Clinical trials have demonstrated that ChEIs positively affect cognition in AD and other conditions, but few studies have examined where effects occur in the brain. The objective of this study was to identify neural correlates of cognitive improvement after three months of donepezil treatment in subjects with mild AD. Our main hypothesis was that donepezil would improve the HFC network, which would significantly correlate with cognitive improvement. Specifically, in nonhuman primates and humans, cholinergic fibers primarily originate from the cholinergic nuclei located in the basal forebrain and pons, and densely innervate the thalamus, limbic regions and neocortical regions (35
). The hippocampus, in particular, is one of the limbic areas with the highest concentration of cholinergic innervation. In AD, a decrease in cortical and hippocampal cholinergic neurotransmission is observed (1
). In vivo
visualization of donepezil binding in the brains of AD patients reveals a significant reduction in the hippocampus (36
). Recent neuroimaging approaches, in particular resting-state R-fMRI studies in AD, have shown hippocampal disconnection with the limbic, thalamic, and neocortical areas (18
). In addition, the hippocampal network is involved in the default mode network, which showed alterations in the AD subjects (20
Currently, very little is known regarding how donepezil affects functional brain networks in patients with mild AD. After 12 weeks of donepezil treatment, subjects demonstrated significant enhancement of the positively connected hippocampal functional networks, especially in the regions of the right PCC, and the left insula and thalamus. It is hypothesized that donepezil treatment significantly increases cholinergic activity, and improves hippocampal network activity. Recent studies demonstrated that the PCC is an important region as a part of the connectivity found in the backbones and brain module (38
). The improved connectivity between the PCC and the hippocampus after the cholinergic treatment could improve the information flow, resulting in cognitive improvement.
The improved HFC network seen after donepezil treatment involves similar regions that have shown improvement in previous acute and prolonged ChEI-challenged fMRI task-activation studies reported in the literature. For example, during a working-memory task paradigm, a single dose of rivastigmine increased activation in the right prefrontal cortical regions in mild AD subjects (8
). Similarly, after only five days of galantamine treatment, MCI patients demonstrated increased left hippocampal, prefrontal, anterior cingulate, and occipital cortical activation during face encoding, and right precuneus and middle frontal gyrus activity with working-memory tasks (9
). MCI patients also have shown increased DLPFC, superior frontal gyrus, temporal lobe, and occipital region activity during working-memory tasks (10
) after an average of 10 weeks of donepezil treatment. Other fMRI task-specific studies have focused on the effects of prolonged administration (20 weeks) of rivastigmine (13
) and galantamine (12
) in mild AD. During various cognitive tasks, increased activation was found in the frontal lobe, inferior parietal lobe and basal ganglia structures. In this study, using resting-state R-fMRI and a shorter duration of donepezil treatment, we found similar results and have extended the findings to include other brain regions, such as the insula, thalamus and pontine regions.
Unlike fMRI studies where the brain responses to treatment are dependent on the type of cognitive task, R-fMRI studies can identify brain networks in which changes in the network activity correlate with changes in cognitive function. In the present study, we employed the changes in the MMSE and ADAS-cog scores as independent variables to identify their neural correlates. As shown in , changes in the parahippocampus activity (Δm) significantly correlated with changes in MMSE scores, such that the stronger the connectivity between the parahippocampus and the hippocampus, the greater the improvement in the MMSE. It is plausible that improvement of the network connectivity in this important cognitive pathway is resultant of the cholinergic enhancement after donepezil treatment.
The regions of the left DLPFC, left IFG and left precentral gyrus in the anticorrelated HFC network showed a negative correlation with changes in MMSE scores. The negative slope represented the increase
or recovery in anticorrelation activity. This is because the sign of functional connectivity in these regions is negative in these anticorrelation networks in the CN group, as shown in . The better the recovery of this anticorrelation network activity is, the greater the cognitive improvement is in these subjects after treatment. The better recovery of anticorrelation connectivity in the DLPFC-hippocampus network may help memory reconsolidation, thereby facilitating signal processing from the hippocampus to the DLPFC (39
Similar neural correlates related to changes in the ADAS-cog score were found in the regions of the left DLPFC and the left MFG, as shown in . Because the DLPFC and MFG regions belonged in the anticorrelated HFC network, and the negative values of ΔADAS-cog represent improvement of cognitive function, the slopes in are positive. This indicates that greater cognitive improvement (represented by negative ΔADAS-cog scores) is associated with better connectivity recovery in these anticorrelated networks (represented by negative Δm values). Clearly, connectivity recovery between the temporal and frontal networks in mild AD could result in cognitive improvement, as measured by ADAS-cog scores. To our knowledge, this is the first study in which neural correlates of cognitive improvement, as evidenced by MMSE and ADAS-cog scores, are revealed and can be employed to monitor or assess therapeutic responses to AD treatment.
It is intriguing that the brain regions with altered HFC network connectivity after treatment (, ADB vs. ADT) did not overlap with those regions that had reduced HFC network connectivity (, ADB vs. CN). In other words, donepezil treatment in patients with AD did not improve the originally reduced HFC connectivity. Instead, the donepezil treatment boosted HFC connectivity in those regions () where the HFC connectivity was normal in the CN group. It is plausible that because the oral donepezil treatment increases the cholinergic activity in the whole brain, this medication increases activity in some regions (as listed in ), even though they were relatively normal before treatment. It is suggested that brain reorganization may occur after treatment. However, such increased connectivity in certain regions () after donepezil treatment could induce some of the imbalances in the neural network, resulting in modest improvement of the cognitive performance.
Justification for Selecting the “Seed” Region
For the determination of functional connectivity, the “seed” method is an original and simple technique, which extracts the intrinsic time course from a region of interest, and then determines the temporal correlation between this extracted signal and the time courses from all other voxels (26
). Although this method requires a priori
definition of a seed region, it is widely employed, because this approach provides high sensitivity and easy interpretation of results. In the related research topic, a majority of previous studies averaged all voxel time courses in the hippocampus as the “seed” signal (18
), while others selected only five voxel time courses as the “seed” signal (23
). Other studies employed the independent component analysis (ICA) method without requiring a priori
specification of a seed region to calculate functional connectivity (19
). There is no consensus, as of yet, on how to choose the optimal number of components, or the manner by which to assign each component with specific neurophysiological meaning (27
). In the present study, we selected 30 voxel time courses with the highest averaged m
-values from each subject as a “seed” region because all but one AD subject (16 voxels
), had a minimum of 30 hippocampal voxels. Our rationales are: 1) the number of hippocampal voxels in each individual subject is different. Some have bigger hippocampi than others; 2) the mean number of hippocampal voxels in AD subjects is significantly smaller than that of the CN subjects; 3) each subject has the same number of voxels with the best functional connectivity as the seed region. We compared results from the literature and found that the selection of different numbers of voxels in the hippocampus could affect the results of functional connectivity (19
). In the present study, we reanalyzed the entire dataset with “averaged voxel time courses over all hippocampus voxels.” The results of the hippocampal functional connectivity differences between mild AD and control subjects are consistent with previous publications (22
), but the differences in functional connectivity are less than the 30-voxel approach. It is possible that the method of averaging all voxel time courses to generate the seed signal may desensitize the difference in the functional connectivity. Although a comprehensive investigation on how the seed selection would affect the hippocampal connectivity is beyond the scope of this study, a sophisticated design, such as clustering analysis, could address this question in a future study.
This study is not without limitations. The study duration was relatively brief compared to some other pharmacological fMRI studies, although we were able to measure the responses of HFC activity to donepezil treatment in AD study subjects, and show the cognitive significance of hippocampal functional alterations in this population. Second, the sample size was relatively small, because the population was limited to mild AD subjects, who never received AD-related medication. Third, this was an open-label study instead of a scientifically ideal double-blind placebo-controlled study. As a result, there is a possibility that the observed results may represent a placebo effect. However, a randomized, placebo-controlled trial of donepezil relative to AD is no longer considered ethical, as the Food and Drug Administration approved this medication for clinical use in mild to severe AD. In practice, such a possibility is low, because previous studies showed that during the clinical trials of donepezil, there was no significant placebo effect observed in AD subjects in three months after treatment (4
). Fourth, another limitation in our study design is that the controls were only scanned at baseline and not at 12 weeks. Finally, while our data analysis was limited to the HFC network, the neural network changes in AD are clearly not limited to the HFC network; we will perform a global functional connectivity analysis with this cohort that will consider all pairs of anatomically defined regions of interest from the whole-brain template in Talairach space.