Neurobiology of Learning and Memory 116 (2014) 14–26

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Retrograde and anterograde memory following selective damage to the dorsolateral entorhinal cortex Nicole J. Gervais a,⇑, Meagan Barrett-Bernstein a, Robert J. Sutherland b, Dave G. Mumby a a b

Center for Studies in Behavioral Neurobiology (CSBN), Department of Psychology, Concordia University, 7141 Sherbrooke Street West (SP-244), Montreal, Quebec H4B 1R6, Canada Department of Neuroscience, Canadian Centre for Behavioural Neuroscience, University of Lethbridge, 4401 University Drive, Lethbridge, Alberta T1K 3M4, Canada

a r t i c l e

i n f o

Article history: Received 10 April 2014 Revised 8 July 2014 Accepted 31 July 2014 Available online 7 August 2014 Keywords: Retrograde amnesia Anterograde memory Entorhinal cortex Allocentric spatial memory Lesion Rat

a b s t r a c t Anatomical and electrophysiological evidence suggest the dorsolateral entorhinal cortex (DLEC) is involved in processing spatial information, but there is currently no consensus on whether its functions are necessary for normal spatial learning and memory. The present study examined the effects of excitotoxic lesions of the DLEC on retrograde and anterograde memory on two tests of allocentric spatial learning: a hidden fixed-platform watermaze task, and a novelty-preference-based dry-maze test. Deficits were observed on both tests when training occurred prior to but not following n-methyl D-aspartate (NMDA) lesions of DLEC, suggesting retrograde memory impairment in the absence of anterograde impairments for the same information. The retrograde memory impairments were temporally-graded; rats that received DLEC lesions 1–3 days following training displayed deficits, while those that received lesions 7–10 days following training performed like a control group that received sham surgery. The deficits were not attenuated by co-infusion of tetrodotoxin, suggesting they are not due to disruption of neural processing in structures efferent to the DLEC, such as the hippocampus. The present findings provide evidence that the DLEC is involved in the consolidation of allocentric spatial information. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction The entorhinal cortex (EC), which is functionally subdivided into the medial (MEC) and lateral (LEC) entorhinal cortex, provides the hippocampus (HPC) with cortical sensory information both directly (Dickson, Magistretti, Shalinsky, Hamam, & Alonso, 2000; Witter, 1993) and indirectly via the perirhinal and postrhinal cortices (Burwell, 2000). Visuospatial information processed by the association cortices is projected to the MEC (Burwell, 2000), which then projects to the dorsal portion of the dentate gyrus via the medial perforant path (MPP; Canto, Wouterlood, & Witter, 2008; Dolorfo & Amaral, 1998). The MEC has been further subdivided, with the dorsolateral band (DLEC) projecting most of the visuospatial input to the dHPC (Fyhn, Molden, Witter, Moser, & Moser, 2004). The MEC receives reciprocal projections from dHPC, and also projects back to different sensory and association regions. These connections form what is believed to be a loop allowing visuospatial input to enter via the MEC, and once processed by the HPC, return to cortical regions via the MEC. ⇑ Corresponding author. Fax: +1 514 848 2817. E-mail addresses: [email protected] (N.J. Gervais), [email protected] (M. Barrett-Bernstein), [email protected] (R.J. Sutherland), [email protected] (D.G. Mumby). http://dx.doi.org/10.1016/j.nlm.2014.07.012 1074-7427/Ó 2014 Elsevier Inc. All rights reserved.

Electrophysiological data indicate the MEC is involved in processing spatial information. MEC neurons fire when the subject is in different locations of the arena, such that a hexagonal, grid-like pattern of activity is produced by one neuron if plotted using discharge maps (Hafting, Fyhn, Molden, Moser, & Moser, 2005). Different neurons in the same region process head direction, while others fire selectively in a grid-like pattern when the head faces a particular direction (i.e. conjunctive cells; Sargolini et al., 2006). Although the foregoing anatomical and electrophysiological evidence support a role of the MEC in spatial learning and memory, the reported effects of MEC lesions on anterograde memory have been inconsistent. In one study, bilateral neurotoxic lesions produced performance deficits on a spatial reference and workingmemory task, but no impairments on all other tasks (Pouzet et al., 1999). Similarly, combined lesions of MEC and postrhinal cortex failed to impair performance on a hidden-platform task (Burwell, Saddoris, Bucci, & Wiig, 2004). However, one study reported that lesions to the MPP produced performance deficits on the hidden-platform task (Ferbinteanu, Holsinger, & McDonald, 1999). Thus, the available data do not clearly suggest a role of the MEC in anterograde memory for spatial information. DLEC damage occurring soon after training produces retrograde amnesia. For example, rats were trained on the hidden-platform task approximately 36 h before receiving either bilateral n-methyl

N.J. Gervais et al. / Neurobiology of Learning and Memory 116 (2014) 14–26 D-aspartic

acid (NMDA) lesions to the DLEC or sham surgery. On a subsequent probe trial, rats with DLEC lesions were impaired, suggestive that they had retrograde amnesia for the platform location (Steffenach, Witter, Moser, & Moser, 2005). Rats were then trained to locate the platform after it was placed in a new location. Although the DLEC-lesioned animals displayed longer escape latencies, they performed similarly to sham-surgery rats on the probe trial given immediately following training. Thus, long-term retention for spatial information was disrupted, but acquisition of spatial information following damage was not reliably impaired, suggesting a limited role for the DLEC in spatial memory. Since memory can be broken down into separate processes (e.g. acquisition, consolidation, and retrieval), determining which of these are interrupted following such damage is an appropriate next step. For example, impaired anterograde memory and temporallygraded retrograde amnesia are both suggestive of disrupted memory consolidation. Unlike investigations into the behavioural effects of DLEC damage, studies of HPC lesions illustrate severe anterograde and retrograde memory impairments for spatial information. Using the hidden-platform task, HPC lesions produce performance deficits in escape latencies and heading direction during acquisition and platform crossings during subsequent probe trials relative to cortical-lesioned and control rats (Morris, Garrud, Rawlins, & O’Keefe, 1982; Sutherland, Whishaw, & Kolb, 1983). Rats with HPC lesions are also impaired during re-acquisition of a hidden-platform task learnt either 1 week or 13 weeks before surgery, with no evidence of a temporal gradient to the impairment (Mumby, Astur, Weisend, & Sutherland, 1999). These and other studies (Aggleton, Hunt, & Rawlins, 1986; Olton & Samuelson, 1976; Sutherland & McDonald, 1990) suggest the dHPC is necessary for acquisition, consolidation, and storage of spatial information necessary for tasks involving place navigation. Experiment 1 of the present study assessed the effects of DLEC damage on retrograde memory for spatial information. Two spatial tasks were used: A hidden-platform water-maze task and noveltydetection based dry-maze test in which rats learn the location of two objects within an open-field arena, and are later returned to the arena after one of the objects has been moved to a different location. Based on our previous experience with this procedure (Gaskin, Gamliel, Tardif, Cole, & Mumby, 2009; Gaskin, Tardif, & Mumby, 2009; Mumby, Gaskin, Glenn, Schramek, & Lehmann, 2002; Wartman et al., 2012), it was anticipated that rats with sham lesions would notice that one object was now in a new location, and they would demonstrate this by displaying an exploratory preference for either the displaced object relative to the non-displaced object, or for the maze quadrants in which a change occurred (an object was displaced to or from) relative to the unchanged quadrants. The main question of interest is whether rats with damage to the DLEC show similar exploratory preferences as sham rats during the test session with the displaced object. The lesions in the Steffenach et al. (2005) study were produced by local injection of NMDA into the DLEC. When NMDA is infused into a target region, it binds to its receptor, which opens calcium ion (Ca2+) channels, creating excessive influx of Ca2+. This results in overproduction of enzymes that damage the neuron. Overstimulation of these glutamatergic neurons by NMDA also results in excessive firing, which can lead to downstream neural damage. Thus, in addition to damaging neurons in the DLEC, the excitotoxicity produced by the NMDA used by Steffenach et al. (2005) may have also overstimulated neurons downstream from the DLEC, including those located in the dHPC. In other words, the observed impairment may be due to damage occurring in either the DLEC, the dHPC, or both. A second goal of the first experiment in the present study was to determine whether blocking downstream damage

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prevents the observed memory impairments. Co-infusion of tetrodotoxin (TTX) with NMDA has previously been used to produce excitotoxic lesions in the HPC while preventing damage downstream in extra-hippocampal structures (Sparks, Lehmann, Hernandez, & Sutherland, 2011) by blocking excessive firing of neurons in the target region. The final goal of Experiment 1 was to determine whether temporally-graded retrograde amnesia (TGRA) is observed following lesions to the DLEC and if so, whether impairments are prevented when NMDA is co-infused with TTX. 2. Experiment 1 2.1. Materials and methods 2.1.1. Subjects Subjects were 40 male Long-Evans rats (Charles River, SaintConstant, Quebec) weighing between 360 and 690 g at the time of surgery. Following arrival at the housing colony, rats were housed in pairs in transparent shoebox cages under 12:12 reverse light cycle (i.e. lights on at 8:00 pm) with ad libitum access to water and food, and after one week of acclimation, were switched to a restricted feeding schedule (25 g/day). A subset of animals (n = 14) underwent environmental enrichment for an 8 week period prior to the start of the experiment. These rats were distributed evenly across conditions. All behavioural procedures were carried out during the dark phase of the light cycle. Following surgery, rats were singly housed for the remaining duration of the experiment. For the first 7 post-operative days, ad libitum access to food and water was given before returning to the same restricted feeding schedule. The recovery period lasted 10 days before recommencing behavioural testing. Concordia University’s Animal Research Ethics Committee approved all procedures in accordance with the guidelines by the Canadian Council on Animal Care. 2.1.2. Apparatuses The water-maze task was conducted in a water-maze pool (137 cm in diameter, 46 cm high), which was located in the same position within the same room for each task. For the hidden-platform task, the apparatus was filled with water (23 ± 1 °C) to a depth of approximately 30 cm and made opaque by adding skim milk powder. A movable escape platform made of Plexiglas (10 cm  10 cm  28 cm) was hidden about 2 cm below the surface of the water. Several extra-maze cues (i.e. two doors, filing cabinet, black shelves, etc.) were made visible from the water surface. The dry-maze test was conducted in a circular arena (122 cm in diameter) made from plywood (2.3 cm thick) and covered in white lacquer and fixed to a table-top 76 cm above the floor. Four transparent Lexan sheets (98.5 cm  0.2 cm  10.5 cm) were fixed together and to the floor of the maze forming a wall. Two metal figurines, approximately 20 cm in height were fixed in place, each at the center of adjacent quadrants. 2.1.3. Surgery Twenty-six rats were anesthetized with isoflurane gas (Jaansen, Toronto, Ontario, Canada) before receiving either bilateral lesions of the dorsolateral band of the medial entorhinal cortex (DLEC) by infusing NMDA (7.5 lg/ll in 0.1 M PBS; n = 20), or NMDA coinfused with Tetrodotoxin citrate (TTX, 4 ng/ll in 0.9% saline; Cedarlane Laboratories, Burlington, ON; n = 6). The remaining (n = 14) received sham surgery. For those in the recent groups (NMDA-Recent: n = 13; NMDA + TTX-Recent: n = 6 Sham-Recent: n = 7), surgery was performed within 3 days following the final training session, and following 7–10 days for the remote groups

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(NMDA-Remote: n = 7, Sham-Remote: n = 7). The stereotaxic coordinates, which are modified from those reported by Steffenach et al. (2005) are shown in Table 1. The NMDA solution (7.5 lg/ll; dissolved in 0.1 M phosphate buffered saline; PBS) was infused at a rate of 0.1 ll/min until 0.06 ll was delivered. Infusions were made via a 26G stainless steel injector attached to PE-20 tubing and a 10 ll Hamilton syringe, which was mounted on a microinjection pump (KD Scientific). Following each infusion, metal tubing was kept in place for an additional 2 min before being retracted. Rats in the sham-surgery group received the same treatment, except that no solution was infused. Three rats in the sham groups (recent: n = 2; remote: n = 1) died during the procedure. 2.1.4. Behavioural procedures 2.1.4.1. Water-maze task – hidden platform. The hidden platform remained stationary at the centre of one of the quadrants (location 1 of Fig. 1) for 8 trials per day for 6 consecutive days before surgery. At the beginning of each trial, a rat was placed into the pool facing the wall at one of 4 starting locations based on the cardinal compass points (N, S, E, W). The starting location varied in a pseudorandom sequence across trials. The trial terminated 10 s after the rat successfully climbed onto the platform, or 10 s after the experimenter guided the rat to the platform, which occurred after 60 s had elapsed. An inter-trial interval of 5–6 min was used for each rat. Following the final pre-surgery fixed-platform trials, a 30 s probe trial (with the hidden platform removed) was given. A video camera was mounted above the arena and was used to record behaviour during all trials. Following surgery, a second probe trial was given to assess retrograde memory for the platform location. The platform was then placed back in location 1, and 8 re-acquisition trials were given. On the subsequent day, the hidden platform was moved to location 2 of Fig. 1 and eight trials were given. On the last post-surgery day, a final probe trial was given. These acquisition and probe trials were used for anterograde memory tests. 2.1.4.2. Water-maze task – visible-platform. Following the final probe trial for the hidden platform task, four trials were given on which the platform was made visible by raising it 2 cm above the water surface and wrapping black tape around the top. The platform was placed in location 3 (see Fig. 1), and the sequence of starting locations was pseudorandom. 2.1.4.3. Dry-maze test. The pre-surgery familiarization phase consisted of daily 7-min sessions over 6 consecutive days. Rats were placed in the center of the arena facing away from the two stimulus objects, which were located in the centre of two adjacent quadrants. The stimulus objects remained in the same location on all 6 sessions. Following surgery, the retrograde memory test consisted of placing the rat back in the arena for 7 min with one of the

Table 1 Stereotaxic coordinates (mm) of NMDA lesions of the DLEC for Experiment 1. Anterioposterior (AP)a 5.2 6.4 7.0 7.6 8.2 8.2 8.6 8.6 9.0

Mediolateral (ML) ±5.5 ±5.5 ±5.5 ±5.5 ±5.5 ±5.0 ±5.5 ±5.0 ±5.5

Note: Angle of the rat’s head set at 0°. a Relative to Bregma. b Relative to skull adjacent to site.

a

Dorsoventral (DV)

Angle

10 a Bottom + 0.5 Bottom + 0.5 Bottom + 0.5 7.5b 6.5 7.0 6.0 5.5

10° 10° 10° 10° 0 0 0 0 0

Fig. 1. Schematic diagram of the water-maze pool depicting platform locations used in Experiments 1 and 2.

objects displaced to a different quadrant. This test also served as the familiarization phase for the anterograde memory test that occurred 24 h later. During the anterograde memory test, the object that had been displaced during the previous session was again displaced to a third quadrant. The other object remained in the same location it had been in since the training phase. During all phases of this test, rats were able to investigate the two stimulus objects, arena, and surrounding extramaze cues. 2.1.5. Statistical and histological analyses Results presented in all figures are expressed as mean (±SEM). All statistical analyses were conducted using SPSS version 20 for Windows (IBM, Chicago, IL) and type I error rate was set at a = .05. 2.1.5.1. Water-maze task. Escape latencies (time in seconds to climb fully onto the platform) were recorded live for acquisition trials on both the hidden- and visible-platform tests. For pre-surgery acquisition trials of the hidden-platform test, latencies across trials of each session were averaged, resulting in 6 averages for each rat. These 6 averages were then used in the analysis of the rats’ presurgery performance. Latencies obtained from the 8 post-surgery acquisition trials (for the retrograde-memory test) were averaged in pairs for each rat (i.e. escape latencies of trial 1 and 2 were averaged) yielding 4 pairs. The latencies for the 8 trials given the following day with the platform in a novel location (for the anterograde memory test) were also averaged in pairs. Latencies for the 4 visible-platform trials were not averaged. For all 3 probe trials (pre- and post-surgery probe trial for the retrograde-memory test, and the final probe trial for the anterograde-memory test), the time (s) spent in each quadrant and number of platform crossings (i.e. swimming over location of arena that previously contained the platform) was scored using ODLog version 2.7.2 (Macropod, software). The percentage of time in the target quadrant (i.e. quadrant that previously contained the platform) was then calculated for each rat. 2.1.5.2. Dry-maze test. Time (s) spent investigating each object during the final pre-surgery familiarization session, the first 5-min of the retrograde memory test, and the 5-min of the anterograde memory test was scored for each rat using ODLog software. A rat was considered to be investigating an object when his head was oriented within a 45° and 3 cm from it, when rearing with at least

N.J. Gervais et al. / Neurobiology of Learning and Memory 116 (2014) 14–26

one forepaw making contact while head oriented upwards, but not while climbing on top of the object. A preference ratio for the displaced object during each test was calculated as the proportion of total investigation time spent exploring the displaced object (tdisplaced/(tdisplaced + tnot displaced)). A rat was considered to be in a particular quadrant of the arena when his entire head was within its boundaries. Time (s) spent in each quadrant was also scored for both post-surgery tests using ODLog. The quadrants that contained the displaced and non-displaced object were referred to as New and Same respectively. A preference ratio for the New quadrant was calculated as the time in the New quadrant over the total time in quadrants containing objects (tNew/(tNew + tSame)). 2.1.5.3. Histology. Following all behavioural procedures, rats received a lethal dose of sodium pentobarbital before transcardial perfusion took place with 0.9% saline (250 ml) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH = 7.4, 250 ml). The brains were excised and stored in 4% paraformaldehyde solution for 4 h before being transferred to 30% sucrose solution overnight. The next day, the brains were placed on dry ice to freeze tissue before storing in a 80 °C freezer until sectioning. Using a cryostat microtome, 40 lm coronal sections through the EC were sliced and mounted on glass microscope slides. Cresyl violet staining was performed. Volume of spared tissue of the EC was estimated according to principles of the Cavalieri method (Schmitz & Hof, 2005). Images were captured of stained sections depicting the EC at the following coordinates relative to Bregma: 5.6 mm, 6.30 mm, 7.04 mm, and 8.00 mm. Each image was 1344  1024 pixels taken at 1.25 magnification using a Hamamatsu ORCA-ER camera mounted on a Leica DM 5000 B microscope. Images were then analyzed using Image J software (http://rsb.info.nih.gov/ij/) with a sampling grid with an area of 0.02 mm2 superimposed randomly over each image. The number of points in contact with intact EC tissue was obtained per image and a total for all images depicting sections per rat was obtained. This total was then divided by the average number of hits obtained from 3 sham rats, which provides a proportion of intact DLEC tissue in each rat. 3. Results 3.1. Histology and lesion quantification Five rats in the NMDA groups (NMDA-Recent: n = 3, NMDA Remote: n = 2) and 1 in the NMDA + TTX group received incomplete lesions and so were removed from all analyses. Fig. 2A shows the

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extent of damage to the DLEC resulting from the smallest and largest DLEC lesion damage, and B shows representative photomicrographs of stained sections from a rat that received NMDA infusions. In addition to the bilateral DLEC damage, 3 animals also received unilateral damage to the perirhinal cortex and/or vHPC. Unbiased estimation revealed that 58.68% of the EC (SEM = 8.32; range: 46.97–75.39%) for the NMDA-Recent group, 36.98% (SEM = 6.41; range: 30.57–43.38%) for the NMDA + TTX-Recent group, and 60.67% (SEM = 13.70; range: 46.97–74.37%) for the NMDA-Remote group was damaged. Three independent-samples t-test were conducted to determine whether there were differences between the 3 lesion groups in terms of the extent of DLEC damage. No differences were found, NMDA-Recent/NMDA + TTX-Recent: t(5) = 1.51, p = .191, Hedge’s g = 1.26, NMDA-Recent/NMDA-Remote: t(5) = 0.13, p = .904, Hedge’s g = 0.11, NMDA + TTX-Recent/NMDARemote: t(5) = 1.57, p = .258, Hedge’s g = 1.57. 3.2. Water-maze task – hidden platform 3.2.1. Pre-surgery acquisition Fig. 3A presents av erage latencies across the 6 pre-surgery acquisition sessions. A mixed-factorial ANOVA was conducted with group (i.e. Sham-Recent, Sham-Remote, NMDA-Recent, NMDA + TTX-Recent, NMDA-Remote) as the between-groups factor and training session as the within-groups factor. A main effect of session, F(5, 130) = 180.89, p < .001, partial g2 = .87 but no main effect of group, F(4, 26) = 1.77, p = .17, partial g2 = .21, and no interaction, F(20, 125) = 0.68, p = .84, partial g2 = .10, was observed. Follow-up t-tests (Bonferroni-corrected) revealed statistically significant decreases in latencies across training sessions 1 through 4, t(30) = 2.92–13.27, p < .05, Hedge’s g = 0.66–2.94, with no further decrease for subsequent sessions, t(30) = 1.18–2.52, p > .05, Hedge’s g = 0.20–0.45. 3.2.2. Retrograde memory test Percent time in target quadrant and platform crossings during the pre- and post-surgery probe trials are presented separately by group in Fig. 4A and B respectively. One-sample t-tests (onetailed) were conducted comparing the percent time in the target quadrant of each group within each probe trial (pre- and postsurgery) to chance (25%). For the pre-surgery trial, four of the 5 groups demonstrated an above-chance preference, NMDA-Recent: t(9) = 5.95, p < .001, Hedge’s g = 1.89, NMDA + TTX-Recent: t(4) = 7.18, p < .01, Hedge’s g = 3.19, Sham-Remote: t(5) = 9.66, p < .001, Hedge’s g = 3.91, and NMDA-Remote: t(4) = 7.19, p < .01, Hedge’s g = 3.23. The Sham-Recent group showed a non-significant

Fig. 2. Representations of damage resulting from NMDA infusions to the DLEC – Experiment 1. (A) Illustration of the extent of damage resulting from NMDA infusions (±TTX) with the smallest (dark grey) and largest (light grey) lesion to the DLEC observed bilaterally. (B) Representative photomicrographs of coronal sections of the DLEC following damage resulting from NMDA infusions (±TTX). Values indicate distance from Bregma (Paxinos & Watson, 1998). Blue dashed-line indicates the borders of the DLEC and the red dashed-line indicates the borders of the lesion. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. Water-maze task-hidden-platform: Experiment 1 – Pre- and post-surgery acquisition trials of retrograde memory test. (A) Average escape latencies (s) from each session are presented separately by group. A decrease was observed across sessions 1 through 4 regardless of group. (B) Latencies from four paired re-acquisition trials given post-surgery are presented for each group. Latencies from the first trial pair were longer for the NMDA-Recent group than any other group (p < .05; independent-samples ttest).

Fig. 4. Water-maze task – hidden-platform: Experiment 1 – Pre- and post-surgery probe trial of retrograde memory test. (A) Percent (%) time in target quadrant during the pre-surgery and post-surgery probe presented for all groups. During the pre-surgery probe, all groups spent more time in the target quadrant than would be expected from chance (25%; p < .01; one-sample t-test), except for the Sham-Recent group. All groups spent similar amount of time in the target quadrant. Following surgery, The 2 sham groups (Sham-Recent and Sham-Remote) and the NMDA-Remote group spent more time in the target quadrant than would be expected from chance (p < .05; one-sample ttest). The 2 NMDA-recent groups (NMDA-Recent and NMDA + TTX-Recent) were at chance level. (B) Number of platform crossings during the pre- and post-surgery probe trials.

preference, t(4) = 2.05, p = .06, Hedge’s g = 0.93. For the post-surgery trial, three of the 5 groups; Sham-Recent: t(4) = 4.65, p < .01, Hedge’s g = 2.06, Sham-Remote: t(5) = 2.46, p < .05, Hedge’s g = 1.00, NMDA-Remote: t(4) = 4.22, p < .01, Hedge’s g = 1.87 demonstrated a preference, whereas the remaining two groups, NMDARecent, t(9) = 1.29, p = .12, Hedge’s g = 0.40, and NMDA + TTXRecent groups, t(4) = 1.37, p = .24, Hedge’s g = 0.62, did not. A repeated-measures ANOVA was conducted for each condition (Recent and Remote) comparing performance on the two probe trials. Within the Recent condition, a main effect of trial (pre- and post-surgery) and a trial by group interaction was found, Trial: F(1, 17) = 6.44, p = .021 partial g2 = .28, Interaction: F(2, 17) = 4.12, p = .035 partial g2 = .33, but no effect of group, F(2, 17) = 1.22, p = .32 partial g2 = .13. Follow-up comparisons comparing preand post-surgery performance within each group demonstrated statistically significant decreases in the percent time spent in the target quadrant from pre- to post-surgery for both the NMDARecent, t(9) = 4.21, p = .002, Hedge’s g = 1.39, and NMDA + TTXRecent, t(4) = 2.84, p = .047, Hedge’s g = 2.03, groups. There was no change in the Sham-Recent group, t(4) = 0.62, p = .57, Hedge’s g = 0.48. Within the Remote condition a statistically significant main effect of trial, F(1, 9) = 35.17, p = .0002 partial g2 = .80, but no effect of group, F(1, 9) = 0.37, p = .56 partial g2 = .04, and no interaction, F(1, 9) = 0.29, p = .601 partial g2 = .03, was found. Separate repeated-measures ANOVAs were also conducted on the number of platform crossings. For the Recent condition, a statistically significant main effect of trial F(1, 17) = 8.22, p = .011 partial g2 = .33, but no effect of group, F(2, 17) = 0.10, p = .91 partial g2 = .01, and no interaction, F(1, 17) = 0.21, p = .813 partial g2 = .02 was found. A main effect of trial, F(1, 9) = 10.02, p = .011 partial g2 = .53, but no effect of group, F(2, 9) = 0.00, p = 1.00, partial g2 = .00, and no interaction, F(1, 9) = 5.11, p = .05 partial g2 = .36 was also found for the Remote condition. Fig. 3B depicts performance of each group during the re-acquisition trials that followed the post-surgery probe trial.

A mixed-factorial ANOVA was conducted and revealed a main effect of trial pair, F(3, 78) = 6.27, p < .01, partial g2 = .19, group, F(4, 26) = 2.84, p < .05, partial g2 = .30, and interaction, F(12, 78) = 3.45, p < .001, partial g2 = .35. Follow-up one-way ANOVAs were conducted on each trial pair followed by Tukey’s post hoc tests. A statistically significant group difference was found on the first trial pair, F(4, 30) = 5.74, p < .01, g2 = .47, with escape latencies being higher in the NMDA-Recent group than any other group (p < .05; Hedge’s g = 1.33–1.73). No other main effects were found for the remaining trial pairs, F(4, 30) = 0.66–1.55, p = .22–.62, g2 = .09–.19. 3.2.3. Anterograde memory test Fig. 5A presents escape latencies for 3 groups (NMDA, NMDA + TTX, Sham) on paired acquisition trials with the hidden platform in a new location. A mixed-factorial ANOVA was conducted and revealed a main effect of trial pair F(3, 84) = 6.19, p > .01, partial g2 = .18, but no main effect of group, F(2, 28) = 0.89, p = .42, partial g2 = .06, or interaction, 2 F(6, 84) = 1.96, p = .08, partial g = .12. Fig. 5B and C depicts performance of the same three groups on the probe trial given 24-h later. The time spent in the target quadrant was no different from chance (25%) for the 3 groups, Sham: t(10) = 0.22, p = .42, Hedge’s g = 0.07, NMDA: t(14) = 0.21, p = .42, Hedge’s g = 0.05, NMDA + TTX: : t(4) = 0.18, p = .43, Hedge’s g = 0.08 A statistically significant group difference was found on the number of platform crossings, F(2, 28) = 3.78, p < .05, g2 = .21, with the Sham group crossing the platform location more often than the NMDA + TTX group (p < .05, Hedge’s g = 1.26). No other group differences were found (p > .05, Hedge’s g = 0.42–1.38). 3.3. Water-maze task – visible-platform Fig. 6 presents the escape latencies for the visible platform trials. A mixed-factorial ANOVA was conducted and revealed a main

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the Sham-Recent group only, t(4) = 2.85 p < .05, Hedge’s g = 1.27. The object investigation ratios were no different from chance for the other groups, Sham-Remote: t(5) = 1.22 p = .14, Hedge’s g = 0.50, NMDA-Recent: t(8) = 0.70 p = .26, Hedge’s g = 0.23, NMDA + TTX-Recent: t(4) = 1.09 p = .18, Hedge’s g = 0.49, and NMDA-Remote: t(4) = 0.86 p = .22, Hedge’s g = 0.38. Separate oneway ANOVAs were conducted on the investigation ratios for the displaced object and New quadrant for the Recent and Remote condition. For the displaced object in the Recent condition, a statistically significant group difference was found for the displaced object, F(2, 16) = 3.81, p = .044, g2 = .32. Follow-up Tukey’s HSD tests revealed a higher preference ratio for the Sham compared to the NMDA + TTX condition (p = .038, Hedge’s g = 1.83). No other comparisons were significant (p = .167–.48, Hedge’s g = 0.66–1.02). There was also no statistically significant group difference for the Remote condition, F(1, 9) = 0.05, p = .83, g2 = .01. The Sham-Recent group also demonstrated a preference for the New quadrant, t(4) = 3.03 p < .05, Hedge’s g = 1.36, whereas the other groups did not, Sham-Remote: t(5) = 1.30 p = .13, Hedge’s g = 0.53, NMDA-Recent: t(9) = 1.50 p = .09, Hedge’s g = 0.18, NMDA + TTX-Recent: t(4) = 0.81 p = .23, Hedge’s g = 0.26, NMDA-Remote: t(4) = 0.28 p = .40, Hedge’s g = 0.27. One-way ANOVAs were conducted separately by condition (Recent and Remote) and neither revealed statistically significant group differences, Recent: F(2, 17) = 2.08, p = .16, g2 = .20, Remote: F(1, 9) = 0.21,

Fig. 5. Water-maze task – hidden-platform: Experiment 1 – Anterograde memory test. (A) Latencies from 4 paired acquisition trials with the platform in a new location are presented separately for 3 groups (Sham, NMDA, and NMDA + TTX). No group differences were observed during any of the trial pairs. (B) Percent (%) time spent in the target quadrant during a probe trial given 24 h later is presented separately for the 3 groups. (C) The number of platform crossings during the probe trial are presented separately for the 3 groups. The Sham group crossed the platform location more often than the NMDA-TTX group (p < .05).

effect of trial, F(3, 81) = 3.96, p < .05, partial g2 = .13, but no effect of group, F(2, 27) = 0.52, p = .60, partial g2 = .04, or interaction, F(6, 81) = 0.63, p = .70, partial g2 = .05. Follow-up comparisons revealed no differences in latencies across trials, t(31) = 0.50– 1.95, p > .05, Hedge’s g = 0.14–.44. 3.4. Dry-maze test 3.4.1. Pre-surgery familiarization and retrograde memory test Fig. 7 presents the total investigation time for each group during the final pre-surgery familiarization session (A) and the investigation ratios for the displaced object and New quadrant for the retrograde memory test (B–C). Investigation ratios of each group were compared to chance (0.5) using one-sample t-tests (one-tailed). Above-chance ratios for the displaced object were observed for

Fig. 6. Water-maze task – visible-platform: Experiment 1. Latencies for the three groups on the 4 visible-platform trials are presented separately.

Fig. 7. Dry-maze test: Experiment 1 – Retrograde memory test. (A) Mean investigation time (s) for the final 7-min pre-surgery familiarization phase presented separately for the 5 groups, who spent equal time investigating objects. (B) Exploratory preference for the displaced object was calculated for each group based on object investigation (tdisplaced/(tdisplaced + tnot displaced)) obtained during the first 5 min of the retrograde memory test. Rats in the Sham-Recent group demonstrated a preference (p < .05, one-sample t-test), whereas the other 4 groups did not. (C) Exploratory preference for the New quadrant object was calculated for each group based on time spent in quadrants containing objects (tNew/ (tNew + tSame)). The Sham-Recent group was the only group to demonstrate an abovechance preference (p < .05, one-sample t-test).

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p = .66, g2 = .02. Total time spent investigating objects during both the final pre-surgery familiarization session and test were analyzed using one-way ANOVAs and no group difference was found for either phase, Pre-surgery familiarization: F(4, 30) = 0.54, p = .71, g2 = .08, Test: F(4, 29) = 1.22, p = .33, g2 = .16. 3.4.2. Anterograde memory test Fig. 8 depicts the preference ratios for the displaced object and New quadrant obtained during the anterograde memory test (A–B). As was the case for the retrograde test, all ratios were compared to chance (0.5) using one-sample t-tests (one-tailed). None of the groups displayed a preference for the displaced object, Sham: t(10) = 1.36 p = .10, Hedge’s g = 0.41, NMDA: t(15) = 0.55 p = .30, Hedge’s g = 0.14, NMDA + TTX: t(4) = 1.74 p = .08, Hedge’s g = 0.78. A one-way ANOVA was conducted and no group difference was found, F(2, 28) = 0.65, p = .53, g2 = .04. There was also no group difference for time spent investigating objects during this test, F(2, 30) = 0.75, p = .48, g2 = .05. For the New quadrant, only the Sham group demonstrated an above-chance preference, t(10) = 1.93 p < .05, Hedge’s g = 0.58, whereas the other 2 groups showed no preference, NMDA: t(14) = 0.57 p = .29, Hedge’s g = 0.15, NMDA + TTX: t(4) = 0.05 p = .48, Hedge’s g = 0.02. A one-way ANOVA was conducted and revealed no statistically significant group difference, F(2, 28) = 1.22, p = .31, g2 = .08. 3.5. Correlations Pearson correlations were conducted to determine the association between the extent of lesion damage and performance on the hidden-platform and dry-maze tests. A statistically significant correlation was found between extent of lesion damage and escape latencies on the first 2 post-surgery re-acquisition trials (r = .82, p < .05). All other correlations were non-significant. Additional correlations were conducted to determine the association between performance on the two allocentric spatial tests. Analyses were conducted separately for retrograde and

anterograde memory tests. For the retrograde memory tests, statistically significant positive correlations were found between the percent time in target quadrant during the post-surgery probe trial of the water-maze task and investigation ratios for both the displaced object (r = .45, p = .012) and New quadrant (r = .38, p = .034) during the dry-maze test. A statistically significant negative correlation was found between the investigation ratio for the New quadrant of the dry-maze test and the escape latencies for the first trial pair of re-acquisition trials of the water-maze task (r = .40, p = .027). All other correlations between the two retrograde memory tests were not statistically significant (r = .002– .253, p > .05). For the anterograde memory tests, statistically significant negative correlations were found between the third trial pair of the acquisition trials of the water-maze task and the investigation ratios for the displaced object (r = .44, p = .013) and New quadrant (r = .43, p = .017). All other correlations between the two anterograde memory tests were not statistically significant (r = .046– .216, p > .05). 4. Experiment 2 The second experiment investigated whether anterograde memory impairments for spatial information are observed following damage to the DLEC. Although the previous experiment also involved an assessment of anterograde memory ability, one important limitation is that rats were well trained prior to surgery and had likely already acquired information about the extra-maze cues present in the testing room. Thus, for Experiment 2, rats naïve to the testing procedures were used to examine whether damage to the DLEC would produce impairments in both tests used in Experiment 1. 4.1. Materials and methods 4.1.1. Subjects Thirty-four male Long Evans rats (Charles River, Saint-Constant, Quebec) weighing between 370 and 560 g at the time of surgery served as subjects in the experiment. Rats were singly housed in transparent shoebox cages under 12:12 reverse light cycle (i.e. lights on at 8:00 pm) with ad libitum access to water. Access to food was ad libitum during the first 7 post-operative days before being reduced to approximately 25 g daily access for the remaining recovery period and duration of experiment. All behavioural procedures, which began either 12 days or 20 weeks post-surgery, were conducted during the dark phase of the light cycle (lights on at 8 pm). All procedures were approved by Concordia University’s Animal Research Ethics Committee, and were in accordance with guidelines of the Canadian Council on Animal Care. 4.1.2. Apparatus For the water-maze task, the same apparatus described in Experiment 1 was used. For the dry-maze test, circular corrugated-plastic flooring was placed at the bottom of the water-maze pool. Two identical ceramic stimulus objects measuring approximately 20 cm in height were fixed in place, each at the center of adjacent quadrants.

Fig. 8. Dry-maze test: Experiment 1 – Anterograde memory test. (A) An exploratory preference for the displaced object of each group (Sham, NMDA, and NMDA + TTX) was obtained during the anterograde memory test. None of the groups demonstrated an above-chance preference. (B) A preference for the New quadrant based on time spent in quadrants containing objects (tNew/(tNew + tSame)) is presented separately for each group. The Sham-group demonstrated a preference for the New quadrant (p < .05; one-sample t-test), whereas the 2 NMDA groups did not.

4.1.3. Surgery Rats received either bilateral lesions to the DLEC (n = 18) or sham surgery (n = 16). Surgery was performed under isoflurane gas (Jaansen, Toronto, Ontario, Canada) and DLEC lesions were performed by infusing NMDA (Sigma Chemical Co., St Louis, MO, USA) into nine sites, bilaterally. The stereotaxic coordinates, which are modified from those presented in Steffenach et al. (2005) are

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shown in Table 2. The same surgical procedure described in Experiment 1 was used.

and was used to determine whether rats demonstrated a preference for the Former quadrant.

4.1.4. Behavioural procedures 4.1.4.1. Water-maze task – hidden platform. During the acquisition trials of this task, the hidden platform remained at the centre of one of the quadrants (location 1 of Fig. 1). A total of 16 hidden-platform trials were given over two consecutive days (8/session) and were administered in the same manner described in Experiment 1. Prior to the first trial on Day 2, a 30 s probe trial was given. A video camera was mounted above the arena and was used to record behaviour for both the water-maze and dry-maze tests.

4.1.5.3. Histology. Following all behavioural procedures, rats received a lethal dose of sodium pentobarbital before transcardial perfusion took place with 0.9% saline (250 ml) followed by 10% formalin buffered acetate (250 ml; Fisher Scientific). The brains were excised and stored in formalin solution for 48 h at 4 °C before being transferred to 80 °C freezer until sectioning. Using a cryostat microtome, 40 lm coronal sections through the EC were sliced and mounted on gel-coated glass microscope slides. The slides were stained with Cresyl violet (Sigma–Aldrich) for microscopic evaluation of the extent of damage to the EC and adjacent regions. Volume of spared tissue of the EC was estimated using the same technique as that described in Experiment 1. Images were captured of stained sections depicting the EC at the following coordinates relative to Bregma: 5.8, 6.80, 7.64, 8.30, and 8.72 mm. Each image was a composite of 9 images taken at 4 magnification using a Photometrics Coolsnap Kino camera mounted on a Nikon T1 microscope. The composite image was created using a 3  3 grid using NIS elements at 5238  3942 pixels and allowed for a complete visual of the EC and adjacent regions. Images were analyzed using the same technique as that described in Experiment 1 and an estimate of proportion of intact tissue was obtained for each rat that received lesions.

4.1.4.2. Water-maze task – visible platform. Following all hiddenplatform trials, the platform was made visible by raising it 2 cm above the water surface and wrapping the exposed section with black tape. The platform remained in location 1 (see Fig. 1) for six trials, which were given in the same manner as that described for the hidden-platform task. 4.1.4.3. Dry-maze test. Rats were placed in the dry-maze for three 7-min familiarization sessions spaced 24 h apart. Similar to Experiment 1, the stimulus objects remained in the same location during the familiarization phase. Twenty-four hours following the third familiarization session, rats returned for the 5 min test phase, which was identical to the familiarization sessions except one object was displaced to the center of another quadrant of the pool. 4.1.5. Statistical and histological analyses 4.1.5.1. Water-maze task. As in Experiment 1, results are expressed in figures as mean (±SEM). Escape latencies were recorded live for acquisition trials on both the hidden- and visible-platform tests. For the hidden-platform test, the 16 trials were averaged in pairs for each rat yielding 4 pairs per session. The percentage of time each rat spent in the target quadrant and the number of platform crossings during the probe trial were obtained in the same manner as that described in Experiment 1. 4.1.5.2. Dry-maze test. Time (s) spent investigating each object during both phases of the test, and time spent in each quadrant of the open field during the retention test was scored for each rat using ODLog software. Preference ratios for the displaced object and the New quadrant were obtained in the same manner as that described in Experiment 1. A third investigation ratio was obtained based on time spent in the quadrant that contained an object during familiarization but was empty during the test (i.e. Former) and the quadrant that was empty during both phases (i.e. Never). This ratio was calculated as the time spent in the Former quadrant over total time in quadrants without objects (tFormer/(tFormer + tNever)), Table 2 Stereotaxic coordinates (mm) of NMDA lesions of the DLEC for Experiment 2. Anterioposterior (AP)a 5.2 5.8 6.4 7.0 7.6 8.2 8.2 8.6 8.6

Mediolateral (ML) ±6.8 a ±6.8 ±6.7 ±6.4 ±6.4 ±5.8b ±5.0 ±5.6 ±5.0

Note: Angle of the rat’s head set at 0°. a Relative to Bregma. b Relative to dura at AP = 1.0, ML = 1.0.

Dorsoventral (DV) 9.5 8.5 8.3 7.7 7.5 7.4 6.8 7.1 6.6

a

5. Results 5.1. Histology and lesion quantification Fig. 9 presents illustrations of coronal sections representing the extent of damage resulting from NMDA infusions (A) and photomicrographs of representative DLEC sections from a rat that received NMDA infusions (B). A total of six animals in the NMDA group were removed from subsequent analyses due to incomplete (i.e. unilateral) damage to the DLEC. Of the remaining rats, 6 received unilateral damage to the perirhinal cortex and 2 to the vHPC. Unbiased estimation revealed that on average, 36.87% (SEM = 4.82, range: 13.62–71.06%) of EC was damaged by NMDA infusions. 5.2. Water-maze task – hidden platform Fig. 10A presents escape latencies for the 8 trial pairs during the two acquisition sessions. A mixed-factorial ANOVA was conducted on the latencies from the first session, with surgery group (NMDA and Sham) as the between-groups factor and trial as the withingroups factor. A main effect of trial was found, F(3, 72) = 25.90, p < .001, partial g2 = .52, but there was no main effect of group F(1, 24) = 0.54, p = .47, partial g2 = .02, and no interaction, F(3, 72) = 2.17, p = .1, partial g2 = .08. Follow-up analyses (Bonferroni-corrected) revealed longer latencies for the first trial pair (i.e. 1–2) than subsequent ones, t(27) = 3.30–7.19, p < .05, Hedge’s g = 0.82–1.93. The percentage of time each group spent in the target quadrant and the number of platform crossings during the 30-s probe trial given 24 h later are presented in Fig. 10B and C respectively. One-sample t-tests (one-tailed) were conducted within each group and revealed that both spent significantly more time in the target quadrant than chance (25%) SHAM: t(15) = 1.93, p < .05, Hedge’s g = 0.48; NMDA: t(11) = 3.03, p < .01, Hedge’s g = 0.87. No group differences were observed on percent time in target quadrant, t(26) = 1.60, p = .12, Hedge’s g = 0.61, nor on the number of platform crossings, t(26) = 1.23, p = .23, Hedge’s g = 0.47. A mixed-factorial ANOVA was conducted on the latencies from the second acquisition session There was a statistically significant

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Fig. 9. Representations of damage resulting from NMDA infusions to the DLEC – Experiment 2. (A) Illustration of the extent of damage resulting from NMDA infusions with the smallest (dark grey) and largest (light grey) lesion to the DLEC observed bilaterally. (B) Representative photomicrographs of coronal sections of the DLEC following damage resulting from NMDA infusions. Values indicate distance from Bregma (Paxinos & Watson, 1998). Blue dashed-line indicates the borders of the DLEC and the red dashed-line indicates the borders of the lesion. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 10. Water-maze task – hidden-platform: Experiment 2. (A) Escape latencies (s) for both groups across the 8 pairs of acquisition trials given over 2 sessions of the hiddenplatform task. A decrease was observed between the first and second trial pair of the first acquisition session (p < .05, dependent-samples t-test). (B) Percent (%) time in target quadrant during the 30 s probe trial was above chance (25%) for both groups (p < .05; one-sample t-test). No differences emerged on this measure, nor (C) on the number of platform crossings.

main effect of trial, F(3, 75) = 6.51, p < .01, partial g2 = .21, and trial by group interaction, F(3, 75) = 3.32, p < .05, partial g2 = .12, but no main effect of group F(1, 25) = 1.24, p = .28, partial g2 = .05. Follow-up analyses (Bonferroni-corrected) revealed no statistically-significant group differences during any of the trial pairs, t(26) = 0.40–2.18, p > .05, Hedge’s g = .83–.48.

5.3. Water-maze task – visible platform Fig. 11 depicts escape latencies during the 6 visible-platform trials. A mixed-factorial ANOVA was conducted and a main effect of trial was observed, F(5, 110) = 5.15, p < .001, partial g2 = .19. No effect of group, F(1, 22) = 1.43, p = .24, partial g2 = .06, and no interaction, F(5, 110) = 0.56, p = .73, partial g2 = .03, was found. Follow-up dependent-samples t-tests (Bonferroni-corrected) demonstrated no statistically significant decreases in latencies, t(25) = 0.42–2.39, p > .05, Hedge’s g = .06–.77.

Fig. 11. Water-maze task – visible-platform: Experiment 2. Latencies for both groups on the 6 visible-platform trials are presented separately.

5.4. Dry-maze test Fig. 12 presents the total investigation time for each group during the familiarization session (A) and the investigation ratios for the displaced object and both New and Former quadrants obtained from the test (B–C). All ratios were compared to chance (0.5) using

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Fig. 12. Dry-maze test: Experiment 2. (A) Mean investigation time (s) for the final 7-min familiarization phase presented separately by group. Sham rats spent more time investigating objects than lesioned rats (p < .05, independent-samples t-test). (B) Preference ratios for the displaced object (tdisplaced/(tdisplaced + tnot displaced)) of both groups obtained during the 5 min of the retention test given 24 h later. (C) Investigation ratio calculated based on time spent in quadrants containing objects (tNew/(tNew + tSame)) is presented separately by group. (D) Investigation ratio calculated based on time spent in quadrants containing no objects (tFormer/(tFormer + tNever)) is presented separately by group. Sham rats demonstrated a preference for the Former quadrant (p < .05; one-sample t-test), whereas the NMDA did not.

one-sample t-tests. Neither group demonstrated a preference for the displaced object: SHAM: t(15) = 1.58, p = .07, Hedge’s g = 0.40; NMDA: t(10) = 0.99, p = .18, Hedge’s g = 0.30, or New quadrant, Sham: t(15) = 0.36, p = .36, Hedge’s g = 0.09 NMDA: t(10) = 0.55, p = .30, Hedge’s g = 0.17. However, the Sham group demonstrated a preference for the Former quadrant, t(15) = 1.99, p < .05, Hedge’s g = 0.50, whereas the NMDA group did not, t(10) = 0.79, p = .23, Hedge’s g = 0.23. No group difference was found on any of the investigation ratios, Displaced object: t(25) = 0.18, p = .86, Hedge’s g = 0.07, New: (25) = 0.69, p = .50, Hedge’s g = 0.27, Former: (25) = 1.89, p = .07, Hedge’s g = 0.74, or on the total time spent investigating objects during the test, t(25) = 0.82, p = .42, Hedge’s g = 0.32. However, the Sham group spent a statistically significantly greater amount of time investigating objects during the third familiarization session than the NMDA group, t(25) = 2.09, p < .05, Hedge’s g = 0.81. 5.5. Correlations Pearson correlations were conducted to determine the association between extent of lesion damage and performance on the hidden-platform and dry-maze tests. A statistically significant correlation was found between extent of lesion damage and escape latencies on the third pair of acquisition trials (trials 5–6, r = .62, p < .05). All other correlations were non-significant. Correlations between performance on the two allocentric spatial memory tests were also conducted. Statistically significant correlations were found between the investigation ratio of the New quadrant and the percent time spent in the target quadrant during the probe trial for the water-maze task (r = .48, p = .011), and between the investigation ratio for the Former quadrant of the dry-maze test and the escape latencies from the second trial pair of the water-maze task (r = .51, p = .007). All other correlations between the two tests were not statistically significant (r = .006–.38, p > .05). 6. Discussion DLEC damage occurring within 3 days of training (i.e. for the NMDA-Recent and NMDA + TTX-Recent groups) resulted in

retrograde memory impairments. On the probe trial of the hidden-platform task given post-surgery, the Sham-Recent group spent more time swimming in the target quadrant than would be expected by chance, but the lesion groups did not. The time spent in the target quadrant did not change from pre- to post-surgery for the Sham-Recent group, but decreased for both lesion groups. On the first 2 re-acquisition trials that followed, the NMDA-Recent (but not the NMDA + TTX-Recent) group continued to display a deficit, as escape latencies for these rats were longer than those in the other groups. Escape latencies on the subsequent trials were equal across groups, suggesting that rats in both lesion groups were able to re-learn the platform location. Taken together, these results suggest retrograde amnesia for both lesion groups in the Recent condition. Impaired performance following DLEC damage in the Recent condition was also observed on the dry-maze test. Whereas the Sham-Recent group demonstrated exploratory preferences for both the displaced object and New (i.e. quadrant that contained an object during the test, but not during familiarization) quadrant, the NMDA-Recent and NMDA + TTX-Recent groups did not. The rats in the Sham-Recent group, but not the two lesion groups, appear to remember the location of the objects presented during pre-surgery familiarization and recognized that one of them had been displaced during the post-surgery test. Taken together, the results from both tests reflect retrograde amnesia for allocentric spatial information following DLEC damage that occurs within 3 days of training. This is consistent with previous studies reporting retrograde amnesia for spatial information following lesions to the EC (Cho, Beracochea, & Jaffard, 1993; Cho & Kesner, 1996; Mitchell, Browning, Wilson, Baxter, & Gaffan, 2008; Steffenach et al., 2005). In the present study, co-infusion of TTX did not prevent retrograde amnesia. TTX is a toxin that binds to voltage-gated sodium (Na+) channels and prevents influx of Na+ into cells, blocking neural firing. Since NMDA infusion triggers the overproduction of enzymes that lead to neural damage and excessive firing, co-infusion of TTX is thought to prevent firing while having no effect on cell damage. Thus, lesions produced by combined infusion of NMDA and TTX results in damage to the target region with the intention of preventing downstream damage that may occur from

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NMDA administration alone. The similar performance of the NMDA-Recent and NMDA + TTX-Recent groups suggests that damage to the DLEC alone and not downstream damage to the dHPC is responsible for the retrograde amnesia. Co-infusion of TTX has previously been shown to prevent seizure activity resulting from intra-HPC infusion of NMDA (Sparks et al., 2011). Behavioural comparisons between the NMDA and NMDA + TTX groups in that study confirmed seizure activity was suppressed by TTX. In a more recent study, blocking Na+ channels attenuated neural activation in the hippocampus following electroconvulsive shock treatment (Gulbrandsen & Sutherland, 2014). Comparisons between NMDA infused alone or in combination with TTX on seizure activity and neural activation were not made in the present study, and so downstream damage to the dHPC may not have been prevented by TTX administration. However, it seems unlikely that dHPC damage is responsible for the retrograde impairments in the present study, as the amnesia observed following dHPC damage is typically more severe (Aggleton et al., 1986; Morris, Garrud, Rawlins, & O’Keefe, 1982; Mumby et al., 1999; Olton & Samuelson, 1976; Sutherland & McDonald, 1990; Sutherland et al., 1983). Therefore, the amnesia reported in the present study is likely due to DLEC damage alone. Unlike the NMDA-Recent and NMDA + TTX-Recent groups, no impairments were observed when DLEC damage occurred within 7–10 days following training (i.e. for the NMDA-Remote group). For example, the NMDA-Remote group demonstrated a preference for the target quadrant during both the pre- and post-surgery probe trials of the water-maze task. During the re-acquisition trials, escape latencies for the NMDA-Remote group were similar to the other groups. Although the results from the water-maze task demonstrate intact retrograde memory in this group, the data from the dry-maze test are less clear. Neither the Sham-Remote nor NMDA-Remote demonstrated exploratory preferences. Given that the delay between the final familiarization session and retrograde memory test was longer for the two groups in the Remote condition (17–20 days) compared to what was experienced by the 3 Recent groups (11–14 days), it is possible that some degree of forgetting may have occurred. Evidence of retrograde amnesia in the NMDA-Recent and NMDA + TTX-Recent groups, but not in the NMDA-Remote group is consistent with TGRA. TGRA following damage to the EC has also been reported in other studies (i.e. Cho & Kesner, 1996; Cho et al., 1993). According to the Standard Model of Systems Consolidation (SMSC), TGRA reflects a re-organization of long-term memories, whereby retrieval of long-term memories initially requires activation of medial temporal lobe structures, but following an extended period of time, retrieval becomes independent of these structures (for a review, see Sutherland, Sparks, & Lehmann, 2010). The exact length of time required for systems consolidation is unknown and also appears to be task-dependent, as TGRA is observed with training-to-surgery delays as long as 14 weeks for contextual fear conditioning (Maren, Aharonov, & Fanselow, 1997), or as brief as 24 h for flavour/odour conditioning task (Tse et al., 2007). Although TGRA in the present study is consistent with SMSC, it is also consistent with cellular consolidation. Cellular consolidation involves neural processes including signaling cascades and protein synthesis whereby acquired information is transferred from a labile to a more stable, long-lasting state (Kandel, 2001). This process of memory stabilization is thought to take between 24 and 100 h (Sutherland et al., 2010). In the present study, damage to the DLEC occurred within this interval for the two Recent groups, but not for the Remote group. Thus, the performance deficits observed in the two Recent groups could have occurred by disrupting either form of consolidation. Although TGRA was observed, anterograde memory was intact. In Experiment 1, escape latencies in the water-maze task were

similar across the NMDA, NMDA + TTX and Sham groups. On the probe trial given 24 h later, the 3 groups spent a similar amount of time in the target quadrant, and although the Sham group crossed the platform location more often than the NMDA + TTX group, it crossed a similar amount of times as the NMDA group. On the dry-maze test, none of the groups demonstrated a preference for the displaced object during the anterograde dry-maze test; the Sham group did, however, demonstrate a preference for the New quadrant, whereas the two lesion groups did not. In Experiment 2, no group differences were observed on the water-maze task. On the dry-maze test, neither group displayed a preference for the displaced object or New quadrant. The Sham group did, however, demonstrate a preference for the Former (i.e. quadrant that contained an object during familiarization, but not test) quadrant, whereas the NMDA group did not. Taken together, the results from the water-maze and dry-maze tests in both experiments do not support a role for the DLEC in the acquisition of allocentric spatial information acquired after damage occurs. Previous studies typically report either no impairment (Burwell et al., 2004) or mild anterograde memory impairments (Ferbinteanu et al., 1999; Pouzet et al., 1999; Steffenach et al., 2005) when spatial information is acquired following selective damage to the MEC. However, when damage extends to the perirhinal cortex, anterograde amnesia has been observed (Kaut & Bunsey, 2001; Nagahara, Otto, & Gallagher, 1995). In the present study, unilateral damage to posterior portions of the perirhinal cortex (PRh) was observed in three rats in Experiment 1 and six in Experiment 2. In addition, unilateral damage to the vHPC was also observed (three in Experiment 1, and two in Experiment 2). The PRh is implicated in allocentric spatial learning and memory (Liu & Bilkey, 1998, 1999; Mumby & Glenn, 2000; Ramos, 2013), whereas the vHPC is not (Gaskin, Gamliel, et al., 2009; Morris, Garrud, Rawlins, & O’Keefe, 1982; Olton & Papas, 1979). Additional analyses were run on both experiments with data from rats with PRh damage removed and the pattern of results did not change. Thus, it seems unlikely that partial damage to these regions is responsible for the observed retrograde amnesia. The extent of damage to the EC in the present study is similar to what is described by Steffenach et al. (2005). Although a large portion of the region remained intact (Experiment 1: 50%; Experiment 2: 60%), retrograde amnesia was observed. Since the amnesia was not extensive, additional analyses were conducted after excluding the rat with the least damage in each lesion group. The pattern of results remained unchanged for both experiments. Although it is possible that more extensive damage to the DLEC may produce more severe impairments, amnesia following selective damage is still evident and consistent with a role of this region in allocentric spatial memory. Although the retrograde amnesia and intact anterograde memory observed in the present study is consistent with the DLEC having no role in consolidation, there are important considerations that need to be addressed before such conclusions can be made. Gaskin, Tremblay, and Mumby (2003) proposed that amnesia occurs when the affected region participates in the encoding of information acquired during training. When the integrity of the region being investigated is compromised during acquisition, other structures compensate allowing for successful encoding and consolidation. In addition to the current study, retrograde amnesia and intact anterograde memory has been observed following damage to the EC (Cho et al., 1993; Mitchell et al., 2008; Thornton, Rothblat, & Murray, 1997), HPC (for a review, see Sutherland et al., 2010), PRh (Mumby & Glenn, 2000; Ramos, 2013), and in studies of patients with medial temporal lobe damage (Squire, Stark, & Clark, 2004). Although the DLEC provides direct visuospatial input to the dHPC, the dHPC also receives such input from other regions, including the perirhinal and postrhinal cortices

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(Burwell et al., 2004). Thus, if damage to the DLEC occurs after encoding takes place, consolidation may be interrupted. However, if encoding occurs after the integrity of the DLEC is already compromised, then the dHPC can maintain intact spatial memory via connections with the perirhinal and postrhinal cortices. This suggestion is consistent with the multiple parallel memory systems proposed by White and McDonald (2002), which describes a competitive interaction between brain regions that process the same information, albeit differently. When both regions function normally, one is likely more dominant than the other and has a greater influence on consolidation. When the functions of the dominant region are compromised, the other region is still capable of encoding the information and the animal is able to retain the information necessary to perform the task. Since the DLEC provides the majority of visuospatial input to the dHPC, it is possible that it serves as the dominant region and when involved in encoding allocentric spatial information, amnesia will be observed if its functions are compromised. However, if the functions of the DLEC are already compromised, the perirhinal and/or posthinal cortices are capable of encoding the allocentric spatial information, preventing amnesia. Although the present study does not directly test the validity of this theory, the pattern of results are consistent with its predictions. Therefore, the retrograde amnesia and intact anterograde memory observed in the present study are entirely consistent with a role of the DLEC in consolidation in so far as it plays a role in the encoding of allocentric spatial information. Acknowledgments We are grateful to Dr. Stéphane Gaskin for his help with the surgical procedures, and to Cynthia Di Giandomenico, Donato Ercolani-Arts, Sophie Duranceau, Ramzi Houdeib, and Jessica Starck for their help with data collection. We are indebted to Gabriel Lapointe and the Center for Microscopy at Concordia for providing the training and equipment necessary for tissue analysis. We are thankful to Aileen Murray and the Animal Care Facility staff for helping in the long-term care of the animals. This work was supported by the National Science and Engineering Research Council of Canada Discovery grants to DGM (156937212). NJG was the recipient of a doctoral training award from le Fonds de recherche du Québec – Santé (FRQS). The CSBN is funded by FRQS. References Aggleton, J. P., Hunt, P. R., & Rawlins, J. N. (1986). The effects of hippocampal lesions upon spatial and non-spatial tests of working memory. Behavioural Brain Research, 19, 133–146. Burwell, R. D. (2000). The parahippocampal region: Corticocortical connectivity. Annals of the New York Academy of Sciences, 911, 25–42. Burwell, R. D., Saddoris, M. P., Bucci, D. J., & Wiig, K. A. (2004). Corticohippocampal contributions to spatial and contextual learning. The Journal of Neuroscience, 24(15), 3826–3836. Canto, C. B., Wouterlood, F. G., & Witter, M. P. (2008). What does the anatomical organization of the entorhinal cortex tell us? Neural Plasticity, 2008, 381243. Cho, Y. H., Beracochea, D., & Jaffard, R. (1993). Extended temporal gradient for the retrograde and anterograde amnesia produced by ibotenate entorhinal cortex lesions in mice. Journal of Neuroscience, 13(4), 1759–1766. Cho, Y. H., & Kesner, R. P. (1996). Involvement of entorhinal cortex or parietal cortex in long-term spatial discrimination memory in rats: Retrograde amnesia. Behavioral Neuroscience, 110(3), 436–442. Dickson, C. T., Magistretti, J., Shalinsky, M., Hamam, B., & Alonso, A. (2000). Oscillatory activity in entorhinal neurons and circuits. Mechanisms and function. Annals of the New York Academy of Sciences, 911, 127–150. Dolorfo, C. L., & Amaral, D. G. (1998). Entorhinal cortex of the rat: Topographic organization of the cells of origin of the perforant path projection to the dentate gyrus. Journal of Comparative Neurology, 398(1), 25–48. Ferbinteanu, J., Holsinger, R. M. D., & McDonald, R. J. (1999). Lesions of the medial or lateral perforant path have different effects on hippocampal contributions to place learning and on fear conditioning to context. Behavioural Brain Research, 101(1), 65–84.

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