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A new automated method to assess the rat recognition memory: Validation of the method
Behavioural Brain Research 222 (2011) 151–157
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Research report
A new automated method to assess the rat recognition memory: Validation of the method Caroline Chambon ∗ , Nico Wegener 1 , Andreas Gravius 2 , Wojciech Danysz 3 In Vivo Pharmacology, Merz Pharmaceuticals GmbH, Eckenheimer Landstrasse 100, D-60318 Frankfurt am Main, Germany
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Article history: Received 19 January 2011 Received in revised form 10 March 2011 Accepted 14 March 2011 Keywords: Rat Memory Object recognition Y-maze Scopolamine Natural forgetting BQCA
a b s t r a c t During the last decade, the rodent object recognition memory task had gained popularity in the field of pharmacological studies, and has been proposed as a useful method to evaluate the efficacy of memory enhancing compounds. In this context, it is important to establish reliable and automated methods with high throughput to evaluate recognition memory in the rat. When performed in a Y-maze apparatus, object recognition has been described to be less dependent on spatial information, and we here report a new method to assess novelty discrimination in a Y-maze apparatus recorded via automated video tracking. During the development of the method, many parameters were recorded and some were selected as being the most reliable, i.e. time spent in the distal half-arms during the first 2 min of the test. The method was then validated under memory deficit conditions produced by a pharmacological treatment (scopolamine) and a long retention delay (72 h) between the sample and the test. The time-induced deficit was prevented by administration of a M1 positive allosteric modulator, BQCA at a dose of 10 mg/kg. Altogether, our data show that the novelty discrimination task performed in a Y-maze apparatus is a reliable method to assess the recognition memory of the rat. This new automated method is as sensitive as the classical object recognition test, and provides advantages such as easily definable parameters and the possibility to use an automated video tracking system that makes the measurement independent of the investigator. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Recognition memory, as a subdivision of episodic memory, is of particular interest for research in the field of Alzheimer’s disease. Indeed, episodic memory capacities have been described as being altered during the early stage of the disease [12,16]. Recognition memory was first studied in humans, for example, as part of the aging process [7,25] or dementia [1,24,32], and in non-human primates [20,21,26] using so-called recognition tasks. Developed in the 1980s [10], the classical recognition task for rodents is based on their natural tendency to explore novelty. The task is built in three phases, first a trial with two identical objects (sample phase), then an inter-trial interval (ITI) or retention delay, and finally a second
∗ Corresponding author at: In Vivo Pharmacology, R&D CNS, Merz Pharmaceuticals GmbH, Alfred-Wegener-Strasse 2, 60438 Frankfurt am Main, Germany. Tel.: +49 0 69 1503 2393, fax: +49 0 69 1503 735. E-mail addresses: [email protected] (C. Chambon), [email protected] (N. Wegener), [email protected] (A. Gravius), [email protected] (W. Danysz). 1 Tel.: +49 0 69 1503 8798; fax: +49 0 69 1503 735. 2 Tel.: +49 0 69 1503 262; fax: +49 0 69 1503 735. 3 Tel.: +49 0 69 1503 564; fax: +49 0 69 1503 735. 0166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.03.032
trial (test phase) performed in presence of a familiar object, i.e. an object encountered during the first trial, and a novel object. Results are analyzed by comparing the length of time spent exploring the novel object compared to the time spent exploring the familiar one. Any discrimination between the objects is explained by the fact that the rodent remembers the object previously encountered during the sample phase and thus spends more time exploring the novel object [11]. In recent years, this task has become more popular in the field of pharmacological studies in rodents [for reviews see [8]] and has been proposed as a useful method to evaluate the efficacy of compounds targeting learning processes or memory [5]. There are two main reasons for the popularity of this test. Firstly, the object recognition task demands no food or water deprivation [10] and secondly, this task presents a high sensitivity to compounds acting on memory. In addition, its design (sample, ITI and test phases) allows the investigation of different memory processes, i.e. acquisition, retention and retrieval [34]. However, the manual scoring of the test is time consuming and scientists are currently in agreement that even though video tracking systems exist, there is no reliable automated version of the test. Video tracking systems have some limitations and cannot evaluate object exploration in the same way as a trained experimenter. Unfortunately, manual scoring, currently considered to be the most
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reliable way to measure object exploration, presents a main inconvenience. There is natural variability between the experimenters’ scoring methods and the lack of 100% objectivity makes it difficult to compare data and results. There is thus a real need to improve data acquisition in the object recognition task. Based on these observations, we explored a different way to assess object discrimination using a video tracking system while preserving the sensitivity of the test. In this study we performed object recognition in a Y-maze apparatus, which has been proposed to be less dependent on spatial information [35]. In the present paradigm, we thus used a maze that prevents animals from using external cues by use of high maze walls. Moreover, the position of the objects at the end of each arm does not allow the rats to use the spatial organization of objects to build a “picture” of the objects as a unit that could be disrupted by changing one of these objects during the test. Such a Y configuration also allows the association of each object to the area defined around it for measurement. In the present automated version of the task, we show the validity of a parameter different from the object exploration. An additional advantage of such a Y-maze apparatus is its size, which forces the rat to move from one arm to another in order to explore the objects. Combined with a video tracking, this enables the assessment of additional parameters related to locomotor activity while the rat is performing the task. In the present study we performed four experiments. In experiment 1, we wanted to investigate the novelty recognition behaviour of naive rats with a 1 h retention delay while using automated video tracking. The collected data were used to select the time frame and the most reliable parameters with which to assess rat memory performance in the Y-maze apparatus. The sensitivity of the test was verified using the selected parameters in experiments 2 and 3. In experiment 2, we validated the choice of the previously selected parameters in a pharmacological model of memory deficit. In experiment 3, the retention delay was prolonged in order to allow the animals to naturally forget the previous sample phase. In experiment 4, we tested whether this time-induced deficit could be pharmacologically prevented by modulation of M1 receptor activity. Altogether, these experiments were aimed at validating the Y-maze version of the object recognition task as a reliable method combining the sensitivity of the classical object recognition task with the advantages of automatization, i.e. higher throughput and observer-independent results.
Fig. 1. Schematic description of the Y-maze apparatus. The maze is made of black coated wood. Arm dimensions are as follows: 60 cm for the length; 26 cm for the width and 56 cm for the height. The figure shows the different compartments defined for the data acquisition by the video tracking software, i.e. arms, distal half-arms, and objects.
2.3. Procedure
Experimentally naive adult male Sprague-Dawley rats (250–300 g; Janvier, France) were housed in groups of four per cage. Colony room temperature and humidity were maintained at 20 ± 1 ◦ C and 50 ± 3%, respectively. Food and water were available ad libitum and animals were kept under an alternating 12 h/12 h day–night cycle (lights on at 07.00) for at least 6 days before the experiments were started. All experiments were conducted during the light period of the day–night cycle and each animal was used only once. The study was approved by the Ethical Committee, Regierungspräsidium Darmstadt, Hessen, and performed in accordance with the recommendations and policies of the U.S. National Institute of Health Guidelines for the Use of Animals.
Prior to testing memory performance, rats were habituated to the maze for 2 consecutive days. Each day, single rats were placed in a transparent cylinder in the centre of the maze for 5 s, then the cylinder was lifted and animals were allowed to freely explore the empty maze for 10 min. Following habituation, the rat’s memory performance was tested on the third day. On this day, 3 similar objects were placed at the far end (10 cm from the wall) of each arm of the Y-maze. The rat was introduced into the cylinder at the centre of the maze and allowed to explore the maze and the objects for 5 min (sample phase). Note that in experiment 3, different sample times were used, i.e. 4, 3 and 2 min, and that in experiment 4, the sample time was reduced to 2 min to make the task more challenging. During the retention delay, the rat was returned to its home cage and one of the familiar objects was replaced by a new object. In experiments 1 and 2, the retention delay was 1 h. In experiment 3, in order to evaluate at which time point a natural forgetting process could be observed, the retention delay was increased up to 48 h. In experiment 4, a retention delay of 72 h was used in order to test the natural forgetting process. The test phase lasted for 5 min. Both sample and test phases were recorded. The video recording started when the rat entered one of the arms. The maze was cleaned with water in between rats as well as between the sample and test phases. Recorded parameters were: time spent (s) in each arm; time spent (s) in each distal half-arm; and time spent (s) to explore each object. Entry into an arm or half-arm was counted when the centre point of the body (defined by the video tracking) entered into one of the defined areas. Object exploration was counted when the rat’s nose point (defined by the video tracking) was in the defined area around the object (2 cm around the object). To exclude place preference, the novel object was pseudo-randomly placed in 1 of the 3 arms. During the 5 min test phase, each parameter was analyzed in 1 min steps. In experiment 1, exploration times were provided for the novel object, familiar object 1 and familiar object 2. Note that in other experiments, the time given for the familiar exploration corresponds to the time spent to explore the two familiar objects divided by two. The following inclusion criteria were used to ensure that the rats could perform the task properly: during the sample phase, animals had to visit each arm at least once; during the test phase, each animal had to visit the arm where the novel object was placed and 1 arm with a familiar object.
2.2. Apparatus
2.4. Treatments
The object recognition task took place in a black wooden Y-maze (arm length: 60 cm; width: 26 cm; height: 56 cm; Fig. 1). The size of the arms fully allowed the rat to explore all around the objects. A digital video camera (Panasonic, USA) mounted above the maze was connected to a computer running the video tracking software EthoVision XT (Noldus, Netherlands). Light sources were placed around the maze providing indirect light with an intensity of 20 lux within the maze. From inside the maze, no external cues were visible. The objects used were 20 cm high bottles, heavy enough to ensure that the rats could not move them. We used a set of white plastic and dark glass bottles. Each object was randomly used as novel or familiar. As far as could be ascertained, the objects had no significance for the animals and they have never been associated to reinforcement.
Scopolamine hydrobromide (Sigma–Aldrich, Steinheim, Germany) was dissolved in sterile physiological saline (vehicle). Benzylquinolone carboxylic acid (BQCA) (synthesized by Merz Pharmaceuticals GmbH, Frankfurt am Main, Germany) was dissolved in a solution of 10% Tween 80 in water (vehicle). Both compounds were injected i.p. 30 min prior to the sample phase in a volume of 2 ml/kg for scopolamine and 4 ml/kg for BQCA.
2. Materials and methods 2.1. Animals
2.5. Statistical analysis Data are expressed as mean ± S.E.M. One way repeated measure analyses of variance (ANOVA) was performed on data from experiment 1. Significant one way
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One way repeated measure ANOVA performed on the time spent in arms during the sample phase showed that naive animals did not display any arm preference (F2,92 = 0.92, p > 0.05). Results from the individual exploration of the novel, familiar 1 and familiar 2 arms during the 5 min of the test phase are presented in Fig. 2A. A one way repeated measure ANOVA performed on the data from each minute of the test revealed an effect of the arm for the 1st minute (F2,92 = 34.92, p < 0.05) and the 2nd minute (F2,92 = 5.98, p < 0.05) but not for the 3rd (F2,92 = 0.02, p > 0.05), 4th (F2,92 = 0.52, p > 0.05) and 5th minutes (F2,92 = 1.19, p > 0.05). Results from the individual exploration of the novel, familiar 1 and familiar 2 half-arms during the 5 min of the test phase are presented in Fig. 2B. A one way repeated measure ANOVA performed on the data from each minute of the test revealed an effect of the half-arm for the 1st minute (F2,92 = 34.81, p < 0.05) and the 2nd minute (F2,92 = 8.44, p < 0.05) but not for the 3rd (F2,92 = 0.13, p > 0.05), 4th (F2,92 = 1.96, p > 0.05) and 5th minutes (F2,92 = 0.51, p > 0.05). Results from the individual exploration of the novel, familiar 1 and familiar 2 objects during the 5 min of the test phase are presented in Fig. 2C. A one way repeated measure ANOVA performed on the data from each minute of the test revealed an effect of the object for the 1st minute (F2,92 = 50.71, p < 0.05) and the 2nd minute (F2,92 = 7.03, p < 0.05) but not for the 3rd (F2,92 = 1.41, p > 0.05), 4th (F2,92 = 2.07, p > 0.05) and 5th minutes (F2,92 = 0.34, p > 0.05). Subsequent post-hoc Holm–Sidak’s t-tests performed to compare exploration of novel, familiar 1 and familiar 2 objects showed that, in naive animals, it is possible to observe a novelty discrimination during the 1st and 2nd minutes of the test with each of the 3 parameters measured (p < 0.05). Based on these data, we chose the first 2 min of the test to measure novelty discrimination in the Y-maze apparatus. Results from the individual exploration of the novel, familiar 1 and familiar 2 for the 3 parameters measured during the first 2 min of the test phase are presented in Fig. 3A. A one way repeated measure ANOVA revealed a different exploration for the arms (F2,92 = 30.59, p < 0.05), the half-arms (F2,92 = 34.94, p < 0.05) and the objects (F2,92 = 47.01, p < 0.05). Subsequent post-hoc Holm–Sidak’s t-tests performed to compare exploration of the novel, familiar 1 and familiar 2 showed that, in naive animals, it is possible to observe a novelty discrimination during the first 2 min of the test with each of the 3 parameters measured (p < 0.05). These data also show that rats explored the familiar arms, half-arms or objects at the same level (p > 0.05). However, the comparison of object exploration measured via manual scoring and video tracking (Fig. 3B) showed an effect of the method used for measurement independent of the object (2 way repeated measure ANOVA: F2,92 = 10.26, p < 0.05 for interaction measurement x object). Subsequent post-hoc Holm–Sidak’s t-tests showed a novelty discrimination with both methods (p < 0.05),
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repeated measure ANOVA was followed by Holm–Sidak’s post-hoc t-tests. A paired t-test was performed on data from experiment 1. Unpaired t-tests were performed on data from experiment 2. One way ANOVA was performed on the distance and velocity data from experiment 4. Significant one way ANOVAs were followed by Dunnett’s post-hoc t-tests. Two way repeated measure ANOVAs were performed in experiment 1 to compare object exploration measured via manual scoring and video tracking, and in experiment 2, 3 and 4 on the time spent in distal half-arms. Significant two way repeated measure ANOVAs were followed by Holm–Sidak’s post-hoc t-tests. Statistical analysis was performed using SigmaPlot 11 software (Systat Software, Erkracht, Germany).
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Fig. 2. Time course of the exploration of the novelty and familiarity in the Y-maze during the test phase for naive rats. The test session was cut in 5 intervals of 1 min. (A) Exploration of the novel, familiar 1 and familiar 2 arms measured during the 5 min of the test. (B) Exploration of the novel, familiar 1 and familiar 2 half-arms measured during the 5 min of the test. (C) Exploration of the novel, familiar 1 and familiar 2 objects measured during the 5 min of the test. Naive animals (n = 47) were able to discriminate the novel with each parameter during the 1st and 2nd minutes of the test. *p < 0.05, one way repeated measure ANOVA followed by Holm–Sidak’s test. The first 2 min of the test were chosen as the most pertinent time frame to measure novelty discrimination.
but also a significantly lower exploration time measured with the video tracking compared to the manual scoring for all 3 objects (p < 0.05). Based on data presented in Fig. 3A and B, we chose the time spent in distal half-arms to measure novelty discrimination during the first 2 min of the test. Moreover, since no difference was observed between the familiar half-arms, in experiments 2, 3 and 4 we chose to pool the exploration times of familiar half-arms 1 and 2.
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Fig. 3. Comparison of the three parameters measured during the first 2 min of the test. (A) Individual exploration of the novel, familiar 1 and familiar 2 for each of the 3 parameters measured (time spent in arms, time spent in distal half-arms and time spent to explore objects) during the first 2 min of the test. A clear novelty preference is observed with each parameter. *p < 0.05, one way repeated measure ANOVA followed by Holm–Sidak’s test. (B) Comparison of levels of object exploration measured via manual scoring and video tracking. #p < 0.05, manual scoring vs. video tracking, two way repeated measure ANOVA followed by Holm–Sidak’s test. Results are expressed as mean ± S.E.M., n = 47. The time spent in distal half-arms was chosen as the best parameter to reflect object discrimination.
3.2. Experiment 2: scopolamine-induced recognition memory deficits in the Y-maze apparatus Unpaired t-tests performed on the distance travelled (t22 = −0.79, p > 0.05) and on the velocity (t22 = −1.05, p > 0.05) showed that scopolamine (0.25 mg/kg) had no effect on the locomotor activity measured during the sample phase. Results from the individual distal half-arm exploration measured during the first 2 min of the test phase are presented in Fig. 4. A two way repeated measure ANOVA revealed a treatment effect (F1,22 = 8.25, p < 0.05) as well as an object effect (F1,22 = 25.38, p < 0.05) and a treatment x object interaction (F2,47 = 11.55, p < 0.05). Subsequent post-hoc Holm–Sidak’s t-tests performed to compare exploration of the novel to exploration of the familiar showed that the group injected with scopolamine 0.25 mg/kg was not able to discriminate the novel (p > 0.05), whereas the vehicle group showed significant discrimination (p < 0.05). In addition, the exploration of the novel was significantly decreased in the scopolamine group compared to the vehicle group (p < 0.05). 3.3. Experiment 3: establishment of the retention delay in the Y-maze apparatus Results from the individual distal half-arm exploration measured during the first 2 min of the test phase after different
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Fig. 4. Effect of scopolamine on novelty discrimination during the test phase. Individual exploration of the novel and the familiar distal half-arms is expressed as mean ± S.E.M. Two way repeated measure ANOVA followed by Holm–Sidak’s t-tests *p < 0.05 novel vs. familiar, #p < 0.05 vs. vehicle, n = 12 per group. Scopolamine at a dose of 0.25 mg/kg induced a novelty discrimination deficit when administered before the sample phase.
retention delays are presented in Fig. 5A. A two way repeated measure ANOVA performed on these data revealed no retention delay effect (F3,43 = 0.42, p > 0.05) but an object effect (F1,43 = 48.40, p < 0.05) and no retention delay x object interaction (F3,93 = 1.27, p > 0.05). Subsequent post-hoc Holm–Sidak’s t-tests performed to compare exploration of the novel to exploration of the familiar showed that naive animals were able to discriminate the novel after all the retention delays used, i.e. 1 h, 4 h, 24 h, and 48 h (p < 0.05). Results from the individual distal half-arm exploration measured during the first 2 min of the test phase after a 48 h retention delay and with different sample lengths are presented in Fig. 5B. A two way repeated measure ANOVA performed on these data revealed no sample length effect (F2,33 = 2.02, p > 0.05) but an object effect (F1,33 = 11.81, p < 0.05) and no sample length x object interaction (F2,71 = 0.627, p > 0.05). Subsequent post-hoc Holm–Sidak’s t-tests performed to compare exploration of the novel to exploration of the familiar showed that naive animals were able to discriminate the novel after a 48 h retention delay when the sample phase was 4 or 3 min (p < 0.05) but not 2 min long. Results from the individual distal half-arm exploration measured during the repetition of an experiment performed with a 48 h retention delay and a sample length of 2 min are presented in Fig. 5C. A paired t-test performed to compare exploration of the novel to exploration of the familiar showed that naive animals were able to discriminate the novel (p < 0.05). These data show that the deficit observed with a 48 h retention delay and a 2 min long sample is not stable through experiments. 3.4. Experiment 4: BQCA-induced reversal of natural forgetting in the Y-maze apparatus A one way ANOVA performed on the distance travelled during the sample phase showed no treatment effect (F2,32 = 2.99, p > 0.05), and one way ANOVA performed on the velocity measured during the sample phase showed no group effect (F2,32 = 2.963, p > 0.05). Together these data indicate that BQCA had no effect on locomotion under these experimental conditions. Results from the individual distal half-arm exploration measured during the first 2 min of the test phase are presented in Fig. 6. A two way repeated measure ANOVA performed on these data revealed no treatment effect (F2,32 = 0.63, p > 0.05) but an object effect (F1,32 = 5.16, p < 0.05) and no treatment x object interaction
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Fig. 6. Effect of BQCA on novelty discrimination during the test phase. Individual exploration of the novel and the familiar distal half-arms is expressed as mean ± S.E.M. *p < 0.05, two way repeated measure ANOVA followed by Holm–Sidak’s t-tests novel vs. familiar, n = 11–12 per group. BQCA at a dose of 10 mg/kg prevents the novelty discrimination deficit induced by a 72 h retention delay.
* BQCA used at 10 mg/kg was able to prevent the natural forgetting induced by a 72 h delay between the sample and the test phase.
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2 min Sample length Fig. 5. Establishment of the natural forgetting protocol. (A) Individual exploration of novel and familiar distal half-arms after different retention delays. (B) Individual exploration of novel and familiar distal half-arms after different sample lengths and a delay of 48 h. *p < 0.05, two way repeated measure ANOVA followed by Holm–Sidak’s t-tests novel vs. familiar (C) Individual exploration of the novel and familiar distal half-arms after a 2 min sample length and a delay of 48 h (Repetition). *p < 0.05, paired t-test. Results are expressed as mean ± S.E.M. n = 12 per group. The deficit observed after a 48 h retention delay and a 2 min sample length is not stable through experiments.
(F2,69 = 2.52, p > 0.05). Subsequent post-hoc Holm–Sidak’s t-tests performed to compare exploration of the novel to exploration of the familiar showed that the group BQCA 10 mg/kg was able to discriminate the novel (p < 0.05) whereas both the vehicle group and the BQCA 5 mg/kg group could not (p > 0.05). These data show that
The present study was performed to validate an automated Ymaze version of the object recognition task. Although this task is used more and more in pharmaceutical studies in rodents, no automated and observer-independent versions currently exist. Here we show that using a Y-maze apparatus instead of an open arena allows the use of a different parameter, i.e. the time spent in the distal half-arm where the object is placed, to assess rats object exploration. Our results also show that this parameter is sensitive enough to detect a pharmacologically-induced recognition memory deficit, the scopolamine-induced deficit, as well as the natural forgetting observed after a 72 h retention delay. Furthermore, our present data show that the M1 receptor positive allosteric modulator (PAM) BQCA is able to prevent the forgetting process induced by a 72 h retention delay in the Y-maze apparatus. Experiment 1 was performed to define a new parameter, which could allow evaluation of novelty discrimination when the object recognition task is performed in a Y-maze apparatus and when exploration is recorded via video tracking. Results from the sample phase show that naive rats display no arm preference in the Y-maze apparatus, proof that this maze-configuration allows a complete isolation from external cues and thus a less spatial-dependent object exploration. Results from the test phase show good novelty discrimination during the first 2 min of the test for the 3 parameters measured, i.e. time spent in the arms, time spent in the distal half-arms, and time spent to explore the objects (Fig. 2A, B and C). This 2 min time frame is in the range of what is normally used in the classical object recognition test. Indeed, in the classical version of the test, the most often used time frames are 2 or 3 min [10,23]. Based on these results, we decided to focus on the first 2 min of the test to choose the parameter to be used to measure novelty discrimination. Several aspects were evaluated to make this choice. First, the parameter had to be easily measurable via video tracking. Second, the exploration had to be high enough to allow a measurable decrease due to the effect of an administered compound, and third, the parameter had to be specific enough to reflect the real object exploration. Taking into consideration the above criteria and the size of the 3 zones measured to express the novelty discrimination in naive animals, we chose the time spent in the distal half-arms
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(an area of approximately 10 cm2 around the object) as the best parameter. To record this measure, the video tracking tracks the body’s centre point, which we believe is more reliable than tracking the nose point as is often used for the object exploration. Indeed, in our experimental conditions, the nose point may jump from the nose to the extremity of a front paw or even be exchanged with the tail point. The instability of the nose point measurement leads to a reduction of the recorded object exploration, a problem which was made clear when comparing manual scoring to the video tracking (Fig. 3B). Under these conditions, we regard the time spent in the distal half-arms as a good compromise between the time spent to explore the objects, which is low and provides only a small time window to measure the effect of a compound, and the time spent to explore the arms, which is not specific enough. Moreover, and in order to provide a double validation of the parameter chosen, i.e. time in half-arm, we verified that the time spent in the proximal half-arm (time in the arm-time in the half-arm) was identical for the 3 arms, even during the test phase (can be checked by comparing data in Fig. 3A). This shows that the time spent into the distal half-arms is dependent on the object placed in it and not on any place preference. Additionally, results from the exploration of the individual half-arms, i.e. familiar 1 and familiar 2, clearly show no exploration difference between the two familiar half-arms (Fig. 3A). Therefore, the following experiments were analyzed using the time spent in the distal half-arms during the first 2 min of the test; and the exploration of the familiar was expressed as the mean of the time taken to explore the 2 familiar half-arms. Experiment 2 was performed to validate the parameters chosen in experiment 1 by using a model of pharmacologically-induced memory deficit. Scopolamine (a muscarinic receptor antagonist) is widely used as a reference compound to induce a memory deficit in various tasks [2,6,29] including the object recognition task. Moreover, the muscarinic system is known to be involved in learning processes [14,15,27] but has also been shown to be altered during normal aging [4,9,13] as well as in pathologies such as Alzheimer’s disease [33]. After a dose-response experiment (data not shown), the scopolamine dose of 0.25 mg/kg was selected as the most relevant to induce a recognition deficit while leaving the rat’s locomotion unaffected. The aim of this experiment was to evaluate the sensitivity of the object recognition task performed in the Ymaze apparatus and to compare our results with data obtained in the classical version of the test. Results from experiment 2 show that administration of scopolamine (0.25 mg/kg) before the sample phase induced an object discrimination deficit when the test is performed after a retention delay of 1 h (Fig. 4). These results are in accordance with data obtained in the classical version of the object recognition test. Indeed, a memory deficit was recently shown [22] after injection of scopolamine at a dose of 0.5 mg/kg, which is in the dose range used in the present study, and a loss of the object discrimination has been reported in animals injected with 0.2 mg/kg of scopolamine [3]. In both studies, the retention delay between the sample and the test phase was also similar to the one used here, i.e. 1 h. Taken together these data indicate that the Y-maze version of the object recognition task used here exhibits the same sensitivity to a scopolamine-induced memory deficit as the classical version. The data also suggest that the memory deficit observed after scopolamine administration is not due to any alterations of locomotion. Furthermore, our data show that scopolamine (0.25 mg/kg) can be utilized to model a pharmacologically-induced memory deficit in the automated Y-maze version of the object recognition test. Experiment 3 was performed to establish a natural forgetting procedure while using the parameters chosen in experiment 1. Again, the aim of this experiment was to evaluate the sensitivity of the object recognition task performed in the Y-maze apparatus and to compare our results with data obtained in the classical version of the test. Results from experiment 3 show that naive rats can
display natural forgetting when the sample length is shortened to 2 min and the retention delay is 48 h long (Fig. 5B), although the deficit obtained under these conditions was not stable throughout the experiments (Fig. 5C). We thus decided to extend the retention delay to 72 h to ensure a stable deficit across experiments. The procedures used in previous studies to induce natural forgetting in the classical object recognition task vary in many aspects, e.g. length of the habituation, length of the sample phase, and length of the retention delay, and the most often used delays are only 4 h [5,17,31] and 24 h [22]. Data obtained in experiment 3, where the effect of increasing the retention delay was tested (Fig. 5A), revealed that after a 4 h retention delay, naive rats performed worse than after a 1 h delay, which would speak in favour of using a 4 h delay to measure impaired performance. But these data also show that, after a 24 h retention delay, the novelty discrimination is back to the control group level (1 h delay). Altogether, these data suggest that processes leading to recall after a 1 h retention delay, which would correspond to short term memory, are not sufficient after a 4 h retention delay; but that other processes, which would correspond to long term memory, lead to recall after a 24 h retention delay. Indeed, if animals are able to clearly discriminate the novel after a 24 h retention delay, it means that the memory could not be lost after a 4 h delay but rather that some processes, which we cannot as yet clearly explain, enabled the rats to access the information in order to perform the task properly. Therefore, we believe that the commonly used 4 h retention delay may not be sufficient to investigate natural forgetting processes. Moreover, it is important to note that the habituation length in experiment 3 was unchanged, to ensure that the stress level of the animals was low enough to allow a memory formation that could then be altered by a longer retention delay. Indeed, we believe that in some previously published natural forgetting experiments, an impairment was obtained after short retention delays, such as 4 h, partly because of a quasi inexistent habituation phase, e.g. 2 min the day before testing [22]. It has been shown that stress can negatively influence explicit memory formation (for review see [28]), and more specifically, it has been shown that in the object recognition task, non-habituated (just handled) rats were unable to discriminate the novel from the familiar after a 24 h retention delay, whereas habituated rats showed no deficit after such a delay [19]. With the conditions we selected in experiment 4, i.e. 2 days of habituation, a sample of 2 min and a retention delay of 72 h, we can be sure that the rats display a stable natural forgetting which can be attributed to the retention delay and not to any stress factor. These results thus show that the model of time-induced memory impairment can also be performed in the automated Y-maze version of the object recognition task. Such a protocol is of particular interest since it provides a non-pharmacological model of a memory deficit, which can be potentially used to evaluate all types of memory improving compounds. Experiment 4 was performed to show that the recognition memory deficit observed after a 72 h retention delay could be prevented by a cognition-enhancing compound. We selected BQCA, a selective M1 receptor PAM, which has recently been described to have such properties [18,30]. BQCA acts at an allosteric site of the M1 receptor enhancing the effects of orthosteric agonists such as acetylcholine. In the present experiment, we showed that BQCA is able to prevent the recognition memory deficit induced by a 72 h retention delay when injected at a dose of 10 mg/kg before the sample phase (Fig. 6). Up to now, the memory-improving capacity of BQCA was only evaluated in two paradigms. The first was the contextual fear conditioning task, in which BQCA reversed the scopolamine-induced deficit in rats when administered before the acquisition phase of the task [18]. The second was a discrimination learning task performed in Tg2576 mice, in which BQCA improved memory performance of 12 months old mice over-expressing amyloid species [30]. Inter-
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estingly, the memory-improving effect of BQCA was obtained at the dose of 30 mg/kg in the discrimination learning test [30] and at doses of 15 and 20 mg/kg in the fear conditioning test [18] but not at 10 mg/kg. Procedural differences between these paradigms and the memory impairment models used may explain the difference in the minimal effective dose however, our data show a memory improving effect of BQCA at the lower dose of 10 mg/kg. Altogether, the recognition memory deficit obtained in experiment 3 and the reversal of this deficit by BQCA in experiment 4 demonstrate the validity of the natural forgetting model performed in the automated Y-maze version of the object recognition task. Taken together, results from the present study show that the object recognition task performed in a Y-maze apparatus displays the same sensitivity as the classical version proposed by Ennaceur and Delacour [10]. Despite using a new parameter, time spent in the distal half-arm where the object is placed, the data presented here are similar to those found in literature. Two models of memory impairment frequently used in pharmacological studies were validated in the Y-maze version, first the pharmacologicallyinduced and second the time-induced memory deficit. There are clear advantages to this new version of the object recognition test using video tracking, such as automated and immediate data acquisition, the assurance of experimenter-independent measurements, and reproducibility of results, as well as the additional measurement of locomotor activity. In conclusion, this automated Y-maze version of the object recognition task appears to be a valuable tool to screen for the effects of compounds believed to act on the rat memory capacity. Acknowledgments We would like to thank Caroline Barberi and Ralph Gross for their support during the experiments. References [1] Alescio-Lautier B, Michel BF, Herrera C, Elahmadi A, Chambon C, Touzet C, Paban V. Visual and visuospatial short-term memory in mild cognitive impairment and Alzheimer disease: role of attention. Neuropsychologia 2007;45(8):1948–60. [2] Baron SP, Wright D, Wenger GR. Effects of drugs of abuse and scopolamine on memory in rats: delayed spatial alternation and matching to position. Psychopharmacology (Berl) 1998;137(1):7–14. [3] Bartolini L, Casamenti F, Pepeu G. Aniracetam restores object recognition impaired by age, scopolamine, and nucleus basalis lesions. Pharmacol Biochem Behav 1996;53(2):277–83. [4] Baskerville KA, Kent C, Nicolle MM, Gallagher M, McKinney M. Aging causes partial loss of basal forebrain but no loss of pontine reticular cholinergic neurons. Neuroreport 2006;17(17):1819–23. [5] Bertaina-Anglade V, Enjuanes E, Morillon D, Drieu la Rochelle C. The object recognition task in rats and mice: a simple and rapid model in safety pharmacology to detect amnesic properties of a new chemical entity. J Pharmacol Toxicol Methods 2006;54(2):99–105. [6] Besheer J, Short KR, Bevins RA. Dopaminergic and cholinergic antagonism in a novel-object detection task with rats. Behav Brain Res 2001;126(1–2):211–7. [7] Boyle Jr E, Aparicio AM, Kaye J, Acker M. Auditory and visual memory losses in aging populations. J Am Geriatr Soc 1975;23(6):284–6. [8] Dere E, Huston JP, De Souza Silva MA. The pharmacology, neuroanatomy and neurogenetics of one-trial object recognition in rodents. Neurosci Biobehav Rev 2007;31(5):673–704. [9] Dunbar GL, Rylett RJ, Schmidt BM, Sinclair RC, Williams LR. Hippocampal choline acetyltransferase activity correlates with spatial learning in aged rats. Brain Res 1993;604(1–2):266–72. [10] Ennaceur A, Delacour J. A new one-trial test for neurobiological studies of memory in rats. 1: Behavioral data. Behav Brain Res 1988;31(1):47–59.
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[11] Ennaceur A. One-trial object recognition in rats and mice: methodological and theoretical issues. Behav Brain Res 2010;215(2):244–54. [12] Ergis AM, Eusop-Roussel E. Early episodic memory impairments in Alzheimer’s disease. Rev Neurol 2008;164(Suppl. 3):S96–101. [13] Gallagher M, Colombo PJ. Ageing: the cholinergic hypothesis of cognitive decline. Curr Opin Neurobiol 1995;5(2):161–8. [14] Ghoneim MM, Mewaldt SP. Studies on human memory: the interactions of diazepam, scopolamine, and physostigmine. Psychopharmacology (Berl) 1977;52(1):1–6. [15] Givens B, Olton DS. Bidirectional modulation of scopolamine-induced working memory impairments by muscarinic activation of the medial septal area. Neurobiol Learn Mem 1995;63(3):269–76. [16] Greene JD, Baddeley AD, Hodges JR. Analysis of the episodic memory deficit in early Alzheimer’s disease: evidence from the doors and people test. Neuropsychologia 1996;34(6):537–51. [17] King MV, Sleight AJ, Woolley ML, Topham IA, Marsden CA, Fone KC. 5-HT6 receptor antagonists reverse delay-dependent deficits in novel object discrimination by enhancing consolidation – an effect sensitive to NMDA receptor antagonism. Neuropharmacology 2004;47(2):195–204. [18] Ma L, Seager MA, Wittmann M, Jacobson M, Bickel D, Burno M, Jones K, Graufelds VK, Xu G, Pearson M, McCampbell A, Gaspar R, Shughrue P, Danziger A, Regan C, Flick R, Pascarella D, Garson S, Doran S, Kreatsoulas C, Veng L, Lindsley CW, Shipe W, Kuduk S, Sur C, Kinney G, Seabrook GR, Ray WJ. Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation. Proc Natl Acad Sci USA 2009;106(37):15950–5. [19] Maroun M, Akirav I. Arousal and stress effects on consolidation and reconsolidation of recognition memory. Neuropsychopharmacology 2008;33: 394–405. [20] Mishkin M, Delacour J. An analysis of short-term visual memory in the monkey. J Exp Psychol Anim Behav Process 1975;1(4):326–34. [21] Moss MB, Rosene DL, Peters A. Effects of aging on visual recognition memory in the rhesus monkey. Neurobiol Aging 1988;9(5–6):495–502. [22] Pascoli V, Boer-Saccomani C, Hermant JF. H3 receptor antagonists reverse delay-dependent deficits in novel object discrimination by enhancing retrieval. Psychopharmacology (Berl) 2009;202(1–3):141–52. [23] Pitsikas N, Boultadakis A, Sakellaridis. Effects of sub-anesthetic doses of ketamine on rats’ spatial and non-spatial recognition memory. Neuroscience 2008;154:454–60. [24] Reed JM, Squire LR. Impaired recognition memory in patients with lesions limited to the hippocampal formation. Behav Neurosci 1997;111(4):667–75. [25] Riege WH, Inman V. Age differences in nonverbal memory tasks. J Gerontol 1981;36(1):51–8. [26] Rolls ET, Perrett DI, Caan AW, Wilson FA. Neuronal responses related to visual recognition. Brain 1982;105(Pt 4):611–46. [27] Rupniak NM, Steventon MJ, Field MJ, Jennings CA, Iversen SD. Comparison of the effects of four cholinomimetic agents on cognition in primates following disruption by scopolamine or by lists of objects. Psychopharmacology (Berl) 1989;99(2):189–95. [28] Sandi C, Pinelo-Nava MT. Stress and memory: behavioral effects and neurobiological mechanisms. Neural Plast 2007;2007:78970–90. [29] Saucier D, Hargreaves EL, Boon F, Vanderwolf CH, Cain DP. Detailed behavioral analysis of water maze acquisition under systemic NMDA or muscarinic antagonism: nonspatial pretraining eliminates spatial learning deficits. Behav Neurosci 1996;110(1):103–16. [30] Shirey JK, Brady AE, Jones PJ, Davis AA, Bridges TM, Kennedy JP, Jadhav SB, Menon UN, Xiang Z, Watson ML, Christian EP, Doherty JJ, Quirk MC, Snyder DH, Lah JJ, Levey AI, Nicolle MM, Lindsley CW, Conn PJ. A selective allosteric potentiator of the M1 muscarinic acetylcholine receptor increases activity of medial prefrontal cortical neurons and restores impairments in reversal learning. J Neurosci 2009;29(45):14271–86. [31] Schreiber R, Vivian J, Hedley L, Szczepanski K, Secchi RL, Zuzow M, van Laarhoven S, Moreau JL, Martin JR, Sik A, Blokland A. Effects of the novel 5-HT(6) receptor antagonist RO4368554 in rat models for cognition and sensorimotor gating. Eur Neuropsychopharmacol 2007;17(4):277–88. [32] Snodgrass JG, Corwin J. Pragmatics of measuring recognition memory: applications to dementia and amnesia. J Exp Psychol Gen 1988;117(1):34–50. [33] Whitehouse PJ, Price DL, Struble RG, Clark AW, Coyle JT, Delon MR. Alzheimer’s disease and senile dementia: loss of neurons in the basal forebrain. Science 1982;215(4537):1237–9. [34] Winters BD, Saksida LM, Bussey TJ. Object recognition memory: neurobiological mechanisms of encoding, consolidation and retrieval. Neurosci Biobehav Rev 2008;32(5):1055–70. [35] Winters BD, Saksida LM, Bussey TJ. Paradoxical facilitation of object recognition memory after infusion of scopolamine into perirhinal cortex: implications for cholinergic system function. J Neurosci 2006;26(37):9520–9.
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Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Research report
A new automated method to assess the rat recognition memory: Validation of the method Caroline Chambon ∗ , Nico Wegener 1 , Andreas Gravius 2 , Wojciech Danysz 3 In Vivo Pharmacology, Merz Pharmaceuticals GmbH, Eckenheimer Landstrasse 100, D-60318 Frankfurt am Main, Germany
a r t i c l e
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Article history: Received 19 January 2011 Received in revised form 10 March 2011 Accepted 14 March 2011 Keywords: Rat Memory Object recognition Y-maze Scopolamine Natural forgetting BQCA
a b s t r a c t During the last decade, the rodent object recognition memory task had gained popularity in the field of pharmacological studies, and has been proposed as a useful method to evaluate the efficacy of memory enhancing compounds. In this context, it is important to establish reliable and automated methods with high throughput to evaluate recognition memory in the rat. When performed in a Y-maze apparatus, object recognition has been described to be less dependent on spatial information, and we here report a new method to assess novelty discrimination in a Y-maze apparatus recorded via automated video tracking. During the development of the method, many parameters were recorded and some were selected as being the most reliable, i.e. time spent in the distal half-arms during the first 2 min of the test. The method was then validated under memory deficit conditions produced by a pharmacological treatment (scopolamine) and a long retention delay (72 h) between the sample and the test. The time-induced deficit was prevented by administration of a M1 positive allosteric modulator, BQCA at a dose of 10 mg/kg. Altogether, our data show that the novelty discrimination task performed in a Y-maze apparatus is a reliable method to assess the recognition memory of the rat. This new automated method is as sensitive as the classical object recognition test, and provides advantages such as easily definable parameters and the possibility to use an automated video tracking system that makes the measurement independent of the investigator. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Recognition memory, as a subdivision of episodic memory, is of particular interest for research in the field of Alzheimer’s disease. Indeed, episodic memory capacities have been described as being altered during the early stage of the disease [12,16]. Recognition memory was first studied in humans, for example, as part of the aging process [7,25] or dementia [1,24,32], and in non-human primates [20,21,26] using so-called recognition tasks. Developed in the 1980s [10], the classical recognition task for rodents is based on their natural tendency to explore novelty. The task is built in three phases, first a trial with two identical objects (sample phase), then an inter-trial interval (ITI) or retention delay, and finally a second
∗ Corresponding author at: In Vivo Pharmacology, R&D CNS, Merz Pharmaceuticals GmbH, Alfred-Wegener-Strasse 2, 60438 Frankfurt am Main, Germany. Tel.: +49 0 69 1503 2393, fax: +49 0 69 1503 735. E-mail addresses: [email protected] (C. Chambon), [email protected] (N. Wegener), [email protected] (A. Gravius), [email protected] (W. Danysz). 1 Tel.: +49 0 69 1503 8798; fax: +49 0 69 1503 735. 2 Tel.: +49 0 69 1503 262; fax: +49 0 69 1503 735. 3 Tel.: +49 0 69 1503 564; fax: +49 0 69 1503 735. 0166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.03.032
trial (test phase) performed in presence of a familiar object, i.e. an object encountered during the first trial, and a novel object. Results are analyzed by comparing the length of time spent exploring the novel object compared to the time spent exploring the familiar one. Any discrimination between the objects is explained by the fact that the rodent remembers the object previously encountered during the sample phase and thus spends more time exploring the novel object [11]. In recent years, this task has become more popular in the field of pharmacological studies in rodents [for reviews see [8]] and has been proposed as a useful method to evaluate the efficacy of compounds targeting learning processes or memory [5]. There are two main reasons for the popularity of this test. Firstly, the object recognition task demands no food or water deprivation [10] and secondly, this task presents a high sensitivity to compounds acting on memory. In addition, its design (sample, ITI and test phases) allows the investigation of different memory processes, i.e. acquisition, retention and retrieval [34]. However, the manual scoring of the test is time consuming and scientists are currently in agreement that even though video tracking systems exist, there is no reliable automated version of the test. Video tracking systems have some limitations and cannot evaluate object exploration in the same way as a trained experimenter. Unfortunately, manual scoring, currently considered to be the most
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reliable way to measure object exploration, presents a main inconvenience. There is natural variability between the experimenters’ scoring methods and the lack of 100% objectivity makes it difficult to compare data and results. There is thus a real need to improve data acquisition in the object recognition task. Based on these observations, we explored a different way to assess object discrimination using a video tracking system while preserving the sensitivity of the test. In this study we performed object recognition in a Y-maze apparatus, which has been proposed to be less dependent on spatial information [35]. In the present paradigm, we thus used a maze that prevents animals from using external cues by use of high maze walls. Moreover, the position of the objects at the end of each arm does not allow the rats to use the spatial organization of objects to build a “picture” of the objects as a unit that could be disrupted by changing one of these objects during the test. Such a Y configuration also allows the association of each object to the area defined around it for measurement. In the present automated version of the task, we show the validity of a parameter different from the object exploration. An additional advantage of such a Y-maze apparatus is its size, which forces the rat to move from one arm to another in order to explore the objects. Combined with a video tracking, this enables the assessment of additional parameters related to locomotor activity while the rat is performing the task. In the present study we performed four experiments. In experiment 1, we wanted to investigate the novelty recognition behaviour of naive rats with a 1 h retention delay while using automated video tracking. The collected data were used to select the time frame and the most reliable parameters with which to assess rat memory performance in the Y-maze apparatus. The sensitivity of the test was verified using the selected parameters in experiments 2 and 3. In experiment 2, we validated the choice of the previously selected parameters in a pharmacological model of memory deficit. In experiment 3, the retention delay was prolonged in order to allow the animals to naturally forget the previous sample phase. In experiment 4, we tested whether this time-induced deficit could be pharmacologically prevented by modulation of M1 receptor activity. Altogether, these experiments were aimed at validating the Y-maze version of the object recognition task as a reliable method combining the sensitivity of the classical object recognition task with the advantages of automatization, i.e. higher throughput and observer-independent results.
Fig. 1. Schematic description of the Y-maze apparatus. The maze is made of black coated wood. Arm dimensions are as follows: 60 cm for the length; 26 cm for the width and 56 cm for the height. The figure shows the different compartments defined for the data acquisition by the video tracking software, i.e. arms, distal half-arms, and objects.
2.3. Procedure
Experimentally naive adult male Sprague-Dawley rats (250–300 g; Janvier, France) were housed in groups of four per cage. Colony room temperature and humidity were maintained at 20 ± 1 ◦ C and 50 ± 3%, respectively. Food and water were available ad libitum and animals were kept under an alternating 12 h/12 h day–night cycle (lights on at 07.00) for at least 6 days before the experiments were started. All experiments were conducted during the light period of the day–night cycle and each animal was used only once. The study was approved by the Ethical Committee, Regierungspräsidium Darmstadt, Hessen, and performed in accordance with the recommendations and policies of the U.S. National Institute of Health Guidelines for the Use of Animals.
Prior to testing memory performance, rats were habituated to the maze for 2 consecutive days. Each day, single rats were placed in a transparent cylinder in the centre of the maze for 5 s, then the cylinder was lifted and animals were allowed to freely explore the empty maze for 10 min. Following habituation, the rat’s memory performance was tested on the third day. On this day, 3 similar objects were placed at the far end (10 cm from the wall) of each arm of the Y-maze. The rat was introduced into the cylinder at the centre of the maze and allowed to explore the maze and the objects for 5 min (sample phase). Note that in experiment 3, different sample times were used, i.e. 4, 3 and 2 min, and that in experiment 4, the sample time was reduced to 2 min to make the task more challenging. During the retention delay, the rat was returned to its home cage and one of the familiar objects was replaced by a new object. In experiments 1 and 2, the retention delay was 1 h. In experiment 3, in order to evaluate at which time point a natural forgetting process could be observed, the retention delay was increased up to 48 h. In experiment 4, a retention delay of 72 h was used in order to test the natural forgetting process. The test phase lasted for 5 min. Both sample and test phases were recorded. The video recording started when the rat entered one of the arms. The maze was cleaned with water in between rats as well as between the sample and test phases. Recorded parameters were: time spent (s) in each arm; time spent (s) in each distal half-arm; and time spent (s) to explore each object. Entry into an arm or half-arm was counted when the centre point of the body (defined by the video tracking) entered into one of the defined areas. Object exploration was counted when the rat’s nose point (defined by the video tracking) was in the defined area around the object (2 cm around the object). To exclude place preference, the novel object was pseudo-randomly placed in 1 of the 3 arms. During the 5 min test phase, each parameter was analyzed in 1 min steps. In experiment 1, exploration times were provided for the novel object, familiar object 1 and familiar object 2. Note that in other experiments, the time given for the familiar exploration corresponds to the time spent to explore the two familiar objects divided by two. The following inclusion criteria were used to ensure that the rats could perform the task properly: during the sample phase, animals had to visit each arm at least once; during the test phase, each animal had to visit the arm where the novel object was placed and 1 arm with a familiar object.
2.2. Apparatus
2.4. Treatments
The object recognition task took place in a black wooden Y-maze (arm length: 60 cm; width: 26 cm; height: 56 cm; Fig. 1). The size of the arms fully allowed the rat to explore all around the objects. A digital video camera (Panasonic, USA) mounted above the maze was connected to a computer running the video tracking software EthoVision XT (Noldus, Netherlands). Light sources were placed around the maze providing indirect light with an intensity of 20 lux within the maze. From inside the maze, no external cues were visible. The objects used were 20 cm high bottles, heavy enough to ensure that the rats could not move them. We used a set of white plastic and dark glass bottles. Each object was randomly used as novel or familiar. As far as could be ascertained, the objects had no significance for the animals and they have never been associated to reinforcement.
Scopolamine hydrobromide (Sigma–Aldrich, Steinheim, Germany) was dissolved in sterile physiological saline (vehicle). Benzylquinolone carboxylic acid (BQCA) (synthesized by Merz Pharmaceuticals GmbH, Frankfurt am Main, Germany) was dissolved in a solution of 10% Tween 80 in water (vehicle). Both compounds were injected i.p. 30 min prior to the sample phase in a volume of 2 ml/kg for scopolamine and 4 ml/kg for BQCA.
2. Materials and methods 2.1. Animals
2.5. Statistical analysis Data are expressed as mean ± S.E.M. One way repeated measure analyses of variance (ANOVA) was performed on data from experiment 1. Significant one way
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3. Results
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One way repeated measure ANOVA performed on the time spent in arms during the sample phase showed that naive animals did not display any arm preference (F2,92 = 0.92, p > 0.05). Results from the individual exploration of the novel, familiar 1 and familiar 2 arms during the 5 min of the test phase are presented in Fig. 2A. A one way repeated measure ANOVA performed on the data from each minute of the test revealed an effect of the arm for the 1st minute (F2,92 = 34.92, p < 0.05) and the 2nd minute (F2,92 = 5.98, p < 0.05) but not for the 3rd (F2,92 = 0.02, p > 0.05), 4th (F2,92 = 0.52, p > 0.05) and 5th minutes (F2,92 = 1.19, p > 0.05). Results from the individual exploration of the novel, familiar 1 and familiar 2 half-arms during the 5 min of the test phase are presented in Fig. 2B. A one way repeated measure ANOVA performed on the data from each minute of the test revealed an effect of the half-arm for the 1st minute (F2,92 = 34.81, p < 0.05) and the 2nd minute (F2,92 = 8.44, p < 0.05) but not for the 3rd (F2,92 = 0.13, p > 0.05), 4th (F2,92 = 1.96, p > 0.05) and 5th minutes (F2,92 = 0.51, p > 0.05). Results from the individual exploration of the novel, familiar 1 and familiar 2 objects during the 5 min of the test phase are presented in Fig. 2C. A one way repeated measure ANOVA performed on the data from each minute of the test revealed an effect of the object for the 1st minute (F2,92 = 50.71, p < 0.05) and the 2nd minute (F2,92 = 7.03, p < 0.05) but not for the 3rd (F2,92 = 1.41, p > 0.05), 4th (F2,92 = 2.07, p > 0.05) and 5th minutes (F2,92 = 0.34, p > 0.05). Subsequent post-hoc Holm–Sidak’s t-tests performed to compare exploration of novel, familiar 1 and familiar 2 objects showed that, in naive animals, it is possible to observe a novelty discrimination during the 1st and 2nd minutes of the test with each of the 3 parameters measured (p < 0.05). Based on these data, we chose the first 2 min of the test to measure novelty discrimination in the Y-maze apparatus. Results from the individual exploration of the novel, familiar 1 and familiar 2 for the 3 parameters measured during the first 2 min of the test phase are presented in Fig. 3A. A one way repeated measure ANOVA revealed a different exploration for the arms (F2,92 = 30.59, p < 0.05), the half-arms (F2,92 = 34.94, p < 0.05) and the objects (F2,92 = 47.01, p < 0.05). Subsequent post-hoc Holm–Sidak’s t-tests performed to compare exploration of the novel, familiar 1 and familiar 2 showed that, in naive animals, it is possible to observe a novelty discrimination during the first 2 min of the test with each of the 3 parameters measured (p < 0.05). These data also show that rats explored the familiar arms, half-arms or objects at the same level (p > 0.05). However, the comparison of object exploration measured via manual scoring and video tracking (Fig. 3B) showed an effect of the method used for measurement independent of the object (2 way repeated measure ANOVA: F2,92 = 10.26, p < 0.05 for interaction measurement x object). Subsequent post-hoc Holm–Sidak’s t-tests showed a novelty discrimination with both methods (p < 0.05),
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repeated measure ANOVA was followed by Holm–Sidak’s post-hoc t-tests. A paired t-test was performed on data from experiment 1. Unpaired t-tests were performed on data from experiment 2. One way ANOVA was performed on the distance and velocity data from experiment 4. Significant one way ANOVAs were followed by Dunnett’s post-hoc t-tests. Two way repeated measure ANOVAs were performed in experiment 1 to compare object exploration measured via manual scoring and video tracking, and in experiment 2, 3 and 4 on the time spent in distal half-arms. Significant two way repeated measure ANOVAs were followed by Holm–Sidak’s post-hoc t-tests. Statistical analysis was performed using SigmaPlot 11 software (Systat Software, Erkracht, Germany).
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Fig. 2. Time course of the exploration of the novelty and familiarity in the Y-maze during the test phase for naive rats. The test session was cut in 5 intervals of 1 min. (A) Exploration of the novel, familiar 1 and familiar 2 arms measured during the 5 min of the test. (B) Exploration of the novel, familiar 1 and familiar 2 half-arms measured during the 5 min of the test. (C) Exploration of the novel, familiar 1 and familiar 2 objects measured during the 5 min of the test. Naive animals (n = 47) were able to discriminate the novel with each parameter during the 1st and 2nd minutes of the test. *p < 0.05, one way repeated measure ANOVA followed by Holm–Sidak’s test. The first 2 min of the test were chosen as the most pertinent time frame to measure novelty discrimination.
but also a significantly lower exploration time measured with the video tracking compared to the manual scoring for all 3 objects (p < 0.05). Based on data presented in Fig. 3A and B, we chose the time spent in distal half-arms to measure novelty discrimination during the first 2 min of the test. Moreover, since no difference was observed between the familiar half-arms, in experiments 2, 3 and 4 we chose to pool the exploration times of familiar half-arms 1 and 2.
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Fig. 3. Comparison of the three parameters measured during the first 2 min of the test. (A) Individual exploration of the novel, familiar 1 and familiar 2 for each of the 3 parameters measured (time spent in arms, time spent in distal half-arms and time spent to explore objects) during the first 2 min of the test. A clear novelty preference is observed with each parameter. *p < 0.05, one way repeated measure ANOVA followed by Holm–Sidak’s test. (B) Comparison of levels of object exploration measured via manual scoring and video tracking. #p < 0.05, manual scoring vs. video tracking, two way repeated measure ANOVA followed by Holm–Sidak’s test. Results are expressed as mean ± S.E.M., n = 47. The time spent in distal half-arms was chosen as the best parameter to reflect object discrimination.
3.2. Experiment 2: scopolamine-induced recognition memory deficits in the Y-maze apparatus Unpaired t-tests performed on the distance travelled (t22 = −0.79, p > 0.05) and on the velocity (t22 = −1.05, p > 0.05) showed that scopolamine (0.25 mg/kg) had no effect on the locomotor activity measured during the sample phase. Results from the individual distal half-arm exploration measured during the first 2 min of the test phase are presented in Fig. 4. A two way repeated measure ANOVA revealed a treatment effect (F1,22 = 8.25, p < 0.05) as well as an object effect (F1,22 = 25.38, p < 0.05) and a treatment x object interaction (F2,47 = 11.55, p < 0.05). Subsequent post-hoc Holm–Sidak’s t-tests performed to compare exploration of the novel to exploration of the familiar showed that the group injected with scopolamine 0.25 mg/kg was not able to discriminate the novel (p > 0.05), whereas the vehicle group showed significant discrimination (p < 0.05). In addition, the exploration of the novel was significantly decreased in the scopolamine group compared to the vehicle group (p < 0.05). 3.3. Experiment 3: establishment of the retention delay in the Y-maze apparatus Results from the individual distal half-arm exploration measured during the first 2 min of the test phase after different
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Fig. 4. Effect of scopolamine on novelty discrimination during the test phase. Individual exploration of the novel and the familiar distal half-arms is expressed as mean ± S.E.M. Two way repeated measure ANOVA followed by Holm–Sidak’s t-tests *p < 0.05 novel vs. familiar, #p < 0.05 vs. vehicle, n = 12 per group. Scopolamine at a dose of 0.25 mg/kg induced a novelty discrimination deficit when administered before the sample phase.
retention delays are presented in Fig. 5A. A two way repeated measure ANOVA performed on these data revealed no retention delay effect (F3,43 = 0.42, p > 0.05) but an object effect (F1,43 = 48.40, p < 0.05) and no retention delay x object interaction (F3,93 = 1.27, p > 0.05). Subsequent post-hoc Holm–Sidak’s t-tests performed to compare exploration of the novel to exploration of the familiar showed that naive animals were able to discriminate the novel after all the retention delays used, i.e. 1 h, 4 h, 24 h, and 48 h (p < 0.05). Results from the individual distal half-arm exploration measured during the first 2 min of the test phase after a 48 h retention delay and with different sample lengths are presented in Fig. 5B. A two way repeated measure ANOVA performed on these data revealed no sample length effect (F2,33 = 2.02, p > 0.05) but an object effect (F1,33 = 11.81, p < 0.05) and no sample length x object interaction (F2,71 = 0.627, p > 0.05). Subsequent post-hoc Holm–Sidak’s t-tests performed to compare exploration of the novel to exploration of the familiar showed that naive animals were able to discriminate the novel after a 48 h retention delay when the sample phase was 4 or 3 min (p < 0.05) but not 2 min long. Results from the individual distal half-arm exploration measured during the repetition of an experiment performed with a 48 h retention delay and a sample length of 2 min are presented in Fig. 5C. A paired t-test performed to compare exploration of the novel to exploration of the familiar showed that naive animals were able to discriminate the novel (p < 0.05). These data show that the deficit observed with a 48 h retention delay and a 2 min long sample is not stable through experiments. 3.4. Experiment 4: BQCA-induced reversal of natural forgetting in the Y-maze apparatus A one way ANOVA performed on the distance travelled during the sample phase showed no treatment effect (F2,32 = 2.99, p > 0.05), and one way ANOVA performed on the velocity measured during the sample phase showed no group effect (F2,32 = 2.963, p > 0.05). Together these data indicate that BQCA had no effect on locomotion under these experimental conditions. Results from the individual distal half-arm exploration measured during the first 2 min of the test phase are presented in Fig. 6. A two way repeated measure ANOVA performed on these data revealed no treatment effect (F2,32 = 0.63, p > 0.05) but an object effect (F1,32 = 5.16, p < 0.05) and no treatment x object interaction
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Fig. 6. Effect of BQCA on novelty discrimination during the test phase. Individual exploration of the novel and the familiar distal half-arms is expressed as mean ± S.E.M. *p < 0.05, two way repeated measure ANOVA followed by Holm–Sidak’s t-tests novel vs. familiar, n = 11–12 per group. BQCA at a dose of 10 mg/kg prevents the novelty discrimination deficit induced by a 72 h retention delay.
* BQCA used at 10 mg/kg was able to prevent the natural forgetting induced by a 72 h delay between the sample and the test phase.
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2 min Sample length Fig. 5. Establishment of the natural forgetting protocol. (A) Individual exploration of novel and familiar distal half-arms after different retention delays. (B) Individual exploration of novel and familiar distal half-arms after different sample lengths and a delay of 48 h. *p < 0.05, two way repeated measure ANOVA followed by Holm–Sidak’s t-tests novel vs. familiar (C) Individual exploration of the novel and familiar distal half-arms after a 2 min sample length and a delay of 48 h (Repetition). *p < 0.05, paired t-test. Results are expressed as mean ± S.E.M. n = 12 per group. The deficit observed after a 48 h retention delay and a 2 min sample length is not stable through experiments.
(F2,69 = 2.52, p > 0.05). Subsequent post-hoc Holm–Sidak’s t-tests performed to compare exploration of the novel to exploration of the familiar showed that the group BQCA 10 mg/kg was able to discriminate the novel (p < 0.05) whereas both the vehicle group and the BQCA 5 mg/kg group could not (p > 0.05). These data show that
The present study was performed to validate an automated Ymaze version of the object recognition task. Although this task is used more and more in pharmaceutical studies in rodents, no automated and observer-independent versions currently exist. Here we show that using a Y-maze apparatus instead of an open arena allows the use of a different parameter, i.e. the time spent in the distal half-arm where the object is placed, to assess rats object exploration. Our results also show that this parameter is sensitive enough to detect a pharmacologically-induced recognition memory deficit, the scopolamine-induced deficit, as well as the natural forgetting observed after a 72 h retention delay. Furthermore, our present data show that the M1 receptor positive allosteric modulator (PAM) BQCA is able to prevent the forgetting process induced by a 72 h retention delay in the Y-maze apparatus. Experiment 1 was performed to define a new parameter, which could allow evaluation of novelty discrimination when the object recognition task is performed in a Y-maze apparatus and when exploration is recorded via video tracking. Results from the sample phase show that naive rats display no arm preference in the Y-maze apparatus, proof that this maze-configuration allows a complete isolation from external cues and thus a less spatial-dependent object exploration. Results from the test phase show good novelty discrimination during the first 2 min of the test for the 3 parameters measured, i.e. time spent in the arms, time spent in the distal half-arms, and time spent to explore the objects (Fig. 2A, B and C). This 2 min time frame is in the range of what is normally used in the classical object recognition test. Indeed, in the classical version of the test, the most often used time frames are 2 or 3 min [10,23]. Based on these results, we decided to focus on the first 2 min of the test to choose the parameter to be used to measure novelty discrimination. Several aspects were evaluated to make this choice. First, the parameter had to be easily measurable via video tracking. Second, the exploration had to be high enough to allow a measurable decrease due to the effect of an administered compound, and third, the parameter had to be specific enough to reflect the real object exploration. Taking into consideration the above criteria and the size of the 3 zones measured to express the novelty discrimination in naive animals, we chose the time spent in the distal half-arms
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(an area of approximately 10 cm2 around the object) as the best parameter. To record this measure, the video tracking tracks the body’s centre point, which we believe is more reliable than tracking the nose point as is often used for the object exploration. Indeed, in our experimental conditions, the nose point may jump from the nose to the extremity of a front paw or even be exchanged with the tail point. The instability of the nose point measurement leads to a reduction of the recorded object exploration, a problem which was made clear when comparing manual scoring to the video tracking (Fig. 3B). Under these conditions, we regard the time spent in the distal half-arms as a good compromise between the time spent to explore the objects, which is low and provides only a small time window to measure the effect of a compound, and the time spent to explore the arms, which is not specific enough. Moreover, and in order to provide a double validation of the parameter chosen, i.e. time in half-arm, we verified that the time spent in the proximal half-arm (time in the arm-time in the half-arm) was identical for the 3 arms, even during the test phase (can be checked by comparing data in Fig. 3A). This shows that the time spent into the distal half-arms is dependent on the object placed in it and not on any place preference. Additionally, results from the exploration of the individual half-arms, i.e. familiar 1 and familiar 2, clearly show no exploration difference between the two familiar half-arms (Fig. 3A). Therefore, the following experiments were analyzed using the time spent in the distal half-arms during the first 2 min of the test; and the exploration of the familiar was expressed as the mean of the time taken to explore the 2 familiar half-arms. Experiment 2 was performed to validate the parameters chosen in experiment 1 by using a model of pharmacologically-induced memory deficit. Scopolamine (a muscarinic receptor antagonist) is widely used as a reference compound to induce a memory deficit in various tasks [2,6,29] including the object recognition task. Moreover, the muscarinic system is known to be involved in learning processes [14,15,27] but has also been shown to be altered during normal aging [4,9,13] as well as in pathologies such as Alzheimer’s disease [33]. After a dose-response experiment (data not shown), the scopolamine dose of 0.25 mg/kg was selected as the most relevant to induce a recognition deficit while leaving the rat’s locomotion unaffected. The aim of this experiment was to evaluate the sensitivity of the object recognition task performed in the Ymaze apparatus and to compare our results with data obtained in the classical version of the test. Results from experiment 2 show that administration of scopolamine (0.25 mg/kg) before the sample phase induced an object discrimination deficit when the test is performed after a retention delay of 1 h (Fig. 4). These results are in accordance with data obtained in the classical version of the object recognition test. Indeed, a memory deficit was recently shown [22] after injection of scopolamine at a dose of 0.5 mg/kg, which is in the dose range used in the present study, and a loss of the object discrimination has been reported in animals injected with 0.2 mg/kg of scopolamine [3]. In both studies, the retention delay between the sample and the test phase was also similar to the one used here, i.e. 1 h. Taken together these data indicate that the Y-maze version of the object recognition task used here exhibits the same sensitivity to a scopolamine-induced memory deficit as the classical version. The data also suggest that the memory deficit observed after scopolamine administration is not due to any alterations of locomotion. Furthermore, our data show that scopolamine (0.25 mg/kg) can be utilized to model a pharmacologically-induced memory deficit in the automated Y-maze version of the object recognition test. Experiment 3 was performed to establish a natural forgetting procedure while using the parameters chosen in experiment 1. Again, the aim of this experiment was to evaluate the sensitivity of the object recognition task performed in the Y-maze apparatus and to compare our results with data obtained in the classical version of the test. Results from experiment 3 show that naive rats can
display natural forgetting when the sample length is shortened to 2 min and the retention delay is 48 h long (Fig. 5B), although the deficit obtained under these conditions was not stable throughout the experiments (Fig. 5C). We thus decided to extend the retention delay to 72 h to ensure a stable deficit across experiments. The procedures used in previous studies to induce natural forgetting in the classical object recognition task vary in many aspects, e.g. length of the habituation, length of the sample phase, and length of the retention delay, and the most often used delays are only 4 h [5,17,31] and 24 h [22]. Data obtained in experiment 3, where the effect of increasing the retention delay was tested (Fig. 5A), revealed that after a 4 h retention delay, naive rats performed worse than after a 1 h delay, which would speak in favour of using a 4 h delay to measure impaired performance. But these data also show that, after a 24 h retention delay, the novelty discrimination is back to the control group level (1 h delay). Altogether, these data suggest that processes leading to recall after a 1 h retention delay, which would correspond to short term memory, are not sufficient after a 4 h retention delay; but that other processes, which would correspond to long term memory, lead to recall after a 24 h retention delay. Indeed, if animals are able to clearly discriminate the novel after a 24 h retention delay, it means that the memory could not be lost after a 4 h delay but rather that some processes, which we cannot as yet clearly explain, enabled the rats to access the information in order to perform the task properly. Therefore, we believe that the commonly used 4 h retention delay may not be sufficient to investigate natural forgetting processes. Moreover, it is important to note that the habituation length in experiment 3 was unchanged, to ensure that the stress level of the animals was low enough to allow a memory formation that could then be altered by a longer retention delay. Indeed, we believe that in some previously published natural forgetting experiments, an impairment was obtained after short retention delays, such as 4 h, partly because of a quasi inexistent habituation phase, e.g. 2 min the day before testing [22]. It has been shown that stress can negatively influence explicit memory formation (for review see [28]), and more specifically, it has been shown that in the object recognition task, non-habituated (just handled) rats were unable to discriminate the novel from the familiar after a 24 h retention delay, whereas habituated rats showed no deficit after such a delay [19]. With the conditions we selected in experiment 4, i.e. 2 days of habituation, a sample of 2 min and a retention delay of 72 h, we can be sure that the rats display a stable natural forgetting which can be attributed to the retention delay and not to any stress factor. These results thus show that the model of time-induced memory impairment can also be performed in the automated Y-maze version of the object recognition task. Such a protocol is of particular interest since it provides a non-pharmacological model of a memory deficit, which can be potentially used to evaluate all types of memory improving compounds. Experiment 4 was performed to show that the recognition memory deficit observed after a 72 h retention delay could be prevented by a cognition-enhancing compound. We selected BQCA, a selective M1 receptor PAM, which has recently been described to have such properties [18,30]. BQCA acts at an allosteric site of the M1 receptor enhancing the effects of orthosteric agonists such as acetylcholine. In the present experiment, we showed that BQCA is able to prevent the recognition memory deficit induced by a 72 h retention delay when injected at a dose of 10 mg/kg before the sample phase (Fig. 6). Up to now, the memory-improving capacity of BQCA was only evaluated in two paradigms. The first was the contextual fear conditioning task, in which BQCA reversed the scopolamine-induced deficit in rats when administered before the acquisition phase of the task [18]. The second was a discrimination learning task performed in Tg2576 mice, in which BQCA improved memory performance of 12 months old mice over-expressing amyloid species [30]. Inter-
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estingly, the memory-improving effect of BQCA was obtained at the dose of 30 mg/kg in the discrimination learning test [30] and at doses of 15 and 20 mg/kg in the fear conditioning test [18] but not at 10 mg/kg. Procedural differences between these paradigms and the memory impairment models used may explain the difference in the minimal effective dose however, our data show a memory improving effect of BQCA at the lower dose of 10 mg/kg. Altogether, the recognition memory deficit obtained in experiment 3 and the reversal of this deficit by BQCA in experiment 4 demonstrate the validity of the natural forgetting model performed in the automated Y-maze version of the object recognition task. Taken together, results from the present study show that the object recognition task performed in a Y-maze apparatus displays the same sensitivity as the classical version proposed by Ennaceur and Delacour [10]. Despite using a new parameter, time spent in the distal half-arm where the object is placed, the data presented here are similar to those found in literature. Two models of memory impairment frequently used in pharmacological studies were validated in the Y-maze version, first the pharmacologicallyinduced and second the time-induced memory deficit. There are clear advantages to this new version of the object recognition test using video tracking, such as automated and immediate data acquisition, the assurance of experimenter-independent measurements, and reproducibility of results, as well as the additional measurement of locomotor activity. In conclusion, this automated Y-maze version of the object recognition task appears to be a valuable tool to screen for the effects of compounds believed to act on the rat memory capacity. Acknowledgments We would like to thank Caroline Barberi and Ralph Gross for their support during the experiments. References [1] Alescio-Lautier B, Michel BF, Herrera C, Elahmadi A, Chambon C, Touzet C, Paban V. Visual and visuospatial short-term memory in mild cognitive impairment and Alzheimer disease: role of attention. Neuropsychologia 2007;45(8):1948–60. [2] Baron SP, Wright D, Wenger GR. Effects of drugs of abuse and scopolamine on memory in rats: delayed spatial alternation and matching to position. Psychopharmacology (Berl) 1998;137(1):7–14. [3] Bartolini L, Casamenti F, Pepeu G. Aniracetam restores object recognition impaired by age, scopolamine, and nucleus basalis lesions. Pharmacol Biochem Behav 1996;53(2):277–83. [4] Baskerville KA, Kent C, Nicolle MM, Gallagher M, McKinney M. Aging causes partial loss of basal forebrain but no loss of pontine reticular cholinergic neurons. Neuroreport 2006;17(17):1819–23. [5] Bertaina-Anglade V, Enjuanes E, Morillon D, Drieu la Rochelle C. The object recognition task in rats and mice: a simple and rapid model in safety pharmacology to detect amnesic properties of a new chemical entity. J Pharmacol Toxicol Methods 2006;54(2):99–105. [6] Besheer J, Short KR, Bevins RA. Dopaminergic and cholinergic antagonism in a novel-object detection task with rats. Behav Brain Res 2001;126(1–2):211–7. [7] Boyle Jr E, Aparicio AM, Kaye J, Acker M. Auditory and visual memory losses in aging populations. J Am Geriatr Soc 1975;23(6):284–6. [8] Dere E, Huston JP, De Souza Silva MA. The pharmacology, neuroanatomy and neurogenetics of one-trial object recognition in rodents. Neurosci Biobehav Rev 2007;31(5):673–704. [9] Dunbar GL, Rylett RJ, Schmidt BM, Sinclair RC, Williams LR. Hippocampal choline acetyltransferase activity correlates with spatial learning in aged rats. Brain Res 1993;604(1–2):266–72. [10] Ennaceur A, Delacour J. A new one-trial test for neurobiological studies of memory in rats. 1: Behavioral data. Behav Brain Res 1988;31(1):47–59.
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