Age-related memory deficits linked to circuit-specific disruptions in the

Michael A. Yassaa,b,c,1, Aaron T. Mattfeldb,c, Shauna M. Starkb,c, and Craig E. L. Starkb,c,1

aDepartment of Psychological and Brain Sciences, The Johns Hopkins University, Baltimore, MD 21218; bDepartment of Neurobiology and Behavior, University of California, Irvine, CA 92697; and cCenter for Neurobiology of Learning and Memory, Irvine, CA 92697

Edited by Edward E. Smith, Columbia University, New York, NY, and approved April 15, 2011 (received for review January 27, 2011) Converging data from rodents and humans have demonstrated an have more difficulties learning new information and may only age-related decline in pattern separation abilities (the ability to encode the “gist” and not as many event details as young adults. discriminate among similar experiences). Several studies have pro- We (20) as well as others (21) have tested this hypothesis more posed the dentate and CA3 subfields of the hippocampus as the explicitly in older adults by using an object discrimination task. potential locus of this change. Specifically, these studies identified Consistent with the predictions from the rodent work, both rigidity in place cell remapping in similar environments in the CA3. studies found that older adults tended to be biased more toward We used high-resolution fMRI to examine activity profiles in the completion at the expense of separation. We also demonstrated and CA3 in young and older adults as stimulus that these behavioral impairments were correlated with hippo- similarity was incrementally varied. We report evidence for “repre- campal DG/CA3 network hyperactivity (20), consistent with the sentational rigidity” in older adults’ dentate/CA3 that is linked to finding in the rodent that aged CA3 place cells exhibit generally behavioral discrimination deficits. Using ultrahigh-resolution diffu- elevated firing rates across novel and familiar environments (22). sion imaging, we quantified both the integrity of the perforant In this investigation, we hypothesized that the pattern separa- path as well as dentate/CA3 dendritic changes and found that both tion signals exhibited by the DG/CA3 would be diminished with were correlated with dentate/CA3 functional rigidity. These results age for highly similar, but not dissimilar inputs (our techniques do highlight structural and functional alterations in the hippocampal not allow us to isolate DG and CA3 activity). Consistent with our network that predict age-related changes in memory function and predictions, we found no differences in DG/CA3 fMRI activity

present potential targets for intervention. between young and older adults when stimuli were made very NEUROSCIENCE different. However, as stimuli were made more similar, the DG/ medial temporal lobe | diffusion tensor imaging | functional MRI | CA3 response was weakened in older adults (consistent with generalization | pattern completion pattern completion) but remained high in young adults, indicating that older adults’ DG/CA3 had an attenuated separation response ong-term memory function is commonly known to deteriorate to the lures. We refer to this change as a representational rigidity, fi Lwith increasing age. One of the sites that undergo the earliest which is operationally de ned as the requirement for increased changes is the hippocampus (1, 2), which has a well-known role in dissimilarity before stimuli can be orthogonalized, thus showing learning new facts and remembering events (3). Recently, electro- greater resistance to change. Critically, the extent of this rigidity fi physiological recording studies in aged rodents have shed light on predicted performance de cits in a behavioral discrimination task some of the possible neural mechanisms in the hippocampus that similar to the one used in our previous work (20). underlie this decline (1). These studies have demonstrated “rigidity” Further, to evaluate the structural correlates of the rigidity we in aged CA3 place cell firing patterns in similar environments. In observed in the DG/CA3 functional network, we used ultrahigh contrast to young CA3 place cells, which readily remap and shift resolution microstructural diffusion tensor imaging (msDTI) their representations in these environments, aged CA3 place cells techniques developed in our laboratory (23) to assess potential changes in diffusion properties within hippocampal subfield gray retain their original fields despite the changes in the environment. matter. We found a correlation between left DG/CA3 functional These data strongly suggest that aging is associated with a dimin- rigidity and the same region’s fractional anisotropy (a measure of ished capacity for pattern separation (learning new information by directional diffusion). Directional diffusion in gray matter is decorrelating similar inputs to avoid interference) and an increased thought to be an index of dendritic integrity (23, 24). Thus, these propensity for pattern completion (retrieval of previously stored results suggest that structural dendritic changes in this region may information from a partial cue), and further suggest that this shift contribute to the functional impairments observed, consistent with could be the result of a functional imbalance in the hippocampal the idea that the dentate and CA3 in particular are selectively vul- dentate gyrus (DG) and CA3 network. nerable to the aging process, because no such relationship was The role of hippocampal subfields in these key processes has long fi – found in other sub elds. been hypothesized in computational models (4 7). The models Next, we tested another key prediction of the model proposed suggest that the DG granule cells are capable of performing es- by Wilson et al. (1), namely that the degraded perforant path pecially strong pattern separation on the distributed representa- input to the DG and CA3 is linked to pattern separation deficits. tions arriving from layer II entorhinal neurons, projecting this The perforant path provides the primary afferent input to the fi signal onto the CA3 sub eld of the hippocampus via the strong dentate and CA3 from layer II entorhinal cortical neurons (25). fi mossy ber pathway. Empirical evidence for the involvement of the Studies in the rodent have shown that this pathway is essential DG and CA3 in pattern separation has been demonstrated by using electrophysiological recordings (8–10), immediate-early genes (11),

and high-resolution functional MRI in humans (12, 13). Ablation Author contributions: M.A.Y. and C.E.L.S. designed research; M.A.Y., A.T.M., S.M.S., and studies using DG-specific lesions (14), as well as ge- C.E.L.S. performed research; M.A.Y., A.T.M., S.M.S., and C.E.L.S. analyzed data; and netic NMDA receptor knockouts (15), have additionally shown M.A.Y., A.T.M., and C.E.L.S. wrote the paper. that the DG is critical for, and the likely locus of pattern separation. The authors declare no conflict of interest. Recent evidence also indicates that neurogenesis in the DG may be This article is a PNAS Direct Submission. particularly important for pattern separation (16–19). 1To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. The shift from pattern separation to pattern completion with This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. age is hypothesized to be one of the reasons why older adults 1073/pnas.1101567108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1101567108 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 for normal hippocampal function (26) and documented its deg- regarding circularity and double dipping (32), we used data from two radation with advancing age (23, 27–31). Here, we tested the previously collected samples to define repetition-sensitive regions of hypothesis that the network imbalance we observed in the fMRI interest (ROIs) in the hippocampus and then applied these ROI data may be the result of an age-related degradation in the per- masks to our new, independent sample to investigate the effect of forant path. To accomplish this goal, we used msDTI to visualize varying the degree of stimulus similarity on activity for lures. and quantify the perforant path diffusion signal as an in vivo Based on extensive behavioral investigations (20), lures were correlate of this pathway’s integrity. We found that perforant path binned according to their mnemonic similarity, allowing us to integrity was tightly correlated with the extent of left DG/CA3 conduct a parametric investigation of DG/CA3 input/output rigidity. In addition, perforant path integrity itself was predictive transfer functions. In our recent work (13), we found that in a of performance of older adults on a behavioral discrimination population of young adults, activity in a repetition-sensitive por- task. A potential functional consequence of a degraded perforant tion of DG/CA3 increased markedly with even small amounts of path is a decrement in the functional correlations between the change in the input (highly similar lures) and remained constantly (EC) and the DG/CA3. To evaluate this possi- elevated as lure similarity increased. We observed this constant bility, we performed a seed-style functional connectivity analysis activity consistent with strong separation again here in young at the subfield level. The functional coupling of the DG/CA3 with adults in the left DG/CA3 (Fig. 2B). Older adults, however, had the entorhinal cortex was highly correlated with the rigidity in the an attenuated left DG/CA3 separation response to the lures such CA3, suggesting that the level of rigidity in the DG/CA3 may be that separation signals were only noted on the highly dissimilar C related to degradation in the signaling between the entorhinal lures (Fig. 2 ). This representational rigidity was consistent with A cortex and the hippocampus. the theoretical predictions shown in Fig. 2 . × These data support the notion that age-related changes in the We conducted a 2 5 ANOVA with group (old vs. young) and fi perforant path and the DG/CA3 network bias this network against condition (L1, L2, L3, L4, L5) as xed factors and subject as separation and contribute to the mnemonic deficits observed in a random factor nested within group. A number of studies (33, older adults. These findings provide converging evidence in sup- 34) have shown that increases or decreases in BOLD response port of a well-characterized model of memory loss in aged rodents between young and old individuals could easily arise as artifacts (1) and contribute to our understanding of the effect of aging on of basal state factors such as metabolic rate and neurovascular the hippocampal network. coupling. With this caveat in mind, testing a main effect of group would have been difficult to interpret (this difference was sig- Results Twenty young adults and 20 older adults underwent a high-resolu- tion (1.5-mm isotropic) BOLD fMRI scanning session while viewing pictures of everyday objects (Fig. 1) and making an indoor/outdoor judgment. In our previous work (12, 13), we identified voxels in the hippocampus that signaled a repetition (i.e., changed their response between a first presentation and a repetition) and then examined activity for similar lure items in these voxels. Lure activity similar to that of a first presentation is consistent with pattern separation. However, if activity for a lure is similartothatofarepetition,this activity is more consistent with pattern completion. To increase our power for assessing activity for lure items and avoid potential issues

Fig. 2. Representational rigidity in older adults DG/CA3. (A) Hypothetical predictions for young and aged DG/CA3 transfer functions. Older adults are expected to exhibit rigidity where more dissimilarity is required for separation to occur (thus a positive slope across lures is expected for older adults and a near-zero slope across lures is expected for young adults). Inset shows actual data from our previous work (13) showing no change in young adults between “high similarity” lures and “low similarity” lures, i.e., DG/CA3 responds equally Fig. 1. Task design. Pictures were shown one at a time and could either be high to both types of lures, consistent with the above predictions. (B) A sample novel, repeated, or similar lures (participants are not informed of the item ROI of repetition-sensitive voxels (novel minus repeat) isolated in the left DG/ status). Different trials have been color-coded with blue (novel), green (re- CA3. (C) A comparison of young and aged left DG/CA3 fMRI activity across lures peated), and red (lure) for the reader’sbenefit; however, participants only saw showing a pattern remarkably consistent with the predictions shown in A. items on a white background. The only task instruction given was to press Percent change in signal is change from the novel foil baseline. The contrast of a button for indoor items and another button for outdoor items. The two lures activity for novel versus repeat items based on an orthogonal dataset was used shown (pineapple and piano) provide examples of the lure stimuli used. to select the voxels averaged here.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1101567108 Yassa et al. Downloaded by guest on September 25, 2021 nificant nevertheless (F1,38 = 5.31, P < 0.05). Thus, we opted to look for a significant interaction, which we observed in the data (F4,152 = 2.13, P < 0.05). Although the young adults showed no difference in left DG/CA3 activity across lure bins [consistent with our previous data (13) and with the idea that separation had occurred even with small amounts of change in the input], the older adults showed a clear positive slope across lure bins (Fig. 2C). This finding suggests that compared with young adults, the older adults’ DG/CA3 required more dissimilarity before pat- terns of activity shifted to reflect a transition from completion to separation. One important question is whether the extent of this rigidity reliably predicted behavioral discrimination abilities. Because the incidental task used inside the scanner could not be used to assess behavior, we tested a number of older participants (n = 15) who underwent this fMRI task in a separate session outside the scanner in an explicit recognition memory task designed to tax pattern separation (with a different set of stimuli). In this task, participants saw novel, repeated and similar lure items and were asked to indicate whether the item was “old,”“similar,” or “new.” Our previous work (20) has shown that older adults have a di- Fig. 4. Relationship between functional and structural measures of in- “ ” tegrity. (A) We found a negative relationship between the left DG/CA3 slope minished separation bias on this task compared with young of activity in older adults and fractional anisotropy in this region (an in- adults (i.e., they were less likely to call lure items similar and more dication of dendritic integrity), providing evidence that intact synaptic func- likely to call them old). We replicated this effect here (Fig. S1), tion in this region is necessary for successful pattern separation. (B) We found and we also observed an inverse correlation between the behav- a negative relationship between the left DG/CA3 slope of activity in older ioral separation bias in older adults and the slope of left DG/CA3 adults and their perforant path integrity, suggesting that degradation in this activity (Pearson’s r = −0.53, P < 0.05; Fig. 3A). In addition, there pathway may underlie the network’s representational rigidity. (C) We also NEUROSCIENCE was also a marginally significant correlation (Pearson’s r = −0.46, found a positive relationship between perforant path integrity in older adults P B and their separation bias on the explicit recognition task, providing further = 0.09; Fig. 3 ) between another behavioral measure, delayed evidence that the perforant path is necessary for successful discrimination. recall performance by participants on the Rey Auditory Verbal (D) We found a negative relationship between the left DG/CA3 slope of ac- Learning Test (RAVLT) (35), and the slope of left DG/CA3 ac- tivity in older adults and degree of functional coupling between the left tivity. Both of these correlations suggest that the degree of rep- entorhinal cortex and the left DG/CA3 suggesting that communication failure resentational rigidity in DG/CA3 predicts mnemonic deficits. in this network is linked to the representational rigidity observed. In addition, we performed ultrahigh-resolution msDTI scans (23) on 15 of the 20 older participants who underwent the fMRI scan. We used these scans to quantify fractional anisotropy (FA) We then sought to determine whether perforant path integrity throughout hippocampal subfields after removing all was related to the ability of the DG/CA3 to engage in pattern signals. These gray matter signals are thought to be a reflection of separation. Perforant path degradation with age is hypothesized the integrity of pyramidal dendrites (23, 24). We found a signifi- to be a catalyst for the shift in the information processing balance in the hippocampal DG/CA3 network that biases this network cant correlation between FA in the left DG/CA3 and the slope of away from pattern separation and toward pattern completion. fMRI signal in the same region taken to indicate the extent of To test this prediction, we examined the msDTI we collected. representational rigidity (Pearson’s r = −0.55, P < 0.05; Fig. 4A). Using this method in the same participants, we have demonstrated No age-related changes in FA or correlations with any other our ability to visualize and quantify diffusion signals specificto measure were observed in any of the other hippocampal sub- the perforant path and showed that these signals were degraded regions (CA1, Sub, EC). in the course of aging and predicted delayed recall performance on the RAVLT (23). In this investigation, we found a significant negative correlation between perforant path integrity and the slope of left DG/CA3 fMRI activity (Pearson’s r = −0.59, P < 0.05, Fig. 4B). Additionally we found a positive relationship between perforant path integrity and the separation bias of participants on the explicit memory task administered outside the scanner in a subset of participants (n = 11) who received both behavioral and DTI sessions (Pearson’s r = 0.73, P < 0.05; Fig. 4C). Finally, to assess the functional impact of structural perforant path degradation, we used functional connectivity analyses to investigate the correlation in hemodynamic activity between the EC and DG/CA3 regions. Here, we found that the degree of functional connectivity between these regions (thought to reflect Fig. 3. Relationships between representational rigidity and behavior. (A) the function of the perforant path) also predicted the extent of We found a negative relationship between the left DG/CA3 slope of activity functional rigidity in the DG/CA3 in older adults (Pearson’s in older adults and their separation bias on an explicit recognition task r = −0.40, P < 0.05; Fig. 4D). designed to tax their pattern separation abilities, suggesting that processing in this region plays a key role in discrimination and that its dysfunction with Discussion age may underlie mnemonic deficits. (B) We also found a marginally sig- nificant relationship between the left DG/CA3 slope of activity in older Converging evidence from animals (1) and humans (20, 21, 36) adults and their performance on a delayed recall task (verbal list learning), demonstrates that, on a behavioral level, pattern separation abil- which is a general index of their hippocampal function. ities are reduced with aging. This deficit is hypothesized to underlie

Yassa et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 many of the memory problems reported with increasing age. Aging Although we hesitate to overinterpret these dendritic diffusion is associated with a decline in episodic memory formation (37), data, we should note that this finding is the first of its kind in humans spatial memory and navigation (38), contextual source memory to our knowledge. Thus, subfield-specific dendritic integrity is a crit- (39), and recollection (40, 41). These functions require intact ical future avenue of investigation. Assessing dendritic integrity pattern separation circuitry (7, 42). Here, we examined the po- could offer important clues as to the neural basis of changes in net- tential neural locus for pattern separation deficits and uncovered work function. Reductions in dendritic integrity specific to the DG/ both a functional change in the DG/CA3 network as well as a re- CA3 region may occur before or subsequent to the loss in afferent lated structural change in the perforant path, both of which pre- fibers observed with age and may be an important feature of the dicted pattern separation abilities. neurocognitive aging process. However, at this time, this relationship Previous work in our laboratory isolated an elevated pattern of remains unclear. We should emphasize, however, that high-resolu- BOLD activity in the region during an explicit recognition task tion DTI allows us to examine gray matter anisotropy in a way that (20); however, we could not make inferences regarding the traditional DTI methods do not. This finding extends the utility of ability of this region to perform the separation computation DTI to investigating gray matter microstructure and not just per se. The explicit approach we previously used, although in- white matter. formative in terms of behavior, was quite limiting in terms of The results presented herein provide compelling evidence for interpretation. Activity during many conditions could be con- an age-related reduction in pattern separation in human DG/ fi taminated by a “recall to reject” strategy, which would prevent us CA3 and point to structural and functional de cits in the per- from making strong claims about the relationship between ac- forant path and the DG/CA3 as potential contributors to this tivity levels and pattern separation/completion (20, 43). How- shift. Our results are largely consistent with data showing that the ever, by using the incidental encoding approach used here, and in perforant path is particularly vulnerable to age-related changes. our previous work in young healthy volunteers (12, 13), we were For example, studies in rodents have shown that the perforant able to parametrically assess changes in activity in the DG/CA3 path input to the hippocampus is reduced in aged rats compared region as a function of mnemonic similarity of stimuli and avoid with young rats (29, 30). It should come as no surprise also that this pitfall. one of the primary targets of perforant path input, the dentate gyrus, also appears vulnerable to the aging process (46–50). We found that relative to the young, the aged DG/CA3 requires fi more dissimilarity (a greater change in the input) to shift its rep- Electrophysiological data in aged rats show reductions in eld fl EPSPs recorded in the dentate (27, 51) as well as presynaptic resentation to re ect a novel learning experience. Instead of place fi cell firing patterns (which we could not assess with fMRI) we used ber potential at the perforant path-dentate synapse (28, 31). the slope of lure bin activity in the DG/CA3 as our in vivo proxy to Recent evidence in aged rodents also suggests that molecular measure representational rigidity (resistance to change). In this and synaptic changes occur in the entorhinal cortex (EC) where scheme, a more positive slope suggests a requirement for greater the perforant path originates (52). One interesting possibility is that the some of the impairments dissimilarity to “remap,” thus showing greater resistance to change. observed in the dentate gyrus could be the result of diminished This pattern was characteristic of the slopes identified in older neurogenesis with increasing age (53, 54). Although there is still adults, but not in the young adults whose activity was consistent with a debate about the exact role of newborn versus immature remapping even for very small changes in the input. Critically, the neurons (18, 19), it is possible that the reduced capacity for extent of this representational rigidity predicted the performance of neurogenesis with increasing age interferes with dentate gyrus older adults on an explicit recognition task that directly tested their function, although evidence for this possibility is not straight- pattern separation abilities. This task was used to demonstrate age- forward (e.g., ref. 55). Although studying neurogenesis directly in related impairments in pattern separation (20, 21). Further, there humans may not be possible, recent work by Small and col- was evidence for a relationship between the rigidity in DG/CA3 leagues (56) using neuroimaging of cerebral blood volume fMRI activity and a simple standardized test of delayed recall (CBV) has isolated an in vivo correlate of neurogenesis, which performance, a memory measure that is known to be sensitive can be coupled with high-resolution fMRI methods in the future to hippocampal damage (44, 45). We have also shown that it is to study the link between DG neurogenesis and its pattern correlated with performance on discrimination tasks sensitive to separation capabilities. fi pattern separation de cits (20). Overall, these brain-behavior Taken together, the data presented here provide converging evi- correlations indicate that representational rigidity in the aged dence from multiple imaging modalities that age-related changes, fi DG/CA3 underlies the behavioral de cit in discriminating among manifest in the hippocampus, establish a permissive environment for very similar experiences. potentially pathological changes that affect memory function. In this Next, we turned to the question of whether structural changes in work, we uncover several of these conditions, namely perforant path perforant path integrity would contribute to the DG/CA3 network degradation, loss of functional connectivity between the entorhinal changes observed in aging. We used ultrahigh-resolution msDTI to cortex and DG/CA3, and functional rigidity as well as decreased examine the potential relationship between the integrity of the anisotropy in DG/CA3. These conditions may collectively contribute perforant path and DG/CA3 function in older adults. We found to the age-related shift in mnemonic function from encoding new that the extent of DG/CA3 rigidity in older adults was inversely memories to retrieval of preexisting memories. correlated with perforant path integrity. Furthermore, we also In summary, we combined two advanced high-resolution im- observed a direct correlation between perforant path integrity and aging modalities to investigate structural and functional changes performance on the explicit recognition task taxing the discrimi- in the hippocampal system with age. These results provide unique nation abilities of participants. We also used a functional assay to empirical demonstration of reduced pattern separation-related investigate the connectivity between the EC and DG/CA3 and activity in human DG/CA3 with age as well as evidence linking found that the extent of this functional coupling also predicted the structural changes in the perforant path and the DG/CA3 to in- degree of DG/CA3 rigidity. Using the same msDTI techniques we formation processing in the same network. The observed changes used for perforant path quantification, we also found a negative may contribute to the mnemonic deficits observed with age by correlation between fractional anisotropy in the DG/CA3 region weakening the processing of novel information and strengthening and its functional rigidity. These results suggest that microstruc- the processing of previously stored information. These results tural changes in the DG/CA3 region, possibly reflecting a reduc- bring us one step closer to a more complete understanding of age- tion in dendritic integrity, may contribute to the functional deficits related changes in hippocampal structure and function. Future observed. studies should continue to make attempts to link across techni-

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1101567108 Yassa et al. Downloaded by guest on September 25, 2021 Table 1. Older adults demographic and neuropsychological higher-order shims and SENSE imaging techniques. Functional EPI images variables were collected by using a high-speed echoplanar single-shot pulse se- quence with a field of view of 230 × 230 mm, echo time of 25 ms, flip angle Measurement Value (SD) of 70°, SENSE factor of 2, a repetition time (TR) of 1,500 ms, a 4 TR initial n 20 skip, and a resolution of 1.5 mm, isotropic. In each run, 26 triple-oblique axial slices were acquired aligned to the principal axis of the hippocampus M:F 8:12 bilaterally. Additional structural scans were also collected (details in Age 71 (4) SI Methods). Years of education 17 (2) Data analysis was performed by using Analysis of Functional NeuroImages MMSE 28 (1) (AFNI). Images were motion corrected and normalized to standard Talairach RAVLT 5-trial total 51 (9) space to provide an initial rough alignment. They were then aligned by using RAVLT immediate recall 11 (2) a ROI-based method we developed in our laboratory that maximizes our RAVLT delayed recall 10 (3) sensitivity to local changes in the medial temporal lobes (20). Behavioral Digit span 18 (5) vectors based on trial type and condition were used to model the data by WAIS full-scale IQ 118 (7) using a deconvolution approach based on multiple linear regression. The Trails A 28 (10) resultant fit coefficients (betas) estimated activity versus baseline (novel Trails B 61 (15) foils) for a given time point and trial type in a voxel. The sum of the fit Verbal fluency 48 (11) coefficients over the expected hemodynamic response (3–12 s after trial ’ Category fluency 21 (5) onset) was taken as the model s estimate of the response to each trial type fi Letter-number sequencing 10 (3) (relative to baseline). Beta estimates were generated for rst presentations and each of the lure bins (L1, L2, L3, L4, L5; L1 being the most similar lures Scores are shown as mean (SD). MMSE, Mini-Mental State Examination; and L5 being the least similar). Sorting the lure trials into different similarity RAVLT, Rey Auditory Verbal Learning Test; WAIS, Wechsler Adult Intelli- bins was conducted based on similarity ratings derived from a large nor- gence Scale. All scores were within norms. mative study conducted in our laboratory (20). Mean beta coefficients were converted to relative signal maps (percent signal change) by using the mean of each run from the betas. We created an a priori mask (Fig. S2) for repe- ques and species to enhance our understanding of this complex tition-sensitive voxels based on data from two studies (12, 13) and applied system and how it changes in the course of aging and disease. this mask to the lure bin data. The mask included several clusters in the DG/ CA3, CA1, and . Voxels within each of these clusters were col- fi fi Methods lapsed for further analysis. Clusters within a speci csub eld were also col- NEUROSCIENCE Participants. We collected data from 20 young adults (8M:12F, mean age 21 y, lapsed to increase signal-to-noise ratio. Collapsed subfields maps are shown SD 3) and 20 older adults (8M:12F, mean age 71 y, SD 4). Written informed in Fig. S3. Additional details on fMRI preprocessing and analysis as well as consent was obtained from all participants. Participants were screened for ROI mask creation are provided in SI Methods. any health conditions that may interact with their neurological status. Young The functional connectivity analysis was based on a seed style correlation adults were recruited from the University of California, Irvine community by analysis. First, we removed all task-related activity and nuisance vector ac- using IRB-approved flyers. Older adults were referred directly through a large tivity from our raw data, leaving a residual time series. We extracted a mean longitudinal study of healthy aging people at the University of California, residual time series from each of our hand-drawn ROIs used for alignment. Irvine. Exclusion criteria included any major medical conditions, (e.g., di- We then correlated the isolated mean residual time series with the residual abetes, heart disease), any neurologically active medication use, any history activity of all voxels in the brain. The resulting correlation coefficients were of mental or psychiatric disorder and any contraindications for MRI. All older Fisher’s z transformed. We then assessed the degree of correlation with the adults scored ≥27 on the Mini-Mental State Examination (MMSE: mean score seed time series for each region by using an anatomical ROI analysis. See 28, SD 1). All participants also received a battery of neuropsychological tests SI Methods for a more detailed description of this analysis. including measures of IQ, memory, attention, and general cognitive function (Table 1). Details of the administration of the RAVLT are included in Ultrahigh-Resolution Microstructural DTI Methods. Diffusion weighted scans SI Methods. were acquired on the same scanner by following the methods we described (23). Twelve 15-slice coronal single shot echo-planar imaging (EPI) scans were Behavioral Tasks. To assess behavior, we used an out-of-scanner explicit three- acquired with a 256 × 256 matrix, a field of view (FOV) of 170, voxel size of alternative recognition task in which participants viewed novel, repeated, 0.664 mm × 0.664 mm in the plane of acquisition, a slice thickness of 3 mm “ ” and similar stimuli (i.e., lures ) and were asked to indicate whether items (1 mm gap), a repetition time of 2,717 ms and an echo time (TE) of 67 ms, a flip were old, similar, or new. Lures were used to test the ability of participants angle of 90° and a SENSE reduction factor of 2.5. Diffusion weighting was to successfully separate. Details of the task are described in SI Methods. applied with b = 1,200 s/mm2 along 32 independent, noncollinear orien- Behavioral data are shown in Fig. S1. For the in-scanner version of the task, tations. One additional image with no diffusion weighting (b = 0) was also we used a different stimulus set of novel, repeated, and lure items and acquired. In addition to the diffusion-weighted scans, we also collected an participants were only asked to indicate whether items were “indoor” or ultrahigh-resolution Fast Spin Echo scan centered on the medial temporal outdoor” objects and their memory for these objects was never explicitly lobe with identical geometry and resolution (FOV = 230, TR/TE = 3,000/80, flip tested as in ref. 12. In our previous work (12, 13), we used half as many lures angle = 90°). These scans were used because they provide detailed anatomical as we used in this study. This limitation hindered our ability to detect information inside hippocampal subfields for each diffusion-weighted slice. changes across lures in more than two bins (i.e., high similarity vs. low sim- ilarity). However, in the current design, we were able to double the number Diffusion weighted images were corrected for head motion and eddy ’ — of lure items by removing the repetitions, thereby increasing the number of current distortions by using FMRIB s Diffusion Toolbox FDT v. 2.0 (57). items in each lure bin from 20 to 40 stimuli. This change was done to ensure Motion and distortion-corrected volumes were merged into a single volume, that we had enough power to detect effects across different lure bins. This and tensor solving was accomplished by calculating the six elements at each procedure was essential because we hypothesized that the DG/CA3 of older pixel by using multivariate linear fitting, allowing us to construct fractional adults will shift to a pattern consistent with a first presentation (i.e., pattern anisotropy (FA) maps as well as images for each of the three eigenvalues and separate) at greater levels of dissimilarity compared with the young adults. eigenvectors. In each participant, we identified the location of the perforant Thus, whereas young adults should readily shift DG/CA3 representation to path on the in-plane inverted T2-weighted scan (SI Methods) by using the reflect a new item as early as the Lure bin 1 (most similar), older adults may boundary between gray matter and white matter in the entorhinal cortex as not do so until Lure bin 3 or 4 (less similar). Because we used an orthogonal a guide. We calculated the amount of diffusion signal that is parallel to the mask from independent data sets to define the required repetition-sensitive canonical orientation of the perforant path by using a method we have voxels, including repeated items in this design was not necessary. Detailed described and validated in young and older adults (23). Area under the curve parameters of the task are described in SI Methods. (Fig. S4) was calculated for each participant’s perforant path diffusion pro- file and used as the key measure of perforant path integrity in this study. High-Resolution fMRI Methods. Functional MRI data were collected by using Additional details on these procedures as well as details on subfield-level a 3-Tesla Philips scanner equipped with a SENSE head coil using both quantification of FA are discussed in SI Methods.

Yassa et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 ACKNOWLEDGMENTS. We thank Ms. Joyce Lacy, Ms. Samantha Rutledge, for Memory Impairments and Neurological Disorders at the University of Mr. Marlo Asis, and Ms. Gianna O’Hara for help with data collection and California, Irvine (Alzheimer’s Disease Research Center National Institute of analysis; Dr. Michela Gallagher for helpful discussions and feedback regard- Aging Grant P50-AG016573) for help with participant recruitment. The study ing this manuscript; the Research Imaging Center at the University of Cal- was supported by National Institute on Aging Grants R03-AG032015 ifornia, Irvine for providing resources for use in this project; and the Institute and R01-AG034613.

1. Wilson IA, Gallagher M, Eichenbaum H, Tanila H (2006) Neurocognitive aging: Prior 30. Smith TD, Adams MM, Gallagher M, Morrison JH, Rapp PR (2000) Circuit-specific memories hinder new hippocampal encoding. Trends Neurosci 29:662–670. alterations in hippocampal synaptophysin immunoreactivity predict spatial learning 2. Burke SN, Barnes CA (2006) Neural plasticity in the ageing brain. Nat Rev Neurosci 7: impairment in aged rats. J Neurosci 20:6587–6593. 30–40. 31. Dieguez DJ, Jr., Barea-Rodriguez EJ (2004) Aging impairs the late phase of long-term 3. Squire LR, Stark CE, Clark RE (2004) The medial temporal lobe. Annu Rev Neurosci 27: potentiation at the medial perforant path-CA3 synapse in awake rats. Synapse 52: 279–306. 53–61. 4. Marr D (1971) Simple memory: A theory for . Philos Trans R Soc Lond B Biol 32. Kriegeskorte N, Simmons WK, Bellgowan PS, Baker CI (2009) Circular analysis in Sci 262:23–81. systems neuroscience: The dangers of double dipping. Nat Neurosci 12:535–540. 5. Treves A, Rolls ET (1994) Computational analysis of the role of the hippocampus in 33. Ances BM, et al. (2009) Effects of aging on cerebral blood flow, oxygen metabolism, memory. Hippocampus 4:374–391. and blood oxygenation level dependent responses to visual stimulation. Hum Brain – 6. McClelland JL, McNaughton BL, O’Reilly RC (1995) Why there are complementary Mapp 30:1120 1132. learning systems in the hippocampus and neocortex: Insights from the successes and 34. Fleisher AS, et al. (2009) Cerebral perfusion and oxygenation differences in ’ – failures of connectionist models of learning and memory. Psychol Rev 102:419–457. Alzheimer s disease risk. Neurobiol Aging 30:1737 1748. ’ ’ 7. Norman KA, O’Reilly RC (2003) Modeling hippocampal and neocortical contributions 35. Rey A (1941) L examen psychologique dans les cas d encéphalopathie traumatique. (Les problems.). Arch Psychol 28:286–340. to recognition memory: A complementary-learning-systems approach. Psychol Rev 36. Stark SM, Yassa MA, Stark CE (2010) Individual differences in spatial pattern 110:611–646. separation performance associated with healthy aging in humans. Learn Mem 17: 8. Leutgeb JK, Leutgeb S, Moser MB, Moser EI (2007) Pattern separation in the dentate 284–288. gyrus and CA3 of the hippocampus. Science 315:961–966. 37. Craik FIM, Simon E (1980) Age differences in memory: The roles of attention and 9. Leutgeb S, Leutgeb JK, Treves A, Moser MB, Moser EI (2004) Distinct ensemble codes depth of processing. New directions in memory and aging: Proceedings of the in hippocampal areas CA3 and CA1. Science 305:1295–1298. George A Talland Memorial Conference (Lawrence Erlbaum Associates, Hillsdale, NJ), 10. Lee I, Yoganarasimha D, Rao G, Knierim JJ (2004) Comparison of population pp 95–112. coherence of place cells in hippocampal subfields CA1 and CA3. Nature 430:456–459. 38. Newman MC, Kaszniak AW (2000) Spatial memory and aging: Performance on 11. Vazdarjanova A, Guzowski JF (2004) Differences in hippocampal neuronal population a human analog of the Morris water maze. Aging, Neuropsychology, and Cognition 7: fi responses to modi cations of an environmental context: Evidence for distinct, yet 86–93. – complementary, functions of CA3 and CA1 ensembles. J Neurosci 24:6489 6496. 39. Henkel LA, Johnson MK, De Leonardis DM (1998) Aging and source monitoring: 12. Bakker A, Kirwan CB, Miller M, Stark CE (2008) Pattern separation in the human Cognitive processes and neuropsychological correlates. J Exp Psychol Gen 127: – hippocampal CA3 and dentate gyrus. Science 319:1640 1642. 251–268. 13. Lacy JW, Yassa MA, Stark SM, Muftuler LT, Stark CE (2011) Distinct pattern separation 40. Jennings JM, Jacoby LL (1997) An opposition procedure for detecting age-related related transfer functions in human CA3/dentate and CA1 revealed using high- deficits in recollection: Telling effects of repetition. Psychol Aging 12:352–361. resolution fMRI and variable mnemonic similarity. Learn Mem 18:15–18. 41. Robitsek RJ, Fortin NJ, Koh MT, Gallagher M, Eichenbaum H (2008) Cognitive aging: A 14. Hunsaker MR, Rosenberg JS, Kesner RP (2008) The role of the dentate gyrus, CA3a,b, common decline of episodic recollection and spatial memory in rats. J Neurosci 28: and CA3c for detecting spatial and environmental novelty. Hippocampus 18: 8945–8954. 1064–1073. 42. Norman KA (2010) How hippocampus and cortex contribute to recognition memory: 15. McHugh TJ, et al. (2007) Dentate gyrus NMDA receptors mediate rapid pattern Revisiting the complementary learning systems model. Hippocampus 20:1217–1227. separation in the hippocampal network. Science 317:94–99. 43. Kirwan CB, Stark CE (2007) Overcoming interference: An fMRI investigation of 16. Clelland CD, et al. (2009) A functional role for adult hippocampal neurogenesis in pattern separation in the medial temporal lobe. Learn Mem 14:625–633. spatial pattern separation. Science 325:210–213. 44. Aggleton JP, Shaw C (1996) Amnesia and recognition memory: A re-analysis of 17. Aimone JB, Deng W, Gage FH (2010) Adult neurogenesis: Integrating theories and psychometric data. Neuropsychologia 34:51–62. separating functions. Trends Cogn Sci 14:325–337. 45. Mayes AR, Holdstock JS, Isaac CL, Hunkin NM, Roberts N (2002) Relative sparing of 18. Aimone JB, Deng W, Gage FH (2010) Put them out to pasture? What are old granule item recognition memory in a patient with adult-onset damage limited to the cells good for, anyway...? Hippocampus 20:1124–1125. hippocampus. Hippocampus 12:325–340. 19. Alme CB, et al. (2010) Hippocampal granule cells opt for early retirement. 46. Gazzaley AH, Siegel SJ, Kordower JH, Mufson EJ, Morrison JH (1996) Circuit-specific Hippocampus 20:1109–1123. alterations of N-methyl-D-aspartate receptor subunit 1 in the dentate gyrus of aged – 20. Yassa MA, et al. (May 20, 2010) Pattern separation deficits associated with increased monkeys. Proc Natl Acad Sci USA 93:3121 3125. hippocampal CA3 and dentate gyrus activity in nondemented older adults. 47. Penner MR, et al. (2010) Age-related changes in Arc transcription and DNA methylation Hippocampus, 10.1002/hipo.20808. within the hippocampus. Neurobiol Aging, 10.1016/j.neurobiolaging.2010.01.009. 21. Toner CK, Pirogovsky E, Kirwan CB, Gilbert PE (2009) Visual object pattern separation 48. Moreno H, et al. (2007) Imaging the Abeta-related neurotoxicity of Alzheimer disease. Arch Neurol 64:1467–1477. deficits in nondemented older adults. Learn Mem 16:338–342. 49. West MJ (1993) Regionally specific loss of neurons in the aging human hippocampus. 22. Wilson IA, Ikonen S, Gallagher M, Eichenbaum H, Tanila H (2005) Age-associated Neurobiol Aging 14:287–293. alterations of hippocampal place cells are subregion specific. J Neurosci 25: 50. Small SA, Tsai WY, DeLaPaz R, Mayeux R, Stern Y (2002) Imaging hippocampal 6877–6886. function across the human life span: Is memory decline normal or not? Ann Neurol 51: 23. Yassa MA, Muftuler LT, Stark CE (2010) Ultrahigh-resolution microstructural diffusion 290–295. tensor imaging reveals perforant path degradation in aged humans in vivo. Proc Natl 51. Barnes CA, McNaughton BL (1980) Physiological compensation for loss of afferent Acad Sci USA 107:12687–12691. synapses in rat hippocampal granule cells during senescence. J Physiol 309:473–485. 24. Jespersen SN, Kroenke CD, Østergaard L, Ackerman JJ, Yablonskiy DA (2007) 52. Stranahan AM, Haberman RP, Gallagher M (2010) Cognitive decline is associated with Modeling dendrite density from magnetic resonance diffusion measurements. reduced reelin expression in the entorhinal cortex of aged rats. Cereb Cortex 21: – Neuroimage 34:1473 1486. 392–400. 25. Witter MP (2007) The perforant path: Projections from the entorhinal cortex to the 53. Kuhn HG, Dickinson-Anson H, Gage FH (1996) Neurogenesis in the dentate gyrus of – dentate gyrus. Prog Brain Res 163:43 61. the adult rat: Age-related decrease of neuronal progenitor proliferation. J Neurosci 26. Hyman BT, Van Hoesen GW, Kromer LJ, Damasio AR (1986) Perforant pathway 16:2027–2033. changes and the memory impairment of Alzheimer’s disease. Ann Neurol 20:472–481. 54. Seki T, Arai Y (1995) Age-related production of new granule cells in the adult dentate 27. Barnes CA (1979) Memory deficits associated with senescence: A neurophysiological gyrus. Neuroreport 6:2479–2482. and behavioral study in the rat. J Comp Physiol Psychol 93:74–104. 55. Bizon JL, Gallagher M (2003) Production of new cells in the rat dentate gyrus over the 28. Barnes CA, Rao G, Houston FP (2000) LTP induction threshold change in old rats at the lifespan: Relation to cognitive decline. Eur J Neurosci 18:215–219. perforant path—granule cell synapse. Neurobiol Aging 21:613–620. 56. Pereira AC, et al. (2007) An in vivo correlate of exercise-induced neurogenesis in the 29. Geinisman Y, deToledo-Morrell L, Morrell F, Persina IS, Rossi M (1992) Age-related loss adult dentate gyrus. Proc Natl Acad Sci USA 104:5638–5643. of axospinous synapses formed by two afferent systems in the rat dentate gyrus as 57. Behrens TE, et al. (2003) Characterization and propagation of uncertainty in revealed by the unbiased stereological dissector technique. Hippocampus 2:437–444. diffusion-weighted MR imaging. Magn Reson Med 50:1077–1088.

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