The mean percent correct in the encoding phase was 96 ± 4 % (mean ± s.e.m; n=20) for the living/non-living judgment task, and 97 ± 3 % for the detection task. They were not significantly different (p >0.05; two-tailed t-test). Reaction times were 1.17 ± 0.05 sec for the living/non-living judgments task, 1.07 ± 0.05 sec for the detection task, and 0.80 ± 0.05 sec for the visuo-motor control task (mean ± s.e.m). These reaction times were significantly different (F(2,54)=13.8, p<10-4, one-way ANOVA). In the post-hoc Tukey’s t-test, the visuo-motor control task was significantly different from both the living/non-living judgments and the detection tasks (p <0.05), but the living/non-living and the detection tasks were not significantly different (p >0.05).
In the retrieval phase, subjects made yes/no recognition memory judgments for previously studied and new words (see Materials and Methods for details). The percent correct for words encoded with living/non-living judgment task was 77 ± 4% (mean ± s.e.m; n=20), which was significantly higher than the percent correct for words encoded with detection task (69± 3%; p < 0.005; two-tailed paired t-test), thus confirming that the words were more deeply encoded in living/non-living judgment task than in the detection task. The reaction time was not significantly different between deeply encoded words (1.23 ± 0.04 sec) and shallowly encoded words (1.21 ± 0.05 sec; p = 0.3; two-tailed paired t-test).
Functional MRI results
shows activated brain areas (n=20 subjects) found in the encoding phase (“deep encoding” versus “visuo-motor control”) superimposed on T1-weighted anatomical slices () and on 3D-transparent brain images (). Left dominant prefrontal, parietal, and temporal activation clusters were found as well as a small right prefrontal cluster (see also ). This pattern is consistent with previous studies (Buckner and Koutstaal, 1998
; Wagner et al., 1998
; Buckner et al., 1999
; Habib et al, 2003
). The left dorsal prefrontal activation (a) was largely located in Brodmann’s Area (BA) 46, BA9/46, BA9 on the middle frontal gyrus (MFG). Only the most ventral part was extended into BA45 on the inferior frontal gyrus (IFG). In this study, we use the term DLPFC for this activated cluster, but note that the most ventral part of the cluster includes IFG. The left VLPFC activation (b) was located in BA 45/47. The temporal cortex activation was in the superior temporal sulcus (STS) and extended into the fusiform gyrus (FG) (BA 21/22/37). shows the mean percent signal changes from baseline (fixation) for the three conditions in the encoding phase (n=20, across subject ± standard error). Our results confirmed the load-dependent activation (Kapur et al. 1994
; Otten et al. 2001
) in the frontal and the temporal encoding-related areas.
(A-D) Activation and fiber tracking from the activated areas in the encoding phase in the comparison “deep encoding versus visuo-motor control”.
Table 1 Regions activated in the memory tasks. Only clusters with a significant activity of p<0.05 corrected for whole-brain multiple comparisons are reported. The coordinates and their T-values are at the peak voxels in each cluster, and the coordinates (more ...)
Fiber tracking from activated brain areas
Fiber tracking was performed from all the cortical activated areas (but not from cerebellum and brain stem) in all the 20 subjects. shows all the reconstructed fibers from the left DLPFC (left) and left VLPFC (right) activation in single subject. The fibers from left DLPFC and VLPFC extended to other left prefrontal regions, as well as the left parietal and temporal cortices. The fibers from the left temporal cortex activation were found to connect with the left frontal, parietal and occipital cortices. For retrieval data, please see Supplementary Figure S1 and Table S1
Group study of DTI fiber tracking
shows the distribution of terminal points for reconstructed fibers in 20 subjects. The fibers were tracked from areas in the left DLPFC (top row), VLPFC (middle row) and temporal cortex (bottom row) that were activated during the encoding phase. The terminal points converged on a large number of memory-related areas also activated during the encoding phase. These areas include: left VLPFC (h), right VLPFC (k), left medial frontal (o, s), left superior frontal (t) and left temporal cortex (f) (see also Table S1). In addition, some fibers terminated in areas activated during the retrieval phase (e.g. i and p). We also found connections to the left hippocampus/parahippocampal region (c). The activated areas in the left DLPFC, VLPFC, and the left temporal cortex were all connected with the left hippocampus/parahippocampal region. We performed this group analysis for all the other activated areas. The results in suggest that activated areas in the left DLPFC and VLPFC both have connections with the left temporal cortex activation (see Figure S2 and S3 for complete continuous slices of this group study for the encoding and retrieval clusters). The activation in the left DLPFC, VLPFC, STS and intraparietal sulcus (IPS) each connected with the hippocampus/ parahippocampal region (, S2, S3).
Connections between memory-related areas
We examined connections between pairs of activation clusters in each subject (, ). Fibers were obtained using any voxel in the starting cluster (as a seed) to any voxel within the end cluster, going in either direction. shows the connections found between the activated areas in the left DLPFC and the left temporal cortex for the encoding phase. shows the connections between the left VLPFC and the left temporal cortex activation for the encoding phase. We performed this analysis on the data obtained from all 20 subjects. The most dorsal part (BA 9) of the left DLPFC cluster connected with the dorsal part (BA21, 22) of the left temporal cortex cluster. In some subjects, there were also connections between the ventral part of the DLPFC cluster and the temporal cortex cluster. Fibers between VLPFC and STS also exhibited different pathways passing through more ventral parts. These fiber pathways were consistent across subjects.
Figure 4 Fibers between two activation clusters in five different subjects. (A) Reconstructed fibers between the left DLPFC (BA9/8/46/45) and left STS to FG (BA21/22/37) in the encoding phase. (B) Reconstructed fibers between the left VLPFC and the left STS to (more ...)
Table 2 Connection patterns that show how many subjects have connections between each pair of activated clusters in encoding (deep encoding versus control). The streamline algorithm (numbers outside of the parenthesis) and the tensor deflection algorithm (numbers (more ...)
summarizes the connectivity data between each pair of clusters for the entire group of subjects (For retrieval, see Supplementary Table S2
). In obtaining the data for , we used both the streamline and tensor deflection algorithms. The connections between the left DLPFC and the left temporal cortex, and between the left VLPFC and the left temporal cortex were found in more than 10 out of 20 subjects with both the streamline (from the left DLPFC to the left temporal cortex: 18 subjects; from the left VLPFC to the left temporal cortex: 14 subjects) and tensor deflection algorithms (from the left DLPFC to the left temporal cortex: 20 subjects; from the left VLPFC to the left temporal cortex: 18 subjects) in encoding. (For other connections, see Supplementary Figure S6
Estimations of tractography error
We estimated the error of fiber tracking between the left DLPFC and the left temporal cortex using a bootstrap method (Lazar and Alexander, 2005
) (see Materials and Methods). First, we made histograms of the probability of a connection to DLPFC for each and every voxel in STS/FG (; see Materials and Methods). Thirty-three continuous seed points in the temporal cortex (a green region in ) showed greater than 50 % probability of a connection to DLPFC. The seed cluster volume was 33 mm3
(1mm sampling of seed points), corresponding to 0.7 % of the entire temporal cortex activation (4848 mm3
). It is clear that these seed points did not distribute diffusively across the whole activated cluster, but were rather confined in a specific region. The Talairach coordinates of these 33 voxels were located in STS (x, y, z = -54 ± 5, -40 ± 2, -1 ± 2). These results indicate that the locations in temporal cortex that had connections with DLPFC were very specific.
We showed the results of boot-trac from a single seed point (Talairach: x, y, z = -49, -41, 0; from a green region in ) in two ways. First, all the fibers for 100 bootstraps are shown in . Most of the fibers (78 %) went to the DLPFC, gradually diverging by the distance from the seed point in the temporal cortex activation. To assess uncertainty of this probability, we performed boot-trac analysis 1000 times. By shuffling the orders of the 1000 boot-trac samples 1000 times, an error for each iteration was obtained (). At 100 iterations of boot-trac, the error was less than 5%. Second, the boot-trac from the same seed is shown as a probabilistic map (; see Materials and Methods). This figure shows how many times, out of 100, the boot-trac fibers passed through each voxel. The probabilities were almost 100 % around the seed point in the temporal cortex activation, but soon went down less than 50 %. This is because of the divergence of the tracked fibers, which depends on the distance from the seed point, as displayed in .
Figure 6 (A) A histogram of the numbers of the fibers between the DLPFC cluster and each of the 100 random clusters for one subject. (B) Histograms of actual numbers of fibers from DLPFC to the temporal cortex (black bars) and median numbers of fibers from DLPFC (more ...)
We obtained a probabilistic map of connections from all 33 seed points with a connection probability greater than 50% (; see Materials and Methods). These seed points are located in restricted locations, so they could compensate for the low probability of one seed point. The results indicate that the fiber tracking from those 33 seed points reached the DLPFC activation with a probability of more than 80 %. The terminal location was the posterior end of the DLPFC cluster (around x, y, z = -49, 3, 36 in the Talairach coordinate), around the precentral sulcus to the middle frontal gyrus. This result indicates that not only the seed points, but also the terminal points were in very restricted locations within the activation. shows the probabilities of fibers from 33 voxels in the temporal cortex to anywhere in the whole brain. Although we did not selectively show the fibers that reached the DLPFC activation, high probabilities were found only along the pathway from the temporal cortex to DLPFC. Thus, the specificity of this pathway was remarkable. We also obtained probabilistic maps (Supplementary Figure S7
, upper row
) from all the voxels in the temporal cortex activation, similar to . All subjects’ results (n=20) were averaged after normalization (Supplementary Figure S7
, lower row
The boot-tracked fibers were diverging by the distance from the seed points, but in many cases, as shown in , most of the fibers reached the DLPFC activation. To see this observation quantitatively, we examined the probability of connections between any voxel in the temporal cortex and any voxel in the DLPFC (see Materials and Methods). We found the connection with a probability of 100% in this subject.
We performed this latter analysis for 18 subjects who showed connections between these regions. The existence of this pathway was crucial to distinguishing between the two models (the Serial and Parallel Pathway Models) mentioned above. Ten subjects out of the 18 subjects showed more than 50% probabilities for the connections between the left DLPFC and the left temporal cortex. The probability for the existence of this connection was very high in these 10 subjects (86.3 ± 18.0%, n=10). This result validates our major finding on the direct pathway between DLPFC and the temporal cortex activation.
Estimations of the connection specificity using arbitrary defined areas
We also examined the specificity of the connections between DLPFC and the temporal cortex. Arbitrary regions satisfying the selection criteria (see Materials and Methods) were used 100 times for each subject. Then, the number of fibers between the DLPFC and those arbitrarily defined regions were obtained (see the histogram in ). There was no connection between the DLPFC cluster and 55% of the random clusters. The median of the number of fibers between the DLPFC cluster and the random clusters was found to be 0, which is significantly different from the observed number of fibers (57 fibers; P<10-16; Wilcoxon signed rank test). The random clusters that had more fibers than the actual number of fibers (57 fibers) were only 5%, and all of them were found in the precuneus in this particular subject.
We performed the same kind of analyses in all 18 subjects who had fibers between DLPFC and temporal cortex clusters. Eleven out of 18 subjects showed a significantly larger number of actual fibers relative to fibers generated from random clusters (P<0.001; Wilcoxon signed rank test). At the population level, we made histograms of the number of actual fibers (black bars in ) and the median number of fibers from random clusters (white bars in ) for 18 subjects, and found a significant difference (P < 0.001, Wilcoxon Signed Rank Test, n=18). These analyses confirmed that the connections between DLPFC and temporal cortex activation were specific. Only some restricted areas (14.0 ± 3.6 %; average ± standard error; n=18) located in the right PFC (BA9/46) and precuneus (BA7) showed more connections with the left DLPFC cluster.
Anatomical network among memory-related areas
A summary of the fiber tracking results among the activation clusters in 20 subjects is shown in . Fibers found in more than half of all 20 subjects with both the streamline and tensor deflection algorithms were shown as arrows in solid lines. The dotted arrow indicates the pathways found in more than 10 subjects only by tensor deflection. (Some arrows are omitted. For a complete table of connections, see and Supplementary Table S2
Figure 7 Summary of the fiber tracking results between the activation clusters. (A) Encoding, (B) Retrieval. The pathways found in more than 10 subjects both by streamline and tensor deflection algorithms are shown as solid arrows, while a dotted arrow indicates (more ...)