Functional Neuroanatomy of Successful Paired Associate Learning in Alzheimer’s Disease

Twelve patients who met the National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer’s Disease and Related Disorders Association criteria (11) for mild probable Alzheimer’s disease and 12 age-matched healthy comparison subjects were recruited. Seven patients were receiving treatment with cholinesterase inhibitors. Subjects were screened for concomitant serious medical diagnoses and previous psychiatric history and were assessed with a comprehensive battery of neuropsychological tests, including the Mini-Mental State Examination, Geriatric Depression Scale (12), Dementia Rating Scale-2 (13), logical memory test from the Wechsler Memory Scale (14), and National Adult Reading Test (15) (Table 1). All subjects provided written informed consent before participating in the study. The study had been approved by the Research Ethics Committee of the South London and Maudsley Trust.

During the visuospatial paired associate learning task, subjects were required to remember the locations of objects that appeared on a computer screen (Figure 1). An object appeared in a white box for 5 seconds and was replaced after 500 msec by another object in a different location until all of the objects in that problem were presented (encoding phase). During encoding subjects heard the instruction “remember.” After 6 seconds, a previously presented object appeared in one of the boxes with the question “Was this here?” (retrieval phase). Subjects were required to make a recognition decision (yes/no) by pressing one of two response keys. If the subject made no response, then the object remained on the screen for 5 seconds. If a response was made, the object disappeared and all locations remained blank until 5 seconds had passed. After 500 msec another object was presented, and the process continued until all objects presented during encoding had been tested. At the end of each retrieval phase, there was a baseline rest period (8 seconds if unsuccessful, 11 seconds if successful or if five failed attempts had been made) before the next encoding phase. After an incorrect attempt, the same object-location pairings were repeated in a different order in the next attempt at the problem. This procedure continued until there was correct identification of all object-locations, or until five successive attempts had been failed, after which subjects were presented with new object-location pairings.

Before scanning, all subjects received off-line testing to identify individual levels of increasing difficulty that could be successfully performed in the scanner. Difficulty was manipulated by varying the number of objects and locations in each problem (Table 2). Problems were pseudorandomly presented at each difficulty level so that no more than two problems at the same level appeared consecutively. The comparison subjects completed three practice sessions, which took place 3–4 days before the first scan, 1 hour before the first scan, and 1 hour before the second scan. The patients with Alzheimer’s disease completed five practice sessions at 6–7 days, 3–4 days, and 1 hour before the first scan and at 3–4 days and 1 hour before the second scan. After scanning, the subjects rated the subjective difficulty at each level of the task.

Data were acquired on the 1.5-T General Electric Neuro-optimized Signa LX Horizon system (General Electric, Milwaukee) at the Maudsley Hospital. In each functional series, 150 T₂*-weighted images depicting BOLD contrast were acquired by using an interleaved echo planar sequence at 16 axial slices (TR=2000 msec, TE=40 msec, flip angle=90°, matrix=64×64, field of view=240 mm², slice thickness=7 mm, interslice distance=0.7 mm). Subjects received eight to 10 functional series in two scanning sessions (separated by 1 week), except for two patients who received five series in one session. A high-resolution, three-dimensional axial image that included the whole brain was also collected for each subject (TE=5.8 msec, TR=17.1 msec, flip angle=20°, matrix=256×256, thickness=1.5 mm).

Two-way mixed analyses of variance with task difficulty as a within-subjects factor and group as a between-subjects factor were used to assess behavioral data. Mean response times to incorrect trials at each subject’s hardest level of difficulty were submitted to an independent samples t test. Neuropsychological test performance was assessed by using Mann-Whitney U tests.

By using statistical parametric mapping (16), functional data were slice-timing corrected, realigned to the first echo-planar imaging volume and unwarped to correct for motion-related variance, coregistered to the high resolution T₁-weighted image, normalized by using affine transformations into standard space (17) based on the Montreal Neurological Institute reference brain, and spatially smoothed with an 8 mm full width at half maximum Gaussian kernel (18). Data were then parametrically analyzed to identify regions displaying differential responses to increasing task difficulty. Within the general linear model, task difficulty was regressed onto the BOLD response to the onset of stimuli, and data were modeled with an epoch design convolved with a canonical hemodynamic response function. Encoding epochs were calculated from the first object’s presentation onset to the last object’s offset, and retrieval epochs were calculated from the first object’s presentation onset to the last object’s response time onset (or presentation offset if no response was made). Epochs corresponding to two, three, four, and five objects lasted 11, 16.5, 22, and 27.5 seconds, respectively. A 128-second high-pass filter was used to remove low-frequency noise. Successful encoding and retrieval epochs were modeled as separate covariates of interest, and unsuccessful epochs and the 6-second interval between encoding and retrieval phases were covariates of no interest.

Load-independent and load-dependent relationships between task difficulty and BOLD responses were assessed by using polynomial regression in which zero-order (boxcar), first-order (linear), and second-order (nonlinear quadratic) terms were compared to a resting baseline. Using this method, we were able to identify both increases in activation and deactivation with increasing task difficulty. Contrast images generated from parameter estimates were entered into one- and two-sample t tests (accounting for subject-to-subject response variability at the random-effects level) to form statistical parametric maps of the z statistic. Voxel-level contrasts were thresholded at p<0.05 (false discovery rate corrected for multiple comparisons), unless stated otherwise, and activations were reported only if the clusters consisted of at least five contiguous voxels. Statistical parametric map coordinates were converted from Montreal Neurological Institute coordinates to standard space to better localize activations by using the Talairach and Tournoux atlas (19).

Encoding and retrieval epochs associated with success, compared to failure, were also assessed. Data were examined as for the parametric analyses with the following exceptions. Only epochs relating to the hardest level of difficulty were assessed to ensure approximately equal success versus failure epochs (in which there were 1 plus incorrect retrieval decisions). This procedure resulted in exclusion of data from two patients and two comparison subjects because of insufficient data associated with failure. All other epochs at easier levels of task difficulty and the encoding/retrieval interval were modeled as covariates of no interest. Contrast images of the difference between correct and incorrect epochs were generated for each subject. The statistical parametric map of this contrast was then masked with a statistical parametric map of the contrast (corresponding to correct plus incorrect attempts > rest) to separate differential activations from suppressions with respect to baseline.

Behavioral data are presented in Table 1, Table 2, and Table 3. Subjective task difficulty ratings were collected only from comparison subjects because the patients with Alzheimer’s disease did not provide reliable reports. For the mean number of attempts per problem, all main effects and interaction terms were significant (task difficulty: F=20.08, df=3, 66, p<0.0005; group: F=11.12, df=1, 22, p<0.005; interaction: F=3.88, df=3, 66, p<0.05). Post hoc Tukey’s pairwise comparisons revealed that the patients with Alzheimer’s disease made a greater mean number of attempts per problem at the hardest level of difficulty. For the mean number of successful problems, the main effect of task difficulty was significant (task difficulty: F=3.11, df=3, 66, p<0.05), while the main effect of group and the interaction of task difficulty and group were not significant (group: F=0.54, df=1, 22, p=0.47; interaction: F=0.49, df=3, 66, p=0.81). The patients and the comparison subjects completed the same number of problems correctly at each level of difficulty. For subjective difficulty ratings, the main effect was significant (F=41.69, df=3, 33, p<0.0005), whereby subjective task difficulty ratings increased systematically across the four levels.

For mean correct response times (adjusted for mean number of attempts across all levels of difficulty) (Table 3), all main effects and the interaction of group and task difficulty were nonsignificant (task difficulty: F=0.69, df=3, 63, p=0.56; group: F=1.30, df=1, 21, p=0.27; interaction: F=0.40, df=3, 63, p=0.75). For incorrect response times, no significant difference was found between the patients (2972.8 msec, SD=278.5) and the comparison subjects (2859.9 msec, SD=374.0) (t=0.65, df=12, p=0.53).

Lateral parietal activations reported during episodic tasks are thought to reflect recognition processes (21) and retrieval processing of spatial information (22). Medial parietal activity has been proposed to underlie imagery (23) and retrieval success (24), and activity in the occipital cortex has been related to perceptual and recognition processes (25). Of the activations within the frontal regions, those in the anterior prefrontal cortex have been attributed to retrieval mode (26) and to postretrieval monitoring (27), while dorsolateral prefrontal cortex activations have been related to monitoring and verification processes and semantic production operations during episodic retrieval (28). Dorsolateral prefrontal cortex activity has also been associated with increasing task difficulty rather than mnemonic processes (29). Other studies have demonstrated increases in anterior cingulate activation during cognitively demanding tasks (30), and such activation has been attributed to inhibition of inappropriate responses (31) and initiation or willed control of behavior (32). Cerebellar activity has been commonly reported in studies of episodic retrieval and has been related to retrieval effort and mode (33) and self-initiation of retrieval processes (34). Finally, frontoparietal activity observed in the current study may also reflect practice-related effects. This interpretation is supported by a recent functional magnetic resonance imaging (fMRI) study in which activation in the lateral prefrontal, superior, and inferior parietal cortices increased after extensive training of visuospatial working memory over a 5-week period (35).

Differences in activation between the patients with Alzheimer’s disease and the comparison subjects were found to result from activation in one group and deactivation in the other. Such differential patterns of activity may reflect the use of different mnemonic strategies across the two groups or may reflect functional compensation for neuropathological changes associated with Alzheimer’s disease. However, they may also reflect differences in effort between the patients and the comparison subjects at the hardest level of difficulty. Although we took as many steps as possible to control for relative difficulty of the task across four levels of difficulty included in the design, the behavioral data indicated that, on average, the patients needed an extra 0.71 attempt to successfully complete problems at the hardest level of difficulty. Given this difference, the findings of increased activation in the comparison subjects and decreased activation in the patients may have resulted from increasing item familiarity in the patient group (because of the additional attempts), while findings of activation in the patients and activation and deactivation in the comparison subjects may reflect increased effort at the hardest level of task difficulty in the patients with Alzheimer’s disease. This interpretation, however, is not supported by our observation that areas of increased activation in the patients and in the comparison subjects match neither brain regions previously identified as being involved in effort nor item familiarity. Instead, the greater signal changes in the lateral and medial parietal regions in the patients, relative to the comparison subjects, during encoding and retrieval appear to reflect additional activity within regions found to be active in comparison subjects during visuospatial paired associate learning. Thus, these small increases in activity in the patients with Alzheimer’s disease may reflect compensatory reallocation of neural resources to compensate for Alzheimer’s disease-related neuropathology. It has previously been argued that increases in activity in the right middle frontal gyrus, right precentral gyrus, and left cerebellum during overt rehearsal of word lists are associated with engagement of the articulatory loop within the white matter (36). Therefore, differential activations observed in the comparison group may be attributable to strategic effects such as greater engagement of subvocalization during encoding.

In the current study seven patients were receiving acetylcholinesterase inhibitor treatment for cognitive impairment. Little is known about the effect of such treatment on the BOLD response in patients with Alzheimer’s disease, although findings of positron emission tomography and fMRI studies of cholinergic enhancement in healthy adults who performed explicit memory tasks suggest that cholinergic stimulation enhances activation in extrastriate regions (37), while cholinergic blockade decreases activation in these regions (38). The effect of cholinergic stimulation or blockade is less consistent in the frontal cortices, with frontal decreases in activation being reported with cholinergic stimulation and blockade during performance of explicit memory tasks (37, 38). Some evidence suggests that a single dose of rivastigmine increased activation in the fusiform gyrus bilaterally during face encoding, in the left superior frontal gyrus during a 1-back working memory task, and in the right inferior and superior frontal gyri during a 2-back working memory task in patients with Alzheimer’s disease (39). Decreases in activation were found in the right middle and superior frontal gyri in the 2-back working memory task alone. Therefore, it is possible that increased activation in frontal-occipital regions in patients receiving acetylcholinesterase treatment in the current study may have reduced any possible activation differences in these regions between the patients and comparison subjects. However, a report of decreases in right frontal activation with increasing memory load under rivastigmine administration (39) suggests that any possible differences in activation within this region between the patients and comparison subjects should have been enhanced. Therefore, the minimal activation differences between the patients and the comparison subjects in the current study are most likely attributable to our attempts to make adjustments for performance and relative task difficulty rather than to the effect of treatment.