Anim Cogn DOI 10.1007/s10071-014-0755-y
SHORT COMMUNICATION
Retrieval-induced forgetting in rats Kazuo Yamada • Masaharu Ueno • Etsushi Takano Yukio Ichitani
•
Received: 3 February 2014 / Revised: 23 April 2014 / Accepted: 25 April 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract ‘‘Retrieval-induced forgetting’’ in rats was evaluated using a modified spontaneous object recognition test. The test consisted of a sample phase, retrieval or interference phase, and a test phase with 60-min delay period inserted between the phases. Rats were randomly assigned to one of three groups (control, retrieval and interference) and allowed to explore the field in which two different objects (A, B) were placed in the sample phase. In the retrieval phase, two identical objects (B, B), which were the same as one of the objects presented in the sample phase, were placed again. In the interference phase, two identical objects (C, C), which were novel for animals, were placed. In the test phase, two different objects (A, D), one of which was identical to that presented in sample phase (familiar object) and the other was novel, were placed and the time spent exploring each object was analyzed. While the exploration of the novel object was significantly longer than that of the familiar object in rats subjected to the interference phase, rats subjected to the retrieval phase could not discriminate between the familiar and the novel objects at the test phase. These results demonstrate the ‘‘retrieval-induced forgetting’’ phenomenon in a spontaneous object recognition test in rats. Keywords Retrieval-induced forgetting Spontaneous object recognition Retroactive interference Rat
K. Yamada (&) M. Ueno E. Takano Y. Ichitani Institute of Psychology and Behavioral Neuroscience, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan e-mail: [email protected]
Introduction The act of remembering can cause forgetting. Successful retrieval not only facilitates later recall of the retrieved items, but also impairs later recall of the related or competing items in memory. This memory phenomenon has been demonstrated with the retrieval-practice paradigm introduced by Anderson et al. (1994). In this paradigm, participants typically learn lists of category–exemplar pairs (e.g., fruits-banana, fruits-orange) from several categories (e.g., fruits, drinks, fish) and then practice retrieval of half of the exemplars from half of the categories (e.g., retrieving banana given the cue fruits-ba____). Finally, they receive a recall test in which they are encouraged to recall all studied exemplars provided with the category names as recall cues. While it is not surprising that retrieval practice improves later recall of the practiced material (fruitsbanana), an intriguing finding is that retrieval practice impairs recall of the unpracticed material (fruits-orange), relative to a control condition in which no retrieval practice occurs at all. This detrimental effect of retrieval is referred to as ‘‘retrieval-induced forgetting (RIF)’’ (Anderson et al. 1994). RIF has been shown to occur on tests of both episodic and semantic memory (Blaxton and Neely 1983; Ba¨uml 2002; Ciranni and Shimamura 1999; Johnson and Anderson 2004). However, the biological mechanisms responsible for this memory phenomenon have not yet been elucidated. One possible explanation of RIF is that there are inhibitory control mechanisms, which are recruited to overcome interference caused by competing memory traces (Anderson et al. 1994; Anderson and Spellman 1995). When a cue is associated with several memory traces, selective retrieval of the desired memory is facilitated by inhibiting other memory traces associated with the same
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cue, thereby attenuating the interference caused by these competitors. In order for memory to function effectively, organisms need to inhibit related but unnecessary memory traces. We had previously investigated the other kind of inhibitory control mechanism in memory, ‘‘directed forgetting (DF),’’ by testing rats in a radial maze task (Kaku et al. 2013). DF manifests as poor memory for information we are instructed to forget compared to information we are instructed to remember. We found the possibility that several brain regions, including the medial prefrontal cortex and the hippocampal CA3 area, were involved in DF. DF reflects the ability to suppress memory retention, or to forget previously acquired information, which is analogous to RIF. Although rodent models are clearly useful to investigate neural mechanisms underlying the psychological phenomenon, to the best of our knowledge, there are not any rodent models of RIF. In the present study, we attempted to demonstrate RIFlike phenomenon in rats using a modified spontaneous object recognition test. Spontaneous recognition is one of the memory tests which utilizes the rodents’ innate tendency to explore novel stimuli longer than familiar stimuli. This test typically consists of a ‘‘sample phase’’ and a ‘‘test phase’’, and a delay is inserted between them. In the sample phase, rats or mice explore the familiar arena in which two identical objects are presented, and after the delay period, they are introduced into the arena again. In the test phase, two objects are presented in the same position as in the sample phase, but one of the objects is novel. In order to make this rodent memory test look as much like the retrieval-practice paradigm used in RIF study in humans (Anderson et al. 1994), we partly modified the procedure. First, two different objects (but not identical objects) were presented during the sample phase. This allowed animals to memorize more than one object in a specific context, which was similar to the study phase in the retrieval-practice paradigm. Second, a retrieval phase was set during the delay period in accordance with three phases (study, retrieval and test) found in the retrieval-practice paradigm. Third, an interference phase was employed as a control for the retrieval phase. While animals were reunited with familiar objects in the retrieval phase, they met novel objects in the interference phase.
Materials and methods Twenty-five male Long–Evans rats (8 weeks old, purchased from Institute for Animal Reproduction, Ibaraki, Japan) were used as subjects and divided into three groups; control (n = 8), retrieval (n = 9) and interference (n = 8) groups. They were housed in groups (2–3/cage) under constant temperature and humidity conditions on a 12:12-h
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light–dark cycle (light on 8:00–20:00) with food and water available ad libitum. All behavioral experiments were conducted in the light phase and were approved by the University of Tsukuba Committee on Animal Research. An open field (90 cm 9 90 cm 9 45 cm; OF) made of polyvinylchloride was used, and its walls were black and the floor gray. The objects employed were white and black triangular cast metal (5 cm on a side, 12 cm in height), white cylinder of cast metal (4 cm in diameter, 12 cm in height), yellow rubber duck (7 cm 9 6 cm 9 8 cm), small glass bottle (5 cm in diameter, 6 cm in height), white plastic bottle (6 cm in diameter, 10 cm in height), can of juice (5 cm in diameter, 13 cm in height) and plastic soup case (10 cm 9 7 cm 9 4 cm). All of them were heavy enough so that the rat could not move them, or they were fixed on the metal plate (6 cm 9 8 cm) to prevent them from being moved by the rat. Three or four objects used in the object recognition test were chosen for each animal in a counterbalanced way. A video camera was suspended above the OF, and the image was projected onto a screen. Handling and habituation to the apparatus preceded the recognition task. Rats received 5-min handling and 10-min habituation to the OF for 3 days. During the habituation, rats individually explored the OF freely. The object recognition test consisted of a sample phase (5 min), retrieval or interference phase (5 min), and a test phase (5 min) with a delay period (60 min) inserted between the phases (Fig. 1). In the sample phase, two different objects (A, B) were placed in the corners of the OF, and the rat explored the field freely. After the rat was taken to its home cage (delay period), the rat that belonged to either retrieval or interference groups was introduced into the field again (retrieval or interference phase). Animals of the control group were kept in the home cage. In the retrieval phase for the retrieval group, two identical objects (B, B), which were the same as one of the objects in sample phase, were placed at the same positions as in the sample phase. In the interference phase for the interference group, two identical objects (C, C), which were novel for the animals, were placed at the same position as in the sample phase. In the test phase, two different objects (A, D) were placed at the same positions as in the sample phase, and one object was identical to one of the objects in the sample phase (familiar object) and the other was a novel object. After each phase, the floor of the OF was wiped with a damp cloth, and the objects were sprayed with 70 % alcohol and then wiped. Time spent exploring each object during the test phase was scored by the experimenter viewing the rats’ behavior on a monitor screen. Exploration was defined as the rat’s nose being directed toward the object within 2 cm. High interrater reliability (a = 0.835) of scoring by two independent judges based on a subset (ten samples) of video recorded behavior meant that the assessment procedure was reliable.
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Fig. 1 Schematic diagram of the modified spontaneous object recognition test. This test consisted of a sample phase (5 min) and a test phase (5 min) with a delay period (125 min). In the sample phase, two different objects (A, B) were placed in the field, and then, in the test phase, one of the objects was displaced by a novel object (A, D). A retrieval or an interference phase was inserted during the delay period for retrieval and interference groups, respectively. While two identical objects (B, B), which were the same as one of the objects in sample phase, were placed at the field in the retrieval phase, two identical objects (C, C), which were novel for animals, were placed in the interference phase
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The time spent exploring each object in the sample phase and the test phase was analyzed by a mixed-design analysis of variance (ANOVA) (Group 9 Phase 9 Object). Individual comparisons were evaluated using a simple main effect test and a post hoc Fisher’s PLSD test with alpha set at 0.05. A discrimination ratio (DR), which was calculated by dividing the amount of exploration of the novel object by the total amount of object exploration in the test phase, was analyzed by oneway ANOVA (group factor), followed by post hoc comparisons using a Fisher LSD test with alpha set at 0.05. DR data were also compared to chance level (50 %) using a one-sample t test.
Results Exploration time of each object in the sample phase and the test phase are shown in Fig. 2a. Animals in all groups explored both objects equally during the sample phase. In the test phase, animals in the control and interference groups explored the novel object longer than the familiar
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Fig. 2 a Time spent exploring each object in the sample phase and the test phase. In the test phase, rats in the retrieval group explored both objects almost equally, while rats in the control and interference groups explored the novel object longer than the familiar object. b Discrimination ratio (DR) in the test phase, calculated by dividing the amount of exploration of the novel object by the total amount of object exploration. DR of the retrieval group was significantly lower than those of the control and interference groups. In both panels, mean ± SEM is shown. *p \ .05, **p \ .01. ?p \ .05, ??p \ .01 above chance (50 %) level. Cont control group, Ret retrieval group, Int interference group
object. In contrast, rats in retrieval group explored both objects almost equally. Three-way ANOVA showed a significant main effect of object [F(1, 22) = 14.86, p \ .01], and also significant interactions between group and phase [F(2, 22) = 4.17, p \ .05], between phase and object [F(1, 22) = 11.81, p \ .01]. An analysis of the interaction between group and phase indicated that the group effect was significant only in the test phase [F(2, 22) = 6.05, p \ .01]. A post hoc comparison revealed that the exploration time in the retrieval group was significantly
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shorter than that in both control and interference groups (p \ .05). On the other hand, the phase effect was not significant in each group. Thus, there were no differences in the total amount of object exploration between the sample phase and the test phase. An analysis of the interaction between phase and object indicated that the phase effect was significant in both objects [familiar F(1, 22) = 7.79, p \ .05; novel F(1, 22) = 5.53, p \ .05]. The object effect was also significant in the test phase [F(1, 22) = 43.02, p \ .01]. With regard to DR (Fig. 2b), there was a significant main effect of group [F(2, 22) = 4.70, p \ .05]. A post hoc comparison revealed that DR of the retrieval group was significantly lower than that of the control (p \ .01) and interference groups (p \ .05). Furthermore, DRs of the control and interference groups were significantly above chance level (50 %) (control t(7) = 7.57, p \ .01; interference t(7) = 2.92, p \ .05), but that of the retrieval group was not. The total amount (mean ± SEM) of object exploration was 27.8 ± 17.7 during the retrieval phase and 37.3 ± 13.4 during the interference phase. A one-way ANOVA revealed no significant difference in the exploration time between the retrieval phase and the interference phase.
Discussion In the present study, we demonstrated, for the first time, the RIF phenomenon in rats using a modified spontaneous object recognition test. Only rats subjected to the retrieval phase, in which rats reunited with (retrieved) one of the objects presented in the sample phase, could not discriminate between the familiar (other object presented in the sample phase) and the novel objects in the test phase. Since rats that were subjected to the interference phase, in which rats met a novel object, were able to discriminate between them in the test phase, the detrimental effect of the insertion of a retrieval phase could not be attributed to proactive or retroactive interference. So, did rats forget the non-retrieved objects? In the test phase, the exploration time of the novel object in the retrieval group was shorter than in the control and interference groups, while there were no differences in the exploration time of the familiar object among the three groups. This decreased the total amount of object exploration in the retrieval group. The present findings that the retrieval phase disrupted the following object recognition seem to be attributed to the decreased exploration of the novel object. If rats subjected to the retrieval phase forgot a familiar object, or falsely recognized the familiar object as a novel object, they would have explored the familiar object longer than control rats (McTighe et al. 2010).
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A pioneering study on the spontaneous object recognition in rats, on the other hand, reported that there were no differences in total time of object exploration between two conditions—one condition in which two familiar objects were presented, the other in which two novel objects were presented (Ennaceur and Delacour 1988). In the present study, there were also no significant differences in the total amount of object exploration between the retrieval phase (two familiar objects) and the interference phase (two novel objects). In addition, the difference in the total amount of object exploration between the sample phase and the test phase in retrieval group was not significant. Given that rat’s tendency to explore a novel object more can be observed only under a specific condition, in which novel and familiar objects are presented simultaneously, the lack of increment of exploring non-retrieved object could not necessarily exclude the possibility that rats in the retrieval group forgot a familiar object, or falsely recognized the familiar object as a novel object. A possibility that the insertion of the retrieval phase decreased the motivational aspect of performance in the following test phase was ruled out, because the interference phase did not affect the performance in the test phase. Thus, we suggest tentatively that retrieval of an object impairs later recall of the related but non-retrieved object, which is like a RIF phenomenon, in spontaneous object recognition test. Recently, Whitt et al. (2012) conducted a similar experiment examining associative processes, but not RIF, in the recognition memory of rats. They found that reunion with one of the objects was able to activate the memory of the other object, if two different objects were simultaneously presented during the sample phase. Such a phenomenon in the object recognition task, which is essentially opposite to the present findings, has been explained in terms of a model of memory proposed by Brandon et al. (2003). Understanding our results through Brandon et al.’s (2003) model implies that excitatory associations could be formed between two different objects (A, B) during the sample phase. When an animal then encountered one of the objects (B, B) during the retrieval phase, memory of the other object (A) was activated indirectly. These processes should enhance the difference in familiarity between the objects (A vs. D) in the test phase, provoking a relatively strong approach to novel object (D); however, this was not the case. This discrepancy may be explained by the difference in the length of delay period inserted between the sample phase and the retrieval phase. While Whitt et al. (2012) used a 10-min delay, we used a relatively long-term delay period (60 min). Since the level of associative activation is dependent on the strength of the association between stimuli (Sanderson and Bannerman 2011), a decay of the association with time might have caused the conflicting findings.
Anim Cogn
With regard to the neural mechanism responsible for RIF, Johansson et al. (2007) demonstrated that prefrontal event-related potentials were involved in RIF, or an inhibitory control of memory, in humans. A number of previous studies have suggested a critical role of the prefrontal cortex in the control of competitor memory traces during memory retrieval (Schnider 2003; Shimamura 2000; Thompson-Schill et al. 2005). Recently, we also reported the possibility that the medial prefrontal cortex and hippocampus are related to DF of spatial information, which is analogous to RIF (Kaku et al. 2013). Thus, the prefrontal cortex appears to be a prime candidate for involvement in RIF. The rodent’s RIF model using spontaneous object recognition test is undoubtedly useful to investigate the neural correlates of RIF. Acknowledgments The authors would like to thank Dr. Constantine Pavlides for critical reading and valuable comments on the manuscript. This study was supported by grants from Japan Society for the Promotion of Science (24653209, 24530909).
References Anderson MC, Spellman BA (1995) On the status of inhibitory mechanisms in cognition: memory retrieval as a model case. Psychol Rev 102:68–100 Anderson MC, Bjork RA, Bjork EL (1994) Remembering can cause forgetting: retrieval dynamics in long-term memory. J Exp Psychol Learn Mem Cogn 20:1063–1087 Ba¨uml KH (2002) Semantic generation can cause episodic forgetting. Psychol Sci 13:356–360 Blaxton TA, Neely JH (1983) Inhibition from semantically related primes: evidence of a category-specific inhibition. Mem Cognit 11:500–510
Brandon SE, Vogel EH, Wagner AR (2003) Stimulus representation in SOP: I Theoretical rationalization and some implications. Behav Processes 62:5–25 Ciranni MA, Shimamura AP (1999) Retrieval-induced forgetting in episodic memory. J Exp Psychol Learn Mem Cogn 25:1403–1414 Ennaceur A, Delacour J (1988) A new one-trial test for neurobiological studies of memory in rats. 1: behavioural data. Behav Brain Res 31:47–59 Johansson M, Aslan A, Ba¨uml KH, Ga¨bel A, Mecklinger A (2007) When remembering causes forgetting: electrophysiological correlates of retrieval-induced forgetting. Cereb Cortex 17:1335–1341 Johnson SK, Anderson MC (2004) The role of inhibitory control in forgetting semantic knowledge. Psychol Sci 15:448–453 Kaku M, Yamada K, Ichitani Y (2013) Can rats control previously acquired spatial information? Evidence of ‘‘directed forgetting’’ phenomenon in delay-interposed radial maze behavior. Behav Brain Res 248:1–6 McTighe SM, Cowell RA, Winters BD, Bussey TJ, Saksida LM (2010) Paradoxical false memory for objects after brain damage. Science 330:1408–1410 Sanderson DJ, Bannerman DM (2011) Competitive short-term and long-term memory processes in spatial habituation. J Exp Psychol Anim Behav Process 37:189–199 Schnider A (2003) Spontaneous confabulation and the adaptation of thought to ongoing reality. Nat Rev Neurosci 4:662–671 Shimamura AP (2000) The role of the prefrontal cortex in dynamic filtering. Psychobiology 28:207–218 Thompson-Schill SL, Bedney M, Goldberg RF (2005) The frontal lobes and the regulation of mental activity. Curr Opin Neurobiol 15:219–224 Whitt E, Haselgrove M, Robinson J (2012) Indirect object recognition: evidence for associative processes in recognition memory. J Exp. Psychol Anim Behav Process 38:74–83
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SHORT COMMUNICATION
Retrieval-induced forgetting in rats Kazuo Yamada • Masaharu Ueno • Etsushi Takano Yukio Ichitani
•
Received: 3 February 2014 / Revised: 23 April 2014 / Accepted: 25 April 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract ‘‘Retrieval-induced forgetting’’ in rats was evaluated using a modified spontaneous object recognition test. The test consisted of a sample phase, retrieval or interference phase, and a test phase with 60-min delay period inserted between the phases. Rats were randomly assigned to one of three groups (control, retrieval and interference) and allowed to explore the field in which two different objects (A, B) were placed in the sample phase. In the retrieval phase, two identical objects (B, B), which were the same as one of the objects presented in the sample phase, were placed again. In the interference phase, two identical objects (C, C), which were novel for animals, were placed. In the test phase, two different objects (A, D), one of which was identical to that presented in sample phase (familiar object) and the other was novel, were placed and the time spent exploring each object was analyzed. While the exploration of the novel object was significantly longer than that of the familiar object in rats subjected to the interference phase, rats subjected to the retrieval phase could not discriminate between the familiar and the novel objects at the test phase. These results demonstrate the ‘‘retrieval-induced forgetting’’ phenomenon in a spontaneous object recognition test in rats. Keywords Retrieval-induced forgetting Spontaneous object recognition Retroactive interference Rat
K. Yamada (&) M. Ueno E. Takano Y. Ichitani Institute of Psychology and Behavioral Neuroscience, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan e-mail: [email protected]
Introduction The act of remembering can cause forgetting. Successful retrieval not only facilitates later recall of the retrieved items, but also impairs later recall of the related or competing items in memory. This memory phenomenon has been demonstrated with the retrieval-practice paradigm introduced by Anderson et al. (1994). In this paradigm, participants typically learn lists of category–exemplar pairs (e.g., fruits-banana, fruits-orange) from several categories (e.g., fruits, drinks, fish) and then practice retrieval of half of the exemplars from half of the categories (e.g., retrieving banana given the cue fruits-ba____). Finally, they receive a recall test in which they are encouraged to recall all studied exemplars provided with the category names as recall cues. While it is not surprising that retrieval practice improves later recall of the practiced material (fruitsbanana), an intriguing finding is that retrieval practice impairs recall of the unpracticed material (fruits-orange), relative to a control condition in which no retrieval practice occurs at all. This detrimental effect of retrieval is referred to as ‘‘retrieval-induced forgetting (RIF)’’ (Anderson et al. 1994). RIF has been shown to occur on tests of both episodic and semantic memory (Blaxton and Neely 1983; Ba¨uml 2002; Ciranni and Shimamura 1999; Johnson and Anderson 2004). However, the biological mechanisms responsible for this memory phenomenon have not yet been elucidated. One possible explanation of RIF is that there are inhibitory control mechanisms, which are recruited to overcome interference caused by competing memory traces (Anderson et al. 1994; Anderson and Spellman 1995). When a cue is associated with several memory traces, selective retrieval of the desired memory is facilitated by inhibiting other memory traces associated with the same
123
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cue, thereby attenuating the interference caused by these competitors. In order for memory to function effectively, organisms need to inhibit related but unnecessary memory traces. We had previously investigated the other kind of inhibitory control mechanism in memory, ‘‘directed forgetting (DF),’’ by testing rats in a radial maze task (Kaku et al. 2013). DF manifests as poor memory for information we are instructed to forget compared to information we are instructed to remember. We found the possibility that several brain regions, including the medial prefrontal cortex and the hippocampal CA3 area, were involved in DF. DF reflects the ability to suppress memory retention, or to forget previously acquired information, which is analogous to RIF. Although rodent models are clearly useful to investigate neural mechanisms underlying the psychological phenomenon, to the best of our knowledge, there are not any rodent models of RIF. In the present study, we attempted to demonstrate RIFlike phenomenon in rats using a modified spontaneous object recognition test. Spontaneous recognition is one of the memory tests which utilizes the rodents’ innate tendency to explore novel stimuli longer than familiar stimuli. This test typically consists of a ‘‘sample phase’’ and a ‘‘test phase’’, and a delay is inserted between them. In the sample phase, rats or mice explore the familiar arena in which two identical objects are presented, and after the delay period, they are introduced into the arena again. In the test phase, two objects are presented in the same position as in the sample phase, but one of the objects is novel. In order to make this rodent memory test look as much like the retrieval-practice paradigm used in RIF study in humans (Anderson et al. 1994), we partly modified the procedure. First, two different objects (but not identical objects) were presented during the sample phase. This allowed animals to memorize more than one object in a specific context, which was similar to the study phase in the retrieval-practice paradigm. Second, a retrieval phase was set during the delay period in accordance with three phases (study, retrieval and test) found in the retrieval-practice paradigm. Third, an interference phase was employed as a control for the retrieval phase. While animals were reunited with familiar objects in the retrieval phase, they met novel objects in the interference phase.
Materials and methods Twenty-five male Long–Evans rats (8 weeks old, purchased from Institute for Animal Reproduction, Ibaraki, Japan) were used as subjects and divided into three groups; control (n = 8), retrieval (n = 9) and interference (n = 8) groups. They were housed in groups (2–3/cage) under constant temperature and humidity conditions on a 12:12-h
123
light–dark cycle (light on 8:00–20:00) with food and water available ad libitum. All behavioral experiments were conducted in the light phase and were approved by the University of Tsukuba Committee on Animal Research. An open field (90 cm 9 90 cm 9 45 cm; OF) made of polyvinylchloride was used, and its walls were black and the floor gray. The objects employed were white and black triangular cast metal (5 cm on a side, 12 cm in height), white cylinder of cast metal (4 cm in diameter, 12 cm in height), yellow rubber duck (7 cm 9 6 cm 9 8 cm), small glass bottle (5 cm in diameter, 6 cm in height), white plastic bottle (6 cm in diameter, 10 cm in height), can of juice (5 cm in diameter, 13 cm in height) and plastic soup case (10 cm 9 7 cm 9 4 cm). All of them were heavy enough so that the rat could not move them, or they were fixed on the metal plate (6 cm 9 8 cm) to prevent them from being moved by the rat. Three or four objects used in the object recognition test were chosen for each animal in a counterbalanced way. A video camera was suspended above the OF, and the image was projected onto a screen. Handling and habituation to the apparatus preceded the recognition task. Rats received 5-min handling and 10-min habituation to the OF for 3 days. During the habituation, rats individually explored the OF freely. The object recognition test consisted of a sample phase (5 min), retrieval or interference phase (5 min), and a test phase (5 min) with a delay period (60 min) inserted between the phases (Fig. 1). In the sample phase, two different objects (A, B) were placed in the corners of the OF, and the rat explored the field freely. After the rat was taken to its home cage (delay period), the rat that belonged to either retrieval or interference groups was introduced into the field again (retrieval or interference phase). Animals of the control group were kept in the home cage. In the retrieval phase for the retrieval group, two identical objects (B, B), which were the same as one of the objects in sample phase, were placed at the same positions as in the sample phase. In the interference phase for the interference group, two identical objects (C, C), which were novel for the animals, were placed at the same position as in the sample phase. In the test phase, two different objects (A, D) were placed at the same positions as in the sample phase, and one object was identical to one of the objects in the sample phase (familiar object) and the other was a novel object. After each phase, the floor of the OF was wiped with a damp cloth, and the objects were sprayed with 70 % alcohol and then wiped. Time spent exploring each object during the test phase was scored by the experimenter viewing the rats’ behavior on a monitor screen. Exploration was defined as the rat’s nose being directed toward the object within 2 cm. High interrater reliability (a = 0.835) of scoring by two independent judges based on a subset (ten samples) of video recorded behavior meant that the assessment procedure was reliable.
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Fig. 1 Schematic diagram of the modified spontaneous object recognition test. This test consisted of a sample phase (5 min) and a test phase (5 min) with a delay period (125 min). In the sample phase, two different objects (A, B) were placed in the field, and then, in the test phase, one of the objects was displaced by a novel object (A, D). A retrieval or an interference phase was inserted during the delay period for retrieval and interference groups, respectively. While two identical objects (B, B), which were the same as one of the objects in sample phase, were placed at the field in the retrieval phase, two identical objects (C, C), which were novel for animals, were placed in the interference phase
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The time spent exploring each object in the sample phase and the test phase was analyzed by a mixed-design analysis of variance (ANOVA) (Group 9 Phase 9 Object). Individual comparisons were evaluated using a simple main effect test and a post hoc Fisher’s PLSD test with alpha set at 0.05. A discrimination ratio (DR), which was calculated by dividing the amount of exploration of the novel object by the total amount of object exploration in the test phase, was analyzed by oneway ANOVA (group factor), followed by post hoc comparisons using a Fisher LSD test with alpha set at 0.05. DR data were also compared to chance level (50 %) using a one-sample t test.
Results Exploration time of each object in the sample phase and the test phase are shown in Fig. 2a. Animals in all groups explored both objects equally during the sample phase. In the test phase, animals in the control and interference groups explored the novel object longer than the familiar
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Fig. 2 a Time spent exploring each object in the sample phase and the test phase. In the test phase, rats in the retrieval group explored both objects almost equally, while rats in the control and interference groups explored the novel object longer than the familiar object. b Discrimination ratio (DR) in the test phase, calculated by dividing the amount of exploration of the novel object by the total amount of object exploration. DR of the retrieval group was significantly lower than those of the control and interference groups. In both panels, mean ± SEM is shown. *p \ .05, **p \ .01. ?p \ .05, ??p \ .01 above chance (50 %) level. Cont control group, Ret retrieval group, Int interference group
object. In contrast, rats in retrieval group explored both objects almost equally. Three-way ANOVA showed a significant main effect of object [F(1, 22) = 14.86, p \ .01], and also significant interactions between group and phase [F(2, 22) = 4.17, p \ .05], between phase and object [F(1, 22) = 11.81, p \ .01]. An analysis of the interaction between group and phase indicated that the group effect was significant only in the test phase [F(2, 22) = 6.05, p \ .01]. A post hoc comparison revealed that the exploration time in the retrieval group was significantly
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shorter than that in both control and interference groups (p \ .05). On the other hand, the phase effect was not significant in each group. Thus, there were no differences in the total amount of object exploration between the sample phase and the test phase. An analysis of the interaction between phase and object indicated that the phase effect was significant in both objects [familiar F(1, 22) = 7.79, p \ .05; novel F(1, 22) = 5.53, p \ .05]. The object effect was also significant in the test phase [F(1, 22) = 43.02, p \ .01]. With regard to DR (Fig. 2b), there was a significant main effect of group [F(2, 22) = 4.70, p \ .05]. A post hoc comparison revealed that DR of the retrieval group was significantly lower than that of the control (p \ .01) and interference groups (p \ .05). Furthermore, DRs of the control and interference groups were significantly above chance level (50 %) (control t(7) = 7.57, p \ .01; interference t(7) = 2.92, p \ .05), but that of the retrieval group was not. The total amount (mean ± SEM) of object exploration was 27.8 ± 17.7 during the retrieval phase and 37.3 ± 13.4 during the interference phase. A one-way ANOVA revealed no significant difference in the exploration time between the retrieval phase and the interference phase.
Discussion In the present study, we demonstrated, for the first time, the RIF phenomenon in rats using a modified spontaneous object recognition test. Only rats subjected to the retrieval phase, in which rats reunited with (retrieved) one of the objects presented in the sample phase, could not discriminate between the familiar (other object presented in the sample phase) and the novel objects in the test phase. Since rats that were subjected to the interference phase, in which rats met a novel object, were able to discriminate between them in the test phase, the detrimental effect of the insertion of a retrieval phase could not be attributed to proactive or retroactive interference. So, did rats forget the non-retrieved objects? In the test phase, the exploration time of the novel object in the retrieval group was shorter than in the control and interference groups, while there were no differences in the exploration time of the familiar object among the three groups. This decreased the total amount of object exploration in the retrieval group. The present findings that the retrieval phase disrupted the following object recognition seem to be attributed to the decreased exploration of the novel object. If rats subjected to the retrieval phase forgot a familiar object, or falsely recognized the familiar object as a novel object, they would have explored the familiar object longer than control rats (McTighe et al. 2010).
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A pioneering study on the spontaneous object recognition in rats, on the other hand, reported that there were no differences in total time of object exploration between two conditions—one condition in which two familiar objects were presented, the other in which two novel objects were presented (Ennaceur and Delacour 1988). In the present study, there were also no significant differences in the total amount of object exploration between the retrieval phase (two familiar objects) and the interference phase (two novel objects). In addition, the difference in the total amount of object exploration between the sample phase and the test phase in retrieval group was not significant. Given that rat’s tendency to explore a novel object more can be observed only under a specific condition, in which novel and familiar objects are presented simultaneously, the lack of increment of exploring non-retrieved object could not necessarily exclude the possibility that rats in the retrieval group forgot a familiar object, or falsely recognized the familiar object as a novel object. A possibility that the insertion of the retrieval phase decreased the motivational aspect of performance in the following test phase was ruled out, because the interference phase did not affect the performance in the test phase. Thus, we suggest tentatively that retrieval of an object impairs later recall of the related but non-retrieved object, which is like a RIF phenomenon, in spontaneous object recognition test. Recently, Whitt et al. (2012) conducted a similar experiment examining associative processes, but not RIF, in the recognition memory of rats. They found that reunion with one of the objects was able to activate the memory of the other object, if two different objects were simultaneously presented during the sample phase. Such a phenomenon in the object recognition task, which is essentially opposite to the present findings, has been explained in terms of a model of memory proposed by Brandon et al. (2003). Understanding our results through Brandon et al.’s (2003) model implies that excitatory associations could be formed between two different objects (A, B) during the sample phase. When an animal then encountered one of the objects (B, B) during the retrieval phase, memory of the other object (A) was activated indirectly. These processes should enhance the difference in familiarity between the objects (A vs. D) in the test phase, provoking a relatively strong approach to novel object (D); however, this was not the case. This discrepancy may be explained by the difference in the length of delay period inserted between the sample phase and the retrieval phase. While Whitt et al. (2012) used a 10-min delay, we used a relatively long-term delay period (60 min). Since the level of associative activation is dependent on the strength of the association between stimuli (Sanderson and Bannerman 2011), a decay of the association with time might have caused the conflicting findings.
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With regard to the neural mechanism responsible for RIF, Johansson et al. (2007) demonstrated that prefrontal event-related potentials were involved in RIF, or an inhibitory control of memory, in humans. A number of previous studies have suggested a critical role of the prefrontal cortex in the control of competitor memory traces during memory retrieval (Schnider 2003; Shimamura 2000; Thompson-Schill et al. 2005). Recently, we also reported the possibility that the medial prefrontal cortex and hippocampus are related to DF of spatial information, which is analogous to RIF (Kaku et al. 2013). Thus, the prefrontal cortex appears to be a prime candidate for involvement in RIF. The rodent’s RIF model using spontaneous object recognition test is undoubtedly useful to investigate the neural correlates of RIF. Acknowledgments The authors would like to thank Dr. Constantine Pavlides for critical reading and valuable comments on the manuscript. This study was supported by grants from Japan Society for the Promotion of Science (24653209, 24530909).
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