Impaired Pavlovian cued fear conditioning in Tg2576 mice expressing a human mutant amyloid precursor protein gene

Behavioural Brain Research 157 (2005) 107–117

Research report

Impaired Pavlovian cued fear conditioning in Tg2576 mice expressing a human mutant amyloid precursor protein gene Philip Barnes, Mark Good∗ School of Psychology, Cardiff University, P.O. Box 901, Cardiff CF10 3YG, UK Received 29 March 2004; received in revised form 18 May 2004; accepted 17 June 2004 Available online 27 July 2004

Abstract The processing of emotional and/or fear-related events is abnormal in patients with Alzheimer’s disease. AD is accompanied by a number of neuropathological features, one of which is the deposition of amyloid plaques. The main aim of the present study was to examine the effects of a human amyloid precursor protein mutation on both the acquisition and expression of fear conditioning in Tg2576 mice. Sixteenmonth-old, but not 4-month-old, transgenic mice showed aberrations in post-shock freezing during training. In a retention test carried out 24 h after training, Tg2576 mice showed comparable levels of conditioned fear elicited by contextual cues. However, freezing elicited by a tone conditioned stimulus was impaired in 16-month-old but not 4-month-old Tg2576 mice. The results are discussed with reference to the role of cue competition (overshadowing) in revealing fear conditioning deficits in Tg2576 mice. © 2004 Elsevier B.V. All rights reserved. Keywords: Alzheimer’s disease; Fear conditioning; Tg2576; Context; Cued; Amyloid

1. Introduction One of the clinical features of Alzheimer’s disease is a change in emotional behaviour [1]. Whilst there has been some controversy in the literature surrounding the onset of emotional changes in AD, it is most commonly viewed as a late onset symptom and is taken to reflect the probable progression of pathology within the amygdala [10,23]. Pavlovian fear conditioning, in which an auditory cue and/or context is paired with foot shock, has been used extensively in animal studies to explore the neural substrates of emotion. Two brain areas, in particular, have become closely associated with fear conditioning to either contextual and/or discrete auditory cues. Impairments in freezing elicited by both contextual and auditory CS’s often accompanies cell loss in amygdala [17]. In contrast, cell loss in the hippocampus is associated with a more selective deficit in contextual fear conditioning ([26], but see [19,25]). ∗

Corresponding author. Tel.: +44 2920 874007; fax: +44 2920 874858. E-mail address: [email protected] (M. Good).

0166-4328/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2004.06.014

Context and cued fear conditioning paradigms have been used to examine learning in mice that express an autosomal dominant form of an Alzheimer amyloid precursor protein mutation. Gerlai et al. [11] reported that 11-month-old female mice expressing an amyloid precursor protein mutation (APPV717F ) showed a complex pattern of fear conditioning deficits. Wild type and transgenic mice showed comparable levels of grooming and tail rattling elicited by either contextual cues or a tone CS that was paired with foot shock. However, mutant mice showed a deficit in conditioned freezing elicited by a tone CS and also a (non-significant) trend for reduced freezing elicited by the context. Tg2576 mice express a human Swedish APP mutation and show age-dependent impairments on a number of spatial learning tasks [4,5,14,16,28]. These deficits have been interpreted as reflecting impaired hippocampal function (e.g. [4]). Studies of fear conditioning in Tg2576 mice have provided additional support for this conclusion. More specifically, under certain conditions, mutant mice display impaired Pavlovian contextual fear conditioning but normal auditory

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conditioning [5,6]. This pattern of results is clearly consistent with other evidence that spatial navigation is disrupted in mutant mice and supports the hypothesis that hippocampal processing is aberrant in aged Tg2576 mice. However, none of the studies using Tg2576 mice have provided an assessment of the performance of these mice during conditioning. Therefore, the purpose of the present study was simply to determine whether Tg2576 mice display aberrant conditioned and/or unconditioned behaviour to contextual or auditory events during conditioning. Two experiments are reported with old (16-month-old) and young (3–4-monthold) Tg2576 mice, respectively, and examined conditioned and unconditioned changes in locomotor and freezing behaviours during training and following a 24 h retention interval.

2. Method 2.1. Subjects Male Tg (HuAPP695 SWE)2576 mice in a hybrid strain of C57Bl/6j with SJL were housed in mixed genotype litter groups of two to six animals. Two cohorts of Tg2576 mice and their littermate controls underwent fear conditioning. In each experiment the Tg2576 mice were compared to littermate controls so that age and background strain were comparable. No more than one wild type and one transgenic animal from an individual litter were included in the experimental cohorts to avoid problems of pseudoreplication. Breeding and other details regarding the maintenance of the colony were as described previously (see [4]). In Experiment 1, the subjects were 9, 16-month-old Tg2576 mice and 13 wild type littermate controls. In Experiment 2, the subjects were 8, 3, 4-month-old Tg2576 mice and eight wild type littermate controls. During the experiments, the animals were housed in littermate groups on a 12 h light-dark cycle. All testing was conducted during the light phase of this cycle. The mice were provided with ad lib access to food and water throughout the experiment. The animals were maintained in full compliance with Home Office (UK) guidelines. 2.2. Apparatus The experimental apparatus consisted of two Coulbourn conditioning chambers (Coulbourn Instruments, Allentown, PA, USA), which measured 18 cm wide by 17 cm deep by 21 cm high. The sides of the chambers were made from aluminium panels that slid into rails, thus allowing the inclusion of a speaker in place of one of the top panels. The front and back of the chamber was made of clear Perspex which allowed video recording of the animal whilst it was in the chamber, via a black and white camera mounted on the inside of the rear panel of the sound attenuating box. The front Perspex panel folded downwards to allow access. The roof of

the chamber was made of aluminium and housed an infrared activity monitor (Coulbourn Instruments; Model H24-61MC; set to mouse sensitivity) positioned above a hole in the roof panel. The activity monitor recorded the change in position of the subjects’ infrared body heat signature. The infrared monitor was capable of detecting both lateral and vertical (rearing) movements (see www.coulbourn.com for further information). This method was used to provide a measure of locomotor activity and to provide an independent means of validating the changes in freezing behaviour. A Pearson’s correlation coefficient produced for the freezing and activity measures recorded from the context and tone test stages showed that the two measures were closely negatively correlated with each other in both experiments. Experiment 1: context test, Pearson’s r = −0.81 (r2 = 0.64, P < 0.04); tone test Pearson’s r = −0.73 (r2 = 0.53, P < 0.05); Experiment 2: context test Pearson’s r = −0.72 (r2 = 0.57, P < 0.05); tone test Pearson’s r = −0.68 (r2 = 0.51, P < 0.05). The infrared activity measure therefore provided an independent assessment of changes in locomotor responses that were sensitive to changes in behaviour brought about by the conditioning contingencies. The chamber was constantly illuminated by a single bulb positioned on the top panel of the wall opposite the speaker. The speaker was connected to a Coulbourn integrated tone generator and volume control. The (5 kHz) tone volume was set at 75 dB (A scale) in each of the two chambers. The floor was a Coulbourn Instruments modular shock floor composed of 5 mm steel bars placed 5 mm apart. The floor grids were connected to a Coulbourn precision regulated animal shocker (model number H13–16). The shock amplitude was set at 0.4 mA for 2 s. The chambers were housed in sound attenuating boxes made of white melamine (dimensions 70 cm wide by 50 cm deep by 50 cm high) with a fold-down door at the front. An electric fan set in to the right hand wall ventilated the sound attenuating boxes. The conditioning chambers were linked to a PC computer via a Coulbourn Habitest Universal Linc. This allowed both control of the chambers from a PC, and recording of the activity measurements. The software used to control the chambers and process the data was written using Coulbourn Graphic State Notation. For the tone test carried out on day 2, the contextual cues in the conditioning chamber were altered. The walls were replaced with a black and white striped alternating pattern using Perspex inserts. The floor grid was covered with a Perspex solid floor covered with a black and white checkerboard pattern. The floor was then covered with a thin layer of fresh sawdust. The odour cues were also changed. Whereas on day 1 the grid floor was wiped with 70% alcohol between each subject, during tone testing the Perspex floor was wiped with a damp cloth containing a diluted solution of Hibiscrub (AstraZeneca; 1 part Hibiscrub to 10 parts water) between each subject. Two test chambers were used in the present study and the assignment of the mice to each chamber was fully counterbalanced.

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2.3. Procedure

3. Results

On day 1, all animals received a 12 min conditioning session. This consisted of a habituation period of 330 s followed by a pre-conditioned stimulus (P-CS) period of 30 s. The animals then received three pairings of a 30 s tone and a foot shock during the final 2 s of the tone. There was an ITI period of 120 s between each tone-shock pairing and after the final tone and shock pair. During the 12 min session, the PC recorded activity automatically. The floor grids were cleaned with 70% alcohol between each subject. On Day 2, the mice received two sessions of testing to evaluate fear elicited by the context and the tone CS. The order of presentation of the tone and context tests was counterbalanced across wild type and transgenic animals and there was an interval of approximately 4 h between each session. During the cue test, the mice were placed in a novel chamber and activity measurements were recorded for a total of 12 min. The first 6 min measured exploratory activity elicited by the novel context. During the second 6 min of the session the tone was played constantly. Between each session the floor was cleaned with a diluted concentration of Hibiscrub to remove olfactory contaminants and debris left by the mice during the test. During the context extinction test, the mice were placed in the chamber used for conditioning on day 1. Activity measurements were recorded for 8 min. The floors were cleaned with 70% alcohol between each subject.

The testing of old and young mice was conducted in two separate replications. However, in order to condense the data analysis, the data for young (3–4 months) and aged (16 months) animals is presented together for each phase of testing to aid clarity and for ease of comparison across groups.

2.4. Scoring of freezing

3.1. Locomotor activity: context habituation Fig. 1a (left panel) shows the mean locomotor activity score during the initial period of context exposure for 16month-old animals. The Tg2576 and control mice displayed comparable levels of activity prior to the delivery of the first conditioning trial and this pattern did not change as a function of time in the chamber. An ANOVA confirmed this impression and showed no significant main effect of group, time bin nor interaction between these factors (all F’s < 1). Fig. 1a (right panel) shows the data from the same stage of training from 3 to 4-month-old mice. Inspection of this figure shows that locomotor activity during the initial period of habituation to the conditioning context was comparable between transgenic and littermate control animals. An ANOVA confirmed this impression and revealed no main effect of group, time bin, nor an interaction between these factors (all F’s < 1). 3.2. Locomotor activity: tone conditioning

Freezing behaviour was defined as a complete suppression of spontaneous locomotor activity, and of all movements except those necessary for respiration (see [2] for further details). The behaviour of the mice was recorded using a camera and video recorder (Panasonic Model Number NV-MV20) whilst undergoing the conditioning and test sessions. Freezing was scored by the experimenter by observing the mouse every 5 s and assessing whether or not the animal was showing signs of immobility or freezing. These 5 s observation scores were used to calculate a percentage of total test time spent freezing. A second observer who was blind with respect to the genotype of the animals also scored a randomly selected set of animals from each group. The measures from each observer agreed on at least 89% of the trials.

Fig. 1b (left panel) shows locomotor activity scores for aged mice during the CS on each conditioning trial. There is a numerical trend for greater suppression of activity during the tone by wild type animals relative to transgenic mice. However, this difference was not significant, F < 1. The suppression of locomotor activity during the tone increased across trials, F(2, 40) = 16.3, P < 0.001 and was similar between the two groups, F < 1. Fig. 1b (right panel) shows the mean activity scores of young Tg2576 and littermate controls during the tone on the three conditioning trials on day 1. The overall locomotor activity scores of both control and Tg2576 mice declined systematically during the presentation of the tone CS, F(2, 28) = 20.5, P < 0.0001. There was no significant difference between the groups (F < 1) and no significant interaction involving group and conditioning trial, F(14, 28) = 1.38, P > 0.05.

2.5. Statistical analysis

3.3. Locomotor activity: context conditioning

Statistical calculations were carried out using analysis of variance (CLR ANOVA v2.0, Clear Lake Research Incorporated, USA). Prior to calculation of analyses of variance, the behavioral data were checked to ensure that the assumptions of ANOVA were not violated. Interactions involving group were analysed using tests of simple main effects and the pooled error term. A Type 1 error rate of P < 0.05 was adopted for all statistical tests.

A widely used measure of conditioning to the context is the change in the suppression of activity during the Pre-CS period. Fig. 1c (left panel) shows the mean levels of activity in the 30 s period prior to the presentation of the tone CS by aged Tg2576 and wildtype controls. Inspection of this figure shows that overall locomotor activity declined progressively during the course of conditioning in both Tg2576 and control mice, F(2, 40) = 14.66, P < 0.001 and that there was no

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Fig. 1. Mean infrared locomotor activity scores for 16-month-old (left panel) and 3–4-month-old (right panel) Tg2576 (Tg+) and littermate controls (Tg−) mice on day 1 during initial exposure to the conditioning context (a), during each tone presentation (b), and during the Pre-CS period (c).

significant difference between the groups (F < 1), and this pattern did not vary as function of Pre-CS periods (F < 1). Thus, during conditioning, 16-month-old Tg2576 and control mice showed comparable suppression of locomotor activity to the context. Locomotor activity levels for young animals during the 30 s Pre-CS period is shown in Fig. 1c (right panel). Overall there was no significant difference between the groups (F < 1). The level of activity declined across P-CS period, F(2, 28) = 14.68, P < 0.05, and the rate of decline did not change as a function of genetic status (F < 1). 3.4. Locomotor activity: post-shock activity The level of activity immediately following the presentation of foot shock is commonly used as a measure of the unconditioned response to foot shock. Rats typically show a burst of locomotor activity followed by suppression of ac-

tivity [7]. Fig. 2a (upper panel) shows the mean locomotor activity scores for aged animals during a 10 s period (in 1 s bins) immediately following the offset of each shock presentation. Consistent with previous reports in normal animals, control mice showed a pattern of locomotor activity that was followed by suppression of this activity. This pattern was evident immediately after the first shock and subsequent shock presentations. However, the pattern of activity suppression following shock presentation differed dramatically in the 16 month-old Tg2576 mice. Mutant mice showed a smaller reduction in activity following offset of the foot shock relative to control mice. This interpretation was confirmed by an ANOVA with group, ITI period and (1 s) time bins as the factors. An ANOVA carried out on the first 10 s of each ITI revealed a main effect of group, F(1, 20) = 11.49, P < 0.01, a main effect of time bin (s), F(9, 180) = 9.14, P < 0.001 and a significant interaction between these factors, F(9, 180) = 2.17, P < 0.03. No other main effects nor interaction

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Fig. 2. (a) Mean infrared locomotor activity scores during the first 10 s period of ITI (in 1 s bins) following the offset of the footshock US (upper panel) and during the last 10 s period of the ITI (lower panel) for 16-month-old Tg2576 (Tg+) and control mice (Tg−). (b) Mean infrared locomotor activity scores during the first 10 s period of ITI (in 1 s bins) following the offset of the footshock US (upper panel) and during the last 10 s period of the ITI (lower panel) for 3–4-month-old Tg2576 (Tg+) and control mice (Tg−).

approached significance (all F’s < 1). Tests of simple main effects revealed significant differences between the two groups from bin 2 to bin 10 (min F(1, 57) = 5.07, P < 0.05). The two groups did not differ in the first and fourth one-second time bin after the shock (F < 1). In addition, the simple main effects analysis revealed no main effect of time bin for Tg2576 mice

(F < 1) and a significant main effect of time bin for wild type mice (F(9, 80) = 3.46, P < 0.01). A similar analysis of the last 10 s of each ITI (shown in Fig. 2a, lower panel) revealed a non-significant main effect of group (F < 1), and ITI period, F(2, 28) = 3.11, P > 0.05, a significant interaction between ITI period and time bin, F(18, 252) = 1.74, P < 0.03, which

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reflects higher levels of activity during the final ITI period, and a non significant three-way interaction F(18, 252)=1.33, P > 0.16. The mean levels of locomotor activity in 3–4-month-old Tg2576 and control mice immediately following the offset of the shock US are shown in Fig. 2b (upper panel). An ANOVA with group, ITI period and (1 s) time bins as factors revealed a non-significant main effect of group, F(1, 14) = 2.33, P > 0.14 a main effect of time bin, F(9, 126) = 15.4, P < 0.001, and a non-significant interaction between these factors, F(2, 28) = 1.11, P > 0.34. No other main effects nor interaction were significant (all F’s < 1). Thus, suppression of locomotor activity immediately following the offset of the foot shock US was comparable in 3–4-month-old Tg2576 and wild type mice. A similar analysis carried out on the last 10 s of the ITI, shown in Fig. 2b (lower panel), revealed a similar pattern, with a non-significant main effect of group (F < 1), and a main effect of ITI period, F(2, 40) = 7.46, P < 0.01. No other main effects nor interactions were significant (max group × ITI period interaction, F(2, 40) = 1.45, P > 0.24).

the first 10 s of tone presentation, F(9, 180) = 2.01, P < 0.05, but not for the last 10 s of tone presentation (F < 1). The interaction between group and time bin was non-significant for both the first and last 10 s periods (max F(9, 180) = 1.35, P > 0.20). A similar analysis carried out on the data from the 3–4month-old mice revealed a similar pattern of results. Both ANOVAs revealed no significant main effects of group (max F(1, 14) = 1.96, P > 0.10) or time bin (both F’s < 1). There were main effects of tone presentation (min F(2, 28) = 11.24, P < 0.001) and no significant interactions involving group and tone presentation (max F(2, 28) = 1.61, P > 0.10) nor group and time bin (max F(9, 126) =1.18, P > 0.30. This analysis suggests that the augmented level of activity immediately following the foot shock in 16-month-old Tg2576 mice was not simply a consequence of a general increase in locomotor activity promoted by the presentation of the tone.

3.5. Locomotor activity: cue conditioning

The context retention test revealed that, at 16 months of age, both wild type and transgenic mice were generally less active at the start of the test session and that this dissipated with time across the 8 min of exposure (see Fig. 3a, left panel), F(1, 15) = 2.17, P < 0.008. There was, however, no difference in overall activity levels between transgenic (17.2, S.D. = 20.4) and wild type mice (24.3, S.D. = 27.1) (F < 1), and no significant interaction between these factors (F < 1). An analysis of locomotor activity in 3–4-month-old animals during the context test showed no differences between transgenic and littermate control animals (see Fig. 3a right panel; mean Tg+ = 20.9, S.D. = 19.1; mean Tg− = 17.9, S.D. = 21.2). A two way ANOVA with genotype and time as factors revealed no significant main effects of genotype, F < 1, but a main effect of time, F(1, 15) = 2.87, P < 0.05 and no significant interaction between these factors, F(9, 210) = 1.25, P > 0.05.

To determine whether the post-shock changes in activity in aged Tg2576 mice reflected increased levels of activity during the tone, a similar analysis of activity during the first and last 10 s of each tone CS was carried out. Table 1 shows the mean level of locomotor activity during the first and last 10 s of each tone presentation for both 16 month (Table 1A) and 3–4-month-old transgenic and control mice (Table 1B). Separate ANOVAs with group, tone presentation and 1 s time bins as factors were carried out on activity levels during the first and last 10 s of tone presentation. The ANOVAs carried out on the data from the 16-month-old mice revealed non-significant main effects of group (both F’s < 1), main effects of tone presentation (min F(2, 40) = 7.88, P < 0.01) and non-significant interactions between these factors (both F’s < 1). There was a significant main effect of time bin for

3.6. Retention test: context conditioning.

Table 1 Mean infrared locomotor activity scores for 16-month-old and 3–4-month-old Tg2576 and control mice during the (A) first 10 s of tone; and (B) last 10 s of tone, presentation during day 1 conditioning trials Age (A) First 10 s of tone 16 months 3–4 months (B) Last 10 s of tone 16 months 3–4 months

Genotype

Tone 1

Tone 2

Tone 3

Mean

S.E.M.

Mean

S.E.M.

Mean

S.E.M.

Tg+ Tg−

2.19 1.88

0.57 0.46

1.67 1.24

0.55 0.42

0.78 0.77

0.44 0.38

Tg+ Tg−

1.89 1.60

0.61 0.56

1.0 1.58

0.52 0.67

0.34 0.69

0.27 0.46

Tg+ Tg−

1.92 2.06

0.63 0.50

1.57 1.37

0.57 0.48

0.96 0.91

0.44 0.40

Tg+ Tg−

1.64 1.88

0.62 0.58

0.60 1.70

0.35 0.59

0.14 0.30

0.11 0.26

Tg+ = APP transgenic and Tg− = littermate control mice. S.E.M. = standard error of the mean.

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Fig. 3. Mean infrared locomotor activity scores for 16-month-old (left panel) and 3–4-month-old (right panel) Tg2576 (Tg+) and control (Tg−) mice during the context-fear test (a), during initial exposure to the novel test context (b), and during presentation of the tone CS on day 2 (c).

3.7. Retention test: cue conditioning Fig. 3b (left panel) shows the mean locomotor activity scores during exploration of the novel context prior to delivery of the tone in 16-month-old Tg2576 and littermate control mice. During the first 2 min, there was a trend for lower levels of activity in the transgenic mice relative to littermate controls. A two-way ANOVA with group and time as factors revealed a significant interaction between group and time bin, F(11, 220) = 4.02, P < 0.05, but no significant main effects of group, F(1, 20) = 3.16, P > 0.05, or time bin, F(11, 20) = 2.15, P > 0.05. Tests of simple effects showed that wild type mice were significantly more active than the Tg2576 mice at the 30, 60 and 90 s periods during exposure to the novel context, (min F(1, 12) = 3.68, P < 0.05). During presentation of the tone (see Fig. 3c, left panel), transgenic animals showed higher levels of locomotor activity than wild type control mice. A two-way ANOVA with genotype and time as factors showed a significant main effect of genotype, F(1, 20) = 4.24, P < 0.05, and a significant main effect of time,

F(11, 20) = 5.11, P < 0.0001, but no significant interaction between these factors, F(15, 300) = 1.2, P > 0.05. Fig. 3b (right panel) shows the mean locomotor activity scores for the 3–4-month-old Tg2576 and control mice during exposure to the novel context prior to the delivery of the tone CS. An ANOVA carried out on the data from which these means were derived revealed no main effect of genotype, F(1, 14) = 3.17, P > 0.05, no main effect of time, F < 1, and no interaction between these factors, F(11, 154) = 1.09, P > 0.05. During the first 3 min of the tone presentation (see Fig. 3c, right panel) wild type mice showed a more pronounced decrease in activity relative to transgenic mice. A two-way ANOVA with genotype and time as factors revealed no significant main effect of group, F(1, 14) = 4.03, P > 0.05, a significant main effect of time, F(11, 20) = 3.46, P < 0.05 and a significant interaction between genotype and time, F(11, 154) = 3.87, P < 0.05. Tests of simple main effects revealed that wild type mice were significantly less active than transgenic mice during time bins 14, 15 and 24 (min F(1, 33) = 4.21, P < 0.05).

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3.8. Freezing: context retention test The percentage of observations with a freezing response in 16-month-old animals elicited by the conditioning context is shown in Fig. 4a (left panel). An ANOVA showed no main effect of genotype, F(1, 20) = 1.21, P > 0.05, no main effect of time, F(15, 20) = 2.07, P > 0.05 and no interaction between these factors, F < 1. Young Tg2576 mice and control mice showed comparable levels of freezing elicited by the context (see Fig. 4a, right panel). ANOVA revealed no main effect of genotype, F < 1, no main effect of time, F(7, 14) = 3.28, P > 0.05 and no interaction between these factors, F(14, 98) = 1.76, P > 0.05. 3.9. Freezing: tone retention test Fig. 4b (left panel) shows the mean percentage of trials on which the 16-month-old mice displayed a freezing response during the 6 min of exposure to a novel context prior to presentation of the tone. Inspection of this figure reveals that there was no difference between the two groups, F(1, 20) = 2.41, P > 0.05, no main effect of time bin (F < 1) and that this pattern did not differ between the groups, F(20, 100) = 3.27, P > 0.05. During presentation of the tone (see Fig. 4c, left panel), aged wild type animals showed higher levels of freezing relative to Tg2576 animals. This was confirmed by ANOVA which showed a significant main effect of group, F(1, 20) = 4.55, P < 0.04, but no main effect of time, F(5, 19) = 2.37, P > 0.05, and no interaction between these factors, F(20, 100) = 1.46, P > 0.05. In the first 6 min of the tone test (see Fig. 4b, right panel) 3–4-month-old Tg2576 and littermate control mice displayed no differences in freezing behaviour. A two way ANOVA with genotype and time as factors revealed no main effect of genotype, F(1, 14) = 1.78, P > 0.05, no main effect of time, F(5, 14) = 2.06, P > 0.05 and no interaction between these factors, F < 1. The mean percentage of trials in which a freezing response was recorded during the tone test is shown in Fig. 4c (right panel). Inspection of this figure shows that Tg2576 and control mice performed at a comparable level across the tone test. An ANOVA showed a non-significant main effect of group, F < 1, a non-significant main effect of time bin F(5, 70) = 1.97, P > 0.10 and a non-significant interaction between these two factors, F < 1.

4. Discussion 4.1. Summary Previous studies have shown that Tg2576 mice are impaired in the retention of aversive Pavlovian conditioning. The aim of the present study was to evaluate this proposal further by examining whether Tg2576 mice showed age-

dependent changes in conditioning during training and/or in the retention of aversive conditioning. A summary of the findings for each experimental cohort during the phases of the experiment is shown in Table 2. During training, both 4 and 16-month-old Tg2576 mice showed suppression of locomotor activity to the tone CS and to the context (Pre-CS). However, 16 month-old APPSWE mutants showed an immediate post-shock freezing deficit. This deficit was absent in 4-month-old transgenic mice. During the retention test, both 4 and 16-month-old Tg2576 mice showed normal levels of freezing elicited by the training context. In contrast to previous studies with Tg2576 mice, we found evidence of impaired retention of conditioned responding elicited by the auditory CS. Before considering this pattern of results further we will first consider the implications of the changes in post-shock freezing observed in 16-month-old transgenic mice. 4.1.1. Post-shock freezing Sixteen-month-old, but not 4-month-old, transgenic mice showed elevated locomotor activity immediately following the foot shock during conditioning. This difference declined during the remaining period of the ITI. Furthermore, there was no difference between the groups during the tone CS. An important point to note about the Tg2576 mice is that both age groups showed comparable levels of locomotor activity during initial exposure to the training context. As such, changes in locomotor activity would seem to be an unlikely confounding variable mediating changes in activity and freezing in transgenic mice in the present study (cf., [12]). When a normal animal receives an aversive shock it reacts with a bout of vigorous locomotor activity. The increase in postshock activity persists for a brief period and then declines to inactivity that is usually measured in terms of freezing [7]. Several features of the post shock activity burst are consistent with the view that it is an unconditioned reaction to shock [7–9]. The fact that 16-month-old Tg2576 mice showed comparable levels of activity in the first second following foot Table 2 Summary of behavioural results for old and young Tg2576 mice and littermate controls during each experimental phase Experimental phase

16-month-old

3–4-month-old

Context habituation Tone conditioning Context conditioning Post-shock activity Cue conditioning

× × × ×

× × × × ×

Context retention

Activity: × Freezing: ×

Activity: × Freezing: ×

Tone retention

Activity: Freezing:

Activity: ∗ Freezing: ×

A tick mark indicates the presence of significant differences between Tg2576 and control mice, and a cross indicates no significant difference relative to littermate control mice ((*) small group effect on locomotor activity confined to three time bins).

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Fig. 4. The mean percentage of observations with a freezing response in 16-month-old (left panel) and 3–4-month-old (right panel) Tg2576 (Tg+) and control (Tg−) mice during the context test (a), during exposure to the novel test context (b) and following presentation of the tone CS on day 2 (c).

shock would suggest that the activity burst component of the unconditioned response to shock was normal in transgenic mice. Thus, 16-month-old mice were clearly sensitive to the presentation of the unconditioned stimulus. Freezing (or immobility) that follows a foot shock is generally considered to be a Pavlovian conditioned response to (contextual) cues that are paired with shock (for a detailed discussion of this topic see [26]). There are two potential explanations of the post-shock deficit in old Tg2576 mice. One possibility is that the mutant mice were generally slower to suppress activity than control mice. However, the fact that 16month-old Tg2576 mice showed a comparable suppression of locomotor activity to control animals during the first 10 s periods of each tone presentation argues against a general deficit in activity suppression. An alternative hypothesis is that post-shock deficits in Tg2576 mice reflects that the mice were slower to associate the context with foot shock. By the end of the ITI the levels of locomotor activity were comparable between the two groups. In other words, the expression of freezing in Tg2576 mice required longer exposure to the various elements or features of the context to elicit freezing. Although speculative, such an account could explain the ini-

tial post-shock activity deficit and the comparable levels of suppression seen during the Pre-CS period. Although 16-month-old Tg2576 mice showed aberrations in post-shock activity during training, retention tests showed that both 4 and 16-month-old transgenic mice displayed normal levels of activity and freezing to the context. In contrast, activity and freezing to the tone CS was significantly impaired in 16-month-old but not 4-month-old Tg2576 mice. Before discussing the implications of this pattern of results, we will first consider the evidence that Tg2576 mice are impaired in context conditioning. 4.1.2. Cue competition and fear conditioning in Tg2576 mice Tg2576 mice can show deficits in context fear conditioning [5]. However, there are reports of normal context freezing in Tg2576 mice. For example, Dineley et al. [6] reported that Tg2576 mice showed a context conditioning deficit at 5 months of age that could be rescued by increasing the number of context-shock pairings; although this manipulation failed to retrieve a deficit in 9-month-old transgenic mice. Corcoran et al. ([5]; Experiment 2) reported that 16–18-month-old

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Tg2576 mice showed normal levels of freezing to a context and to an auditory CS when the context elicited higher levels of freezing than the tone CS. In contrast, Tg2576 mice showed a deficit in context conditioning when the level of freezing supported by the tone was greater than that supported by the context. Corcoran et al. ([5]; Experiment 3) suggested therefore that the effect of the APPSWE mutation on contextual conditioning was dependent upon the strength of the context-US association. According to this hypothesis, the extent to which other cues in the environment (e.g., the tone) competed with, or overshadowed, the context for associative strength (cf., [20,24]) was differentially affected by the mutation. A similar analysis has been proposed for the differential effects of hippocampal cell loss on foreground and background contextual fear conditioning [22]. In the present study, the level of freezing elicited by the context in 16-month-old control and Tg2576 mice was generally higher than that elicited by the tone and suggests that the context acquired greater associative strength than the tone CS. The absence of a context freezing deficit in the transgenic mice could therefore be explained by appealing to the idea that the context was sufficiently salient to support conditioning [5]. The novel aspect of our data is that transgenic mice nevertheless showed impaired expression of conditioning to a tone CS. This is unlikely to reflect a simple sensory deficit as both young and old mutant mice showed changes in locomotor activity to the tone during conditioning and locomotor activity was suppressed during the initial presentation of the CS at test. The neural systems responsible for the detection of the auditory cue appear to be intact in mutant mice. Although clearly contradictory to the results reported by Corcoran et al. [5] this pattern of results might be reconciled with those reported in that study by reference to the same principles of overshadowing. In Experiment 1, the context supported higher levels of freezing than the auditory cue during testing. Thus, one could argue that, in aged Tg2576 mice, conditioning to the auditory cue was overshadowed by the presence of the more salient contextual cue (see also [11]). Although, this analysis is speculative, and requires testing by systematic manipulation of the relative salience of the conditioning cues, it does suggest a means by which the results of the present study and those reported by Corcoran et al. [5] may be reconciled. A deficit in tone conditioning is not typically associated with hippocampal dysfunction (but see [19,25]). Cell loss in the amygdala can impair conditioning to both the context and a tone CS [18]. As noted by Corcoran et al. ([5]; p. 248.) the presence of an auditory fear conditioning deficit in mutant mice might be anticipated on the grounds of the dense amyloid pathology observed in this region in Tg2576 mice. The present study has provided some evidence that is consistent with this idea. Clearly, however, the pattern of Pavlovian conditioning deficits in Tg2576 mice does not model the effects of cell loss in the amygdala directly. The conditioning impairment in Tg2576 mice appears to be related to the salience of the cues that are paired with shock [5]. However, if amyloid

deposition does compromise the function of the amygdala it may be at the expense of processing those cues that are relatively weakly associated with foot shock. This idea requires further investigation.

5. Conclusion The main aim of the present study was to examine the effects of the APPSWE mutation on the acquisition of Pavlovian fear conditioned responding to a context and to an auditory CS. Consistent with previous reports, the present study revealed some evidence that the processing of contextual information was disrupted during conditioning in old mutant mice. However, our study also revealed a striking effect of the APPSW mutation on conditioning to an auditory cue that was age-dependent. Although this finding is consistent with similar findings in AD patients [13] it also suggests that further work is required to evaluate the impact of the APP mutation on the functional integrity of neural systems mediating emotional learning, including the amygdala.

Acknowledgements This research was supported by grants from the Wellcome Trust and the BBSRC. Philip Barnes was supported by a Ph.D. studentship from the Medical Research Council (UK). We would like to thank Alison Yates for help in running the transgenic colony.

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