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Perirhinal cortex damage and anterograde object-recognition in rats after long retention intervals
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Behavioural Brain Research 185 (2007) 82–87
Research report
Perirhinal cortex damage and anterograde object-recognition in rats after long retention intervals Dave G. Mumby a,∗ , Pavel Piterkin a , Valerie Lecluse a , Hugo Lehmann b a
Center for Studies in Behavioural Neurobiology, Department of Psychology, SP-244, Concordia University, 7141 Sherbrooke St. W., Montreal, Quebec H4B 1R6, Canada b Canadian Centre for Behavioural Neuroscience, The University of Lethbridge, Canada Received 30 November 2006; received in revised form 10 July 2007; accepted 15 July 2007 Available online 21 July 2007
Abstract Damage to the perirhinal cortex (PRh) in rats impairs anterograde object-recognition memory after retention intervals of up to several hours, but there is little direct evidence to link PRh function to object-recognition abilities after substantially longer intervals that span several days or weeks. We assessed the effects of PRh lesions on anterograde object recognition using a novel-object preference test, with retention intervals lasting 24 h and 3 weeks. The rats received multiple exposures to the sample object during the learning phase—5 min per day on 5 consecutive days. Control rats displayed a significant novel-object preference after both retention intervals, indicating recognition of the sample object, whereas the rats with PRh lesions displayed a significant preference after the 24-h interval, but not after the 3-week interval. When the learning phase of the trial was shortened to a single 5-min session, the PRh group was impaired in the 24-h condition. The findings indicate that the disruptive effects of PRh damage on anterograde object recognition persist over very long postlearning intervals. The results indicate further that object recognition impairments following PRh damage are not ubiquitous, and that learning conditions play a significant role in determining the subsequent recognition performance in rats with PRh damage. © 2007 Elsevier B.V. All rights reserved. Keywords: Nonspatial memory; Object recognition; Novelty preference; Exploratory behavior; Open field
Object-recognition memory is the ability to discriminate between objects that have been previously encountered and objects that have not. This ability is often impaired in patients with medial temporal-lobe damage affecting the hippocampus (HPC) and various parahippocampal cortical areas, including the entorhrinal cortex, parahippocampal gyrus, and perirhinal cortex (PRh). Much evidence suggests the PRh is the most important temporal-lobe structure for object recognition. Damage to the PRh impairs performance on tests of anterograde object-recognition memory in rats and monkeys [1–8], whereas, damage limited to the HPC typically spares anterograde objectrecognition, at least in rats [9]. A significant number of PRh neurons display diminished responses following presentation of familiar objects as compared to novel objects, but such firing characteristics are uncommon among HPC neurons [10–13].
∗
Corresponding author. Tel.: +1 514 848 2424x2233; fax: +1 514 848 4545. E-mail address: [email protected] (D.G. Mumby).
0166-4328/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2007.07.026
Our current understanding of the cellular and molecular mechanisms that underlie memory distinguishes between processes supporting relatively short-lasting versus long-lasting memories. Some of the processes that support short-lived memories are initiated within milliseconds of an experience and persist for only a few seconds, minutes, or hours. Processes that support long-lasting memories take a relatively long time to transpire, requiring several minutes, hours, or days. Most evidence linking the PRh to object recognition comes from studies that examined relatively short-term processes. For example, studies reporting decreased responsiveness of PRh neurons to repeated presentation of objects have generally been limited to recording periods of less than an hour, although some have employed interpresentation intervals as long as 24 h [13]. Most lesion studies have assessed anterograde recognition after retention intervals lasting minutes or hours, and only a few have used intervals as long as 24 h. Accordingly, there is limited data on the role of PRh in supporting object-recognition memory at intervals that unambiguously require long-term (i.e., protein-synthesis dependent) representations—intervals spanning days or weeks. The purpose
D.G. Mumby et al. / Behavioural Brain Research 185 (2007) 82–87
of the present experiment was to assess the effects of PRh damage on object recognition after substantially longer retention intervals than in previous studies. Rats can recognize objects after intervals of up to several weeks, even when those objects lack any innate or acquired biological significance [14–16]. This ability can be demonstrated using a novel-object preference (NOP) test of object recognition [17]. On conventional versions of the NOP test, a subject is placed in an open-field arena and allowed to investigate two identical sample objects for a few minutes. The subject is then removed for a retention interval, after which it is returned to the open field, which now contains two new objects—one object is identical to the sample and the other is novel. If the retention interval is not longer than a few hours, most normal rats spend more time investigating the novel object than the sample during the first few minutes of the preference test, and when this bias is observed it is inferred that the rats recognize the sample object. A simple modification of the NOP test that promotes long-lasting recognition memory involves repeatedly exposing the rat to the sample object during the familiarization phase. We used this method in the present study to assess the effects of PRh lesions on anterograde object recognition after long retention intervals. Rats received bilateral lesions of the PRh or sham surgery, and after recovery, they received repeated exposures to a sample object in an open field—5 min per day on 5 consecutive days. A NOP test was conducted after retention intervals of 24 h and 3 weeks. The rats with PRh lesions displayed normal novel-object preference after the 24-h interval, but not after the 3-week interval. On a subsequent retest using conventional procedures, the rats with PRh lesions that received only a single 5-min sample-familiarization session failed to show a novelty preference after a 24-h interval. 1. Experiment 1 1.1. Method 1.1.1. Subjects Subjects were 32 male Long-Evans rats (Charles River, St. Constant, Quebec), 14–28 week old at the beginning of the experiment. They were housed in pairs, except during the first few days of recovery from surgery, during which time they were housed individually. The rats had continuous access to food and water in their home cages, and were under a 12-h light:12-h dark cycle, with light onset at 8:00 p.m. On most days prior to the start of the present experiment, the rats were allowed to interact in large groups (10–12 rats), for 3–6 h, inside large complex environments. This period of “enrichment” began at the age of approximately 28 days, and continued until the day before rats received surgery for this experiment. 1.1.2. Apparatus The open-field arena was constructed of gray PVC plastic and had dimensions of 60 cm × 70 cm × 70 cm high. A stainless-steel tray served as the floor and was covered with wood shavings. The floor could be removed through a slot at the bottom of one wall to facilitate changing the shavings between each trial. A videocamera was positioned over the arena and familiarization and test phases were videotaped for later analysis. The test stimuli consisted of six different objects made of glass, porcelain, or glazed ceramic. The objects varied in height between approximately 6 and 12 cm, and in width between 6 and 10 cm. Attached with epoxy to the bottom of each object was a small glass jar (6 cm high), and attached to the floor of the arena were two inverted jar lids, each positioned 27 cm from opposing corners
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Table 1 Coordinates (in millimeters) relative to bregma for the sites at which current was delivered for the electrolytic lesions of the perirhinal cortex Anterior–posterior
Medial–lateral
Dorsal–ventral
3.5 4.5 5.5 6.5 7.5
8.5 8.5 8.5 8.5 8.5
9.2 9.2 9.2 9.2 8.4
of the arena. Objects were fixed in place by screwing the jars into the lids. There were four copies of each object, which were used interchangeably. The objects were washed after each use with a solution of household bleach, thinly diluted in water (5% solution). 1.1.3. Surgery Rats received electrolytic lesions of the perirhinal cortex (Group PRh, n = 16), or sham surgery (Group SHAM, n = 16). Surgery was performed under isoflorane anesthesia. A scalp incision was made, and the muscle overlying the temporal skull was displaced. A portion of skull overlying the traget area was removed using a hand-held drill. A bipolar stainless steel electrode insulated with Teflon except for approximately 1 mm at the tip, and angled at 10◦ to the vertical plane, was used to deliver electric current (1.5 mA for 10 s) to five sites per hemisphere through the PRh (coordinates are shown in Table 1). Sham-surgery control rats received similar treatment, except that no damage was made to the skull or brain. All rats were allowed to recover for 2 weeks before behavioral testing began. All procedures were approved by the Concordia University Animal Care and Use Committee, and were in accordance with guidelines of the Canadian Council for Animal Care. 1.1.4. Behavioral procedures After the 2-week postsurgery recovery period, the rats were habituated to the open field by placing them in it, individually, for 20 min, on 2 consecutive days. Two identical objects were present during habituation sessions, but those objects were not used in the subsequent experimental sessions. Testing on the NOP task began for each rat within 3 days of its final habituation session. The six test objects were divided into three object-pairs, which served as the sample and novel objects for the three NOP trials that most subjects received. Rats received a single familiarization session each day, on 5 consecutive days. On each of these sessions, a rat spent 5 min in the open field with two copies of the sample object, and time spent investigating the sample objects was measured. After a retention interval, the rat was returned to the open field, which now contained a copy of the sample object and a novel object. Object investigation was measured during a 3-min preference test. Each rat received two NOP trials—one with a 24-h retention interval between the last familiarization session and the test session, and the other with a 3-week interval (order counterbalanced within each group). A different pair of sample and novel objects were used for the two trials. One object of each pair served as the sample object for half of the rats in each group, and the other object of the pair served as the sample for the other half of the rats. Thus, all rats encountered the same two objects during a particular test session. The main dependent measure was the investigation ratio—the proportion of total object-investigation during the test phase that was spent investigating the novel object [tnovel /(tnovel + tsample )]. A rat was considered to be investigating an object when its head was oriented within 45◦ of the object and within 4 cm of it. Rearing with the head oriented upward was also included if at least one forepaw was on the object. Climbing over the objects or sitting on them was not included. The experimenter who scored the videotapes was blind to the treatment of individual rats. 1.1.5. Histological procedures At the completion of behavioral testing all rats were sacrificed using a lethal dose of sodium pentobarbital (100 mg/kg, i.p.). They were perfused with saline solution followed by 10% formalin solution. Their brains were excised and
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Fig. 1. The location and extent of the largest (gray) and smallest (black) PRh lesions at three coronal planes (relative to bregma, in mm). Arrows indicate the approximate dorsal and ventral boundaries of the perirhinal cortex, based on Burwell et al. [28]. stored in 10% formalin/30% sucrose solution until sectioning. The brains were frozen-sectioned at 40 m, every fifth section through the PRh was mounted on a glass microscope slide, and stained with cresyl violet.
1.2. Results 1.2.1. Histology Fig. 1 illustrates the largest and smallest PRh lesion in each hemisphere. The perirhinal cortex was nearly completely destroyed bilaterally in each PRh rat, and each also had minor bilateral damage to portions of lateral entorhinal cortex. Most of the lesions included slight to moderate damage to ventroposterior portions of area Te2. Each PRh rat also sustained minor damage to anterior portions of the postrhinal cortex, in either one or both hemispheres; estimated damage to postrhinal cortex ranged from 0 to 30%. There was minor unilateral damage to the ventral subiculum and temporal CA1 field in one PRh rat. 1.2.2. Behavioral results Fig. 2 shows the time each group spent investigating the sample objects during the five familiarization sessions for each condition; trials with 24-h and 3-week intervals were averaged for each rat. The groups did not differ significantly at any point during this phase, nor was there a significant difference in the overall amount of object investigation during the familiarization phase. A repeated-measures analysis of variance (ANOVA) revealed a nonsignificant main effect of Group, F(1, 30) = 0.02, p > .10, a nonsignificant main effect of Time, F(4, 120) = 1.35, p > .10 and a nonsignificant interaction, F(4, 120) = 0.53, p > .10.
The left panel of Fig. 3 shows the mean investigation ratios during the 3-min test session for each condition. One-sample t-tests indicated that both the SHAM group and the PRh group displayed a significant novel-object preference after the 24-h interval (for both groups, p < .05, one-tailed). After the 3-week interval, SHAM rats displayed a novelty preference, whereas the PRh rats did not. An ANOVA comparing scores across all conditions revealed a significant main effect of Interval, F(1, 26) = 4.91, p = .036, nonsignificant effect of Group, F(1, 26) < 1, and a significant interaction, F(1, 26) = 6.90, p = .014. Scores in the PRh group were significantly lower after the 3-week interval than after the 24-h interval t(12) = 4.26, p < .01, whereas the
Fig. 2. Left: Time spent investigating the sample objects during each of the five sample-exposure sessions in Experiment 1. Right: Total sample-object investigation combined for all sessions. Error bars represent SEMs.
D.G. Mumby et al. / Behavioural Brain Research 185 (2007) 82–87
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Fig. 3. Left: Mean investigation ratios in each condition of Experiment 1. Right: Time spent investigating objects during the test sessions of Experiment 1. Error bars represent SEMs. Fig. 4. Left: Mean investigation ratios in Experiment 2. Right: Time spent investigating objects during the familiarization session. Error bars represent SEMs.
scores in the SHAM group were not significantly different at the two delays. The right panel of Fig. 3 shows how much time was spent investigating objects during the test session. Increasing the retention interval from 24 h to 3 weeks led to a significant decrease in object investigation, F(1, 26) = 24.99, p < .001. The two groups showed similar levels of object investigation, F(1, 26) < 1, and there was a nonsignificant Group × Interval interaction, F(1,26) = 2.108, p > .10.
tigating the sample objects during the familiarization session, t(20) = .302, p > .05. A dependent-samples t-test revealed that the mean investigation ratios in the PRh group were significantly lower than they were following the 24-h retention interval in Experiment 1, t(13) = 3.03, p < .05), indicating a significant effect of the variation in familiarization procedures across the two experiments. The performance of control rats after the 24-h interval was not significantly different in the two experiments (p > .10).
2. Experiment 2 3. Discussion The results from the 24-h condition in Experiment 1 were unexpected, because there are previous reports of NOP deficits in rats with PRh lesions after much shorter retention intervals (e.g., [15,18,19]). The most obvious procedural difference between Experiment 1 and the previous studies is the number of familiarization sessions—five versus one. The purpose of Experiment 2 was to determine whether our rats’ PRh lesions would produce NOP deficits under conditions that were previously shown to be sensitive to bilateral PRh lesions; namely, after a single 5-min familiarization session and a long retention interval. 2.1. Method This experiment began approximately 2 weeks following the end of Experiment 1. A subset of the SHAM rats from Experiment 1 (n = 7), and all but one of the PRh rats (n = 15) received an additional NOP trial with a 24-h retention interval, but this time they received only a single 5-min familiarization session with the sample object. None of the rats had previously encountered the two objects that were used as the sample and novel objects. The assignment of a particular object from this pair as the sample versus novel object was roughly counterbalanced within each group.
2.2. Results The left panel of Fig. 4 shows the mean investigation ratios during the first minute of the test session. The SHAM group displayed a novel-object preference that fell slightly short of statistical significance (p = .068), whereas the PRh group clearly did not show a novel-object preference. Moreover, investigation ratios were significantly higher in the SHAM group than in the PRh group, t(20) = 2.11, p < .05. The right panel of Fig. 4 shows that the two groups spent a similar amount of time inves-
The main finding of Experiment 1 was that rats with PRh lesions displayed a delay-dependent deficit on the NOP test when multiple exposures to the sample object were provided during the 5-day familiarization phase. They performed like control rats after a 24-h interval, displaying a significant novelobject preference. Unlike the control rats, however, PRh-lesion rats did not display a significant preference after the 3-week interval. This is, arguably, the first demonstration that the disruptive effects of PRh damage on object recognition in rats persist over very long retention intervals—intervals that unambiguously require the operation of mechanisms leading to long-lasting representations. The impaired test performance by the rats with PRh lesions does not appear to be due to some disadvantage those rats had during the learning phase, because the groups did not differ in the amount of time spent investigating the sample object over the familiarization phase. The main finding of Experiment 2 was that limiting the familiarization phase to a single session revealed a deficit after a 24-h retention interval in the rats with PRh lesions. Several studies have reported deficits in novel-object preference after PRh damage with retention intervals of 24 h, or less. Accordingly, the results of Experiment 2, when considered alone, are not surprising. They are informative, however, when compared to the normal performance of the same rats after a 24-h retention interval in Experiment 1. To the best of our knowledge, this is the first demonstration that the object-recognition impairment displayed by rats with PRh damage can be overcome by changing the nature of the learning conditions. In this case, the change was an increase in the number of prior exposures to a sample object, from once, to five times. We cannot argue from our data that
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five exposures enhanced memory of the sample objects, because control rats’ performance after a 24-h interval was not better in Experiment 1 when they had five familiarization sessions than it was in Experiment 2 when they had only a single familiarization session. Still, at least two factors associated with repeated sample exposure are likely to affect the persistence of the representations that underlie object-recognition memory: One factor is the amount of time spent investigating the sample object during the familiarization phase—rats obviously spend more time overall investigating the sample objects when they have five 5min sessions than when they have only one 5-min session. A second factor relates to the well-known phenomenon that information acquired over multiple, distributed training sessions is often better retained than information acquired during massed training [20]. This phenomenon also occurs on tests of Pavlovian conditioning in rats [21–24]. We cannot determine from our data the relative importance of time exposed to the sample objects during familiarization versus the temporal spacing of that exposure, because as the number of sample familiarization sessions was decreased from five to one, so was the total amount of time the rats were exposed to the sample objects. When speculating about potential mechanisms by which object recognition after a 24-h interval in rats with PRh lesions would be facilitated by repeated sample exposure, it is interesting to note that certain molecular events that lead to long-lasting changes in synaptic efficacy show a similar dependence on repetition and appropriate spacing of learning (or induction) episodes; for example, sustained elevation of cAMP-dependent protein kinase is induced by repeated tetanization of the perforant path and leads to long-lasting long-term enhancement and synaptogensis in the hippocampus [25]. The point here is that there are good reasons to suggest that five spaced sample exposures may activate certain plasticity mechanisms that are not activated by a single exposure. The resilience of 24-h performance in rats with PRh damage after multiple sample-exposures cannot be easily explained by supposing that multiple exposures engaged PRh-independent mechanisms that were not engaged by a single exposure, because performance was impaired after a 3-week interval following multiple exposures. One possibility, however, is that such supposed PRh-independent mechanisms support representations that are forgotten at a faster rate than those supported by the PRh-dependent mechanisms that presumably operated normally in the control rats and supported object recognition after the 3-week interval. This is the interpretation we give for our results. The present results in rats with PRh lesions can be contrasted with previous results in rats with HPC lesions. In one study that used familiarization and test procedures that were nearly identical to those of Experiment 1, rats with extensive bilateral HPC lesions displayed normal novelty preference after retention intervals as long as 3 weeks [16]. Normal performance in rats with HPC damage has also been reported in studies that used the conventional procedure of providing only a single 5-min sampleexposure, as we did in Experiment 2 [14,19,26,27]. It should be noted, also, that rats with posttraining PRh lesions display retrograde impairments on the NOP task [15,27]. Interestingly, unlike the different NOP results seen after pretraining lesions of
PRh versus HPC, the results are the same on retrograde NOP tests after postraining lesions of the PRh or HPC; retrograde deficits follow bilateral damage to either the PRh or the HPC [14]. Lesion studies in rats have now shown that the PRh plays an essential role in enabling normal object-recognition memory throughout a considerable range of retention intervals, lasting from a few seconds to a few weeks. The present data indicate further that anterograde object-recognition deficits following PRh damage can be attenuated by altering the learning conditions. They suggest that PRh-independent mechanisms can support object-recognition memory after repeated exposure to an object, but not after a single exposure. One important direction to go from here will be to learn more about the factors that attenuate object-recognition deficits after PRh damage, including the independent contributions of time spent investigating the sample object and the spacing of sample-object exposures during the learning phase. Acknowledgement This research was supported by The Natural Science and Engineering Research Council (NSERC) of Canada, grant number 156937-02. References [1] Aggleton JP, Brown MW. Episodic memory, amnesia, and the hippocampalanterior thalamic axis. Behav Brain Sci 1999;22:425–44. [2] Aggleton JP, Keen S, Warburton EC, Bussey TJ. Extensive cytotoxic lesions involving both the rhinal cortices and area TE impair recognition but spare spatial alternation in the rat. Brain Res Bull 1997;43:279–87. [3] Mumby DG, Pinel JPJ. Rhinal cortex lesions and object recognition in rats. Behav Neurosci 1994;108:11–8. [4] Gaffan D, Murray EA. Monkeys with rhinal cortex lesions succeed in object discrimination learning despite 24-hour intertrial intervals and fail at match to sample despite double sample presentations. Behav Neurosci 1992;106:30–8. [5] Meunier M, Bachevalier J, Mishkin M, Murray EA. Effects on visual recognition of combined and separate ablations of the entorhinal and perirhinal cortex in rhesus monkeys. J Neurosci 1993;13:5418–32. [6] Murray EA. What have ablation studies told us about the neural substrates of stimulus memory? Sem Neurosci 1996;8:13–22. [7] Wiig KA, Bilkey DK. Lesions of rat perirhinal cortex exacerbate the memory deficit observed following damage to the fimbria–fornix. Behav Neurosci 1995;109:620–30. [8] Zola-Morgan S, Squire LR, Amaral DG, Suzuki WA. Lesions of the perirhinal and parahippocampal cortex that spare the amygdala and hippocampal formation produce severe memory impairment. J Neurosci 1989;9:4355–70. [9] Mumby DG. Perspectives on object-recognition memory following hippocampal damage: lessons from studies in rats. Behav Brain Res 2001;127:159–82. [10] Brown MW, Aggleton JP. Recognition memory: what are the roles of the perirhinal cortex and hippocampus? Nat Rev Neurosci 2001;2:51–61. [11] Brown MW, Bashir ZI. Evidence concerning how neurones of the perirhinal cortex may effect familiarity discrimination. Philos Trans R Soc Lond B 2002;357:1083–95. [12] Brown MW, Xiang J-Z. Recognition memory: neuronal substrates of the judgement of prior occurrence. Prog Neurobiol 1998;55:1–41. [13] Xiang JZ, Brown MW. Differential neuronal encoding of novelty, familiarity and recency in regions of the anterior temporal lobe. Neuropharmacol 1998;37:657–76.
D.G. Mumby et al. / Behavioural Brain Research 185 (2007) 82–87 [14] Gaskin S, Tremblay A, Mumby DG. Anterograde and retrograde objectrecognition memory in rats with hippocampal lesions. Hippocampus 2003;13:962–9. [15] Mumby DG, Glenn MJ, Nesbitt CE, Kyriazis DA. Dissociation of object recognition and object discrimination in retrograde memory following lesions of perirhinal cortex in rats. Behav Brain Res 2002;132:215–26. [16] Mumby DG, Tremblay A, Lecluse V, Lehmann H. Hippocampal damage and anterograde object-recognition in rats after long retention intervals. Hippocampus 2005;15:1050–6. [17] Ennaceur A, Delacour J. A new one-trial test for neurobiological studies of memory in rats. I: Behavioral data. Behav Brain Res 1988;31: 47–59. [18] Bussey TJ, Muir JL, Aggleton JP. Functionally dissociating aspects of event memory: the effects of combined perirhinal and postrhinal cortex lesions on object and place memory in the rat. J Neurosci 1999;19: 495–502. [19] Winters BD, Forwood SE, Cowell RA, Saksida LM, Bussey TJ. Double dissociation between hippocampus and perirhinal cortex on tests of spatial and object recognition memory: heterogeneity of function within the medial temporal lobe. J Neurosci 2004;24:5901–8. [20] Keppel G. Facilitation in short- and long-term retention of paired associates following distributed practice in learning. J Verb Learn Verb Behav 1964;3:91–111.
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[21] Fanselow MS, DeCola JP, Young SL. Mechanisms responsible for reduced contextual conditioning with massed unsignaled unconditional stimuli. J Exp Psychol Anim Behav Process 1993;19:121–37. [22] Fanselow MS, Tighe TJ. Contextual conditioning with massed versus distributed unconditional stimuli in the absence of explicit conditional stimuli. J Exp Psychol Anim Behav Process 1988;14:187–99. [23] Williams DA, Frame KA, LoLordo VM. Reexamination of contextual conditioning with massed versus distributed unconditioned stimuli. J Exp Psychol Anim Behav Process 1991;17:202–9. [24] Yin H, Barnet RC, Miller RR. Trial spacing and trial distribution effects in Pavlovian conditioning: contributions of a comparator mechanism. J Exp Psychol Anim Behav Process 1994;20:123–34. [25] Kandel ER. The molecular biology of memory storage: a dialogue between genes and synapses. Science 2001;294:1030–8. [26] Forwood SE, Winters BD, Bussey TJ. Hippocampal lesions that abolish spatial maze performance spare object recognition memory at delays of up to 48 h. Hippocampus 2005;15:347–55. [27] Mumby DG, Gaskin S, Glenn MJ, Schramek TE, Lehmann H. Hippocampal damage and exploratory preferences in rats: memory for objects, places, and contexts. Learn Mem 2002;9:47–57. [28] Burwell RD, Witter MP, Amaral DG. Perirhinal and postrhinal cortices of the rat: a review of the neuroanatomical literature and comparison with findings from the monkey brain. Hippocampus 1995;5:390–408.
Behavioural Brain Research 185 (2007) 82–87
Research report
Perirhinal cortex damage and anterograde object-recognition in rats after long retention intervals Dave G. Mumby a,∗ , Pavel Piterkin a , Valerie Lecluse a , Hugo Lehmann b a
Center for Studies in Behavioural Neurobiology, Department of Psychology, SP-244, Concordia University, 7141 Sherbrooke St. W., Montreal, Quebec H4B 1R6, Canada b Canadian Centre for Behavioural Neuroscience, The University of Lethbridge, Canada Received 30 November 2006; received in revised form 10 July 2007; accepted 15 July 2007 Available online 21 July 2007
Abstract Damage to the perirhinal cortex (PRh) in rats impairs anterograde object-recognition memory after retention intervals of up to several hours, but there is little direct evidence to link PRh function to object-recognition abilities after substantially longer intervals that span several days or weeks. We assessed the effects of PRh lesions on anterograde object recognition using a novel-object preference test, with retention intervals lasting 24 h and 3 weeks. The rats received multiple exposures to the sample object during the learning phase—5 min per day on 5 consecutive days. Control rats displayed a significant novel-object preference after both retention intervals, indicating recognition of the sample object, whereas the rats with PRh lesions displayed a significant preference after the 24-h interval, but not after the 3-week interval. When the learning phase of the trial was shortened to a single 5-min session, the PRh group was impaired in the 24-h condition. The findings indicate that the disruptive effects of PRh damage on anterograde object recognition persist over very long postlearning intervals. The results indicate further that object recognition impairments following PRh damage are not ubiquitous, and that learning conditions play a significant role in determining the subsequent recognition performance in rats with PRh damage. © 2007 Elsevier B.V. All rights reserved. Keywords: Nonspatial memory; Object recognition; Novelty preference; Exploratory behavior; Open field
Object-recognition memory is the ability to discriminate between objects that have been previously encountered and objects that have not. This ability is often impaired in patients with medial temporal-lobe damage affecting the hippocampus (HPC) and various parahippocampal cortical areas, including the entorhrinal cortex, parahippocampal gyrus, and perirhinal cortex (PRh). Much evidence suggests the PRh is the most important temporal-lobe structure for object recognition. Damage to the PRh impairs performance on tests of anterograde object-recognition memory in rats and monkeys [1–8], whereas, damage limited to the HPC typically spares anterograde objectrecognition, at least in rats [9]. A significant number of PRh neurons display diminished responses following presentation of familiar objects as compared to novel objects, but such firing characteristics are uncommon among HPC neurons [10–13].
∗
Corresponding author. Tel.: +1 514 848 2424x2233; fax: +1 514 848 4545. E-mail address: [email protected] (D.G. Mumby).
0166-4328/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2007.07.026
Our current understanding of the cellular and molecular mechanisms that underlie memory distinguishes between processes supporting relatively short-lasting versus long-lasting memories. Some of the processes that support short-lived memories are initiated within milliseconds of an experience and persist for only a few seconds, minutes, or hours. Processes that support long-lasting memories take a relatively long time to transpire, requiring several minutes, hours, or days. Most evidence linking the PRh to object recognition comes from studies that examined relatively short-term processes. For example, studies reporting decreased responsiveness of PRh neurons to repeated presentation of objects have generally been limited to recording periods of less than an hour, although some have employed interpresentation intervals as long as 24 h [13]. Most lesion studies have assessed anterograde recognition after retention intervals lasting minutes or hours, and only a few have used intervals as long as 24 h. Accordingly, there is limited data on the role of PRh in supporting object-recognition memory at intervals that unambiguously require long-term (i.e., protein-synthesis dependent) representations—intervals spanning days or weeks. The purpose
D.G. Mumby et al. / Behavioural Brain Research 185 (2007) 82–87
of the present experiment was to assess the effects of PRh damage on object recognition after substantially longer retention intervals than in previous studies. Rats can recognize objects after intervals of up to several weeks, even when those objects lack any innate or acquired biological significance [14–16]. This ability can be demonstrated using a novel-object preference (NOP) test of object recognition [17]. On conventional versions of the NOP test, a subject is placed in an open-field arena and allowed to investigate two identical sample objects for a few minutes. The subject is then removed for a retention interval, after which it is returned to the open field, which now contains two new objects—one object is identical to the sample and the other is novel. If the retention interval is not longer than a few hours, most normal rats spend more time investigating the novel object than the sample during the first few minutes of the preference test, and when this bias is observed it is inferred that the rats recognize the sample object. A simple modification of the NOP test that promotes long-lasting recognition memory involves repeatedly exposing the rat to the sample object during the familiarization phase. We used this method in the present study to assess the effects of PRh lesions on anterograde object recognition after long retention intervals. Rats received bilateral lesions of the PRh or sham surgery, and after recovery, they received repeated exposures to a sample object in an open field—5 min per day on 5 consecutive days. A NOP test was conducted after retention intervals of 24 h and 3 weeks. The rats with PRh lesions displayed normal novel-object preference after the 24-h interval, but not after the 3-week interval. On a subsequent retest using conventional procedures, the rats with PRh lesions that received only a single 5-min sample-familiarization session failed to show a novelty preference after a 24-h interval. 1. Experiment 1 1.1. Method 1.1.1. Subjects Subjects were 32 male Long-Evans rats (Charles River, St. Constant, Quebec), 14–28 week old at the beginning of the experiment. They were housed in pairs, except during the first few days of recovery from surgery, during which time they were housed individually. The rats had continuous access to food and water in their home cages, and were under a 12-h light:12-h dark cycle, with light onset at 8:00 p.m. On most days prior to the start of the present experiment, the rats were allowed to interact in large groups (10–12 rats), for 3–6 h, inside large complex environments. This period of “enrichment” began at the age of approximately 28 days, and continued until the day before rats received surgery for this experiment. 1.1.2. Apparatus The open-field arena was constructed of gray PVC plastic and had dimensions of 60 cm × 70 cm × 70 cm high. A stainless-steel tray served as the floor and was covered with wood shavings. The floor could be removed through a slot at the bottom of one wall to facilitate changing the shavings between each trial. A videocamera was positioned over the arena and familiarization and test phases were videotaped for later analysis. The test stimuli consisted of six different objects made of glass, porcelain, or glazed ceramic. The objects varied in height between approximately 6 and 12 cm, and in width between 6 and 10 cm. Attached with epoxy to the bottom of each object was a small glass jar (6 cm high), and attached to the floor of the arena were two inverted jar lids, each positioned 27 cm from opposing corners
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Table 1 Coordinates (in millimeters) relative to bregma for the sites at which current was delivered for the electrolytic lesions of the perirhinal cortex Anterior–posterior
Medial–lateral
Dorsal–ventral
3.5 4.5 5.5 6.5 7.5
8.5 8.5 8.5 8.5 8.5
9.2 9.2 9.2 9.2 8.4
of the arena. Objects were fixed in place by screwing the jars into the lids. There were four copies of each object, which were used interchangeably. The objects were washed after each use with a solution of household bleach, thinly diluted in water (5% solution). 1.1.3. Surgery Rats received electrolytic lesions of the perirhinal cortex (Group PRh, n = 16), or sham surgery (Group SHAM, n = 16). Surgery was performed under isoflorane anesthesia. A scalp incision was made, and the muscle overlying the temporal skull was displaced. A portion of skull overlying the traget area was removed using a hand-held drill. A bipolar stainless steel electrode insulated with Teflon except for approximately 1 mm at the tip, and angled at 10◦ to the vertical plane, was used to deliver electric current (1.5 mA for 10 s) to five sites per hemisphere through the PRh (coordinates are shown in Table 1). Sham-surgery control rats received similar treatment, except that no damage was made to the skull or brain. All rats were allowed to recover for 2 weeks before behavioral testing began. All procedures were approved by the Concordia University Animal Care and Use Committee, and were in accordance with guidelines of the Canadian Council for Animal Care. 1.1.4. Behavioral procedures After the 2-week postsurgery recovery period, the rats were habituated to the open field by placing them in it, individually, for 20 min, on 2 consecutive days. Two identical objects were present during habituation sessions, but those objects were not used in the subsequent experimental sessions. Testing on the NOP task began for each rat within 3 days of its final habituation session. The six test objects were divided into three object-pairs, which served as the sample and novel objects for the three NOP trials that most subjects received. Rats received a single familiarization session each day, on 5 consecutive days. On each of these sessions, a rat spent 5 min in the open field with two copies of the sample object, and time spent investigating the sample objects was measured. After a retention interval, the rat was returned to the open field, which now contained a copy of the sample object and a novel object. Object investigation was measured during a 3-min preference test. Each rat received two NOP trials—one with a 24-h retention interval between the last familiarization session and the test session, and the other with a 3-week interval (order counterbalanced within each group). A different pair of sample and novel objects were used for the two trials. One object of each pair served as the sample object for half of the rats in each group, and the other object of the pair served as the sample for the other half of the rats. Thus, all rats encountered the same two objects during a particular test session. The main dependent measure was the investigation ratio—the proportion of total object-investigation during the test phase that was spent investigating the novel object [tnovel /(tnovel + tsample )]. A rat was considered to be investigating an object when its head was oriented within 45◦ of the object and within 4 cm of it. Rearing with the head oriented upward was also included if at least one forepaw was on the object. Climbing over the objects or sitting on them was not included. The experimenter who scored the videotapes was blind to the treatment of individual rats. 1.1.5. Histological procedures At the completion of behavioral testing all rats were sacrificed using a lethal dose of sodium pentobarbital (100 mg/kg, i.p.). They were perfused with saline solution followed by 10% formalin solution. Their brains were excised and
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Fig. 1. The location and extent of the largest (gray) and smallest (black) PRh lesions at three coronal planes (relative to bregma, in mm). Arrows indicate the approximate dorsal and ventral boundaries of the perirhinal cortex, based on Burwell et al. [28]. stored in 10% formalin/30% sucrose solution until sectioning. The brains were frozen-sectioned at 40 m, every fifth section through the PRh was mounted on a glass microscope slide, and stained with cresyl violet.
1.2. Results 1.2.1. Histology Fig. 1 illustrates the largest and smallest PRh lesion in each hemisphere. The perirhinal cortex was nearly completely destroyed bilaterally in each PRh rat, and each also had minor bilateral damage to portions of lateral entorhinal cortex. Most of the lesions included slight to moderate damage to ventroposterior portions of area Te2. Each PRh rat also sustained minor damage to anterior portions of the postrhinal cortex, in either one or both hemispheres; estimated damage to postrhinal cortex ranged from 0 to 30%. There was minor unilateral damage to the ventral subiculum and temporal CA1 field in one PRh rat. 1.2.2. Behavioral results Fig. 2 shows the time each group spent investigating the sample objects during the five familiarization sessions for each condition; trials with 24-h and 3-week intervals were averaged for each rat. The groups did not differ significantly at any point during this phase, nor was there a significant difference in the overall amount of object investigation during the familiarization phase. A repeated-measures analysis of variance (ANOVA) revealed a nonsignificant main effect of Group, F(1, 30) = 0.02, p > .10, a nonsignificant main effect of Time, F(4, 120) = 1.35, p > .10 and a nonsignificant interaction, F(4, 120) = 0.53, p > .10.
The left panel of Fig. 3 shows the mean investigation ratios during the 3-min test session for each condition. One-sample t-tests indicated that both the SHAM group and the PRh group displayed a significant novel-object preference after the 24-h interval (for both groups, p < .05, one-tailed). After the 3-week interval, SHAM rats displayed a novelty preference, whereas the PRh rats did not. An ANOVA comparing scores across all conditions revealed a significant main effect of Interval, F(1, 26) = 4.91, p = .036, nonsignificant effect of Group, F(1, 26) < 1, and a significant interaction, F(1, 26) = 6.90, p = .014. Scores in the PRh group were significantly lower after the 3-week interval than after the 24-h interval t(12) = 4.26, p < .01, whereas the
Fig. 2. Left: Time spent investigating the sample objects during each of the five sample-exposure sessions in Experiment 1. Right: Total sample-object investigation combined for all sessions. Error bars represent SEMs.
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Fig. 3. Left: Mean investigation ratios in each condition of Experiment 1. Right: Time spent investigating objects during the test sessions of Experiment 1. Error bars represent SEMs. Fig. 4. Left: Mean investigation ratios in Experiment 2. Right: Time spent investigating objects during the familiarization session. Error bars represent SEMs.
scores in the SHAM group were not significantly different at the two delays. The right panel of Fig. 3 shows how much time was spent investigating objects during the test session. Increasing the retention interval from 24 h to 3 weeks led to a significant decrease in object investigation, F(1, 26) = 24.99, p < .001. The two groups showed similar levels of object investigation, F(1, 26) < 1, and there was a nonsignificant Group × Interval interaction, F(1,26) = 2.108, p > .10.
tigating the sample objects during the familiarization session, t(20) = .302, p > .05. A dependent-samples t-test revealed that the mean investigation ratios in the PRh group were significantly lower than they were following the 24-h retention interval in Experiment 1, t(13) = 3.03, p < .05), indicating a significant effect of the variation in familiarization procedures across the two experiments. The performance of control rats after the 24-h interval was not significantly different in the two experiments (p > .10).
2. Experiment 2 3. Discussion The results from the 24-h condition in Experiment 1 were unexpected, because there are previous reports of NOP deficits in rats with PRh lesions after much shorter retention intervals (e.g., [15,18,19]). The most obvious procedural difference between Experiment 1 and the previous studies is the number of familiarization sessions—five versus one. The purpose of Experiment 2 was to determine whether our rats’ PRh lesions would produce NOP deficits under conditions that were previously shown to be sensitive to bilateral PRh lesions; namely, after a single 5-min familiarization session and a long retention interval. 2.1. Method This experiment began approximately 2 weeks following the end of Experiment 1. A subset of the SHAM rats from Experiment 1 (n = 7), and all but one of the PRh rats (n = 15) received an additional NOP trial with a 24-h retention interval, but this time they received only a single 5-min familiarization session with the sample object. None of the rats had previously encountered the two objects that were used as the sample and novel objects. The assignment of a particular object from this pair as the sample versus novel object was roughly counterbalanced within each group.
2.2. Results The left panel of Fig. 4 shows the mean investigation ratios during the first minute of the test session. The SHAM group displayed a novel-object preference that fell slightly short of statistical significance (p = .068), whereas the PRh group clearly did not show a novel-object preference. Moreover, investigation ratios were significantly higher in the SHAM group than in the PRh group, t(20) = 2.11, p < .05. The right panel of Fig. 4 shows that the two groups spent a similar amount of time inves-
The main finding of Experiment 1 was that rats with PRh lesions displayed a delay-dependent deficit on the NOP test when multiple exposures to the sample object were provided during the 5-day familiarization phase. They performed like control rats after a 24-h interval, displaying a significant novelobject preference. Unlike the control rats, however, PRh-lesion rats did not display a significant preference after the 3-week interval. This is, arguably, the first demonstration that the disruptive effects of PRh damage on object recognition in rats persist over very long retention intervals—intervals that unambiguously require the operation of mechanisms leading to long-lasting representations. The impaired test performance by the rats with PRh lesions does not appear to be due to some disadvantage those rats had during the learning phase, because the groups did not differ in the amount of time spent investigating the sample object over the familiarization phase. The main finding of Experiment 2 was that limiting the familiarization phase to a single session revealed a deficit after a 24-h retention interval in the rats with PRh lesions. Several studies have reported deficits in novel-object preference after PRh damage with retention intervals of 24 h, or less. Accordingly, the results of Experiment 2, when considered alone, are not surprising. They are informative, however, when compared to the normal performance of the same rats after a 24-h retention interval in Experiment 1. To the best of our knowledge, this is the first demonstration that the object-recognition impairment displayed by rats with PRh damage can be overcome by changing the nature of the learning conditions. In this case, the change was an increase in the number of prior exposures to a sample object, from once, to five times. We cannot argue from our data that
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five exposures enhanced memory of the sample objects, because control rats’ performance after a 24-h interval was not better in Experiment 1 when they had five familiarization sessions than it was in Experiment 2 when they had only a single familiarization session. Still, at least two factors associated with repeated sample exposure are likely to affect the persistence of the representations that underlie object-recognition memory: One factor is the amount of time spent investigating the sample object during the familiarization phase—rats obviously spend more time overall investigating the sample objects when they have five 5min sessions than when they have only one 5-min session. A second factor relates to the well-known phenomenon that information acquired over multiple, distributed training sessions is often better retained than information acquired during massed training [20]. This phenomenon also occurs on tests of Pavlovian conditioning in rats [21–24]. We cannot determine from our data the relative importance of time exposed to the sample objects during familiarization versus the temporal spacing of that exposure, because as the number of sample familiarization sessions was decreased from five to one, so was the total amount of time the rats were exposed to the sample objects. When speculating about potential mechanisms by which object recognition after a 24-h interval in rats with PRh lesions would be facilitated by repeated sample exposure, it is interesting to note that certain molecular events that lead to long-lasting changes in synaptic efficacy show a similar dependence on repetition and appropriate spacing of learning (or induction) episodes; for example, sustained elevation of cAMP-dependent protein kinase is induced by repeated tetanization of the perforant path and leads to long-lasting long-term enhancement and synaptogensis in the hippocampus [25]. The point here is that there are good reasons to suggest that five spaced sample exposures may activate certain plasticity mechanisms that are not activated by a single exposure. The resilience of 24-h performance in rats with PRh damage after multiple sample-exposures cannot be easily explained by supposing that multiple exposures engaged PRh-independent mechanisms that were not engaged by a single exposure, because performance was impaired after a 3-week interval following multiple exposures. One possibility, however, is that such supposed PRh-independent mechanisms support representations that are forgotten at a faster rate than those supported by the PRh-dependent mechanisms that presumably operated normally in the control rats and supported object recognition after the 3-week interval. This is the interpretation we give for our results. The present results in rats with PRh lesions can be contrasted with previous results in rats with HPC lesions. In one study that used familiarization and test procedures that were nearly identical to those of Experiment 1, rats with extensive bilateral HPC lesions displayed normal novelty preference after retention intervals as long as 3 weeks [16]. Normal performance in rats with HPC damage has also been reported in studies that used the conventional procedure of providing only a single 5-min sampleexposure, as we did in Experiment 2 [14,19,26,27]. It should be noted, also, that rats with posttraining PRh lesions display retrograde impairments on the NOP task [15,27]. Interestingly, unlike the different NOP results seen after pretraining lesions of
PRh versus HPC, the results are the same on retrograde NOP tests after postraining lesions of the PRh or HPC; retrograde deficits follow bilateral damage to either the PRh or the HPC [14]. Lesion studies in rats have now shown that the PRh plays an essential role in enabling normal object-recognition memory throughout a considerable range of retention intervals, lasting from a few seconds to a few weeks. The present data indicate further that anterograde object-recognition deficits following PRh damage can be attenuated by altering the learning conditions. They suggest that PRh-independent mechanisms can support object-recognition memory after repeated exposure to an object, but not after a single exposure. One important direction to go from here will be to learn more about the factors that attenuate object-recognition deficits after PRh damage, including the independent contributions of time spent investigating the sample object and the spacing of sample-object exposures during the learning phase. Acknowledgement This research was supported by The Natural Science and Engineering Research Council (NSERC) of Canada, grant number 156937-02. References [1] Aggleton JP, Brown MW. Episodic memory, amnesia, and the hippocampalanterior thalamic axis. Behav Brain Sci 1999;22:425–44. [2] Aggleton JP, Keen S, Warburton EC, Bussey TJ. Extensive cytotoxic lesions involving both the rhinal cortices and area TE impair recognition but spare spatial alternation in the rat. Brain Res Bull 1997;43:279–87. [3] Mumby DG, Pinel JPJ. Rhinal cortex lesions and object recognition in rats. Behav Neurosci 1994;108:11–8. [4] Gaffan D, Murray EA. Monkeys with rhinal cortex lesions succeed in object discrimination learning despite 24-hour intertrial intervals and fail at match to sample despite double sample presentations. Behav Neurosci 1992;106:30–8. [5] Meunier M, Bachevalier J, Mishkin M, Murray EA. Effects on visual recognition of combined and separate ablations of the entorhinal and perirhinal cortex in rhesus monkeys. J Neurosci 1993;13:5418–32. [6] Murray EA. What have ablation studies told us about the neural substrates of stimulus memory? Sem Neurosci 1996;8:13–22. [7] Wiig KA, Bilkey DK. Lesions of rat perirhinal cortex exacerbate the memory deficit observed following damage to the fimbria–fornix. Behav Neurosci 1995;109:620–30. [8] Zola-Morgan S, Squire LR, Amaral DG, Suzuki WA. Lesions of the perirhinal and parahippocampal cortex that spare the amygdala and hippocampal formation produce severe memory impairment. J Neurosci 1989;9:4355–70. [9] Mumby DG. Perspectives on object-recognition memory following hippocampal damage: lessons from studies in rats. Behav Brain Res 2001;127:159–82. [10] Brown MW, Aggleton JP. Recognition memory: what are the roles of the perirhinal cortex and hippocampus? Nat Rev Neurosci 2001;2:51–61. [11] Brown MW, Bashir ZI. Evidence concerning how neurones of the perirhinal cortex may effect familiarity discrimination. Philos Trans R Soc Lond B 2002;357:1083–95. [12] Brown MW, Xiang J-Z. Recognition memory: neuronal substrates of the judgement of prior occurrence. Prog Neurobiol 1998;55:1–41. [13] Xiang JZ, Brown MW. Differential neuronal encoding of novelty, familiarity and recency in regions of the anterior temporal lobe. Neuropharmacol 1998;37:657–76.
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