Delacour J (1988) A new one-trial test for neurobiological studies of memory in rats. 1. Behavioral data. Behav Brain Res 31:47–59. Ennaceur A, Neave N, Aggleton JP (1997) Spontaneous object recognition and object location memory in rats: the effects of lesions in the cingulated cortices, the medialprefrontal cortex, the cingulum bundle and the fornix. Exp Brain Res 113:509 –519. Geyer MA (1998) Behavioural studies of hallucinogenic drugs in animals: implications for schizophrenia research. Pharmacopsychiatry 31:73–79. Grayson B, Idris NF, Neill JC (2007) Atypical antipsychotics attenuate a sub-chronic PCP-induced cognitive deficits in the novel object recognition task in the rat. Behav Brain Res 184:31–38. Imre G, Fokkema DS, Den Boer JA, Ter Horst GJ (2006) Dose-response characteristics of ketamine effect on locomotion, cognitive function and central neuronal activity. Brain Res Bull 69:338–345. Javitt DC, Zukin RS (1991) Recent advances in the phencyclidine model of schizophrenia. Am J Psychiatry 148:1301–1308. Jones KW, Bauerle LM, DeNoble VJ (1990) Differential effects of and phencyclidine receptor ligands on learning. Eur J Pharmacol 179:97–102. Jordan S, Chen R, Fernalld R, Johnson J, Regardie K, Kambayashi J, Tadori Y, Kitagawa H, Kikuchi T (2006) In vitro biochemical evidence that the psychotomimetic phencyclidine, ketamine and dizocilpine (MK-801) are inactive at cloned human and rat dopamine D2 receptors. Eur J Pharmacol 540:53–56. Kos T, Popik P, Pietraszek M, Schafer D, Danysz W, Dravolina O, Blokhina E, Galankin T, Bespalov AY (2006) Effect of 5-HT3 receptor antagonist MDL72222 on behaviours induced by ketamine in rats and mice. Eur Neuropsychopharmacol 16:297–310. Krystal JH, Karper LP, Seibyl JP, Freeman GK, Delaney R, Bremner JD, Heninger GR, Bowers MB, Charney DS (1994) Subanesthetic effects of the noncompetitive NMDA antagonist, ketamine, in humans. Psychotomimetic, perceptual, cognitive, and neuroendocrine responses. Arch Gen Psychiatry 51:199 –214. Lalonde R, Joyal CC (1991) Effects of ketamine and 1-glutamic acid diethyl ester on concept learning in rats. Pharmacol Biochem Behav 39:829 – 833. Liy-Salmeron G, Meneses A (2007) Role of 5HT1-7 receptors in shortand long-term memory for an autoshaping task: Intrahippocampal manipulations. Brain Res 1147:140 –147.
Long JM, Kessner RP (1995) Phencyclidine impairs temporal order memory for spatial location in rats. Pharmacol Biochem Behav 52:645– 648. Lorrain DS, Baccei CS, Bristow LJ, Anderson JJ, Varney MA (2003) Effects of ketamine and N-methyl-D-aspartate on glutamate and dopamine release in the rat prefrontal cortex: modulation by a group II selective metabotropic glutamate receptor agonist LY379268. Neuroscience 117:697–706. Mansbach RS, Geyer MA (1991) Parametric determinants in prestimulus modifications of acoustic startle: interaction with ketamine. Psychopharmacology 105:162–168. Markowska AL, Stone WS, Ingram DK, Reynolds J, Gold PE, Conti LH, Pontecorvo MJ, Wenk GL, Olton DS (1989) Individual differences in aging: behavioural and neurobiological correlates. Neurobiol Aging 10:31– 43. Meneses A (2007) Do serotonin1–7 receptors modulate short and long-term memory? Neurobiol Learn Mem 87:561–572. Moghaddam B, Adams B, Verma A, Daly D (1997) Activation of glutamatergic neurotransmission by ketamine: a novel step in the pathway from NMDA receptor blockade to dopaminergic and cognitive disruptions associated with the prefrontal cortex. J Neurosci 17:2921–2927. Nilsson M, Hansson S, Carlsson A, Carlsson ML (2007) Differential effects of the N-methyl-D- aspartate receptor antagonist MK-801 on different stages of object recognition memory in mice. Neuroscience 149:123–130. Pichat P, Bergis OE, Terranova JP, Urani A, Duarte C, Santucci V, Guedet C, Voltz C, Steinberg R, Stemmelin J, Oury-Donat F, Avenet P, Griebel G, Scatton B (2007) SSR180711, a novel selective alpha7 nicotinic receptor partial agonist: (II) efficacy in experimental models predictive of activity against cognitive symptoms of schizophrenia. Neuropsychopharmacology 32:17–34. Pitsikas N, Zisopoulou S, Sakellaridis N (2006) Nitric oxide donor molsidomine attenuates psychotomimetic effects of the NMDA receptor antagonist MK-801. J Neurosci Res 84:299 –305. Pitsikas N (2007) Effects of scopolamine and L-NAME on rats’ performance in the object location test. Behav Brain Res 179:294 –298. Razoux F, Garcia R, Lena I (2007) Ketamine at a dose that disrupts motor behavior and latent inhibition, enhances prefrontal cortex synaptic efficacy and glutatmate release in the nucleus accumbens. Neuropsychopharmacology 32:719 –727. Robinson JK, Crawley JN (1993) Intraventricular galanin impairs delayed non-matching to sample performance in rats. Behav Neurosci 107:458 – 467. Seeman P, Ko F, Tallerico T (2005) Dopamine receptor contribution to the action of PCP, LSD and ketamine psychotomimetics. Mol Psychiatry 10:877– 883. Steckler T, Drinkenburg WHIM, Sahgal A, Aggleton JP (1998) Recognition memory in rats. I. Concepts and classification. Prog Neurobiol 54:289 –311. Tricklebank MD, Singh L, Oles RJ, Preston C, Iversen SD (1989) The behavioural effects of MK-801: a comparison with antagonists acting non-competitively and competitively at the NMDA receptor. Eur J Pharmacol 167:127–135. Uchihashi Y, Kuribara H, Isa Y, Morita T, Sato T (1994) The disruptive effects of ketamine on passive avoidance learning in mice: involvement of dopaminergic mechanism. Psychopharmacology 116: 40 – 44. Verma A, Moghaddam B (1996) NMDA receptor antagonists impair prefrontal cortex function as assessed via spatial delayed alternation performance in rats: modulation by dopamine. J Neurosci 16:373–379. Willetts J, Balster RL, Leander JD (1990) The behavioural pharmacology of NMDA receptor antagonists. Trends Pharmacol Sci 11:423– 428.
(Accepted 1 April 2008) (Available online 7 April 2008)