Age-related impairments in operant DMTP performance in the PS2APP mouse, a transgenic mouse model of Alzheimer's disease

Behavioural Brain Research 161 (2005) 220–228

Research report

Age-related impairments in operant DMTP performance in the PS2APP mouse, a transgenic mouse model of Alzheimer’s disease M.L. Woolley ∗ , T.M. Ballard PRBD-N, F. Hoffmann-La Roche, CH-4070 Basel, Switzerland Received 30 October 2004; received in revised form 3 February 2005; accepted 10 February 2005 Available online 19 March 2005

Abstract One of the earliest signs of Alzheimer’s disease (AD) is loss of memory for recent events. This deficit in short term memory has been characterised in mild/moderate AD patients as a delay-dependent deficit in a delayed matching to sample (DMTS) task. PS2APP mice coexpressing hPS2mut and hAPPswe exhibit a spatial-temporal elevation in brain amyloid deposition and inflammation associated with temporal cognitive decline. The aim of the current study was to train PS2APP mice (C57BL/6J × DBA/2 mixed background) and appropriate control mice (B6D2F1 background) in a rodent delayed response task, the delayed matching to position (DMTP) task, prior to the onset of plaque formation and subsequently at 2–4 monthly intervals to investigate the effect of aging and increasing plaque load on DMTP performance. At 5 months of age (baseline) DMTP performance was equivalent with both PS2APP and control mice demonstrating a working memory curve across increasing delay intervals of 1–24 s. A comparison of PS2APP and control mice across ages revealed a selective age-related, delay-dependent, impairment on choice accuracy in PS2APP mice, consistent with the cognitive decline and temporal amyloidosis previously described for this mouse model. These data are also relevant for other conditional transgenic mouse models which allow time-sensitive induction or inhibition of gene expression such that mice can be trained to perform the task prior to activation or inactivation of the gene and tested thereafter. © 2005 Elsevier B.V. All rights reserved. Keywords: PS2APP mouse; Delayed matching to position (DMTP); Spatial memory; Working memory; Alzheimer’s disease

1. Introduction Alzheimer’s disease is a chronic neurodegenerative disorder and the major cause of late-onset dementia. Characterised by the deposition of extracellular neuritic plaques, comprising fibrillar amyloid beta (A-␤) peptide, and the intracellular aggregation of neurofibrillary tangles (NFTs), comprising hyperphosphorylated tau, as well as inflammation (reviewed by [40]) to date the only definitive diagnosis of AD can be made post mortem. The pathology shows a distinct spatialtemporal pattern, initially affecting areas of the temporal cortex, notably the transentorhinal region before extending to the ∗ Corresponding author. Present address: Psychiatry CEDD, GlaxoSmithKline plc, Third Avenue, Harlow, Essex, CM19 5AW, UK. Tel.: +44 1279 627912; fax: +44 1279 875389. E-mail address: [email protected] (M.L. Woolley).

0166-4328/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2005.02.007

hippocampus and certain other cortical zones. At the latter stages, a more widespread pathology is seen particularly in corticolimbic regions whilst other areas such as the cerebellum remain unaltered [4]. Accordingly, AD patients have a mild-moderate decline in memory function at the early stages of the disease progressing to a more global cognitive decline, followed by behavioural alterations during the later stages of the disease. Mnemonic impairments in AD patients are observed in a wide variety of tasks involving both short and long-term, episodic and semantic memory [33]. However, most sensitive, and selective for early AD, are tests of forgetting with short-term memory such as the paired associates learning task (PAL [19,30,44]) and delayed response tasks such as the delayed matching to sample task (DMTS [18,30,38,44]). In the latter case when asked to match an object with that of a previously presented sample, AD patients perform identically

M.L. Woolley, T.M. Ballard / Behavioural Brain Research 161 (2005) 220–228

to normal patients following a short delay between the sample and choice phase but exhibit a delay-dependent deficit when compared with normal patients following extended delays. Identification of genetic risk factors for AD (mutations in ␤-amyloid precursor protein (APP), presenilins PS1 and PS2, and apoliprotein E4 (ApOE4), reviewed in [40,41]) favours the ‘amyloid hypothesis’ of AD, i.e. that A␤ aggregation is probably upstream of the formation of TAU filaments [22]. Taken together with the development of transgene technology, rodent models of AD have been revolutionised. Whilst there are relatively few tau-based transgenic mouse models of AD (e.g. JNPL3 [31,32]) there are several transgenic mouse lines based on the ‘amyloid hypothesis’ (reviewed in [24,27]). Mouse lines overexpressing human ␤-APP, i.e. PDAPP [20], Tg2576 [26], APP23 [43], CRND8 [7] all exhibit diffuse neuritic plaques and inflammation with accelerated A␤ deposition seen in the PSAPP mouse which co-expresses mutated human PS1 and ␤-APP [10,3,25]. Despite these advances few studies have thoroughly investigated the temporal development of cognitive decline relative to plaque formation and accompanying inflammation. To address this issue Richards et al. [36] recently generated the PS2APP mouse, which overexpresses human ␤-amyloid precursor protein (␤-APP) with the Swedish mutations K670N/M671L and a mutant form of human Presenilin 2 (PS2, N1411). These mice exhibit an age-related, region-dependent, increase in cerebral amyloidosis and inflammation and a corresponding age-related decline in cognitive function. At 4 months of age PS2APP mice have low levels of insoluble amyloid in the cortex and hippocampus but perform equivalently to control mice when assessed in the Morris water maze and active avoidance. At 8 months of age mice exhibit a 6–12-fold increase in the levels of insoluble amyloid in the cortex and hippocampus and are impaired in spatial and active avoidance learning. By 12 months mice exhibit a 60–100-fold increase in insoluble amyloid in the cortex and hippocampus and no longer learn the position of the hidden platform in the water maze as well as exhibiting further impairments in active avoidance learning. Much work with transgenic mouse models of AD has focused on spatial learning in various maze procedures, i.e. Morris water maze, radial maze, T maze or avoidance learning in passive/active avoidance procedures (reviewed in [27,24]) all of which inevitably involve aspects of both working and long-term memory. Since recent work has shown that delayed response tasks are particularly sensitive to cognitive decline in the early stages of AD (detailed above) we investigated the effect of increasing plaque load in our mouse model of AD in the rodent delayed matching to position task [11]. Unlike the maze and avoidance procedures this enables repeated testing of the same mice thereby reducing variability and represents a similar test to that used as part of the clinical assessment of cognitive decline in AD patients. Given the temporal development of cerebral amyloidosis reported for the PS2APP mice [36], mice were trained to a stable baseline performance

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in the DMTP task prior to the onset of plaque deposition and retested at 2–4 monthly intervals thereafter until 20 months of age.

2. Materials and methods 2.1. Subjects PS2APP mice were generated at F. Hoffmann-La Roche as described previously [36]. The double transgenic mice were obtained by crossing homozygous APPswe males with homozygous PS2mut females, both of which were on a mixed C57BL/6J × DBA/2 background. For this reason it was not possible to use littermate controls so in order to enable a sufficient supply of age- and sex-matched controls B6D2F1 (i.e. C57BL/6J × DBA/2 cross, Charles River, France) mice were used. Unlike the B6D2F1 mice PS2APP transgenics are not isogenic. However, on the basis of the recent published evidence that C57Bl/6J and DBA/2 mice exhibit similar measures of choice accuracy in operant DMTP and DNMTP tasks [15,16,14] we predict that any age-related behavioural alterations would be attributable to the transgene rather than any subtle differences of the genetic background that may emerge with age, although it should be noted that no age-related comparison between mouse strains has been performed. It should also be noted that each transgenic group was derived from multiple litters, preserving random combinations of background C57Bl/6J and DBA/2 genes, which should result in a more accurate assessment of the transgene impact on phenotype [8]. Fourteen male PS2APP and 12 male control mice were transferred to the behavioural unit 2 weeks prior to the onset of testing where they were left to habituate for 1 week and the food deprivation schedule was started the following week. All mice were housed individually in holding rooms at controlled temperature (20–22 ◦ C) on a 12 h light:12 h dark cycle (lights on 06.00 h) and given water ad libitum. Access to food was restricted to 2.5 g food (KLIBA standardised pellets 10 mm diameter, Kaiseraugst, Switzerland) per mouse at the end of the light cycle, plus food earned during the test sessions. 2.2. Test apparatus Testing was performed in eight standard operant chambers (Med Associates, St. Albans, VT, USA) equipped with two retractable levers positioned either side of a central food tray. A single stimulus light was positioned above each lever. The equipment was run by Kestrel software (Conclusive Solutions, Harlow, UK) operating on an IBM-compatible PC. 2.3. Pre-training At 2.5 months of age mice were initially pre-trained to lever press for food reward (20 mg Formula P pellet, Noyes, NH, USA) in 30 min daily sessions under a continuous reinforcement schedule (CRF-1) in which each lever was presented singly an equal number of times (total 30 min run time). Mice were trained at this level until they were consistently pressing the levers (9 days) and thus had associated lever pressing with food reward. At this stage mice continued training under a modified CRF schedule (CRF-2) which served to habituate the animals to repeated presentation and retraction of the levers. During this schedule as soon as the lever was pressed it was

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retracted and a pellet delivered into the central food tray and 5 s after the mouse had collected the food reward the next trial commenced. Mice were again trained until they were consistently pressing the levers under this schedule (11 days) at which point the number of pellets received was restricted to 60 [15,16]. 2.4. Training on DMTP Once mice had attained consistent performance during the CRF2 schedule they were trained on the matching to position rule. This task consisted of a single lever being inserted into the chamber and the illumination of the appropriate stimulus light (sample stage). The mouse was required to press the sample lever, which immediately retracted and to nose poke into the central food tray with a single nose poke resulting in the presentation of both levers and stimulus lights. Pressing the lever previously presented at the sample stage resulted in the delivery of a single food reward (choice stage). If the mouse pressed the other lever it was recorded as an incorrect response and was unrewarded. An incorrect response or failure to respond to either the sample or choice levers during the 20 s limited hold (i.e. an omission) resulted in a time out period of 30 s. The next trial was signalled by illumination of the house light for a 5 s period, after which the sample lever was extended. The number of such trials per session was limited to 60 [15]. Initially the delay between the sample and choice stage was 0 s after the first magazine nose poke. Once the animals had learnt the matching rule (>80% correct, <20% omissions), the delay period was increased in the following way: 20 trials at each of 0, 1, 2 s-delay; 12 trials at each of 0, 1, 2, 3, 4 s-delay; 12 trials at each of 0, 1, 2, 4, 6 s-delay; 12 trials at each of 0, 1, 2, 4, 8 s-delay; 10 trials each of 0, 1, 2, 4, 8, 12 s-delay; 10 trials each of 0, 1, 2, 4, 8,16 s; up to 10 trials each of 1, 2, 4, 8, 16, 24 s-delay. At this stage, the first nose poke after the end of the delay led to the presentation of the two choice levers. Delay intervals were presented in a pseudorandom manner forcing mice to continuously nose poke during the delay period in order to avoid mediating behaviour. Mice were trained daily until they demonstrated a consistent performance across delays for 14 consecutive days. Baseline performance was then assessed over the last three consecutive days. All measures obtained over this period were averaged to provide single values of each performance variable for each mouse.

levers. Number of omissions indicates the total number of missed trials during the session, i.e. no response to the lever or magazine during the delay period. Latency measures include the amount of time (s) the animals take to respond to the sample lever (sample latency), choice levers (choice latency) and to collect the food reward (magazine latency). Nose pokes indicates the number of nose pokes into the magazine tray per second. 2.6. Statistical analysis During acquisition, i.e. pre-training and training on DMTP between genotype comparisons were analysed using one factor (genotype) repeated measures (day) ANOVA. During testing between genotype comparisons of percent correct and bias responses were initially assessed using three factor ANOVA with genotype as the between group comparison and both age and delays as repeated measures. In the event of a significant age × delay × genotype interaction, or an age × genotype interaction, between genotype comparisons at each age group were analysed using one factor (genotype) repeated measures (delay) ANOVA followed where appropriate by a Students unpaired t-test. In all cases latencies, omissions, and total percentage correct responses at each age were collapsed across delays and subsequently analysed using a Students unpaired t-test.

3. Results 3.1. Pre-training At the commencement of training there was no significant difference in the body weight of the two groups of mice (24.8 ± 0.4 and 24.2 ± 0.2 g for control and PS2APP mice, respectively). All mice rapidly learned to collect food pellets from the food tray during pre-training and there was no significant difference between the two groups of mice in the rate of responding during the 9 days of CRF-1 training (ANOVA F(1,24) = 0.9, NS) or during the 11 days of CRF-2 training (ANOVA F(1,24) = 0.06, NS, Fig. 1).

2.5. Testing on DMTP Following the acquisition of baseline performance mice were maintained on the food restricted schedule in their home cages and were tested for DMTP performance at 8, 10, 12, 16 and 20 months. During testing each mouse was re-exposed to the DMTP task in the same box it was previously trained in. Mice were tested until they had achieved stable performance across delays, i.e. six consecutive days in each case. DMTP performance at each time point was assessed over the last three consecutive days of testing. All measures obtained over these last 3 days of testing were averaged to provide single values of each performance variable for each mouse. Parameters measured included percentage correct responses (choice accuracy) shown against the six delay time periods and collapsed across delays. Bias index (BI) is the responses on the preferred lever minus responses on the less-preferred lever divided by the number of responses, i.e. a BI of 1 indicates 100% of responses made on the preferred lever and 0 indicates equal responses on both

Fig. 1. Comparison of control (n = 14,
) and PS2APP (n = 12, 䊉) mice on the number of rewards achieved during CRF-1 and CRF-2 training.

± ± ± ± ± ± 80 0.8 6.2 1.6 0.6 2.0 1 0.3 0.7 0.1 0.0 0.1 ± ± ± ± ± ± 94 1.4 6.4 1.7 0.8 1.8 0.3* 0.5 0.1* 0.0** 0.1

± ± ± ± ± ± 81 0.5 4.5 1.3 0.6 1.9 1 0.6 0.7 0.1 0.0 0.1 ± ± ± ± ± ± 94 2.4 5.4 1.5 0.8 1.7 3 0.5 0.5 0.1 0.0*** 0.1** ± ± ± ± ± ± 89 0.9 5.0 1.5 0.6 2.2 1 0.4 0.6 0.1 0.0 0.1 ± ± ± ± ± ± 94 0.9 5.2 1.7 0.8 1.9 0.3 0.4 0.1* 0.0*** 0.1*

± ± ± ± ± ± Data presented as mean ± SEM. ∗ P < 0.05. ∗∗ P < 0.01. ∗∗∗ P < 0.001 vs. respective control (Students unpaired t-test). Control (n = 14), PS2APP (n = 9).

89 1.0 5.4 1.4 0.6 2.1 1 0.2 0.3 0.1 0.0 0.1 ± ± ± ± ± ± 96 1.2 5.0 1.7 0.9 1.8 0.4 0.3 0.1 0.2*** 0.1

± ± ± ± ± ± 88 1.0 4.1 1.3 0.5 2.3 1 0.4 0.2 0.1 0.4 0.1 ± ± ± ± ± ± 97 1.4 3.7 1.4 0.8 2.1 0.4 0.3 0.1 0.0*** 0.1

± ± ± ± ± ± 92 1.4 4.3 1.0 0.4 2.4 1 0.9 0.6 0.1 0.0 0.1 ± ± ± ± ± ± 96 1.9 4.9 1.2 0.7 2.1 Total percent correct Omission, N Sample latency, s Correct latency, s Magazine latency, s Nose pokes/s

PS2APP Control PS2APP Control PS2APP Control

3***

PS2APP Control PS2APP

12 months 10 months 8 months

The effect of genotype at each age was assessed for both percent correct and bias measures since there was a significant age × genotype interaction for both measures (ANOVA F(5,105) = 7.6, P < 0.001; ANOVA F(5,105) = 4.3, P ≤ 0.001, respectively). However, a significant age × delay × genotype interaction was seen only for percent correct responses (ANOVA F(25,525) = 2.0, P < 0.01) and not on bias measures (ANOVA F(25,525) = 1.14, NS). At 5 months of age, following 2.5 months of training, both groups of mice had achieved a stable baseline performance. PS2APP mice showed a reduction in the total percent correct responses collapsed across delays when compared with control mice suggesting impaired performance in the PS2APP mice (ANOVA F(1,21) = 9.0, P < 0.05, Table 2). However,

5 months

3.3. Testing on DMTP

Table 2 Effect of genotype and age on performance parameters from the DMTP studies

Both groups of mice learned the 0 s-delay matching contingency to the criterion of >80% correct and <20% omissions during the first 2 days of training (Table 1). Over the course of the acquisition phase PS2APP mice required significantly more sessions to reach criterion when compared with control mice as shown by a main effect of genotype (ANOVA F(1,21) = 8.6, P ≤ 0.01) and genotype × delay interaction (ANOVA F(6,126) = 8, P < 0.001). However this reflected an increase in the number of sessions required to reach criteria during an initial short delay, i.e. at 1–2 s (P < 0.001 versus controls) and at the end of acquisition, i.e. 1–24 s (P < 0.05 versus controls, Table 1). It did not reflect an impairment of performance with increasing memory load since no deficit was observed at longer delays of 1–12 or 1–16 s, nor was there a consistent increase in the number of trials required to reach criteria at each delay suggesting that overall PS2APP mice acquired the task equivalently to controls. Notably, three PS2APP mice never achieved criteria at the longest delay, two of which never achieved >80% correct trials and one of which demonstrated >20% omissions. These mice have therefore been excluded from the DMTP acquisition analysis and from the rest of the study.

PS2APP

3.2. Training on DMTP

Control

16 months

Data are presented as mean ± SEM. ∗ P < 0.05. ∗∗∗ P < 0.001 vs. controls.

2***

1.1 ± 0.1 3.7 ± 0.5*** 1.0 ± 0.1 1.2 ± 0.2 1.7 ± 0.3 1.0 ± 0.0 3.2 ± 0.4*

Control

PS2APP (n = 9)

1.2 ± 0.1 1.3 ± 0.2 1.1 ± 1.0 1.0 ± 0.0 1.3 ± 0.2 1.4 ± 0.2 1.4 ± 0.2

2**

Control (n = 14)

0 1–2 1–4 1–8 1–12 1–16 1–24

20 months

Delay (s)

1*

Table 1 Number of days required to reach criteria, i.e. >80% correct and <20% omissions at each delay increment

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4*** 0.4 0.7 0.1 0.0*** 0.1

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Fig. 2. Comparison of control (n = 14,
) and PS2APP (n = 9, 䊉) mice on choice accuracy in the DMTP task. Percent correct responses by delay at (A) baseline (5 months), (B) 8 months, (C) 10 months, (D) 12 months, (E) 16 months, (F) 20 months. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. controls.

further analysis showed that PS2APP and controls performed equivalently on percent correct responses at each of the six delays (genotype × delay interaction: ANOVA F(5,105) = 1.18, NS, Fig. 2A) and did not differ in BI across delays (ANOVA F(5,105) = 0.2, NS, Fig. 3A). Notably magazine latency was significantly reduced in PS2APP mice when compared with control mice (P < 0.05, Table 2) an effect which was consistent throughout testing. However, all other measures were equivalent between the two groups (Table 2). Collectively these data indicate that baseline performance at 5 months of age in PS2APP mice was not grossly different to controls. At 8 months of age PS2APP mice showed a reduction in total percent correct responses collapsed across delays (ANOVA F(1,21) = 17.0, P < 0.001) when compared with controls. Moreover, this was delay-dependent (genotype × delay interaction: ANOVA F(5,105) = 6.2, P = < 0.001) with PS2APP mice performing equivalently to control mice at the shorter delay intervals of 1, 2 and 4 s but

with reduced choice accuracy at the longer delays of 8, 16 and 24 s (Fig. 2B) consistent with impaired working memory [11]. Moreover, there was no effect of genotype on the BI across delays (ANOVA F(5,105) = 2.0, NS, Fig. 3B) nor was there an effect on other measures with the exception of magazine latency, which was reduced in PS2APP mice versus controls (P < 0.001, Table 2). At 10 months of age, PS2APP mice similarly exhibited a reduction in total percent correct responses collapsed across delays (ANOVA F(1,21) = 18.24, P < 0.001). Moreover, the effect was delay-dependent (genotype × delay interaction: ANOVA F(5,105) = 4.03, P < 0.01) with a significant reduction in choice accuracy in PS2APP mice at the longer delays of 4, 16 and 24 s (Fig. 2C). Again, there was no effect of genotype on the BI across delays (ANOVA F(5,105) = 0.5, NS, Fig. 3C). As seen at the earlier ages, magazine latency was significantly attenuated in PS2APP mice (P < 0.001 versus control mice) as was the correct latency (P < 0.05) and

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Fig. 3. Comparison of control (n = 14,
) and PS2APP (n = 9, 䊉) mice on bias responding in the DMTP task. Bias responses by delay at (A) baseline (5 months), (B) 8 months, (C) 10 months, (D) 12 months, (E) 16 months, (F) 20 months. Data are expressed as mean ± SEM. *** P < 0.001 vs. controls.

in addition PS2APP mice displayed an increased number of nose pokes/s (P < 0.05, Table 2). At 12 months of age, performance of PS2APP mice did not differ from control mice. Thus measures of percent correct responses collapsed across delays and percent correct by delay were equivalent between the two genotypes (Fig. 2D) as were bias measures (Fig. 3D). As seen previously magazine latency was significantly attenuated in PS2APP mice (P < 0.001 versus control mice) and PS2APP mice showed a significantly elevated number of nose pokes (P < 0.05), although this latter effect was not a large increase (Table 2). All other measures were equivalent between genotype (Table 2). At 16 months of age, PS2APP mice again showed a reduction in total percent correct responses collapsed across delays (ANOVA F(1,21) = 18.73, P < 0.001) when compared with controls. Moreover this was delay-dependent (genotype × delay interaction: ANOVA F(5,105) = 8.1, P < 0.001) with a significant reduction at longer delays of 2, 4, 8, 16 and 24 s (Fig. 2E). There was a significant effect of genotype on the BI across delays (ANOVA F(5,105) = 3.1, P ≤ 0.01) although post hoc analysis showed that this was due to increased biased responses in PS2APP mice only at the delay of 8 s (Fig. 3E) and was therefore not a delay-dependent ef-

fect. Moreover, the reduction in percent correct responses seen in PS2APP mice was not associated with a reduction in motor performance as PS2APP mice actually displayed a reduction in missed trials when compared with control mice (P < 0.01). Furthermore, the deficit was not associated with a reduction in response rates or motivational performance in PS2APP mice since these mice actually revealed a reduction in magazine (P < 0.01) and correct latencies (P < 0.05) when compared with control mice (Table 2). By 20 months of age, the performance of the PS2APP mice was so poor that these mice were now impaired even at the earliest (1 s) delay. Thus, PS2APP mice exhibited a reduction in total percent correct responses collapsed across delays (ANOVA F(1,21) = 16.3, P < 0.001) and a significant reduction in percent correct responses at all six delays when compared with control mice. However, the magnitude of the impairment increased over delays, as the memory load was increased, such that at the longest delay, i.e. 24 s mice were performing at almost chance level (genotype × delay interaction: ANOVA F(5,105) = 6.3, P < 0.001, Fig. 2F). Notably, the effect of genotype on the BI across delays just failed to reach significance at this age (ANOVA F(5,105) = 2.1, P = 0.07) showing a tendency for increased biasing in PS2APP mice from ITIs of 4 s onwards (Fig. 3F).

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4. Discussion PS2APP mice exhibited an age-related, delay-dependent, reduction in DMTP performance. At 5 months of age, they performed similarly to control mice exhibiting a similar level of choice accuracy (percent correct responses) across the increasing delays, i.e. 1–24 s. Impairments were first observed at 8 months of age with the mice exhibiting a similar level of choice accuracy as controls at shorter delays (i.e. 1–4 s) but reduced choice accuracy as the memory load was increased, i.e. 8–24 s, a profile consistent with an attenuation of working memory [11]. The mice continued to exhibit a delay-dependent reduction in choice accuracy at 10, 16 and 20 months of age which became progressively worse even with reduced memory load across ages. Thus at 20 months of age mice performed poorly even at the shortest delay of 1 s. Whilst such a profile was not seen at 12 months of age the reasons for this are not clear as there was no modification made to the experimental procedure, e.g. feeding schedule. During pre-training, both control and PS2APP mice learnt to associate lever pressing with food reward equivalently and both groups of mice learnt the 0 s-delay contingency within the first 2 days of training. Following the introduction of delays PS2APP mice did show a tendency to acquire the task more slowly but this was not consistent throughout training and was not related to the increasing difficulty of the task suggesting that acquisition of the task was similar between the two genotypes. Moreover at 5 months of age, PS2APP mice performed similarly to controls. These data are consistent with those previously reported in [36] in which 5 month old PS2APP mice rarely exhibited amyloid plaques and performed similarly to control mice when assessed for spatial learning in the Morris water maze and active avoidance learning. It should also be noted that great care was taken not to overtrain the animals to the task. This was done by limiting the retesting phase to the attainment of consistent performance across three consecutive trials for each individual mouse, a response which always occurred within 6 consecutive test days for both genotypes. PS2APP mice exhibited a selective, age-related, delaydependent, reduction in percent correct responses as described above. This could not be accounted by any age-related alteration of motor performance, i.e. missed trials or response rates. Thus whilst PS2APP mice did exhibit a reduction in missed trials and correct latency at 16 months of age, suggestive of faster motor responses at this age, it should be noted that this was not a huge difference in either case (correct latency: 1.5 versus 1.3 s and missed trials: 2.4 versus 0.5 for control and PS2APP mice respectively in both cases). Moreover, the time taken to respond to the correct and sample levers, as well as the number of missed trials, was low in both genotypes across ages and was not age-related. PS2APP mice did exhibit a reduction in magazine latency when compared with controls. It could be argued that this more impulsive nature may itself contribute to the increased errors. Never-

theless, since a similar reduction in magazine latency was observed at each age rather than being an age-related effect this seems unlikely. In contrast, this suggests that despite being more motivated to perform the task at each age the mice continued to develop an age-related reduction in choice accuracy. A final measure considered herein is the bias index (BI), one form of biased responding described by Steckler [42]. Whilst it cannot be excluded that additional types of bias responding may play a small role in the present study, the influence of the BI appears to be minimal. Thus, despite the delay-dependent reduction in choice accuracy seen at 16 months of age PS2APP mice showed a significant increase in the bias index only at the delay of 8 s. Thus, increased bias responding cannot account for the delay-dependent impairment in choice accuracy seen in these mice at this age. At 20 months of age PS2APP did show a tendency towards increased bias measures across delays when compared with controls. However, even at the longest delays when PS2APP mice were performing around chance, the BI was only 0.4, suggesting that side biasing behaviours were not exceptionally high and consequently mice were making correct and incorrect selection choices to both lever positions [39,17]. Thus, whilst the marginal increase in biasing behaviour may contribute towards impaired choice accuracy in PS2APP mice at this age it seems unlikely that this alone can completely account for the larger reduction in choice accuracy seen in PS2APP mice at this age. Collectively these data are therefore consistent with a selective age-related reduction in spatial working memory in PS2APP mice in the operant DMTP task. This temporal decline in cognitive function closely follows the temporal development of amyloidosis and inflammation previously reported for PS2APP mice [36]. The perforant path-subiculum are the major interfaces between the hippocampal formation and cortex representing crucial structures for relaying information between these systems and are some of the earliest affected regions in the AD brain [45,21,5]. Bilateral knife cuts of the perforant pathway have been shown to delay-dependently attenuate spatial working memory in both the operant DMTP and delayed-(non)matching to position (DNMTP) tasks [29,23]. In the current study PS2APP mice first revealed impairments in choice accuracy at 8 months of age, a time point associated with the initial onset of A␤ deposition and inflammation in the subiculum and frontolateral (motor and orbital) cortex [36]. These data are therefore consistent with the hypothesis that AD pathology and degeneration of the perforant pathway and associated temporal lobe structures is likely to contribute to the mnemonic deficits characteristic of early AD. Based on spatial working memory other brain regions have been shown to be involved in the operant DMTP task, e.g. the hippocampus and medial prefrontal cortex. Thus, lesions of the perforant pathway [29,33], the fimbria-fornix pathway [11,17,1,46], hippocampus proper [2], and medial prefrontal cortex [12] selectively, and delay-dependently attenuate choice accuracy in this task. Therefore, as the number

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and size of A␤ deposits progressively increases with age to encompass most of the hippocampal formation and neocortex at 12 and 16 months of age, not surprisingly the magnitude of the deficit in spatial working memory is increased. Notably, the reduction in choice accuracy seen in PS2APP mice at even the shortest delay at 20 months of age suggests that whilst these mice clearly exhibit spatial working memory deficits as indicated by the delay-dependent impairment in choice accuracy, that they also have difficulty in processing the data possibly due to impaired vision or attentional performance. Whilst not assessed in [36] it seems likely that plaque load is further increased at 20 months of age and has spread to additional cortical areas involved in vision or attentional processing. For example the NbM as well as their associated areas has been shown to exhibit amyloid plaques, NFTs and synapse/cell loss [34,4]. In rats NbM lesions produce delayindependent impairments in the operant DMTP and DNMTP [11,17,13] an effect proposed to be at least partially due to impaired attentional performance [37]. Various pharmacological agents have been used to impair performance in choice accuracy in the rat DMTP (or DNMTP) task including cholinergic (scopolamine), glutamatergic (e.g. dizocilpine) and GABAergic (benzodiazepine)based disruptions [35,9,6,28] although to date little pharmacology has been investigated in the mouse DMTP (but see [15,16,14]). Nevertheless, such approaches usually bias the detection towards those of a similar mechanism of action, e.g. a scopolamine based impairment favours the detection of acetylcholinesterase inhibitors (AChEI) such as donepezil (Aricept® ). With increasing interest into disease modifying strategies for AD, i.e. A␤ immunisation, ␥-secretase inhibitors, the use of a transgenic mouse model of AD, such as the PS2APP mouse used in the current study, which permits repeated testing of the mice at increasing ages, would prove suitable not only for assessing symptomatic relief of AD but also for assessing the effects of disease modifying therapies and thus reduced amyloid burden on cognitive function. In conclusion, by training a mouse model of AD, the PS2APP mouse, to perform on operant DMTP prior to the onset of AD pathology we were able to demonstrate an age-related, delay-dependent impairment of spatial working memory consistent with the temporal amyloidosis and inflammation previously reported for this mouse line [36]. These data are also relevant for other conditional transgenic mouse models which allow time-sensitive induction or inhibition of gene expression in which mice can be trained to perform the task prior to activation or inactivation of the gene and tested thereafter.

Acknowledgements We would like to thank Dr. Laurence Ozmen and Patrick Biry for genotyping and breeding the PS2APP transgenic mice.

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