Impaired spatial learning in the APPSwe+ ... - Wiley Online Library

Genes, Brain and Behavior (2007) 6: 54–65

# 2006 The Authors Journal compilation # 2006 Blackwell Munksgaard

Impaired spatial learning in the APPSwe + PSEN1DE9 bigenic mouse model of Alzheimer’s disease R. S. Reiserer†,‡, F. E. Harrison†,‡, D. C. Syverud† and M. P. McDonald*,†,‡,§ †

Department of Pharmacology, ‡ Vanderbilt Brain Institute, and John F. Kennedy Center for Research in Human Development, Vanderbilt University, Nashville, TN, USA *Corresponding author: M. McDonald, PhD, Vanderbilt University, 851 Light Hall, Mail Stop no. 0325, Nashville, TN 37232-0325, USA. E-mail: [email protected] §

Mice co-expressing the Swedish amyloid precursor protein mutation (APPSwe) and exon 9 deletion (DE9) of the PSEN1 gene begin to develop amyloid plaques at 6–7 months of age. We demonstrate here a spatial learning deficit in 7-month-old APPSwe + PSEN1DE9 bigenic mice using an adaptation of the Barnes maze. Mice were first trained on a cued target followed by a hidden-target condition. Although bigenic mice quickly learned the cued-target version of the task, they were significantly impaired when switched to the hidden-target version. In contrast, a separate group of double-transgenic mice trained first on the spatial hidden-target version of the task were unimpaired relative to wild-type controls. We propose that processes such as general rule learning, context learning and exploratory habituation exert a greater influence when the testing environment is novel and overshadow the spatial memory deficit in naive bigenic mice. However, when cued-target training is conducted first, these processes habituate and the spatial learning deficit is unmasked. Seven-month-old APPSwe + PSEN1DE9 mice were unimpaired on tests of memory that did not involve learning the rules governing spatial associations. Keywords: Alzheimer, amyloid, anxiety, behavior, memory, mice, transgenic Received 5 January 2006, revised 9 February 2006, accepted for publication 20 February 2006

The development of amyloid plaques is a well-characterized feature of Alzheimer’s disease, and transgenic mouse models of Alzheimer’s disease have become indispensable tools for the study of amyloid processing, plaque formation and the cognitive decline that accompany neuropathology. Mutant mouse models have focused on two genes that are known to cause Alzheimer’s disease, namely amyloid precursor protein (APP) and presenilin 1 (PSEN1). Transgenic

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mice overexpressing APP recapitulate some of the features of Alzheimer’s disease, including age-related aggregation of amyloid plaques and progressive cognitive impairment (Borchelt et al. 1996; Chapman et al. 1999; Duff et al. 1996; Hsiao et al. 1996; Savonenko et al. 2005). Considerable research shows that cognitive deficits in APP-overexpressing mice are correlated with the degree of Ab aggregation or levels of soluble Ab in the brain (Arendash & King 2002; Arendash et al. 2001; Chapman et al. 1999; Ewers et al. 2005; Gordon et al. 2001; Kobayashi & Chen 2005; Liu et al. 2003; Savonenko et al. 2005). Transgenic mice coexpressing APP and PSEN1 mutant transgenes are also used to model the memory deficits and amyloid-associated neuropathology of Alzheimer’s disease (Arendash et al. 2001; Austin et al. 2003; Gordon et al. 2002; Holcomb et al. 1998; Holcomb et al. 1999; Janus & Westaway 2001; Jankowsky et al. 2005; Savonenko et al. 2005). Age-related amyloid deposition is detectable in hippocampus and neocortex of APP/PSEN1 double-transgenic mice at 6–7 months, earlier than with single APP transgenics. The double-transgenic mice used in the present study incorporate a chimeric human/murine APP construct bearing the Swedish double mutation and the exon-9-deleted PSEN1 mutation (APPSwe þ PSEN1/DE9). These mice were created by co-injection of transgenes, resulting in chromosomal co-localization and co-segregation of APP and PSEN1 (Borchelt et al. 1996; Borchelt et al. 1997; Jankowsky et al. 2001). To date, only a single study has assessed cognition in these co-segregating APPSwe þ PSEN1/DE9 mice. In that study, 7-month-old bigenic mice behaved normally in a T-maze spontaneous alternation test (Lalonde et al. 2004). Using similar double transgenics generated by the crossbreeding of APP and PSEN1 single transgenics, two studies detected impairments in APPSwe þ PSEN1/DE9 mice in repeated-reversal and radial-arm versions of the water-maze task (Jankowsky et al. 2005; Savonenko et al. 2005). In contrast to the spontaneous alternation task used by Lalonde et al. (Lalonde et al. 2004), the cross-bred bigenic mice exhibited deficits on one type of spatial referencememory task, specifically, learning the rule that reinforcement is associated with specific spatial cues. The deficits in those mice were more robust when the rules changed daily, e.g. in the repeated reversal and radial-arm versions of the water maze. These methods differ from standard watermaze testing in important ways. For example, the mice will have already habituated to the testing environment, learned about the testing context and procedures and learned the doi: 10.1111/j.1601-183X.2006.00221.x

Impaired spatial learning in APP/PSEN1 Tg mice

rule that a platform is located somewhere below the surface of the water. In the present study, we adapted the standard Barnes-maze test of spatial memory to control for habituation, context learning and general rule learning and used it to assess learning in APPSwe þ PSEN1/DE9 bigenic mice in which the two transgenes co-segregate.

spent in, each arm were recorded using NIH image software, running a macro specially written to collect elevated-plusmaze data (Miyakawa et al. 2001a). The time in closed arms was calculated as the percent of total time on arms, i.e. excluding time in the central area (File 2001).

Light/dark exploration

Materials and methods Subjects Seven-month-old APPSwe þ PS1/DE9 bigenic mice obtained from Jackson Laboratory (Bar Harbor, ME, USA; stock no. 004462) were maintained as double hemizygotes by crossing with wild-type individuals on a B6C3F1/J background strain (Jackson Labs’ stock no. 100010). The genotype was confirmed by polymerase chain reaction analysis of tail biopsies. The transgenic mice (11 females and 10 males) and their non-transgenic littermates (16 females and 14 males) were group-housed by gender in standard tub cages (26.5 17 12 cm) with fiber bedding under a 12/12-h light/dark cycle (lights on at 0600 h), with free access to food and water except where indicated. Mice were housed in a colony room in the same suite as the behavioral testing rooms and were transported to the testing rooms at least 1 h before testing began. Because mice were needed for additional testing at 2 years of age, they were not sacrificed following behavioral testing for biochemical or histological analysis. All procedures were approved by the Vanderbilt University Institutional Animal Care and Use Committee and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Behavioral procedures All behavioral procedures were conducted in the same order in all mice. The Y-maze and elevated-plus-maze tests were conducted on the same day, followed by the light/dark test the following day. Barnes-maze testing began on the first day of the following week, lasting a total of 12 days. The response acquisition and novel taste neophobia tasks were conducted 1 and 2 weeks following Barnes-maze testing, respectively.

Elevated plus maze A standard elevated plus maze was used to assess differential exploratory tendencies in walled vs. open arms. Greater or lesser anxiety may be inferred from these tendencies (Cryan & Holmes 2005; File 2001). The acrylic maze consisted of four arms, 30 cm long 6 cm wide, elevated 40 cm off the floor. Two of the arms contained walls 15 cm high; the other two arms were open. At the beginning of the 5-min session, mice were gently placed in the 10 10-cm central area and allowed to explore freely for 5 min. The number of entries onto, and the amount of time Genes, Brain and Behavior (2007) 6: 54–65

The tendency of mice to differentially explore light and dark areas was measured using four identical commerciallyavailable activity monitors (MED Associates, Georgia, VT, USA). Each monitor consisted of a 27-cm2 arena with a white acrylic floor and four clear acrylic walls 20.2 cm high. An array of 16 infrared emitters and sensors spanned each horizontal direction 1 cm above the floor, with an additional set elevated 4 cm to detect rearing. A black acrylic insert was used to darken half of each monitor, with a small opening to allow mice free access to both compartments. Each mouse was placed in the lighted compartment of the monitor for a period of 5 min, and its location was recorded every 50 ms and analyzed following the session. The latency to enter the dark, the percent of time spent in each compartment and transitions between the two compartments were calculated.

Y-maze spontaneous alternation Spontaneous alternation is a test of short-term spatial working memory (Lalonde 2002). The spontaneous alternation test used a standard Y-maze made of clear acrylic. Each of the three enclosed arms was 5.1 cm wide and 4.6 cm high and extended 31.9 cm from the center of the maze. The number and sequence of arm entries were scored by hand in 5-min sessions using a remote video monitor that allowed the experimenter to be out of view of the subject. Alternations were counted when a mouse entered each of the three arms in succession, in any order, without re-entering one of the arms. Percent alternation was calculated as the number of alternations divided by the number of total arm entries minus two.

Novel taste neophobia The neophobia test is typically used as a measure of anxiety but can also be used to assess memory for the novel food (Gutierrez et al. 2003; Morilak et al. 1983; Vogt & Rudy 1984). A mouse will typically eat only a small amount of a novel food until it is determined to be safe. On subsequent encounters with the same food, mice will eat a larger amount of it. Mice with poor memory for the initial encounter will eat a smaller amount of food during the second encounter than mice with good memory for the novel food. Mice were placed inside a clean, empty tub cage for a 5-min adaptation period. A preweighed block of mild cheddar cheese (Masters Gallery Foods, Plymouth, WI, USA) was then placed in the center of the cage. After 15 min, the cheese was removed and

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weighed, and the mouse was returned to its home cage. Either 1 or 2 days later, the procedure was repeated in an identical fashion. All sessions were conducted with cheese from a single package. For each session, a control block of cheese was placed in an empty tub cage with no mouse present, to control for evaporation of water from the cheese during the 15-min session. All post-session weights were adjusted for the amount of evaporation in the control cheese block. The amount of cheese consumed by the mouse was calculated as the pre-session weight minus the adjusted post-session weight to the nearest 0.1 mg.

Response acquisition A subset of the mice were tested on an operant responseacquisition task. This task involves learning the operant rule that a response in a nose-poke hole will result in reinforcement (Baron & Meltzer 2001). Mice were placed in commercially available operant chambers (Medical Associates, Georgia, VT, USA) for a period of 100 min. The operant chambers were equipped with a nose-poke hole that was illuminable from the inside, and a food-delivery bay was located approximately 3 cm to the right of the nose-poke hole. Infrared beams spanned the front of the hole to detect nose-poke responses and food-bay entries. Mice were deprived of food for 20 h before the session, and each nose-poke response in the lighted hole resulted in delivery of a sweet liquid reinforcer to the food bay (4 seconds access to 30% sucrose solution). Responses in the nosepoke hole when it was not lit carried no scheduled consequences. During the session, mice learn that reinforcer delivery is contingent upon a nose-poke response, and frequency of responding increases. Slow learners will emit fewer nose-poke responses during the session.

Barnes maze The Barnes maze is a spatial-learning task that allows subjects to use spatial cues to locate a means of escape from a mildly aversive environment. Like the Morris water maze, solving the Barnes-maze task involves reference memory for the rules governing the reinforcement contingencies in the maze. The mice are required to use spatial cues to find an escape location (reinforcement). In the cued version of the task, mice are required to use a discrete visible cue to find the escape location. In each case, the rules are stable from trial to trial, and learning this rule is essential for proficient performance in the maze. Although the Barnes maze is often considered less anxiogenic than the water maze (Holmes et al. 2002; Miyakawa et al. 2001b; Pompl et al. 1999), we know of no specific data demonstrating this. However, the Barnes maze confers a distinct advantage in that it requires the subject to make a choice to demonstrate good memory. In contrast, in unitary tasks such as the water maze, learning is inferred from speed of swimming or

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distance traversed. Because subjects in the Barnes maze are required to make a choice, the sensorimotor requirements for a correct response are identical to those for an incorrect response. Thus differences in speed, anxiety and locomotor ability are automatically controlled for. This is a particular advantage when studying mutant mice, because subtle effects of mutations are not always evident upon casual examination. The Barnes maze consisted of a white acrylic circular disk 90 cm in diameter with 12 equally spaced holes (5 cm in diameter) located 5 cm from the edge. Each of these holes could be opened or closed by means of a sliding, white acrylic door. A black acrylic escape box (8 8 8 cm), to which the mice gained access by way of a ridged, white acrylic ramp (30 incline), could be fitted below any of the holes in place of the door. The maze was raised 56 cm from the floor by a central pedestal that formed a pivot and allowed 360 horizontal rotation. A black acrylic start box consisted of a 13 13 13-cm bottomless cube with a hinged lid and a handle for easy lifting. The testing room was illuminated by two fluorescent lights located on the ceiling (normal room lighting). Trials were recorded using a camera connected to a Macintosh computer and analyzed using the public domain NIH IMAGE software running a macro specially written to collect Barnes-maze data (Miyakawa et al. 2001b). Target zones were defined to include each separate hole and 1 cm around them. The software collected the number of errors, latency to enter the escape hole and the path length to enter the escape hole in cm. Each trial began by placing a mouse inside the start box positioned centrally on the maze. After 30 seconds, the start box was lifted and the video capture software started by remote control. Eleven of the 12 holes were blocked during training trials; the remaining hole provided access to the escape box. The white escape ramp was positioned directly under the hole so that the black escape box was not visible to the mouse except when directly over the escape hole. Each mouse was permitted to explore the maze freely for the 5-min session. If the mouse entered the escape box within the allotted time of 5 min, the hole was immediately covered to prevent re-entry onto the maze. The mouse was left in the escape box for 1 min before being returned to its home cage. If the mouse did not enter the escape box within 5 min, it was picked up gently by the base of the tail, placed in the palm of the hand and let down at the side of the escape hole. The base of the tail was then pulled gently in the direction away from the hole, which induces the mouse to move in the opposite direction of the tail pull and enter the hole. The hole was then covered for 1 min before the subject was returned to its home cage. Although aversive stimuli such as bright lights, loud buzzers and/or fans situated above the surface of the Barnes maze are typically used to motivate animals to move into the escape hole (Bredy et al. 2004; Komater et al. 2005; Paylor et al. 2001; Pompl et al. 1999; Seeger et al. 2004), we wanted to establish a Genes, Brain and Behavior (2007) 6: 54–65


procedure in which escape from the open area into the dark enclosure was the only motivator guiding the subjects’ behavior. Thus by handling the mice gently and giving them a full minute in the escape box followed by immediate removal to the home cage, we were able to establish relatively short asymptotic latencies in the absence of explicit aversive motivators. After each trial, the maze and escape box were cleaned thoroughly with a 10% alcohol solution to remove odors, and the maze was rotated to eliminate the use of non-experimental intra-maze cues. Five training sessions were run on consecutive days, consisting of four trials each. Trials were administered in a spaced fashion, i.e. the first trial for each subject was run before starting the first subject on its second trial. A 5-min probe trial, run 1 h after the fourth training trial on the fifth day of each testing block, was identical to the training trials except that all 12 holes were blocked. All subjects received two consecutive weeks of testing, one of hidden-target trials and the other of cued-target trials. Some mice (14 wild type and seven bigenic) received hiddentarget training first, the standard method of conducting the Barnes-maze test. Although cued-target control trials are seldom conducted in Barnes-maze experiments, we felt that this was an important control for these mice given that so little has been published with them. Thus, the five sessions of hidden-target training were followed by five sessions of cued-target training in these mice. We also conducted a novel version of the Barnes-maze test in a separate group of mice, in which cued-target training was conducted first, followed by hidden-target training (16 wild type and 14 bigenic). In the hidden-target trials, the hole was always located in the same position relative to the extramaze cues around the test room, even though the maze was rotated between trials. In the cued-target trials, the position of the escape hole was varied such that on any given trial the escape hole was never at the same location or at either hole adjacent to where it had been on the previous trial. The location of the escape hole was also marked by a conspicuous polystyrene cone attached to the maze perimeter adjacent to the target hole.

were analyzed within each genotype and condition separately using RMANOVA. Because mice sometimes encountered the escape hole but then wandered away without entering, errors were classified as either primary (occurring before the first encounter with the escape hole) or omission (encounters with the target hole without subsequent entry into the target hole). In addition to total errors, primary and omission errors were analyzed separately when appropriate. Trained observers used computer-generated traces and the order of hole locations visited to classify the search strategies used by the mice as direct (the first hole visited was the escape hole or a hole directly adjacent to the escape hole), serial (the first visit to the target hole was preceded by visits to at least two adjacent holes in a serial manner) or random (no systematic search pattern was evident). Post-hoc comparisons among strategies were conducted using the Scheffe´ test.

Results Light/dark and elevated-plus-maze tests of anxiety Mice were first tested on the elevated plus maze and light/ dark exploration tests of anxiety. In the elevated plus maze, double-transgenic mice spent less time in the closed arms than wild-type control mice [Fig. 1(a); F1,49 ¼ 4.12, P ¼ 0.0478]. There were no genotype differences in the proportion of entries that were made in closed arms [Fig. 1(b); F1,49 ¼ 0.22, P ¼ 0.639], the total number of arm entries [Fig. 1(c); F1,49 ¼ 0.53, P ¼ 0.471] or the distance traversed in the plus maze [Fig. 1(d); F1,49 ¼ 1.40, P ¼ 0.243]. In the light/dark test of anxiety, there were no genotype differences on any measure [Fig. 1(e,f); time spent in dark F1,49 ¼ 0.13, P ¼ 0.718; light/dark transitions F1,49 ¼ 1.02, P ¼ 0.319]. There was also no genotype difference on latency to enter the dark [F1,48 ¼ 0.01, P ¼ 0.934; data not shown]. Although the decreased time spent in the closed arms of the elevated plus maze would indicate decreased anxiety in the APP/ PSEN1 mice, this was not borne out by other measures of anxiety. Nevertheless, the possibility of lower anxiety in the transgenics must be considered when interpreting results from other behavioral tests.

Statistical analyses Elevated plus maze, light/dark exploration and Y-maze spontaneous alternation were analyzed using factorial analysis of variance (ANOVA). Neophobia, response acquisition and Barnes-maze training data were analyzed using two-factor repeated-measures ANOVA (RMANOVA) with genotype as the between-subjects factor and session or time as the repeated measure. Sessions to criterion on the Barnes-maze task was calculated using a mean of one error or less on two consecutive sessions as the criterion. Mice that did not meet the criterion by the fifth session were assigned a score of 6. Sessions to criterion in the Barnes-maze test was analyzed using an unpaired t-test. Probe-trial data and strategy data Genes, Brain and Behavior (2007) 6: 54–65

Spontaneous alternation, neophobia and responseacquisition tests of memory Consistent with the T-maze spontaneous alternation results previously reported (Lalonde et al. 2004), the APP/PSEN1 bigenic mice performed as well as wild-type mice in the Y-maze spontaneous alternation test of memory (Fig. 2a,b). Both percent alternation [F1,49 ¼ 0.12, P ¼ 0.733] and arm choices [F1,49 ¼ 1.44, P ¼ 0.236] were the same between genotypes. Similarly, we did not observe genotype differences in the amount of cheese consumed during the novel taste neophobia test (Fig. 2c). Whether the retention interval was 1 day [F1,13 ¼ 0.09, P ¼ 0.766; Genotype Day

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Figure 1: Anxiety testing in APP/PSEN1 transgenic mice and wild-type controls. (a) APP/PSEN1 mice spent less time in the closed arms of the elevated plus maze, as a percentage of time spent on all four arms. All other measures on the plus maze were normal in bigenic mice, including (b) the percentage of arm entries made into closed arms, (c) total arm entries and (d) locomotor activity on the maze. APP/PSEN1 transgenics were also normal on measures of anxiety assessed in the light/dark exploration test, including (e) the percentage of time spent in the dark compartment and (f) light/dark transitions.

F1,13 ¼ 0.63, P ¼ 0.443] or 2 days [F1,8 ¼ 0.46, P ¼ 0.516; Genotype Day F1,8 ¼ 0.04, P ¼ 0.856], memory for the initial exposure to the novel food was intact in the double transgenic mice. On the response-acquisition test of learning, both groups increased responding significantly during the 100-min session [Fig. 2d F19,399 ¼ 56.83, P < 0.0001]. However, there was no genotype difference in the ability to learn the response–reinforcer association [F1,21 ¼ 0.70, P ¼ 0.412; Genotype 5-min period F19,399 ¼ 1.11, P ¼ 0.336].

Barnes-maze-memory task All mice were tested on both the hidden-target and cuedtarget versions of the Barnes-maze task, with the order randomized across subjects for both genotypes. When

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Figure 2: Memory testing in APP/PSEN1 transgenic mice and wild-type controls. There were no genotype differences in (a) percent alternation or (b) total arm entries in the Y-maze spontaneous alternation task, (c) the amount of a novel food consumed on the reference day or after a 1- or 2-day retention interval in the novel taste neophobia test or (d) the rate at which mice learned a response–reinforcer relationship in the responseacquisition test.

tested in the traditional manner for spatial learning tasks, with the hidden-target version conducted first, mice of both genotypes performed equally well [Fig. 3(a); F1,19 ¼ 0.13, P ¼ 0.728; Genotype Trial, F4,76 ¼ 0.98; P ¼ 0.425]. The total number of errors decreased significantly across the five sessions for both groups, indicating that they had learned to use the spatial cues to find the hidden target [F4,76 ¼ 12.46, P < 0.0001]. There were also no genotype differences in latency to enter the escape hole [F1,19 ¼ 0.69, P ¼ 0.416; Genotype Trial, F4,76 ¼ 1.41; P ¼ 0.240] or path length to find the hole [F1,19 ¼ 0.02, P ¼ 0.905; Genotype Trial, F4,76 ¼ 1.37; P ¼ 0.253], the measures traditionally used to infer learning in spatial-memory tasks (data not shown). Performance on the probe trial, on which the escape hole Genes, Brain and Behavior (2007) 6: 54–65


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Figure 3: Barnes-maze performance in mice trained with the hidden target first. (a) Mice of both genotypes learned to find the hidden escape hole equally well. (b) Memory for the location of the escape tunnel was tested using a probe trial. Both genotypes showed selective search for the target location, indicating good memory for the location of the escape tunnel an hour earlier. (c) When switched to a cued escape-hole location, performance did not significantly improve over the five training sessions in either group. (d) Nevertheless, both wild-type and APP/PSEN1 mice showed selective search on the cued-target probe trial, indicating that they remembered that the escape hole was located adjacent to the visible cue.

was covered and looked identical to the other 11 holes, confirmed that both groups had learned to find the location of the hidden escape hole (Fig. 3b). Both wild-type [F11,143 ¼ 3.23, P ¼ 0.0006] and bigenic [F11,66 ¼ 2.54, P ¼ 0.0097] mice showed selective search for the location at which the escape hole had previously been positioned. Two days after the probe trial, the same mice were trained on the cued-target version of the Barnes-maze task. Although performance in both groups generally improved over the five visible-target training sessions, the change was not statistically significant [Fig. 3(c); F4,76 ¼ 1.56, P ¼ 0.194] and there were no genotype differences [F1,19 ¼ 0.27, P ¼ 0.609; Genotype Trial, F4,76 ¼ 0.56, P ¼ 0.691]. Latency to find the escape hole [F1,19 ¼ 0.10, P ¼ 0.756; Genotype Trial, F4,76 ¼ 0.59; P ¼ 0.673] and path length [F1,19 ¼ 0.27, P ¼ 0.609; Genotype Trial, F4,76 ¼ 0.89; P ¼ 0.476] were also similar between genotypes (data not shown). Despite the lack of significant improvement over the five cued-target training sessions, Genes, Brain and Behavior (2007) 6: 54–65

performance on the probe trial showed that mice of both genotypes showed selective search for the visible cue [wild type F11,143 ¼ 2.89, P ¼ 0.0018; bigenic F11,66 ¼ 4.54, P < 0.0001]. This indicates that mice of both genotypes learned to run to the polystyrene beacon in order to find the escape hole (Fig. 3d). A separate group of mice was run on the cued-target version of the Barnes maze first, followed by the hiddentarget version. Mice of both genotypes quickly learned to associate the polystyrene intramaze cue with the escape hole, as assessed by a significant decrease in total errors [Fig. 4(a); F4,112 ¼ 5.46, P < 0.0001]. There were no significant differences between genotypes in the number of errors made during training [F1,28 ¼ 2.43, P ¼ 0.131; Genotype Trial, F4,112 ¼ 0.26; P ¼ 0.905]. There were also no genotype differences in latency [F1,28 ¼ 1.49, P ¼ 0.233; Genotype Trial, F4,112 ¼ 0.52; P ¼ 0.719] or path length [F1,28 ¼ 0.68, P ¼ 0.415; Genotype Trial, F4,112 ¼ 0.60; P ¼ 0.665] (data not shown). A probe trial

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Figure 4: Barnes-maze performance in mice trained with the cued target first. (a) Both wild-type and APP/PSEN1 mice rapidly learned to associate the discrete polystyrene cue with the escape hole. (b) Probe trial performance shows that mice of both genotypes had good memory that the escape hole had been located near the cue beacon an hour earlier. (c) When switched to the hidden-target location, APP/PSEN1 mice made significantly more errors in their attempts to find the escape tunnel, compared with wild-type mice. (d) Probe-trial performance after hidden-target training showed that both groups had learned to associate the extra-maze cues with the location of the escape hole.

conducted an hour after the last training trial confirmed that mice of both genotypes showed selective search for the visible cue previously associated with the escape tunnel [Fig. 4(b); wild type F11,165 ¼ 7.85, P < 0.0001; bigenic F11,143 ¼ 5.63, P < 0.0001]. After training on the cued-target version of the Barnes maze, the same mice were run on the hidden-target version. Under this version of the task, APP/PSEN1 mice made significantly more errors compared with wild-type mice [Fig. 4(c); F1,28 ¼ 5.13, P ¼ 0.0315; Genotype Trial, F4,112 ¼ 1.57, P ¼ 0.187]. APP/PSEN1 bigenic mice took significantly longer to learn the hidden-target version after pretraining with the cued target, as indicated by their taking more sessions to reach criterion (4.4 0.4) than wild-type mice [3.3 0.3; t(28) ¼ 2.08, P ¼ 0.0467]. Genotype differences in latency [F1,28 ¼ 1.85, P ¼ 0.184; Genotype Trial, F4,112 ¼ 0.71; P ¼ 0.584] and path length [F1,28 ¼ 0.90, P ¼ 0.351; Genotype Trial, F4,112 ¼ 1.29; P ¼ 0.280] were not statistically different (data not shown). Despite the significant spatial learning impairment exhibited by the

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APP/PSEN1 mice, by the fifth training session both genotypes were able to solve the maze proficiently. This was confirmed by probe trial performance showing that mice of both genotypes associated the extra-maze spatial cues with the previous location of the escape hole [Fig. 4(d); wild type, F11,165 ¼ 3.21, P ¼ 0.0006; bigenic, F11,143 ¼ 3.71, P < 0.0001]. To interpret properly the increase in errors in the APP/PSEN1 mice, analysis of a number of control measures is required. Changes in speed or pattern of solving the maze may sometimes be confused as a memory deficit. One of the behaviors we noticed early in training was that some mice would occasionally come across the escape hole but then meander away from it instead of entering the tunnel. We analyzed the incidence of this behavior, which we called an omission error, because it may have affected the number of total errors if there were genotype differences in its expression. However, there were no significant differences in omission errors between the genotypes during the cued-target pre-training phase [Fig. 5(a); F1,28 ¼ 0.01, P ¼ 0.937; Genotype Trial, Genes, Brain and Behavior (2007) 6: 54–65



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Figure 5: Control measures in the Barnes-maze test when the cued-target version was administered first. (a) Encounters with the target hole followed by further exploration of non-target holes were termed ‘omission errors’. Early in training, mice of both genotypes committed equal numbers of omission errors. (b) By the time they switched to the hidden-target version, this behavior had virtually disappeared in mice of both genotypes. On the cued-target version of the task, APP/PSEN1 mice also did not differ from wild-type controls in (c) primary errors (errors committed before the first encounter with the escape hole), as well as in either of the traditional measures of learning in the Barnes maze, (e) latency to enter the escape hole or (g) path length to find the hole. Although (d) primary errors, (f) latency and (h) path length mirrored genotype differences in total errors to some extent, only the difference in primary errors was significant on the spatial version of the task. Genes, Brain and Behavior (2007) 6: 54–65

F4,112 ¼ 0.16; P ¼ 0.956]. Omission errors were virtually nonexistent during the second (spatial) phase of testing, in both genotypes [Fig. 5(b); F1,28 ¼ 0.03, p ¼ 0.871; Genotype Trial F4,112 ¼ 1.01; p ¼ 0.408]. To correct total errors for the number of omission errors, we calculated the number of errors to the first encounter with the escape hole, termed primary errors. By correcting for those instances in which a mouse runs to the escape hole but does not enter immediately, we may get a better measure of what the mouse has learned. As is evident in Fig. 5(c,d), the number of primary errors paralleled the measure of total errors. There were no genotype differences on this measure during the cued-target training [Fig. 5(c); F1,28 ¼ 1.85, P ¼ 0.184; Genotype Trial F4,112 ¼ 0.86; P ¼ 0.492]. However, when switched to the hidden-target version, APP/PSEN1 mice were significantly impaired relative to wild-type mice [Fig. 5(d); F1,28 ¼ 5.12, P ¼ 0.0316; Genotype Trial F4,112 ¼ 1.66; P ¼ 0.164]. Additional control measures were latency and distance traveled before the first encounter with the escape hole (primary latency and primary path length, respectively), regardless of whether the mouse actually entered the hole. As with the other measures, there were no genotype differences during the cued-target pre-training version of the task, in either primary latency [Fig. 5(e); F1,28 ¼ 1.47, P ¼ 0.236; Genotype Trial F4,112 ¼ 1.03; P ¼ 0.396] or primary path length [Fig. 5(g); F1,28 ¼ 0.33, P ¼ 0.572; Genotype Trial F4,112 ¼ 1.18; P ¼ 0.323]. When switched to the hiddentarget version of the task, differences emerged that to some extent paralleled the APP/PSEN1 increase in errors. However, these differences were not statistically significant with respect to either primary latency [Fig. 5(f); F1,28 ¼ 1.49, P ¼ 0.233; Genotype Trial, F4,112 ¼ 1.09; P ¼ 0.366] or primary path length [Fig. 5(h); F1,28 ¼ 0.81, P ¼ 0.376; Genotype Trial, F4,112 ¼ 1.63; P ¼ 0.173]. An analysis of the strategies used by each mouse on the fifth session of each training block revealed interesting differences between the genotypes. There was no effect of order of testing (cued or hidden target first) on the tendency to use one type of strategy over the other; hence, data were collapsed across order for clarity. On the cued-target version of the task, the wild-type mice showed significant differences in strategy use [Fig. 6(a); F2,56 ¼ 17.47, P < 0.0001]. Post-hoc analyses showed that they were more likely to use a direct search strategy than either a serial or random search strategy (Scheffe´ tests, P < 0.0001). There was no difference in the percentage of time that wild-type mice used serial or random search strategies (Scheffe´ test, P ¼ 0.729). In contrast, APP/ PSEN1 transgenic mice used all three search strategies to an equal extent during cued-target trials [F2,42 ¼ 2.96, P ¼ 0.063]. Although the bigenic mice used cued and serial strategies nearly twice as often as random searches, these differences were not significant (Scheffe´ tests, P > 0.086). Wild-type mice also showed significant differences in strategy use on the hidden-target trials [Fig. 6(b); F2,56 ¼ 47.80, P < 0.0001]. Similar to their performance on the cued-target

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trials, wild-type mice tended to run directly to the hole on the hidden-target trials, rather than using a serial or random search strategy (Scheffe´ tests, direct vs. serial or random, P < 0.0001; serial vs. random P ¼ 0.865). In contrast to their performance on the cued-target trials, the APP/PSEN1 bigenic mice showed a distinct search-strategy preference on the hidden-target trials [F2,42 ¼ 17.36, P < 0.0001]. Similar to the performance of wild-type mice, the bigenic mice were more likely to run directly to the escape hole than to search serially or randomly (Scheffe´ tests, P < 0.0001). Serial and random searches were both used with similar frequency in the APP/PSEN1 mice (Scheffe´ test, P ¼ 0.994).

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Mice with co-segregating APP and PSEN1 mutations develop amyloid plaques starting at 6–7 months of age. Their neuropathology has been well characterized, but memory deficits have not yet been detected. Consistent with previous reports (Lalonde et al. 2004), we found that the bigenic mice exhibit reduced anxiety compared with wildtype controls. We also show here for the first time a specific spatial learning deficit in the APPSwe þ PSEN1/DE9 mice. The double-transgenic mice were impaired on a spatial version of the Barnes-maze task only after pre-training with a cued-target version, suggesting that other cognitive and/or non-cognitive processes may mask deficits in the traditional (hidden target) version of this task. In contrast to the spatial-learning deficit exhibited by the APP/PSEN1 transgenics, they were not impaired on the Y-maze spontaneous alternation, novel taste neophobia or response-acquisition tests of memory. The nature of the learned material and the types of memory tapped by these tasks are considerably different than in the Barnes-maze task. In order to understand the nature of the cognitive deficit in the bigenic mice, it is important to examine the behavioral contingencies involved in each of the tasks. The spontaneous alternation test involves spatial exploration, and the tendency to alternate requires working memory for the locations visited in the previous few seconds. There are no rules to learn and no explicit reinforcement, and intra-maze or egocentric cues may be as important to the tendency to alternate as extra-maze cues. The novel taste neophobia task involves memory for a specific event, specifically recognition memory for the novel food encountered a day or two earlier. The specific memory may involve several features of the novel food – the flavor, odor, texture, color and shape of the food were all novel for these mice. Our experiments did not dissociate performance based on these features, and it may be the case that different mice attend to different features. Despite these potential differences in food recognition, the bigenic mice do not have impaired memory for this non-spatial event. The response-acquisition task involves learning of a response–reinforcer relationship, i.e. learning the rule governing the operant contingency. Specifically, the mice must learn the rule that a response in the nose-poke hole results in the delivery of a reinforcer. The Barnes maze also involves reference memory for a response–reinforcer relationship. In this case, the response is entry into the escape hole, and the reinforcer is escape from the open, well-lit area. The differences in the two tasks involve the discriminative stimuli (SD.) that signal operation of the reinforcement contingency. In the response-acquisition task, the SD is a proximal, discrete light emanating from the nose-poke hole. In the cued version of the Barnes maze, the SD is a proximal, discrete polystyrene cone adjacent to the escape hole. In contrast, the SD. in the hidden-target version of the Barnes maze is a configuration of distal spatial stimuli. Thus



the learning deficit observed in the APP/PSEN1 bigenic mice was specific to the spatial reference-memory version of the Barnes-maze task, and they were unimpaired on two reference-memory tasks signaled by proximal, unambiguous visual cues. Although the double-transgenic mice had a specific impairment on the spatial version of the Barnes maze, it was only after pre-training with the cued version of the task. When mice began training on the Barnes maze, the number of errors was considerably higher than chance (compare session 1 in Figs 3a and 4a). As training progressed, the number of errors decreased in all mice and under all conditions. When the cue conditions changed, whether from cued to hidden target or vice versa, the number of errors predictably increased. However, the errors did not increase to the level emitted at the start of training, in either condition. Instead, mice that had been pre-trained in one condition committed less than half the number of errors when switched to the new condition. This improved performance was likely due to factors in the maze that do not involve spatial learning, such as habituation, exploration and rule learning. When initially placed on the maze, mice tend to explore. Evidence for this tendency comes from the number of omission errors committed during training (Fig. 5a,b). During cued-target pretraining, mice of both genotypes occasionally encountered the escape hole but then wandered away without entering. In this case, the appeal of further exploration may have been stronger than the desire to escape from the well-lit, open environment. On later trials, after they had learned about their context and explored it fully, the tendency to explore was reduced and omission errors were virtually non-existent. General rule learning also plays an important role in the Barnes-maze task. Regardless of which version of the task was conducted first (cued or hidden target), the mice must first learn the rule that there is a single escape hole located somewhere on the maze and that the escape hole leads to an enclosed, dark place. The fact that they learned this rule is evident in the decreased number of errors and shorter escape latencies with repeated sessions. Interestingly, rule learning is precisely the type of spatial deficit exhibited by the bigenic mice after cued-target pre-training, as discussed above. Specifically, they had to learn the rule that the escape hole was associated with specific spatial cues. Whether they were also impaired in learning the general rules governing Barnes-maze performance is impossible to determine from our data, because it is confounded with habituation, exploratory activity and other learning processes during training. However, the ability of the double transgenics to learn nonspatial rules in other memory tasks is apparently unimpaired. Thus the evidence supports the notion that the deficit in APP/PSEN1 mice is specific to learning about the rules governing spatial associations and is not a general referencememory deficit. An alternative explanation is one of ‘behavioral flexibility’, operationally defined as the ability to learn a new strategy Genes, Brain and Behavior (2007) 6: 54–65

while restraining performance of a previously learned strategy that is no longer successful (Ragozzino et al. 1999; Seeger et al. 2004). However, if this were the case in the present study, the transgenics would have been impaired when switching from the hidden- to the cued-target versions as well as from cued to hidden. It is possible that the APP/ PSEN1 mice are only impaired in switching from using a recognition strategy to using a spatial strategy. Two studies have shown a spatial deficit in double transgenics after repeated reversals in the water maze (Jankowsky et al. 2005; Savonenko et al. 2005). However, in both cases, it is a change in spatial rules and not a change from cued to spatial. This is consistent both with an interpretation of impaired flexibility and a deficit in learning new spatial rules. However, it does not address the question of cross-modal flexibility in the present study. A number of studies have examined performance of APP or APP/PSEN1 transgenic mice on the hidden- followed by the visible-platform versions of the water maze (Arendash et al. 2001; Arendash et al. 2004; Hsiao et al. 1996; Jensen et al. 2005). Under these conditions, mice are either impaired or unimpaired on both versions or impaired on the spatial version followed by normal cued-platform performance. These results are consistent with our data showing normal behavioral flexibility in transgenics when switching from a spatial to a cued task. However, they do not address the plausibility of impaired behavioral flexibility when switching from a cued to a spatial task in the bigenic mice. Additional studies explicitly examining cross-modal and intra-modal strategy switching are needed to dissociate these processes. Interestingly, mice of both genotypes were more likely to use a direct search strategy on the hidden-target trials than on the cued trials (Fig. 6). This is surprising given that the escape hole was clearly marked by the polystyrene beacon on cued trials. One reason for this difference may be that the mice may treat the cue as part of the environment. In this sense, the cued-target version of the Barnes maze is different than visible-platform training in the water maze. In the water maze, the correct escape location is signaled by either the platform itself or a discrete cue located directly above the platform. In either case, the visible cue is located within the maze. In the Barnes maze, the cue is located adjacent to the escape hole on the outside edge of the maze, and thus from the starting point in the center of the maze it may appear as part of the landscape. Mice have a natural tendency to form configural associations with environmental cues and thus may automatically form a configuration that includes the discrete intra-maze cue along with the landscape cues in the background. Under these conditions, the predictive power of the discrete cue may be reduced, at least in the initial trials, because its location changes from trial to trial and the configuration of cues that signals the escape-hole location changes along with it. The fact that APP/PSEN1 mice used a direct search strategy on only 40% of the cued trials (Fig. 6a) suggests that they may have had more trouble than

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the wild-type mice in learning that the discrete cue, and not a spatial configuration containing the cue, signaled the location of the escape hole. Our results are consistent with other reports showing spatial learning impairments in mice that overexpress APP and Ab. Although many studies have demonstrated cognitive deficits in APP-overexpressing mice, the phenotypes are not as robust as one would expect in a model of severe dementia, and on many memory tasks the transgenics are unimpaired (Hsiao et al. 1996; King & Arendash 2002; King et al. 1999; Savonenko et al. 2003). The present study shows that this may be due in part to mnemonic and non-mnemonic processes associated with exposure to the novel Barnesmaze environment. After the mice had become familiar with the rules and environment of the maze, a robust cognitive impairment was evident. Far from being specific to the Barnes maze, obfuscating factors such as these are nearly ubiquitous in behavioral testing situations with rodents. Thus controlling for the multiple processes involved in ostensibly unsophisticated memory tasks may be a more fruitful approach to detecting cognitive deficits in APP-overexpressing mice.

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Acknowledgments Support was provided by the National Institute of Aging (AG022439) and National Institute of Child Health and Development (HD015052). Some of the behavioral experiments were conducted within the Murine Neurobehavioral Laboratory core facility.

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