Paradigm overview

In the initial learning phase, participants learned to associate binaural auditory scenes (i.e., audio-clips) with the location of an embedded difficult-to-detect auditory target. While half the audio-clips contained a target, allowing a target-context association to be created in memory (memory condition), the other half of audio-clips were not paired with an auditory target (neutral condition), thus serving as a control condition. After a one hour delay, participants were presented with the same audio-clips, but now with all of them having a lateralized target tone, or an infrequent catch tone. While memory trials were associated with a specific spatial memory for the location of the target acquired during the learning task, the neutral trials were equally familiar from the learning task, but were not associated with a specific contextual spatial memory for the target. In a prior study, we showed that participants were more accurate and faster to detect the target on memory cue trials where the audio-clip and the target were combined during the learning phase, compared to neutral cue trials where no memory association existed and thus attention was divided between the left and right auditory fields40. The facilitating effect of the memory cue reflects memory-guided attention.

Participants

The final sample consisted of 60 healthy participants between 40 and 61 years of age recruited through the Oxford Biobank, comprised three groups with varying APOE4 gene doses: E4/E4 (N = 20), E4/E3 (N = 20), and E3/E3 (N = 20). One participant who had scored below the inclusion cut-off for screening with the Addenbrooke’s Cognitive Examination version 3 (ACE-III; cut-off <86/100) was excluded from the sample41. The mean age of the sample (51 years) was well below the mean age of symptom onset in APOE4 homozygotes (68 years) and heterozygotes (76 years)42. APOE2 carriers were not tested due to the relative infrequency of this allele1, and also to preclude potential confounding effects since the APOE2 genotype has been shown to be protective against AD in some studies43,44.

Participants selected from each genotype group were chosen randomly from the Oxford Biobank participant pool based on the age criteria set by the experimenters as well as the requirement for normal hearing and no history of major physical or psychological illness. Letters were mailed to all participants that fit the recruitment criteria (E3/E3 N = 2621; E3/E4: N = 908; E4/E4 N = 107) by an independent administrator at the Oxford Biobank who was otherwise uninvolved in the project. Twenty interested participants from each genotype group were then randomly selected and contacted to participate in the experiment. Genetic results were not divulged to participants at any stage, nor to the experimenter before data collection was completed.

All participants provided informed consent prior to taking part in the study in accordance with the Declaration of Helsinki. The study was approved by the South Central Oxford C Research Ethics Committee (08/H0606/133). All collected data were anonymised and stored securely in accordance with the Data Protection Act 1998.

It should be noted that the Oxford Biobank participant sample used in this experiment showed a higher level of education than that of the general UK population (M = 9.4 years; http://www.nationmaster.com/country-info/stats/Education/), even though education was matched across groups. However, the majority of research suggests that the effects of APOE4 and education on development of dementia are independent45,46.

All participants were right-handed, fluent in English, had no history of psychiatric, neurological, or other major illness and had normal or corrected-to-normal vision and normal hearing. Hearing was assessed using pure tone thresholds for octave frequencies ranging from 250 to 8000 Hz, with the criterion for normal hearing being thresholds lower than or equal to 25 decibels of Sound Pressure Level (dB SPL) and less than or equal to a 15 dB SPL difference between the two ears at each octave frequency.

Groups were matched for age (F(2,57) = 0.576, p = 0.57), gender (X2 (2, N = 60) = 1.01, p = 0.61), years of education (F(2,57) = 1.59, p = 0.21), as well as cognitive ability as per scores on the ACE-III (F(2,57) = 0.24, p = 0.79). No differences in parental history of dementia (F(2,57) = 0.26, p = 0.78), maternal grandparents’ history of dementia (F(2,56) = 0.73, p = 0.49) or paternal grandparents’ history of dementia (F(2,55) = 0.54, p = 0.58) were observed. One participant was excluded in the analysis of maternal grandparents’ history of dementia, and two participants were excluded in the analysis of paternal grandparents’ history of dementia, because the information was not known. Parental history, maternal grandparental history, and paternal grandparental history were calculated as a score between 0 and 2 (no parent with dementia = 0, one parent with dementia = 1, two parents with dementia = 2). A total family history score was also calculated and matched across groups (F(2, 55) = 1.04, p = 0.36). This score comprised a sum of parental history score and both grandparental history scores, with affected parents given a weighting of 2 and grandparents given a weighting of 1, for a maximum score of 8. Demographic variables are presented in Table 1.

Table 1 Demographic characteristics of final sample, by group. Full size table

Cognitive assessment

The ACE-III was administered to screen for cognitive impairment41, since the aim of the current study was to assess the impact of APOE genotype independent of and prior to AD symptomology. The ACE-III is a relatively brief neuropsychological screening test consisting of attention, memory, language, fluency and visuo-spatial ability tests. It has been widely adopted in clinical settings to detect cognitive impairment associated with AD and other forms of dementia. Only participants who scored in the normal range (i.e., greater than the 86/100 cut-off score) were included in the study.

Subjective memory assessment

Subjective memory was evaluated to identify subtle changes in self-perceived memory ability. In prior work, subjective memory concerns have been identified in asymptomatic familial AD carriers, and co-occur with hippocampus-dependent memory decline47. The presence of subjective memory concerns is also related to abnormal changes in beta-amyloid and tau biomarkers in APOE4 carriers48,49,50, and is considered by some researchers to be the earliest symptomatic manifestation of AD51.

Here, subjective memory was assessed using the Everyday Memory Questionnaire (EMQ) consisting of 18 questions relating to the perceived frequency of memory problems and forgetting in everyday situations52. Responses were given on a six point scale ranging from 0 = “not at all” to 5 = “more than once a day”, and the total EMQ score was calculated from 90 points (5 × 18), with higher scores indicating larger subjective memory concerns.

Apoliprotein E genotyping

APOE genotyping was performed by the Oxford Biobank, using Applied Bio-systems TaqMan® SNP Genotyping Assay, C_3084793_20 and C_904973_10 corresponding to APOE SNPs rs429358 and rs7412, respectively, and run with ABI 7900HT Fast Real-Time PCR system. Diplotypes corresponding to APOE E3/E3, APOE E3/E4 and APOE E4/E4 were then identified.

Stimuli, tasks and procedure

92 binaural audio-clips were retrieved from “http://www.freesounds.org/.” The clips were chosen to maintain considerable semantic relevance in order to increase the likelihood that an appropriate association could be formed and labelled in LTM. Semantic relevance was first evaluated based on subjective experience of the experimenter. At this first step, 104 audio-clips were selected by the experimenter, which had been used in a separate experiment. As a second step, in a separate pilot study, 12 audio-clips were eliminated which were judged to have “little to no semantic relevance” on a 5-step scale. Clips were cut to a length of 2500 ms, with a 100 ms rise and fall time, down-sampled to a standard sampling rate of 44100 Hz. All stimuli were presented through BOSE QuietComfort 25 over-ear noise cancelling headphones, at a listening volume of 60 dB SPL on average across stimuli, with some sounds peaking at about 80 dB SPL. Acoustic stimuli and visual cues were presented using Presentation software (version 13, Neurobehavioral Systems, Albany, CA).

The auditory target, which was embedded within the audio-clips, was a 500 Hz pure tone with 200 ms duration. A 200 ms high-pitched 2000 Hz catch tone was embedded within a minority of audio-clips. The target and catch tone had a 20 ms rise and fall time, were sampled at a 44100 Hz frequency, and were presented at a volume that was adjusted for each participant to allow 80% detectability.

Following audiometric screening, cognitive assessment, and subjective memory assessment, participants filled out a basic questionnaire to collect demographic characteristics including age, sex, years of education, and family history of memory impairment (Table 1). Next, they completed the main experimental task, which consisted of four parts.

1. Determining individual signal-to-noise (SNR) thresholds. We used a two alternative forced choice procedure to estimate each participant’s threshold in detecting a pure tone target. On each trial, participants were presented with the same audio-clip twice (each audio-clip was 500 ms in duration) separated by a 500 ms silent interval. The audio-clip was selected from a set of four audio-clips (i.e., bird chirping, sandpaper, people murmuring, industrial machine). None of the audio-clips used to estimate participants’ thresholds were used in the subsequent phases of the study. A pure tone target (500 Hz, 500 ms in duration, 50 ms rise/fall time) was embedded in one of the two audio-clip presentations. Participants were asked to indicate by pressing a button in which of the two audio-clip presentations the target was embedded. The 79% threshold of detectability was estimated using a three-down one-up algorithm53. At the beginning of the test, the target intensity was set at 60 dB SPL and decreased by a factor of 5 after three correct responses using the Attenuation function in Presentation (Neurobehavioral Systems). The intensity was increased by a factor of 5 after a mistake. The threshold was calculated by taking an average of the last 8 of 12 reversals54. The target intensity at the threshold level was then used in the subsequent phases of the experiment. 2. Learning. A total of 92 audio-clips, presented binaurally for 2500 ms, were divided into target-present (memory cue) and target-absent (neutral cue) trials (46 each). In the memory trials, a pure tone target was paired with the audio-clip, presented in the left (23 trials) or right (23 trials) ear at random. In the neutral trials, no target was presented. Each participant was presented with the same 92 audio-clips over six learning blocks (552 trials in total) to promote a strong association between audio-clips and location of the target when present. In addition, to strengthen the target-audio-clip associations, trials for which participants made an incorrect response were repeated immediately until a correct response was made within each block. The order of trials was random within each block, and trials for each condition were counterbalanced across participants and groups. Within memory trials, the target tone was played at each participant’s SNR threshold at 2000 ms after sound onset, and lasted for 200 ms. Participants were instructed to listen for the location of the target within each audio-clip, and pressed the left, right or down arrow key on a keyboard when the target was played from the left side, right side, or if no target was present, respectively. Participants were given 2000 ms to respond following the offset of the audio-clip, and subsequently received visual feedback for 500 ms to indicate hit, incorrect response (i.e., responding ‘no target’ when a target was present, or responding to target at the incorrect location), or no response, followed by a 1000 ms interval before the onset of the next trial. Participants were asked to respond as quickly and as accurately as possible. 3. Memory-guided attention. A one hour retention interval separated learning and the memory-guided attention test. During this time, participants filled in questionnaires (screening, ACE-III, EMQ) and completed a simple verbal memory task relevant to a separate study55. Following the retention interval, participants completed 92 trials in which they had to detect the pure tone target within a binaural audio-clip (80 trials) and withhold responses during a minority of catch trials (12 trials). The intensity of the pure tone target and catch tone was adjusted for each participant according to the 79% SNR threshold established at the beginning of the study40. Figure 1 provides a trial overview. On each trial, the same audio-clip, selected from the learning phase, was presented twice. The first presentation (S1) served as a cue to orient attention and did not include the target nor the catch tone. The second presentation (S2) occurred 1000 ms after S1 offset and included the pure tone target or the catch tone. For memory trials, the target (46 memory target trials) or catch tone (6 memory catch trials) was always presented at the previously learned location. For neutral trials, which did not include a pure tone target during the learning phase, a target (46 neutral target trials) or catch tone (6 neutral catch trials) was presented to either the left or right ear at random. Participants were instructed to press the left or right keyboard arrow button as quickly and as accurately as possible when they heard the pure tone target, and withhold responses on catch trials. A 2500 ms time window was given for responses starting from the onset of the target, and 1000 ms preceded the onset of the next trial. The order of trials was random within each block, and trials for each condition were counterbalanced across participants and groups. Figure 1 On top, an overview of the three main experimental tasks is shown: six block learning task (purpose: to create target-context associations), memory-guided attention task (purpose: to examine whether memory formed in the learning task guides attention) and explicit memory task (purpose: to determine whether and for which audio-clips the target location was consciously accessible). Below is an overview of one trial for each experimental condition, for the learning task and memory-guided attention task. In the memory cue condition, participants learned the location of the target, located in left or right hemispace, and the target was then presented at the learned location in the memory-guided attention task. RT and accuracy to detect the target was compared to neutral cue condition trials, where participants did not learn the location of a target (i.e., no target-context association formed in the learning task), and the target was then presented at a novel location in the memory-guided attention task. Note: S1 and S2 in the memory-guided attention task represent the first and second repetition of the audio-clip, separated by a 1000 ms ISI. Only S2 contained an embedded pure tone target, 2000 ms after S2 onset. Full size image The target was inserted towards the end of S2 (jittered between 1900–2100 ms after the audio-clip onset, 50 ms steps, rectangular distribution). A lengthy exposure to cues was intended to increase the informativeness of the memory cue and allow participants time to activate auditory memory for target location – context associations. 4. Explicit memory. A cued recall task was administered immediately after the memory-guided attention task in order to determine whether participants formed explicit associations between audio-clips and target location. Participants were presented with all 92 audio-clips, but without targets. For each audio-clip, participants indicated with a key-press response whether the target had been presented to the left or right ear or whether no target had been present in the learning phase. Participants were allowed as much time as needed to make their response. Subsequently, confidence in each response was rated using a 4-step scale key-press response, coding “I don’t know” responses as 0, “not very confident” responses as 1, “fairly confident” responses as 2, and “very confident” responses as 3.

Experimental design and statistical analyses

The three APOE genotype groups were compared in a between-subjects design to identify differences in learning, explicit memory, memory-guided attention, and the relationship between explicit memory and memory-guided attention. Differences in explicit memory and memory-guided attention across groups were also analyzed across age.

Rate of auditory associative learning

A 3 × 6 mixed ANOVA was used to compare the rate of associative learning of audio-clip and target pairings across groups, with APOE4 genotype Group (E3/E3, E3/E4, E4/E4) as a between-subject variable, and Learning block (Block 1–6) as a within-subject variable. Response time (RT) and accuracy were input as dependent variables. RT was calculated from the time of target onset, and accuracy was calculated as a percentage of correct responses of all possible responses (incorrect plus too slow). Incorrect trials, trials where RT was faster than 100 ms or slower than 2500 ms (i.e., too slow to respond trials), as well as outliers (RT > 2.5 SD from the block mean) were excluded from the RT analysis. A between-subjects ANOVA was then used to confirm that all Groups reached the same level of performance by the last learning block.

Explicit memory

Accuracy during the explicit memory test and confidence ratings were compared across groups using a mixed ANOVA with APOE genotype Group (E3/E3, E3/E4, E4/E4), Explicit memory (correct, incorrect), and Confidence in responses (1 = not very confidence, 2 = fairly confident, 3 = very confident) as factors. Any “I don’t know” (0) responses were combined with incorrect responses in this analysis. In each group, regression analyses were performed to assess the effect of Age on accuracy. Because the primary aim was to determine whether Group is a moderating variable in the relationship between age and explicit memory, a multiple regression was also performed with Age and Group as regressors and explicit memory accuracy as the dependent variable. The Age × Group interaction term represented the moderating effect (i.e., whether the effect of age changed across groups).

Memory-guided attention

A 2 × 3 mixed ANOVA was used to assess the effects of APOE genotype on memory-guided attention (RT and accuracy), with Memory cue (memory cue vs neutral cue trials) as a within-subject variable, and APOE genotype Group (E3/E3, E3/E4, E4/E4) as a between-subject variable. Performance on the last trial of the learning task was input as a covariate in the memory-guided attention analysis to control for any differences in initial acquisition. In addition to fast response trials (<100 ms) and outliers (RT > 2.5 SD from the mean), error and catch trials were also excluded from the main analysis.

Regressions were set up to identify the effect of age on memory-guided attention for each group. Because we wanted to determine whether group is a moderating variable in the relationship between age and memory-guided attention, a multiple regression was set up with Age and Group as regressors and memory-guided attention as the dependent variable. The latter was calculated as a normalized difference score in RT to detect the target within memory and neutral audio-clip cues (M Neutral − M Memory /M Neutral + M Memory ). The Age × Group interaction term represented the moderating effect. It should be noted that we calculated normalized difference scores in RT as opposed to accuracy because we expected that the ability for memory representations to facilitate speed in younger non-carriers would be pronounced compared to accuracy gains56.

In addition, to examine the relationship between explicit memory and memory-guided attention, a regression coefficient was calculated for each group. Explicit memory accuracy was input as the regressor, and memory-guided attention was operationalized with the normalized RT difference score. To assess whether this relationship changed across groups, a linear regression was set up with Explicit memory and Group as regressors, and normalized RT difference score as the dependent variable. The interaction of Explicit memory and Group reflected changes in the relationship between explicit memory and memory-guided attention across groups. We confirmed that Explicit memory and Group were not collinear (Variance Inflation Factor = 1.01).

We performed two subsidiary analyses to confirm that the benefit for memory cue trials reflected a biasing of spatial attention rather than a general increased readiness to respond. In the first analysis, we assessed whether the memory-guided benefit remains on those trials where participants recalled the target location at the wrong hemispace (e.g., recalled left when the target had been learned on the right), that is, where explicit memory acted as an invalid cue. Within-subject pairwise contrasts were used to compare RT on explicitly remembered memory cue trials, neutral cue trials, and invalid memory cue trials. To note, only those participants that had a sufficient number of invalid cue trials (i.e., 8 or more memory cue trials that were incorrectly recalled) and valid memory cue trials (i.e., 8 or more memory cue trials that were correctly recalled) were included in this analysis. Eight trials from each condition were chosen as the cut off to allow enough power for statistical analysis while maintaining a sufficient sample size (N = 22). As a secondary confirmation that the data cannot be understood as a generic increase in attention, we performed a within-subjects ANOVA comparing ability to withhold responses to catch trials associated with a memory cue and neutral cue. If the effect was simply driven by a generic increase in readiness in memory cue trials, then performance on catch trials that had previously been associated with a target location would also be affected.

Subjective memory assessment

Total EMQ scores (/90) were compared across Groups with a between-subjects ANOVA. To determine the association between subjective memory concerns and explicit memory, a non-parametric Spearman correlation (rho) was calculated between explicit memory accuracy and EMQ scores for each group47. To determine the association between subjective memory concerns and confidence in explicit memory, rho was also calculated between EMQ scores and total confidence scores for each group. Total confidence scores were quantified as the sum of confidence scores across all recall trials for each participant, with confidence scores for each trial being a number between 0 and 3.