Characterizing spatial extinction in an abbreviated version of the Barnes maze

Behavioural Processes 86 (2011) 30–38

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Characterizing spatial extinction in an abbreviated version of the Barnes maze Viviana Vargas-López a,b , Marisol R. Lamprea a,b , Alejandro Múnera a,c,∗ a

Behavioral Neurophysiology Laboratory, Universidad Nacional de Colombia, Bogotá, Colombia Psychology Department, School of Human Sciences, Universidad Nacional de Colombia, Bogotá, Colombia c Physiological Sciences Department, School of Medicine, Universidad Nacional de Colombia, Bogotá, Colombia b

a r t i c l e

i n f o

Article history: Received 8 April 2010 Received in revised form 31 July 2010 Accepted 3 August 2010 Keywords: Spatial learning Extinction Associative learning Spatial memory Barnes maze

a b s t r a c t Adult male Wistar rats were trained to find an escape box in the Barnes maze in order to characterize the extinction process of a learned spatial preference. To do so, once they had fully acquired the spatial task, they were repeatedly exposed to the maze without the escape box. Multiple behavioral measurements (grouped into motor skill and spatial preference indicators) were followed up throughout the complete training process. Animals gained efficiency in finding the escape box during acquisition, as indicated by the reduction in the time spent escaping from the maze, the number of errors, the length of the traveled path, and by the increase in exploration accuracy and execution speed. When their retention and preference were tested 24 h later, all the subjects retained their enhanced performance efficiency and accuracy and displayed a clear-cut preference for the escape hole and its adjacent holes. Almost all motor skill indicators followed an inverse, though not monotonic, pattern during the extinction training, returning to basal levels after three trials without escape box, displaying a transient relapse during the fifth extinction trial. Preference indicators also followed a reverse pattern; however, it took seven trials for them to return to basal levels, relapsing during the eighth extinction trial. The abbreviated Barnes maze acquisition, evaluation, and extinction procedures described herein are useful tools for evaluating the effects of behavioral and/or pharmacological treatment on different stages of spatial memory, and could also be used for studying the neurophysiological and neurobiological underpinnings of this kind of memory. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Rodents, birds and several other species acquire and update knowledge about the spaces they navigate by recognizing, encoding, storing and recovering information about object configuration, spatial location and specific routes. Such experience-based knowledge allows them to locate relevant resources in their environment while preventing risky situations (Thinus-Blanc, 1996; Good, 2002; Carrillo-Mora et al., 2009). A diversity of experimental methods and mazes has been used in assessing spatial learning and memory in animal models, such as the Barnes maze, the radial maze, the T maze, the Y maze and the Morris water maze, the latter being the most widely used nowadays (Patil et al., 2009). Spatial learning is liable to the same behavioral effects as those of conditioned learning, suggesting that associative learning is its basic mechanism (Chamizo, 2002). Behavioral phenomena

∗ Corresponding author. Present address: Laboratorio de Neurofisiología Comportamental, Departamento de Ciencias Fisiológicas, Facultad de Medicina, Universidad Nacional de Colombia, Edificio 471, Oficina 430, Carrera 30 No. 45-03, Ciudad Universitaria, Bogotá, Colombia. Tel.: +57 1 3165000x15058. E-mail address: [email protected] (A. Múnera). 0376-6357/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2010.08.002

observed in spatial learning include blocking (Chamizo et al., 1985; Rodrigo et al., 1997; Roberts and Pearce, 1999; Pearce et al., 2006), overshadowing (March et al., 1992; Sánchez-Moreno et al., 1999; Biegler and Morris, 1999), latent inhibition (Wishaw, 1991; Prados, 1999; Prados and Redhead, 2002) and extinction (Lattal et al., 2003; Prados et al., 2003, 2008; Harloe et al., 2008). Extinction is a process through which an organism adjusts its previously acquired behavior to changing environmental demands (Bouton, 2004). It was described for the first time in classical conditioning procedures (Pavlov, 1927) and was also observed later on in operant conditioning (Skinner, 1963). Repeated non-contingent presentation of conditioned and unconditioned stimuli or the omission of reinforcement diminishes the rate of conditioned responses in such protocols (Neuringer et al., 2001; Quirk, 2006). Theories explaining extinction can be grouped into two broad classes (Falls, 1998). The first class supports the idea that extinction involves destroying the originally learned stimuli association (Rescorla and Wagner, 1972; Mackintosh, 1975; McClelland and Rumelhart, 1985). The second class argues that most original learning survives extinction. In fact, conditioned responses can return after extinction, as indicated by post-extinction phenomena such as spontaneous recovery, rapid reacquisition, reinstatement and renewal effects (Pavlov, 1927; Konorski, 1948; Pearce and

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Hall, 1980; Myers and Davis, 2002; Bouton, 2004; Delamater, 2004; Rescorla, 2004). Furthermore, there is evidence showing that extinction differs from “forgetting” and involves new learning which is partly modulated by the context (Falls, 1998; Bouton, 2004). A dramatic increase in understanding the molecular and neural mechanisms of extinction has been achieved during the last few years (Myers and Davis, 2002; Quirk, 2006; Lattal et al., 2006; Bouton et al., 2006; Quirk and Mueller, 2007). However, much of the research has been focused on the extinction of classical conditioned responses (Delamater, 2004; Maren and Quirk, 2004; Phelps and LeDoux, 2005), and less is known about the extinction of other types of behavior such as spatial learning, mostly having been evaluated using the Morris water maze (Morris, 1984; Lattal et al., 2003, 2004; Morris et al., 2006; Suzuki et al., 2004). Subjects’ performance in spatial memory tasks has been found to be different when evaluated in water or land mazes (Patil et al., 2009). Such differences can be attributed to the amount of stress induced by the specific features of training in each maze. It has been reported that both Morris and Barnes maze training induce adrenal activation and corticosterone release (Roozendaal et al., 1996; Harrison et al., 2009). However, it has been demonstrated in the latter work that Morris maze training induces greater increases in plasma corticosterone than Barnes maze training does (Harrison et al., 2009). The higher the training-induced corticosterone elevation in this study, the worse the performance in the water maze was. By contrast, training-induced corticosterone elevation in the Barnes maze did not impair spatial task performance (Harrison et al., 2009). In this vein, being devoid of intrinsic stress-associated detrimental effects, the Barnes maze affords a crucial advantage for evaluating stress effects on spatial learning and memory (Harrison et al., 2009) and evaluating the effect of memory-enhancing pharmacological treatment (Patil et al., 2009). Even though the Barnes maze has similarities to the Morris water maze (since both allow learning, working memory and spatial reference memory to be evaluated), less physical effort (Williams et al., 2003) is required in the former to perform the task and no strong aversive stimuli or deprivation is used as reinforcement (Barnes, 1979). Instead, weak aversive stimulation can be added to increase the motivation to escape from the circular platform (Sunyer et al., 2007). The Barnes maze thus allows spatial learning to be assessed in a slightly aversive environment without the confounding effects of additional physical exertioninduced fatigue and adrenal activation brought on by swimming (Roozendaal et al., 1996; Williams et al., 2003; Harrison et al., 2009). Taking these methodological issues into account, generalizing knowledge about the neural basis for the extinction of spatial learning and memory should, therefore, be done cautiously. Since each experimental model has advantages or disadvantages, depending on the process to be evaluated (Carrillo-Mora et al., 2009; Harrison et al., 2009), using an alternative standardized model for evaluating different experimental interventions’ effects on spatial learning extinction thus becomes relevant. This paper describes and standardizes experimental and analytical procedures for evaluating the extinction process in an abbreviated version of the Barnes maze in adult male Wistar Rats. Such procedures will be useful for evaluating the effects of different interventions on acquiring, retaining, and, especially, the extinction of learned spatial preferences A subject’s performance in a given memory task results from the interwoven activity of several memory-related neural circuits. Since behavioral or pharmacological interventions eventually cause differential effects on the activity of such memory-related neural circuits, it is plausible that each experimental intervention would modify different aspects of the performance. A group of measurements was thus developed to better detect and characterize

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the acquisition and extinction of the spatial task, since it is desirable to measure various different behavioral aspects, as well as to detect subtle experimentally induced performance modifications in future work. Given the above-mentioned theoretical issues, it was expected that measurements taken into account in this work would differ in their rate of change throughout the acquisition and extinction training. In fact, it was found that most measurements changed faster during acquisition than during extinction. Moreover, hole and sector exploration changed at a slower rate than the rest of the indicators during extinction. 2. Materials and methods 2.1. Subjects Fourteen, 66 ± 4 day old, naïve, male Wistar rats, supplied by the Instituto Nacional de Salud in Bogota, weighing 270 ± 30 g (mean ± SEM), were used as subjects. Animals were housed in groups of four and maintained in a sound-attenuated room with controlled humidity (40 ± 5%) and temperature (20 ± 1 ◦ C) using a 12 h light–dark cycle (lights on at 07:00 a.m). Rats had ad libitum access to water and food. The subjects were kept in the laboratory for one week before any experimental procedure to allow them to become acclimatized to the new housing conditions. All procedures were conducted between 8:00 a.m. and 1:00 p.m. to avoid circadian peak corticosterone secretion and were performed according to local and international guidelines (NIH Guide for the Use and Care of Laboratory Animals). All efforts were made to minimize the number of animals and to avoid unnecessary suffering to the experimental subjects. 2.2. Training set-up and procedure Animals were trained on a 1.22 m diameter, black acrylic circular platform placed 80 cm above the floor. The platform had 18 evenly spaced (every 20◦ ) peripheral holes. Each hole had a 9.5cm diameter and was centered 10 cm from the edge. A randomly chosen hole (escape hole) allowed the subject to escape from the platform to a box (escape box) placed immediately below it. Lids placed on the inferior surface of the platform made it possible to locate the escape box (a 24 cm × 10 cm × 8.5 cm white acrylic rectangular prism) directly underneath any hole (since the floor was white, it was not possible for the animal to use box color to determine its position). At the beginning of any trial, the subject was placed in the center of the platform inside a start box (an opaque, 17 cm diameter, 15 cm high, white acrylic open-ended cylinder). The start box was coupled to a pulley system to raise it quickly to a resting position 2 m above, setting the subject free to explore the maze. The start box provided a standard starting context and ensured the animal’s initial random orientation. The labyrinth (Fig. 5A) was placed at the center of a square (2.3 m side) experimental room having white walls and floor (the door had the same color as the walls, but its borders were not concealed). Highcontrast signals (30-cm high, opaque black geometric figures: a cross, a circle, a square, and a triangle) were fixed in the walls of the experimental room (affixed in the center of each wall, 20 cm above maze level) giving the subject extra-maze visual cues (distal spatial cues or reference cues) that allowed it to learn the position of the target (escape hole; Fig. 5B). A 90-dB white-noise generator and two white-light 150-W bulbs placed in the ceiling of the experimental room provided motivation for escaping from the platform. Whenever the white-light bulbs were switched off, a 20-W red light was switched on to permit the experimenter to handle the rat and to eliminate olfactive clues by cleansing the maze and the boxes with a 10% ethylic alcohol solution. An infrared video cam-

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era linked to a VCR and a TV monitor was mounted 110 cm above the platform center to record the subject’s performance on a DVD recorder. Rats were handled for 5 min daily by the experimenter during the acclimation period to reduce possible stress caused by manipulation during the experimental procedure. After this period, each animal underwent a 9-min habituation session during which it was successively exposed to the experimental context (experimental room, 3 min), to the escape box (EB, 3 min) and to the start box (3 min). The experimental room was lit with the red bulb during this session to reduce possible preexposure effects, such as latent inhibition or perceptual learning (Prados and Redhead, 2002). The acquisition session was performed 24 h later; it consisted of eight trials (A1–A8). Acquisition trials began with the animal inside the start box for 30 s; the start box was then raised, the aversive stimuli (bright light and noise) were switched on and the rat was allowed to freely explore the maze. The trial finished either when the rat entered the EB or after 240 s of maze exploration. If the animal did not enter the escape box by itself after 240 s, the experimenter picked it up gently and placed it over the escape hole allowing the animal to enter the escape box. The aversive stimuli were switched off at the end of each acquisition trial, the dim red light was switched on and the animals were allowed to stay in the EB for 60 s. The evaluation trials and the extinction session were performed 24 h after acquisition. Two evaluation trials were designed to test spatial memory retrieval: (a) one trial identical to those during the acquisition phase (EB(+) ), which allowed retention evaluation; and, (b) one trial without escape box (EB(−) ) to evaluate spatial preference. The need to measure the degree to which the increased efficiency in execution was retained 24 h after acquisition was the main reason for using an evaluation trial (EB(+) ) identical to the acquisition ones. On the other hand, the need to ascertain the knowledge of the escape box location as indicated by the persistent exploration of the goal hole and its neighborhood was the main reason for using an additional evaluation trial in the absence of the escape box. The relevance of using different probe tests and measurements to evaluate spatial memory and extinction has been described in other spatial learning protocols (Markowska et al., 1993; Lattal et al., 2003). Since the EB(−) trial was not reinforced, it also served as the beginning of the extinction training, which consisted of eight nonreinforced trials (EB(−) , X2–X8). Animals were placed in the maze and allowed to explore for 240 s during the extinction trials but, since there was no escape box, they could not escape from the aversive stimulation in the way learned the day before. A 240 s inter-trial interval was used during all phases of this protocol. 2.3. Behavioral measurement and data analysis The subjects’ video-stored performance was measured off-line using X-Plo-Rat 3.3 software (Cardenas et al., 2001). The behavioral measurements analyzed were as follows. (a) First hole latency: time (in s) spent by the animal since it had been released from the start box until it explored a hole in the maze for the first time. An individual hole exploration was defined as being a single downward head deflection made inside such hole. Head deflections were detected using the zenithal video recording, therefore they had to be greater than 30◦ to be detected (unpublished data). (b) Escape latency: time (in s) spent by the animal since it had been released from the start box until it entered the escape box (during the acquisition and retention test trials) or until the first exploration of the escape hole (during the preference test and extinction trials). (c) Path length: distance (in cm) covered by the animal during a given trial, estimated on the basis of reconstructing the route (every

2 s the position of a rat’s body center, expressed in polar coordinates with origin at the maze’s center, was recorded in order to reconstruct the route). (d) Mean velocity: path length divided by escape latency (in cm/s). Path length was divided by trial duration (240 s) during preference test and extinction. This measurement did not discriminate activity and inactivity periods. (e) Non-goal hole exploration: the number of explorations of holes different from the escape one. Such explorations were considered as errors during the acquisition and EB(+) trials. (f) Weighted non-goal exploration: the sum (in cm) of multiplying the number of explorations of a given hole by such hole’s linear distance to the escape hole. This measurement, then, gave a greater weight to errors made far from the escape hole. (g) Mean distance to target: weighted nongoal exploration divided by non-goal hole explorations (in cm). This measurement was compared to the mean distance traveled by the animal when randomly exploring the maze. Random maze exploration was theoretically defined as being indistinct exploration of all maze holes; in such circumstances, mean distance to target was equal to 60 cm. (h) Hole exploration frequency: the number of explorations of each hole during the trial. The escape hole was numbered as hole 0 for graphical normalized representation purpose, the remaining holes being numbered 1 to 9 clockwise, and −1 to −8 counterclockwise (Fig. 5B). (i) Escape-hole exploration frequency: the number of explorations of the escape hole during the trial. (j) All-hole exploration frequency: the total number of hole explorations made by the animal throughout the trial. (k) One-sixth sector exploration frequency: the sum of the number of explorations for three consecutive holes (goal sector: −1, escape, and 1 holes. Non-goal sectors: 2, 3, and 4 holes; 5, 6, and 7 holes; 8, 9, and −8 holes; −7, −6, and −5 holes; −4, −3, and −2 holes. See Fig. 5B for hole numbering and sector limits illustration). (l) One-sixth sector random exploration level: total hole exploration frequency divided by six. In addition to the former measurements, the number and consistency of the stools passed by the subject during a given trial were also recorded as an indirect indicator of emotional arousal. Behavioral measurements per trial were grouped for making repeated-measures intra-group comparisons. Significance levels were set at p < 0.05.

3. Results Time spent to start hole exploration during a given trial significantly changed throughout the training process (Fig. 1A; one-way repeated-measures ANOVA: F(16,208) = 7.188, p < 0.001). Starting on A2, first hole latency was significantly shorter than that observed during A1 (Holm-Sidak post hoc test: p < 0.05) and became progressively shorter throughout the rest of the acquisition trials (Fig. 1A, left). Such latency remained short during the evaluation trials (EB(+) and EB(−) ), became progressively longer throughout the extinction training, and became no different from A1 values from X4 onwards (Fig. 1A, right). A comparable global pattern was found for escape latency (Fig. 1B; one-way repeated-measures ANOVA: F(16,208) = 5.247, p < 0.001). The time taken to reach the escape box became significantly shorter than that for A1 starting on A4 (Holm-Sidak post hoc test: p < 0.05) and became progressively reduced throughout acquisition (Fig. 1B, left). Such latency remained short during the evaluation trials (EB(+) and EB(−) ), became progressively longer throughout the extinction training, and became no different from A1 from X4 onwards (Fig. 1B, right). X5 escape latency returned to being significantly shorter than that for A1. The distance traveled during a given trial significantly changed throughout the training process (Fig. 2A; one-way repeatedmeasures ANOVA: F(16,208) = 23.457, p < 0.001). The A2 path length

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Fig. 1. First hole and escape latency evolution throughout training. (A) First hole latency. (B) Escape latency. All data are represented as mean + SEM. Left panels illustrate acquisition, right panels illustrate extinction. Both latencies diminished throughout acquisition, remained short 24 h later during evaluation (EB(+) and EB(−) ), and progressively returned to basal values throughout extinction. Abbreviations: A1–A8: acquisition trials; EB(+) : retention evaluation trial (with escape box); EB(−) : spatial preference evaluation trial (without escape box); asterisk indicates a significant difference compared to A1.

was significantly shorter than that observed during A1 (p < 0.05); it was no different from A1 during A3–A5, and became progressively shorter during A6–A8 (Fig. 2A, left). This distance remained short during the retention test (EB(+) ). By contrast, animals traveled a longer distance during EB(−) , X2, and X3 than during A1 (HolmSidak post hoc test: p < 0.05), although the path length progressively decreased throughout these three trials. The distance traveled in the maze from X4 onwards became no different from that recorded in A1 (Fig. 2A, right). Mean distance to target, as compared to chance, also changed significantly throughout the training process (Fig. 2B; one-way repeated-measures ANOVA: F(17,220) = 2.113, p < 0.01). Animals progressively tended to explore holes closer to the escape one during acquisition (Fig. 2B, left); mean distance to target was significantly shorter than chance exploration in A2, A4, and A6–A8 (Holm-Sidak post hoc test: p < 0.05). This distance remained shorter than chance during the evaluation trials (EB(+) and EB(−) ), became progressively closer to chance levels throughout the extinction training, and became no different from chance in X3–X4, and X6–X8 (Fig. 2B, right). Mean displacement velocity during a given trial significantly changed throughout the training process (Fig. 2C; one-way repeated-measures ANOVA: F(16,208) = 8.553, p < 0.001). Speed increased throughout the whole acquisition phase, being significantly different from A1 from A6 onwards (Holm-Sidak post hoc test: p < 0.05), remaining increased during the retention test (EB(+) ) (Fig. 2C, left). By contrast, during spatial preference evaluation trial (EB(−) ), and the rest of extinction training, animals traveled around the maze with a mean velocity that was no different from that recorded in A1 (Fig. 2C, right). Total non-goal hole exploration frequency significantly changed throughout the training process (Fig. 3A; one-way repeatedmeasures ANOVA: F(16,208) = 14.026, p < 0.001). Non-goal hole exploration became progressively reduced during the acquisition

training, became significantly lower than A1 from A6 onwards (Holm-Sidak post hoc test: p < 0.05), and remained low during the retention test (EB(+) ) (Fig. 3A, left). By contrast, non-goal exploration frequency (Fig. 3A, right) was significantly higher during EB(−) , X2, and X3 than during A1 (Holm-Sidak post hoc test: p < 0.05), and progressively diminished thereafter towards basal levels. A comparable overall pattern was found for weighted nongoal exploration (Fig. 3B; one-way repeated-measures ANOVA: F(16,208) = 8.208, p < 0.001). Weighted non-goal exploration (an indicator of how far from the escape hole a subject explored during a given trial) became reduced throughout the acquisition phase, became significantly lower than A1 from A6 onwards (Holm-Sidak post hoc test: p < 0.05), and remained short during the retention test (EB(+) ) (Fig. 3B, left). By contrast, distance to the escape hole increased significantly during the spatial preference evaluation trial (EB(−) ) and X2 (Fig. 3B, right), compared to A1, and progressively diminished towards basal levels. Hole exploration during extinction training was compared to basal exploration (hole exploration pattern recorded during A1, when the subjects randomly explored the maze). A two-way repeated-measures ANOVA, followed by a Holm-Sidak post hoc test, showed that hole exploration pattern significantly changed throughout the extinction (Fig. 4); mean exploration per trial became reduced as extinction training went on (trial factor: F(8,1768) = 26.318, p < 0.001); mean exploration of each hole throughout extinction was not homogeneous (hole factor: F(17,1768) = 35.768, p < 0.001) and there was a significant interaction of trial and hole factors (trial x hole: F(136,1768) = 5.946, p < 0.001). The subjects displayed a significant preference for exploring the escape and its adjacent holes during EB(−) , X2, X3, and X5 (p < 0.05), such preference became steadily reduced throughout the extinction training and reached basal levels in X4, and X6–X8 (Fig. 4A). Escape-hole exploration frequency was also significantly higher than basal exploration during EB(−) , X2, and X3 (p < 0.05); this fre-

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Fig. 2. Distance and velocity evolution throughout training. (A) Path length. (B) Mean distance to target. (C) Mean velocity. All data are represented as mean + SEM. Left panels illustrate acquisition, right panels illustrate extinction. Both total and mean distances became reduced throughout acquisition and remained short 24 h later during the retention test; distance traveled in the maze during extinction progressively returned to basal values. On the contrary, mean velocity increased during acquisition and remained increased during the retention test; it was no different to that recorded during A1 during extinction. Abbreviations: as in Fig. 1; asterisk indicates a significant difference compared to basal levels (values in A1 for path length and mean velocity, and random level represented by the dotted line for mean distance to target).

quency became continuously reduced throughout the extinction training and reached basal levels during X4, and X6–X8 (Fig. 4B). The all-holes exploration frequency was significantly higher than control (A1) during EB(−) , X2, and X3 (p < 0.05), and became reduced to basal levels throughout the extinction (Fig. 4C). Goal sector (−1, 0, and 1 holes) exploration was compared to the mean exploration of non-goal sectors (five sectors grouping three consecutive holes each) throughout the extinction trials. A two-way repeated-measures ANOVA followed by a Holm-Sidak post hoc test, showed that the sector exploration pattern significantly changed throughout extinction (Fig. 5C); mean exploration per trial diminished as extinction training went on (trial factor: F(7,223) = 45.942, p < 0.001), mean exploration of goal and non-goal sectors throughout the whole extinction was not homogeneous (hole factor: F(1,223) = 76.023, p < 0.001), and there was a significant interaction of trial and sector factors (trial × hole: F(7,223) = 30.243, p < 0.001). Goal sector exploration frequency decreased significantly throughout the extinction trials (p < 0.001; Fig. 5C, gray bars); by contrast, there were no significant changes in mean non-

goal sector exploration during the extinction trials (Fig. 5C, white bars). Goal sector exploration was significantly higher than mean non-goal sector exploration during EB(−) to X5 (p < 0.001). Such preference became marginal during X6 and X8 (p < 0.05), and disappeared during X7 (p = 0.162). The number of stools passed during a given trial changed significantly throughout training process (one-way repeated-measures ANOVA: F(16,208) = 4.607, p < 0.001). Animals passed less stools during A2–A8 than during A1 (Holm-Sidak post hoc test: p < 0.001). The number of stools passed during EB(+) was no different from that during A1 and became significantly lower during EB(−) (Holm-Sidak post hoc test: p < 0.01). By contrast with that found during acquisition training, the number of stools increased transiently from X2 to X5, becoming no different from A1, and decreased significantly during the last three extinction trials (Holm-Sidak post hoc test: p < 0.01). All subjects’ stools were solid throughout the acquisition, evaluation, and the last extinction trials; however, from X2 to X6, up to half of the subjects passed loose stools (2 (16) = 65.523, p < 0.001).

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Fig. 3. Non-goal hole exploration evolution throughout training. (A) Non-goal hole exploration. (B) Weighted non-goal exploration. All data are represented as mean + SEM. Left panels illustrate acquisition, right panels illustrate extinction. Exploration of holes different to the escape one became reduced during acquisition and remained so 24 h later during the retention test; exploration was higher during extinction than that displayed in A1 at the beginning of the training and then became reduced until it reached basal values. Abbreviations: as in Fig. 1; asterisk indicates a significant difference compared to A1.

Fig. 4. Hole exploration and preference throughout extinction. (A) Hole exploration frequency (escape-hole exploration is represented by a gray bar and is numbered as hole 0, the remaining holes are numbered 1 to 9 clockwise, and −1 to −8 counterclockwise, see Fig. 5B). (B) Escape-hole exploration frequency. (C) All-hole exploration frequency. All data are represented as mean + SEM. All-hole exploration measurements were significantly above basal level during EB(−) , X2, and X3; although they became steadily reduced throughout extinction, they reached basal level thereafter (except by a transient rebound of goal and adjacent holes preference during X5). Abbreviations: as in Fig. 1; an asterisk indicates a significant difference compared to A1.

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Fig. 5. Sector exploration and sector preference during extinction. (A) Perspective view, from above, of the experimental room showing maze position and stimuli used as spatial cues. (B) Top view of the maze illustrating hole numbering and one-sixth sector division (dotted lines). Goal hole, labeled as 0, is highlighted in dark gray, while goal sector is highlighted in light gray. (C) Sector exploration frequency. Goal sector exploration frequency (gray bars) became reduced throughout the whole extinction training, whilst non-goal sector exploration (white bars) remained unchanged. Goal sector exploration was significantly higher than mean non-goal sector exploration during all trials, except for X7. Gray line, mean random exploration; black lines, mean random exploration 95% confidence interval limits. Difference between goal and mean non-goal sector exploration for a given trial: *p < 0.05; **p < 0.001. Difference in goal sector exploration in a given trial respecting exploration during EB(−) : § p < 0.001. All data are represented as mean + SEM. Abbreviations: as in Fig. 1.

4. Discussion The animals’ performance during acquisition changed progressively throughout the training in the present experiment, gaining in efficiency and accuracy to find the escape hole. The time taken to start hole exploration and to enter the escape box became reduced, exploration of holes different to the escape one (especially those farther from the target) decreased, the length of the path became shorter, and mean locomotion speed increased during this training phase. All these changes reached their maximum during the last three acquisition trials. Stool number and consistency suggest that slight emotional arousal occurred during the first acquisition trial which then waned throughout the remaining acquisition trials. Taken together, such behavioral modifications indicate that the animals completely acquired the spatial task through the training. Furthermore, such spatial learning was retained after 24 h as indicated by the lack of significant differences in performance between the retention test (EB(+) ) and the last acquisition trial (A8) and by the clear-cut preference for the escape hole and the goal sector during

the preference test (EB(−) ). These data indicate that this abbreviated Barnes maze acquisition protocol using a single session of eight reinforced trials with a 4-min inter-trial interval was enough to induce spatial learning and memory, as has been previously reported by our group (Claro et al., 2008; Troncoso et al., 2010). This abbreviated Barnes maze training version contrasted with the typical protocol used in the literature, which usually includes several days of training (4–5), fewer trials per day (2–4) and longer intertrial intervals (15 min) (Barnes, 1979; McLay et al., 1998; Williams et al., 2003; Harloe et al., 2008). The efficiency of this short version provides an advantage for simplifying the evaluation of behavioral and pharmacological interventions during different phases of spatial learning and memory. Moreover, a single acquisition session avoids the inconvenience of repeated treatments throughout training. As a matter of fact, repeated drug administration effects could become distorted by sensitization or desensitization, while repeated behavioral treatments could induce undesired associative or non-associative learning effects. On the other hand, spatial learning and memory codification may depend on the way learning was acquired: either massed or spaced (Commins et al., 2003). The effects of treatment should thus be established for both, massed and spaced learning. The protocol presented here, as well as a new one-day version of the Morris water maze (Zheng et al., 2009), provide the opportunity for exploring the effects of behavioral or pharmacological treatments on massed spatial training. The animals’ performance also changed during extinction. Since the experimental conditions rendered the animals unable to avoid the aversive situation, the behavioral pattern they had acquired became progressively reorganized; the time to start exploration and reach the escape hole progressively increased, hole exploration frequency transiently increased above basal levels during the first three extinction trials and the exploration path included holes placed farther from the escape one, the distance traveled during the first extinction trials was longer than during the retention test and thereafter diminished steadily until basal levels were reached, and mean locomotion speed dropped to basal levels and remained so throughout extinction. These changes mainly occurred during the first three extinction trials; however, a transient but slight relapse was observed during the fifth extinction trial. Prolonged path length and increased hole exploration displayed at the beginning of extinction can most likely be explained by the fact that trials no longer finished when the animal found the escape hole, since there was no escape box, thereby inducing longer trial duration. Besides, given that the animals had learned to solve the task by seeking the escape hole, they displayed an intense exploratory pattern during evaluation that revolved around the escape and neighboring holes. The change of these indicators during the first extinction trials should not thus necessarily be interpreted as impaired spatial memory retrieval. Hole by hole and sector preference progressively changed throughout extinction. The escape hole and its adjacent holes were explored above chance level during the first three extinction trials; such hole preference disappeared thereafter, reappearing transiently during the fifth extinction trial. By contrast, when joint exploration of the escape hole and those around it were considered, the preference for the goal sector decreased significantly but persisted throughout extinction training; however, such preference became marginal by the end of the training, and was not manifest during the seventh extinction trial. Conversely, mean non-goal sector preference (which was significantly below chance level at the beginning) caught up to this level during the rest of the extinction trials. The increase in the number of stools passed, as well as the occurrence of loose stools in up to half of the subjects during most extinction trials, indicates that the unexpected change in environmental contingencies elicited a moderate emotional arousal that could have motivated some of the observed behavioral changes. In

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fact, when behavioral measurements indicated that the subjects’ had successfully extinguished the spatial preference, the changes in stool number and consistency also receded, suggesting that emotional arousal waned almost in parallel. Taken together, these findings indicate that animals can reorganize recently learned behavioral patterns in response to changes in environmental demands throughout extinction training. However, such reorganization does not imply unlearning of acquired spatial navigation strategy, as evidenced by the reappearance of short latency to reach the escape hole, short mean distance to target, and escape-hole exploration preference during the fifth extinction trial. Furthermore, goal sector preference (which was not displayed during the seventh extinction trial) slightly reappeared during the last trial. Such evidence suggests (as has been proposed) that extinction of spatial learning preference does not imply a progressive loss of acquired association (Lattal et al., 2003, 2004; Suzuki et al., 2004). Extinction of acquired spatial preference in mice has been described using the Morris water maze (Lattal et al., 2003) and appetitive and aversive versions of the Barnes circular maze (Harloe et al., 2008). Since the platform (in the water maze) or the escape box (in Barnes maze) were absent during the extinction trials in these experiments then the animals reduced the time they spent exploring the goal sector as the training sessions passed by. However, this reduction was not in any case a monotonic function of training length; in fact, there was wide intra-group variability, and animals partially recovered their preference for the goal sector even after several training sessions. All these previously reported extinction-associated features were also observed in the present work, in spite of the brevity of the Barnes maze training protocol used. The parameters used for analyzing behavioral changes during acquisition and extinction in the Barnes maze frequently include motor skill-related measurements (total or primary latency, total or primary path length, and mean speed) as well as spatial preference-related measurements (total or primary errors, number of pokes in each hole, and mean angular error) (Barnes, 1979; McLay et al., 1998; Sunyer et al., 2007). Dissociation in the rate of behavioral change during extinction was found in this work, depending on the type of behavioral indicator considered; whilst rapid changes were observed in motor skill-related measurements, spatial preference-related measurements changed at a slower pace. Such dissociated evolution indicates that even though maze exploration rapidly became reduced, acquired spatial preference was resilient to extinction training. Preference-related parameters could therefore be considered as being more sensitive for describing spatial learning extinction. However, we believe that both types of measurement are complementary and necessary to thoroughly analyze the effects of behavioral or pharmacological interventions on extinction. On the one hand, besides describing spatial extinction, it is also desirable to rule-out possible non-cognitive effects of experimental interventions using this paradigm. On the other hand, if diverse memory traces were involved in spatial memory tasks, then they would have differential susceptibility to different experimental manipulations, and, therefore, using different measurements would increase the chance of finding experimentally induced derailments in their evolution throughout the whole training process. Furthermore, the indicators used here for evaluating spatial preference allow high precision in determining the animals’ exploration accuracy, given that the maze can be dissected into narrow areas. In fact, the navigational precision indicated by one-sixth sector analysis is 40◦ . Moreover, if hole by hole preference is considered, then navigational precision could be further increased (20◦ ). The aforementioned methodological issues, taken jointly with the present results as well as unpublished data from the Behavioral Neurophysiology Laboratory, lead us to suggest that the abbreviated Barnes maze acquisition, evaluation, and extinc-

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