Behavioural Brain Research 292 (2015) 44–49
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Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
The recognition of a novel-object in a novel context leads to hippocampal and parahippocampal c-Fos involvement Arias N. a,c,∗ , Méndez M. b,c , Arias JL. b,c a b c
Department of Experimental Psychology, University of Cambridge, Downing Street, Cambridge CB2 3EB, UK Laboratorio de Neurociencias, Departamento de Psicología, Universidad de Oviedo, Plaza Feijoo s/n, 33003 Oviedo, Spain INEUROPA, Instituto de Neurociencias del Principado de Asturias, Spain
h i g h l i g h t s • • • •
Role of context change in a one-trial-novel object recognition task was studied. Two conditions:OR-NORMAL(sample and probe same context):OR-CONTEXT (different). OR-NORMAL greater exploration of objects than OR-CONTEXT. c-Fos involves hippocampus, entorhinal and temporal association cortices in the task.
a r t i c l e
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Article history: Received 4 March 2015 Received in revised form 4 May 2015 Accepted 6 June 2015 Available online 10 June 2015 Keywords: Novel object-place recognition task C-fos Hippocampus Entorhinal cortex Temporal association cortex Context
a b s t r a c t Contextual memory implies recognition based on the association between past and present events experienced. It is important for daily functioning and dysfunctional in many neuropsychological disturbances. The network related to this memory is still open for debate, even though it has been associated with medial temporal lobe regions, including the perirhinal, entorhinal and temporal association cortices, as well as the hippocampus and prefrontal cortex. Our work tries to elucidate whether a change in the context, such as differences in the amount of stimuli presented on the walls and floor of an open field during object exploration, affects the recognition of an object that has been experienced before, and whether this context manipulation could be linked to changes in c-Fos expression. For this purpose, we used a one-trial novel-object recognition task. The animals were divided into two different experimental conditions; in the OR-NORMAL group, the sample and probe test were performed in the same context. However, in the OR-CONTEXT group, the probe test was performed in a different context. Our results showed that the OR-NORMAL group presented a greater exploration of objects than the OR-CONTEXT group. However, both groups presented significant exploration of the novel object. To label the brain regions involved in novel-object recognition under these conditions, we marked the expression of c-Fos protein. Results suggest that a neural circuit that includes the hippocampus, entorhinal and temporal association cortices is involved in the recognition of the novel-object in a novel context. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Episodic memory is characterized by the ability to recollect past experiences [1]. For this function, it is necessary to remember not only previously encountered information, but also where, when, and how the knowledge about a certain fact was acquired, and what features or attributes were related to the learning situation [2]. Thus, the term contextual memory is related to the spatiotemporal
∗ Corresponding author at: Department of Experimental Psychology, University of Cambridge, Downing Street, Cambridge CB2 3EB, UK. E-mail address: [email protected] (N. Arias). http://dx.doi.org/10.1016/j.bbr.2015.06.012 0166-4328/© 2015 Elsevier B.V. All rights reserved.
and perceptual attributes that facilitate the encoding and retrieval of the episodic memory [3]. Contextual memory is important not only due to its involvement in daily functioning [4], but also because it is altered in euthymic bipolar patients [5], cirrhotic disorder [6] and drug-abuse related syndromes [7]. This type of memory implies recognition of the association between past and present events experienced. This recognition has been associated with a network of medial temporal lobe regions that includes the perirhinal, entorhinal and temporal associative cortex, as well as the hippocampus and prefrontal cortex [8–12]. Several studies have shown that multiple sensory systems related to stimulus recognition activate the perirhinal cortex
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[13–19], supporting the idea that associations between individual features that represent a stimulus as a whole (within-stimulus association of components) are represented in the perirhinal cortex. On the other hand, complex associations between stimuli and environment (context) may be represented in the hippocampal formation [13]. The association of an object with a location is known to play an important role in establishing perceptual continuity within the dynamic environments of everyday experiences [20], and it plays an essential role in the formation of coherent episodic memory representations. However, the differential implications of the brain structures under context changes, as an integrated representation of the events or separate associations between them, have not yet been explored. The role of context change in this type of memory was studied by using a novel-object recognition task in which sample and retention were performed in the same context (OR-NORMAL group) and a novel-object recognition task in which sample and retention were performed in different contexts (OR-CONTEXT group). We hypothesized that: (1) The change in the context would interfere with the exploration of the new object; and (2) the change in the context would reveal a differential brain activation pattern during recognition. To label the brain regions involved in this task, we marked the expression of c-Fos protein. Studying c-Fos can provide information about the neuronal plasticity required for memory processes [21]. The expression of c-Fos protein is induced after learning, and it is indicative of a change in neuronal activity [21–23]. This technique allows the simultaneous examination of the activity of populations of neurons in multiple brain regions [24]. Because the hippocampus does not act as an isolated structure in these learning tasks, other cerebral regions involved were also studied. These regions were the prefrontal cortex and adjacent cortical areas within the temporal lobe, such as the temporal association, entorhinal and perirhinal cortices, as ablation, electrophysiological lesions or dysfunctions suggest that these structures might be involved in this task [25–28]. 2. Material and methods 2.1. Animals We used 20 male Wistar rats from the vivarium of the University of Oviedo. They weighed 250–270 g at the beginning of the study. The rats were housed in groups of three to five, three weeks prior to the beginning of the experiments, and maintained under standard laboratory conditions (20–22 ◦ C, 65–70% relative humidity and a 12 h light/dark cycle (08.00–20.00/20.00–08.00)). Food and water were available ad libitum throughout the experiments, and sessions were performed during the light phase, between 9:00 a.m. and 13:00 p.m. All procedures were carried out according to the European Parliament and the Council of the European Union 2010/63/UE, and they were approved by the Oviedo University committee for animal studies. The animals were distributed in two groups on a one-trial novel-object recognition task (OR-NORMAL group, n = 10 and OR-CONTEXT group, n = 10). 2.2. Apparatus Object exploration was assessed in a square open field (40 × 40 × 33 (height) cm) with an open roof, placed in a rectangular room. The room was illuminated by two diffuse white lights placed at the sides of the room, providing an illumination density of approximately 50 lux. During the habituation of both groups, the walls and ground of the open field were covered by gray fiberglass. During the sample and probe tests, the walls consisted of patterns of dark and white plastic bars (2.5 cm width) placed vertically or
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horizontally, depending on the experimental condition. At the same time, the ground could change from smooth gray plastic to rough black plastic. A video camera, connected to a video recorder, was mounted above the field to store the sample and test trials on video files for off-line analysis. After each trial, the apparatus was thoroughly cleaned with a 75% ethanol solution. Pairs of three different ceramic objects were used. For habituation, we used two identical objects (2.5 × 2.5 × 8 cm); whereas for memory testing, we used two different pairs of objects, Objects A (3 × 3 × 6 cm) and Objects B (3.5 × 3.5 × 6.5 cm), which were made of ceramics and glass. In all the trials, the objects were placed in the center of the open field with a distance of 10.5 cm between them. All the objects were attached to the ground of the open field in order to avoid their displacement during the task. After each trial, the objects were thoroughly cleaned with a 75% ethanol solution to remove odor cues. 2.3. Novel-object recognition task 2.3.1. Habituation Prior to testing, all the animals were handled on four consecutive days for 10 min/day and habituated to the empty arena on three consecutive days. On the first day of habituation, groups of between three and five rats were placed together in the open field and allowed to explore it for 6 min without objects. On the following day, rats were given another 6-min session for exploration, but this time only one rat at a time was placed in the open field. On the third and last habituation day, rats received three daily sessions lasting 6 min each. There was a 20 min inter-trial interval when the two habituation objects were placed opposite each other, 10 cm apart, on two of the walls of the open field. This time, the rats were also brought individually to the testing room and placed in the open field facing the center of the wall where no objects were located. The habituation objects were not used in the assessment of memory for object recognition. 2.3.2. Sample and probe tests In the test phase, rats were first placed individually in the center of the open field. Two copies of an object were located in opposite corners, 7 cm from the walls of the open field. Four minutes of exploration were recorded in this sample trial. After a delay of 50 min, rats received a second 4 min probe test trial where a copy of the original or familiar object and a second novel object were used. For the OR-NORMAL group, the sample and probe test were performed in the same context, where the walls were covered by vertical black and white bars, and smooth gray plastic was placed in the floor. However, the OR-CONTEXT group performed the probe test in a different context, that is, an open field where the black and white bars of the walls were horizontal, and the floor was rough and black (Fig. 1). 2.4. c-Fos immunocytochemistry Coronal sections (30 m) of the brain were cut at −20 ◦ C in a cryostat (Leica CM1900, Germany). The sections were mounted on gelatinized slides post-fixed in buffered 4% paraformaldehyde (0.1 M, pH 7.4) for 30 min and rinsed in phosphate-buffered saline (PBS) (0.01 M, pH 7.4). They were subsequently incubated for 15 min with 3% hydrogen peroxidase in PBS to remove endogenous peroxidase activity, and then washed twice in PBS. After blocking with PBS solution containing 10% Triton X-100 (PBS-T) (Sigma, USA) and 3% bovine serum albumin for 30 min, sections were incubated with a rabbit polyclonal anti-c-Fos solution (1:10,000) (Santa Cruz Biotech, sc-52, USA) diluted in PBS-T for 24 h at 4 ◦ C in a humid chamber. Slides were then washed three times with PBS and incubated in a goat anti-rabbit biotinylated IgG secondary antibody
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Fig. 1. Schematic illustration of the one-trial object-place recognition task. Two experiments were performed using the same or different contexts. The figure shows the different experimental conditions during sample and probe tests in both experimental groups.
(Pierce, USA; diluted 1:200 in incubating solution) for 2 h at room temperature. They were washed three times in PBS and reacted with avidin–biotin peroxidase complex (Vectastain ABC Ultrasensitive Elite Kit, Pierce) for 1 h. After two washes in PBS, the reaction was visualized, treating the sections for about 3 min in a commercial nickel-cobalt intensified diaminobenzidine kit (Pierce). The reaction was finalized by washing the sections twice in PBS. Slides were then dehydrated through a series of graded alcohols cleared with xylene and cover-slipped with Entellan (Merck, USA) for microscopic observation. All immunocytochemistry procedures included sections that served as controls, where the primary antibody was not added. Slides containing sections of a specific brain region were stained at the same time. Slides were coded so that the investigator who performed the entire analysis would have no knowledge about the group of individual subjects. The total number of c-Fos positive nuclei was quantified in three alternate sections 30 m apart. Quantification was done by systematically sampling each of the regions selected, using a microscope (Olympus BH-2, Japan) attached to an analog camera (Sony XC-77, Japan) and a TV monitor (300× total magnification). c-Fos positive nuclei were defined based on homogenous gray–black stained elements with a well-defined border. Finally, the mean count for three sections was calculated for each subject and region (number of positive nuclei/150 m2 ). The regions of interest were anatomically defined according to Paxinos and Watson’s atlas [29]. The regions of interest and their distance in mm from bregma was: +3.20 mm for the infralimbic cortex (IL), the prelimbic cortex (PL), the cingulate cortex (CG); −3.84 mm for the CA1, CA3 and the dentate gyrus (DG) subfields of the dorsal hippocampus; −5.04 mm for the perirhinal (Prh), the entorhinal cortex (Ent) and the temporal association cortex (TeA). 2.5. Statistical analysis 2.5.1. Novel-object recognition data Exploration of an object by the rat was defined as directing the nose toward the object at a distance of less than 2 cm and actively exploring it. Sitting and turning around on the object were not considered exploratory behavior. For each rat, the time spent exploring
the objects in both sample sessions and the test session were scored off-line from video files, using stopwatches. Two researchers who were blind to the groups coded each video. Additionally, a grid of 10 × 10 cm squares was virtually superimposed on the arena floor to record exploratory behaviors during the probe phase as an index of context exploration. “Ambulation” was defined as the crossing of at least 1 floor grid line within a 3 s period, and “rearing” was defined as lifting the forelimbs and sitting back on the haunches [30]. Context exploration data, ambulation and rearing of the groups were compared with a t-test. A number of measurements were taken in order to examine exploration and discrimination of the objects. The proportion of exploration in the sample and test phases was considered as e1 and e2, respectively, and it was calculated by dividing the exploration of all objects in the phase divided by the total time available for exploration. Two measures of discrimination were taken: discrimination index (d1) and discrimination ratio (d2) [31]. The discrimination index, d1, consisted of the difference in time spent exploring the two objects in the probe or test phase, the novel-object minus the familiar object. The discrimination ratio, d2, was also calculated in the test phase. This is the difference in the exploration time of the two objects divided by the total time spent exploring the objects, that is, d1/time spent exploring both objects. Exploration in the sample and the test phase (e1 and e2) was compared between groups with a t-test. Furthermore, paired t-tests were performed on each experimental group in order to appreciate possible differences in the time spent exploring the objects in the sample and probe phases (e1 and e2). Discrimination data, d1 and d2, of the groups were compared with a t-test. In addition, t-tests were performed on d1 and d2 to determine whether the mean scores differed from zero (chance performance indicating equal exploration of the two objects). This would indicate that the groups could successfully discriminate between the objects. A non-parametric Mann–Whitney U test (U) for independent samples was carried out when normality or equal group variances failed. 2.5.2. c-Fos data To determine c-Fos activity, 12 subjects were analyzed (ORNORMAL n = 6, OR-CONTEXT n = 6). For statistical analysis, cell
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Fig. 2. Performance of the experimental groups on the object-place recognition task. Discrimination index and discrimination ratio (d1 and d2) for NORMAL and CONTEXT groups.
counts from the three selected sections for a given brain region in each animal were averaged, and the mean was used for statistical analysis. Student t-tests for independent samples were used to assess whether the number of c-Fos positive nuclei were different between groups in each brain region. Moreover, a non-parametric Mann–Whitney U test (U) for independent samples was carried out when normality or equal group variances failed.
3. Results 3.1. Behavioral results The results showed significant group differences in the exploration index e2: t18 = 2208, P = 0.040, where the OR-NORMAL group employed more time than the OR-CONTEXT group in exploring the objects. However, no differences were found in the exploration index e1: U = 128.000, n1 = 10, n2 = 10, P = 0.089). There were no significant differences in exploration between the sample phase and the test phase for the OR-NORMAL group (e1 = 0.164 ± 0.021, e2 = 0.264 ± 0.0536: t18 = −1.731, P = 0.101) or for the OR-CONTEXT group (e1 = 0.130 ± 0.0112, e2 = 0.137 ± 0.0207: t18 = −0.277, P = 0.785). The results showed that both groups were able to discriminate between a previously-encountered object and a novel object. The d1 and d2 means were both significantly higher than zero for both the OR-NORMAL group (d1: t18 = 4.421, P < 0.001 d2: t18 = 14.823, P < 0.001) and the OR-CONTEXT group (d1: t18 = 4.278, P < 0.001; d2: t5 = 5.722, P < 0.001). Significant differences were found between groups in d1 (U = 144.000, n1 = 10, n2 = 10, P = 0.004) and in d2 (t18 = 3.399, P = 0.003; Fig. 2). Additionally, context exploratory behaviors during the probe phase showed differences between groups. Ambulation and rearing were higher in the OR-CONTEXT group than in the OR-NORMAL group (t18 = −9.241, P < 0.001 and t18 = −6.059, P < 0.001, respectively; Fig. 3).
3.2. c-Fos results Differences were found between the OR-NORMAL and ORCONTEXT groups in the CA1 (t10 = −4.192, P = 0.002), DG (U = 21.000, n1 = 6, n2 = 6, P = 0.002), Ent (U = 25.000, n1 = 6, n2 = 6, P = 0.026) and TeA (t10 = − 7.167, P < 0.001). In these regions, the number of c-Fos positive nuclei was higher in the OR-CONTEXT group compared to the OR-NORMAL group. No differences between groups were found in Cg (t10 = −1.436, P = 0.182), PL (U = 32.000, n1 = 6, n2 = 6, P = 0.310), IL (U = 30.000, n1 = 6, n2 = 6, P = 0.180), CA3 (t10 = − 1.478, P = 0.170) or Prh (t10 = 0.643, P = 0.535; Fig. 4).
Fig. 3. Bar graphs showing the time spent (mean ± SEM) by experimental groups ambulating and rearing during the 4 min object recognition task probe phase. Significant differences were found in the time devoted to each behavior (*p < 0.05). OR-CONTEXT group spent significantly more time ambulating and rearing than OR-NORMAL group.
Fig. 4. Fos counts. Number of c-Fos positive nuclei in OR-NORMAL and OR-CONTEXT groups (mean ± SEM) in the structures where significant differences were found (*p < 0.05). The number of c-Fos was significantly higher in the OR-CONTEXT group compared to the OR-NORMAL group in the hippocampus, entorhinal and temporal association cortices.
4. Discussion Our study revealed that a change in the context where an object is presented can affect novel-object exploration, interfering in object recognition. Behavioral data revealed that the new context affects total object exploration. Animals tend to explore the
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objects more when they are presented in a familiar context. Our data showed the differential contribution of the hippocampal subfields and extra-hippocampal structures to novel-object in novel context recognition. Regarding behavior, the group that recognizes the object in a familiar context, OR-NORMAL, spends more time exploring both objects in the test phase than the group that performs recognition in a novel context, OR-CONTEXT. These results indicate that the animals spend more time exploring the objects when they are in their usual context; therefore, context modification interferes with the time spent on object exploration. However, to confirm whether the groups exhibit a tendency toward further exploration, we proceeded to analyze both indexes in each of the groups, comparing the time spent exploring the objects in both the sample and test phases within each group. We can see that within each group the exploration indexes (e1 and e2) did not differ. However, during the test phase, while in both cases an object is new, the NORMAL group spent even more time exploring the new object (e2). Moreover, these results are supported by the ambulatory and rearing behavior, which increased in the CONTEXT group. This result suggests that the change in context can influence the exploratory behavior of animals, so that they spend more time exploring a new environment that is unfamiliar to them, while paying less attention to specific objects. In addition, the d1 and d2 indexes indicate that the OR-NORMAL group spent more time exploring the novel-object compared to the OR-CONTEXT group. These data show that the animals spend more time exploring the objects when they are in their habitual context. Thus, a change in the context interferes with the time spent exploring novel objects. The novelty judgment is supported by several regions. Traditionally, ablation or electrophysiological evidence has suggested that the hippocampal formation, the entorhinal cortex, and adjacent cortical areas within the temporal lobe, such as the perirhinal cortex, might be involved in recognition memory [25]. Detailed studies using bilateral hippocampal lesions revealed significantly impaired memory for object-place recognition [32], while many object-recognition studies found no apparent effects of hippocampal lesions [33]. Other studies related to the recognition of familiar spatial rearrangements showed significant increases in Fos levels in CA1, and significant decreases in the dentate gyrus (DG) [34], highlighting the long-standing debate about hippocampal involvement in recognition memory. The entorhinal cortex, which is the major cortical input to the hippocampus [35], is involved in the discrimination of familiar objects in certain places, but not in the recognition of either novel objects or novel places [36]. For this reason, the involvement of the hippocampus and entorhinal cortex in novel-object in novelcontext recognition was expected. Our results link novel-object in novel-context recognition to an increase in c-Fos immunoreactivity in the CA1, DG, entorhinal cortex, and temporal association cortex. How can this early gene expression in the RO-CONTEXT group be explained? The increased Fos levels in CA1 and the DG could be related to the mediation of these structures in consolidating new information and representing metric spatial representations [37]. Our data could suggest the involvement of these hippocampal regions in combining the associative recognition of objects with their locations and local contexts, information that is not combined until reaching the hippocampus and that could occur in parallel with the storage of other features of the process. The entorhinal cortex has been involved in binding objects with the contexts in which they are experienced to form a representation of a new contextualized environment [38]. Moreover, the entorhinal cortex is the major cortical input to the hippocampus [35], where increases in the DG and CA1 associated with novelty have been reported [39]. Novel stimuli processing has been associated
with activity in the direct pathway from the entorhinal cortex to the dentate gyrus [39]. The increased Fos levels in the entorhinal cortex could suggest that contextual features of the environment are being integrated with object identity in this region. However, previous studies [40] showed that exploration of a novel environment and performance on a well-learned odor discrimination task elicited a common pattern of c-fos expression in the hippocampus that involved a higher expression in CA1, followed by CA3 and the DG. Along these lines, our results showing a higher implication of the DG, followed by CA1 and the absence of CA3 involvement, may be more related to the exploration of a novel stimuli or the binding between novel-object under novel-context exposure. However, further experiments are needed to understand the role of the DG. The role of the temporal association cortex (TeA) has been paired with the perirhinal cortex, due to its anatomical position adjacent to the perirhinal cortex. Furthermore, electrophysiological recordings in both monkey and rat brains have revealed units in both regions that typically reduce the response when the animals are confronted with familiar stimuli [41]. This raises the possibility that both TeA and the perirhinal cortex may have a role to play in information storage for object-recognition memory [42]. Our results suggest a role that is probably more related to information storage under a novel context condition. Additionally, it is important to highlight the fact that no increases in c-Fos expression were found in the prefrontal cortex. Studies using disconnection approaches [43] revealed that the prefrontal cortex plays a role in recognition memory in cases requiring the integration of the object with spatial location information received from other neural regions. In our study, in the OR-CONTEXT condition, novelty comes from a novel object that should be recognized in a novel context, mainly requiring a retrieval of “what” is presented. The lack of Fos expression in the prefrontal cortex does not necessarily refute the role of this structure in the integration of object and space information [44]. In conclusion, our work revealed that there was interference caused by changing environmental conditions in a novel-object recognition task, suggesting that a neural circuit that includes the hippocampus and entorhinal and temporal association cortices is involved in recognition in a novel context. These results could highlight how a dysfunction in this network may affect contextual memory, which is important not only for daily functioning, but also because it is altered in many disorders.
Acknowledgements This research was supported by Foundation Alfonso Martin Escudero Grants to NA and Project Grants of the Spanish Ministry of Economy and Competitiveness: PSI 2010-19348 and PSI 201345924-P. Authors are grateful to Ldo. Jorge Arias for his technical support and Cynthia Depoy for the English review.
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Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
The recognition of a novel-object in a novel context leads to hippocampal and parahippocampal c-Fos involvement Arias N. a,c,∗ , Méndez M. b,c , Arias JL. b,c a b c
Department of Experimental Psychology, University of Cambridge, Downing Street, Cambridge CB2 3EB, UK Laboratorio de Neurociencias, Departamento de Psicología, Universidad de Oviedo, Plaza Feijoo s/n, 33003 Oviedo, Spain INEUROPA, Instituto de Neurociencias del Principado de Asturias, Spain
h i g h l i g h t s • • • •
Role of context change in a one-trial-novel object recognition task was studied. Two conditions:OR-NORMAL(sample and probe same context):OR-CONTEXT (different). OR-NORMAL greater exploration of objects than OR-CONTEXT. c-Fos involves hippocampus, entorhinal and temporal association cortices in the task.
a r t i c l e
i n f o
Article history: Received 4 March 2015 Received in revised form 4 May 2015 Accepted 6 June 2015 Available online 10 June 2015 Keywords: Novel object-place recognition task C-fos Hippocampus Entorhinal cortex Temporal association cortex Context
a b s t r a c t Contextual memory implies recognition based on the association between past and present events experienced. It is important for daily functioning and dysfunctional in many neuropsychological disturbances. The network related to this memory is still open for debate, even though it has been associated with medial temporal lobe regions, including the perirhinal, entorhinal and temporal association cortices, as well as the hippocampus and prefrontal cortex. Our work tries to elucidate whether a change in the context, such as differences in the amount of stimuli presented on the walls and floor of an open field during object exploration, affects the recognition of an object that has been experienced before, and whether this context manipulation could be linked to changes in c-Fos expression. For this purpose, we used a one-trial novel-object recognition task. The animals were divided into two different experimental conditions; in the OR-NORMAL group, the sample and probe test were performed in the same context. However, in the OR-CONTEXT group, the probe test was performed in a different context. Our results showed that the OR-NORMAL group presented a greater exploration of objects than the OR-CONTEXT group. However, both groups presented significant exploration of the novel object. To label the brain regions involved in novel-object recognition under these conditions, we marked the expression of c-Fos protein. Results suggest that a neural circuit that includes the hippocampus, entorhinal and temporal association cortices is involved in the recognition of the novel-object in a novel context. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Episodic memory is characterized by the ability to recollect past experiences [1]. For this function, it is necessary to remember not only previously encountered information, but also where, when, and how the knowledge about a certain fact was acquired, and what features or attributes were related to the learning situation [2]. Thus, the term contextual memory is related to the spatiotemporal
∗ Corresponding author at: Department of Experimental Psychology, University of Cambridge, Downing Street, Cambridge CB2 3EB, UK. E-mail address: [email protected] (N. Arias). http://dx.doi.org/10.1016/j.bbr.2015.06.012 0166-4328/© 2015 Elsevier B.V. All rights reserved.
and perceptual attributes that facilitate the encoding and retrieval of the episodic memory [3]. Contextual memory is important not only due to its involvement in daily functioning [4], but also because it is altered in euthymic bipolar patients [5], cirrhotic disorder [6] and drug-abuse related syndromes [7]. This type of memory implies recognition of the association between past and present events experienced. This recognition has been associated with a network of medial temporal lobe regions that includes the perirhinal, entorhinal and temporal associative cortex, as well as the hippocampus and prefrontal cortex [8–12]. Several studies have shown that multiple sensory systems related to stimulus recognition activate the perirhinal cortex
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[13–19], supporting the idea that associations between individual features that represent a stimulus as a whole (within-stimulus association of components) are represented in the perirhinal cortex. On the other hand, complex associations between stimuli and environment (context) may be represented in the hippocampal formation [13]. The association of an object with a location is known to play an important role in establishing perceptual continuity within the dynamic environments of everyday experiences [20], and it plays an essential role in the formation of coherent episodic memory representations. However, the differential implications of the brain structures under context changes, as an integrated representation of the events or separate associations between them, have not yet been explored. The role of context change in this type of memory was studied by using a novel-object recognition task in which sample and retention were performed in the same context (OR-NORMAL group) and a novel-object recognition task in which sample and retention were performed in different contexts (OR-CONTEXT group). We hypothesized that: (1) The change in the context would interfere with the exploration of the new object; and (2) the change in the context would reveal a differential brain activation pattern during recognition. To label the brain regions involved in this task, we marked the expression of c-Fos protein. Studying c-Fos can provide information about the neuronal plasticity required for memory processes [21]. The expression of c-Fos protein is induced after learning, and it is indicative of a change in neuronal activity [21–23]. This technique allows the simultaneous examination of the activity of populations of neurons in multiple brain regions [24]. Because the hippocampus does not act as an isolated structure in these learning tasks, other cerebral regions involved were also studied. These regions were the prefrontal cortex and adjacent cortical areas within the temporal lobe, such as the temporal association, entorhinal and perirhinal cortices, as ablation, electrophysiological lesions or dysfunctions suggest that these structures might be involved in this task [25–28]. 2. Material and methods 2.1. Animals We used 20 male Wistar rats from the vivarium of the University of Oviedo. They weighed 250–270 g at the beginning of the study. The rats were housed in groups of three to five, three weeks prior to the beginning of the experiments, and maintained under standard laboratory conditions (20–22 ◦ C, 65–70% relative humidity and a 12 h light/dark cycle (08.00–20.00/20.00–08.00)). Food and water were available ad libitum throughout the experiments, and sessions were performed during the light phase, between 9:00 a.m. and 13:00 p.m. All procedures were carried out according to the European Parliament and the Council of the European Union 2010/63/UE, and they were approved by the Oviedo University committee for animal studies. The animals were distributed in two groups on a one-trial novel-object recognition task (OR-NORMAL group, n = 10 and OR-CONTEXT group, n = 10). 2.2. Apparatus Object exploration was assessed in a square open field (40 × 40 × 33 (height) cm) with an open roof, placed in a rectangular room. The room was illuminated by two diffuse white lights placed at the sides of the room, providing an illumination density of approximately 50 lux. During the habituation of both groups, the walls and ground of the open field were covered by gray fiberglass. During the sample and probe tests, the walls consisted of patterns of dark and white plastic bars (2.5 cm width) placed vertically or
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horizontally, depending on the experimental condition. At the same time, the ground could change from smooth gray plastic to rough black plastic. A video camera, connected to a video recorder, was mounted above the field to store the sample and test trials on video files for off-line analysis. After each trial, the apparatus was thoroughly cleaned with a 75% ethanol solution. Pairs of three different ceramic objects were used. For habituation, we used two identical objects (2.5 × 2.5 × 8 cm); whereas for memory testing, we used two different pairs of objects, Objects A (3 × 3 × 6 cm) and Objects B (3.5 × 3.5 × 6.5 cm), which were made of ceramics and glass. In all the trials, the objects were placed in the center of the open field with a distance of 10.5 cm between them. All the objects were attached to the ground of the open field in order to avoid their displacement during the task. After each trial, the objects were thoroughly cleaned with a 75% ethanol solution to remove odor cues. 2.3. Novel-object recognition task 2.3.1. Habituation Prior to testing, all the animals were handled on four consecutive days for 10 min/day and habituated to the empty arena on three consecutive days. On the first day of habituation, groups of between three and five rats were placed together in the open field and allowed to explore it for 6 min without objects. On the following day, rats were given another 6-min session for exploration, but this time only one rat at a time was placed in the open field. On the third and last habituation day, rats received three daily sessions lasting 6 min each. There was a 20 min inter-trial interval when the two habituation objects were placed opposite each other, 10 cm apart, on two of the walls of the open field. This time, the rats were also brought individually to the testing room and placed in the open field facing the center of the wall where no objects were located. The habituation objects were not used in the assessment of memory for object recognition. 2.3.2. Sample and probe tests In the test phase, rats were first placed individually in the center of the open field. Two copies of an object were located in opposite corners, 7 cm from the walls of the open field. Four minutes of exploration were recorded in this sample trial. After a delay of 50 min, rats received a second 4 min probe test trial where a copy of the original or familiar object and a second novel object were used. For the OR-NORMAL group, the sample and probe test were performed in the same context, where the walls were covered by vertical black and white bars, and smooth gray plastic was placed in the floor. However, the OR-CONTEXT group performed the probe test in a different context, that is, an open field where the black and white bars of the walls were horizontal, and the floor was rough and black (Fig. 1). 2.4. c-Fos immunocytochemistry Coronal sections (30 m) of the brain were cut at −20 ◦ C in a cryostat (Leica CM1900, Germany). The sections were mounted on gelatinized slides post-fixed in buffered 4% paraformaldehyde (0.1 M, pH 7.4) for 30 min and rinsed in phosphate-buffered saline (PBS) (0.01 M, pH 7.4). They were subsequently incubated for 15 min with 3% hydrogen peroxidase in PBS to remove endogenous peroxidase activity, and then washed twice in PBS. After blocking with PBS solution containing 10% Triton X-100 (PBS-T) (Sigma, USA) and 3% bovine serum albumin for 30 min, sections were incubated with a rabbit polyclonal anti-c-Fos solution (1:10,000) (Santa Cruz Biotech, sc-52, USA) diluted in PBS-T for 24 h at 4 ◦ C in a humid chamber. Slides were then washed three times with PBS and incubated in a goat anti-rabbit biotinylated IgG secondary antibody
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Fig. 1. Schematic illustration of the one-trial object-place recognition task. Two experiments were performed using the same or different contexts. The figure shows the different experimental conditions during sample and probe tests in both experimental groups.
(Pierce, USA; diluted 1:200 in incubating solution) for 2 h at room temperature. They were washed three times in PBS and reacted with avidin–biotin peroxidase complex (Vectastain ABC Ultrasensitive Elite Kit, Pierce) for 1 h. After two washes in PBS, the reaction was visualized, treating the sections for about 3 min in a commercial nickel-cobalt intensified diaminobenzidine kit (Pierce). The reaction was finalized by washing the sections twice in PBS. Slides were then dehydrated through a series of graded alcohols cleared with xylene and cover-slipped with Entellan (Merck, USA) for microscopic observation. All immunocytochemistry procedures included sections that served as controls, where the primary antibody was not added. Slides containing sections of a specific brain region were stained at the same time. Slides were coded so that the investigator who performed the entire analysis would have no knowledge about the group of individual subjects. The total number of c-Fos positive nuclei was quantified in three alternate sections 30 m apart. Quantification was done by systematically sampling each of the regions selected, using a microscope (Olympus BH-2, Japan) attached to an analog camera (Sony XC-77, Japan) and a TV monitor (300× total magnification). c-Fos positive nuclei were defined based on homogenous gray–black stained elements with a well-defined border. Finally, the mean count for three sections was calculated for each subject and region (number of positive nuclei/150 m2 ). The regions of interest were anatomically defined according to Paxinos and Watson’s atlas [29]. The regions of interest and their distance in mm from bregma was: +3.20 mm for the infralimbic cortex (IL), the prelimbic cortex (PL), the cingulate cortex (CG); −3.84 mm for the CA1, CA3 and the dentate gyrus (DG) subfields of the dorsal hippocampus; −5.04 mm for the perirhinal (Prh), the entorhinal cortex (Ent) and the temporal association cortex (TeA). 2.5. Statistical analysis 2.5.1. Novel-object recognition data Exploration of an object by the rat was defined as directing the nose toward the object at a distance of less than 2 cm and actively exploring it. Sitting and turning around on the object were not considered exploratory behavior. For each rat, the time spent exploring
the objects in both sample sessions and the test session were scored off-line from video files, using stopwatches. Two researchers who were blind to the groups coded each video. Additionally, a grid of 10 × 10 cm squares was virtually superimposed on the arena floor to record exploratory behaviors during the probe phase as an index of context exploration. “Ambulation” was defined as the crossing of at least 1 floor grid line within a 3 s period, and “rearing” was defined as lifting the forelimbs and sitting back on the haunches [30]. Context exploration data, ambulation and rearing of the groups were compared with a t-test. A number of measurements were taken in order to examine exploration and discrimination of the objects. The proportion of exploration in the sample and test phases was considered as e1 and e2, respectively, and it was calculated by dividing the exploration of all objects in the phase divided by the total time available for exploration. Two measures of discrimination were taken: discrimination index (d1) and discrimination ratio (d2) [31]. The discrimination index, d1, consisted of the difference in time spent exploring the two objects in the probe or test phase, the novel-object minus the familiar object. The discrimination ratio, d2, was also calculated in the test phase. This is the difference in the exploration time of the two objects divided by the total time spent exploring the objects, that is, d1/time spent exploring both objects. Exploration in the sample and the test phase (e1 and e2) was compared between groups with a t-test. Furthermore, paired t-tests were performed on each experimental group in order to appreciate possible differences in the time spent exploring the objects in the sample and probe phases (e1 and e2). Discrimination data, d1 and d2, of the groups were compared with a t-test. In addition, t-tests were performed on d1 and d2 to determine whether the mean scores differed from zero (chance performance indicating equal exploration of the two objects). This would indicate that the groups could successfully discriminate between the objects. A non-parametric Mann–Whitney U test (U) for independent samples was carried out when normality or equal group variances failed. 2.5.2. c-Fos data To determine c-Fos activity, 12 subjects were analyzed (ORNORMAL n = 6, OR-CONTEXT n = 6). For statistical analysis, cell
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Fig. 2. Performance of the experimental groups on the object-place recognition task. Discrimination index and discrimination ratio (d1 and d2) for NORMAL and CONTEXT groups.
counts from the three selected sections for a given brain region in each animal were averaged, and the mean was used for statistical analysis. Student t-tests for independent samples were used to assess whether the number of c-Fos positive nuclei were different between groups in each brain region. Moreover, a non-parametric Mann–Whitney U test (U) for independent samples was carried out when normality or equal group variances failed.
3. Results 3.1. Behavioral results The results showed significant group differences in the exploration index e2: t18 = 2208, P = 0.040, where the OR-NORMAL group employed more time than the OR-CONTEXT group in exploring the objects. However, no differences were found in the exploration index e1: U = 128.000, n1 = 10, n2 = 10, P = 0.089). There were no significant differences in exploration between the sample phase and the test phase for the OR-NORMAL group (e1 = 0.164 ± 0.021, e2 = 0.264 ± 0.0536: t18 = −1.731, P = 0.101) or for the OR-CONTEXT group (e1 = 0.130 ± 0.0112, e2 = 0.137 ± 0.0207: t18 = −0.277, P = 0.785). The results showed that both groups were able to discriminate between a previously-encountered object and a novel object. The d1 and d2 means were both significantly higher than zero for both the OR-NORMAL group (d1: t18 = 4.421, P < 0.001 d2: t18 = 14.823, P < 0.001) and the OR-CONTEXT group (d1: t18 = 4.278, P < 0.001; d2: t5 = 5.722, P < 0.001). Significant differences were found between groups in d1 (U = 144.000, n1 = 10, n2 = 10, P = 0.004) and in d2 (t18 = 3.399, P = 0.003; Fig. 2). Additionally, context exploratory behaviors during the probe phase showed differences between groups. Ambulation and rearing were higher in the OR-CONTEXT group than in the OR-NORMAL group (t18 = −9.241, P < 0.001 and t18 = −6.059, P < 0.001, respectively; Fig. 3).
3.2. c-Fos results Differences were found between the OR-NORMAL and ORCONTEXT groups in the CA1 (t10 = −4.192, P = 0.002), DG (U = 21.000, n1 = 6, n2 = 6, P = 0.002), Ent (U = 25.000, n1 = 6, n2 = 6, P = 0.026) and TeA (t10 = − 7.167, P < 0.001). In these regions, the number of c-Fos positive nuclei was higher in the OR-CONTEXT group compared to the OR-NORMAL group. No differences between groups were found in Cg (t10 = −1.436, P = 0.182), PL (U = 32.000, n1 = 6, n2 = 6, P = 0.310), IL (U = 30.000, n1 = 6, n2 = 6, P = 0.180), CA3 (t10 = − 1.478, P = 0.170) or Prh (t10 = 0.643, P = 0.535; Fig. 4).
Fig. 3. Bar graphs showing the time spent (mean ± SEM) by experimental groups ambulating and rearing during the 4 min object recognition task probe phase. Significant differences were found in the time devoted to each behavior (*p < 0.05). OR-CONTEXT group spent significantly more time ambulating and rearing than OR-NORMAL group.
Fig. 4. Fos counts. Number of c-Fos positive nuclei in OR-NORMAL and OR-CONTEXT groups (mean ± SEM) in the structures where significant differences were found (*p < 0.05). The number of c-Fos was significantly higher in the OR-CONTEXT group compared to the OR-NORMAL group in the hippocampus, entorhinal and temporal association cortices.
4. Discussion Our study revealed that a change in the context where an object is presented can affect novel-object exploration, interfering in object recognition. Behavioral data revealed that the new context affects total object exploration. Animals tend to explore the
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objects more when they are presented in a familiar context. Our data showed the differential contribution of the hippocampal subfields and extra-hippocampal structures to novel-object in novel context recognition. Regarding behavior, the group that recognizes the object in a familiar context, OR-NORMAL, spends more time exploring both objects in the test phase than the group that performs recognition in a novel context, OR-CONTEXT. These results indicate that the animals spend more time exploring the objects when they are in their usual context; therefore, context modification interferes with the time spent on object exploration. However, to confirm whether the groups exhibit a tendency toward further exploration, we proceeded to analyze both indexes in each of the groups, comparing the time spent exploring the objects in both the sample and test phases within each group. We can see that within each group the exploration indexes (e1 and e2) did not differ. However, during the test phase, while in both cases an object is new, the NORMAL group spent even more time exploring the new object (e2). Moreover, these results are supported by the ambulatory and rearing behavior, which increased in the CONTEXT group. This result suggests that the change in context can influence the exploratory behavior of animals, so that they spend more time exploring a new environment that is unfamiliar to them, while paying less attention to specific objects. In addition, the d1 and d2 indexes indicate that the OR-NORMAL group spent more time exploring the novel-object compared to the OR-CONTEXT group. These data show that the animals spend more time exploring the objects when they are in their habitual context. Thus, a change in the context interferes with the time spent exploring novel objects. The novelty judgment is supported by several regions. Traditionally, ablation or electrophysiological evidence has suggested that the hippocampal formation, the entorhinal cortex, and adjacent cortical areas within the temporal lobe, such as the perirhinal cortex, might be involved in recognition memory [25]. Detailed studies using bilateral hippocampal lesions revealed significantly impaired memory for object-place recognition [32], while many object-recognition studies found no apparent effects of hippocampal lesions [33]. Other studies related to the recognition of familiar spatial rearrangements showed significant increases in Fos levels in CA1, and significant decreases in the dentate gyrus (DG) [34], highlighting the long-standing debate about hippocampal involvement in recognition memory. The entorhinal cortex, which is the major cortical input to the hippocampus [35], is involved in the discrimination of familiar objects in certain places, but not in the recognition of either novel objects or novel places [36]. For this reason, the involvement of the hippocampus and entorhinal cortex in novel-object in novelcontext recognition was expected. Our results link novel-object in novel-context recognition to an increase in c-Fos immunoreactivity in the CA1, DG, entorhinal cortex, and temporal association cortex. How can this early gene expression in the RO-CONTEXT group be explained? The increased Fos levels in CA1 and the DG could be related to the mediation of these structures in consolidating new information and representing metric spatial representations [37]. Our data could suggest the involvement of these hippocampal regions in combining the associative recognition of objects with their locations and local contexts, information that is not combined until reaching the hippocampus and that could occur in parallel with the storage of other features of the process. The entorhinal cortex has been involved in binding objects with the contexts in which they are experienced to form a representation of a new contextualized environment [38]. Moreover, the entorhinal cortex is the major cortical input to the hippocampus [35], where increases in the DG and CA1 associated with novelty have been reported [39]. Novel stimuli processing has been associated
with activity in the direct pathway from the entorhinal cortex to the dentate gyrus [39]. The increased Fos levels in the entorhinal cortex could suggest that contextual features of the environment are being integrated with object identity in this region. However, previous studies [40] showed that exploration of a novel environment and performance on a well-learned odor discrimination task elicited a common pattern of c-fos expression in the hippocampus that involved a higher expression in CA1, followed by CA3 and the DG. Along these lines, our results showing a higher implication of the DG, followed by CA1 and the absence of CA3 involvement, may be more related to the exploration of a novel stimuli or the binding between novel-object under novel-context exposure. However, further experiments are needed to understand the role of the DG. The role of the temporal association cortex (TeA) has been paired with the perirhinal cortex, due to its anatomical position adjacent to the perirhinal cortex. Furthermore, electrophysiological recordings in both monkey and rat brains have revealed units in both regions that typically reduce the response when the animals are confronted with familiar stimuli [41]. This raises the possibility that both TeA and the perirhinal cortex may have a role to play in information storage for object-recognition memory [42]. Our results suggest a role that is probably more related to information storage under a novel context condition. Additionally, it is important to highlight the fact that no increases in c-Fos expression were found in the prefrontal cortex. Studies using disconnection approaches [43] revealed that the prefrontal cortex plays a role in recognition memory in cases requiring the integration of the object with spatial location information received from other neural regions. In our study, in the OR-CONTEXT condition, novelty comes from a novel object that should be recognized in a novel context, mainly requiring a retrieval of “what” is presented. The lack of Fos expression in the prefrontal cortex does not necessarily refute the role of this structure in the integration of object and space information [44]. In conclusion, our work revealed that there was interference caused by changing environmental conditions in a novel-object recognition task, suggesting that a neural circuit that includes the hippocampus and entorhinal and temporal association cortices is involved in recognition in a novel context. These results could highlight how a dysfunction in this network may affect contextual memory, which is important not only for daily functioning, but also because it is altered in many disorders.
Acknowledgements This research was supported by Foundation Alfonso Martin Escudero Grants to NA and Project Grants of the Spanish Ministry of Economy and Competitiveness: PSI 2010-19348 and PSI 201345924-P. Authors are grateful to Ldo. Jorge Arias for his technical support and Cynthia Depoy for the English review.
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