Neuropsycholo`ia\ Vol[ 25\ No[ 8\ pp[ 890Ð803\ 0887Þ 0887 Elsevier Science Ltd[ All rights reserved\ Pergamon Printed in Great Britain

9917Ð2821:87 ,08[99¦9[99PII] S9917Ð2821"87#99906Ð6

Memory impairments associated with hippocampalversus parahippocampal!gyrus atrophy]

an MR volumetry study in Alzheimer|s disease

S[ KO� HLER\�$%% S[ E[ BLACK\�$% M[ SINDEN\$ C[ SZEKELY\$\ D[ KIDRON\�$J[ L[ PARKER\$ J[ K[ FOSTER\�$\ M[ MOSCOVITCH\�& G[ WINCOUR\�' J[ P[ SZALAI��

and M[ J[ BRONSKILL$$

� Rotman Research Institute of Baycrest Centre for Geriatric Care\ University of Toronto\ Toronto\ Canada^ $ Cognitive NeurologyUnit\ Sunnybrook Health Science Centre\ Toronto\ Canada^ % Department of Medicine\ University of Toronto\ Toronto\ Canada^

�� Clinical Epidemiology and Health Care Research Program "Sunnybrook Unit#\ University of Toronto\ Toronto\ Canada^$$ Department of Medical Biophysics\ Sunnybrook Health Science Centre\ Toronto\ Canada^ & Department of Psychology\ Erindale

Campus\ University of Toronto\ Toronto\ Canada^ ' Department of Psychology\ Trent University\ Peterborough\ Canada

"Received 05 September 0886^ accepted 17 January 0887#

Abstract*Delayed memory impairments and medial temporal!lobe atrophy are considered to be cardinal features of Alzheimer|sdisease "AD#[ The goal of the present magnetic resonance "MR# volumetry study was to investigate the relationship between bothfeatures[ We determined MR!derived estimates of hippocampal and parahippocampal volume in a sample of 16 AD patients and ina group of 15 healthy control subjects "NCs# of comparable age and education[ We examined the performance of the two groups onimmediate and delayed recall trials of an auditoryÐverbal list!learning task "CVLT#\ a visual non!verbal memory task "VisualReproduction of the WMS!R#\ and a screening procedure that provides an estimate of overall cognitive functioning "DRS#[ Volumesof the hippocampus and the parahippocampal gyrus were signi_cantly smaller in AD patients than in NCs[ AD patients wereimpaired in their overall level of cognitive functioning and showed memory de_cits under immediate and delayed recall conditions[The association between medial temporal!lobe atrophy and cognitive impairments in AD was found to be highly speci_c] Hippo!campal volume correlated positively with delayed but not immediate recall of the verbalÐauditory list learning task[ In contrast\parahippocampal gyrus volume\ speci_cally in the right hemisphere\ was positively related to delayed but not immediate recall ofthe non!verbal visual memory task[ In NCs\ there was a trend towards a negative association between hippocampal volumes anddelayed verbal recall[ Our results suggest that hippocampal and parahippocampal gyrus atrophy in AD are related to distinctaspects of the patients| memory impairments[ Our _ndings have implications for current discussions regarding contributions of thehippocampus and the parahippocampal gyrus to memory in the intact human brain[ Þ 0887 Elsevier Science Ltd[ All rights reserved[

Key Words] anterograde amnesia^ hippocampus^ medial temporal lobes^ human^ episodic memory^ magnetic resonance[

Impairments in episodic memory\ i[e[ memory for eventsdistinct in time and space\ and medial temporal!lobeatrophy are considered to be core features of Alzheimer|sdisease "AD# and play a crucial role in its diagnosis ð09\17\ 65\ 72Ł[ A large body of neuropsychological andneurological research has focused on either one or theother but few studies have investigated both abnor!malities together in the same participants[ Recently\ how!

%% Author to whom all correspondence should be addressed]Stefan Ko�hler\ PhD\ who is now at the Montreal NeurologicalInstitute Cognitive Neuroscience Unit\ McGill University\ 2790University Street\ Montreal\ Quebec\ Canada H2A 1B3[ Fax]¦¦ "403# 287!7439[

890

ever\ research has started to address the relationshipbetween memory impairments and medial temporal!lobeatrophy in AD ð02\ 05\ 51\ 73Ł[ In the present study\ weaim to show how an investigation of this relationshipmay help not only to determine the neural basis of speci_ccognitive impairments in AD but also to contribute tounderstanding of the organization of memory in theintact human brain[

Similar to research on the amnesic syndrome\ muchneuropsychological research on AD has been conductedin order to specify the nature of anterograde episodicmemory impairments[ It has generally become acceptedthat the memory impairment in AD is multifaceted andcan a}ect processes that pertain to encoding\ storage and

S[ Ko�hler et al[:Medial temporal lobes and memory in AD891

retrieval ð13\ 49Ł[ It remains to be determined\ however\whether all facets are caused by pathological changesin a single structure\ conceivably the hippocampus\ orwhether pathological changes in multiple structuresinside and possibly outside the medial temporal!loberegion contribute to these de_cits[

Findings from lesion studies in rats\ non!human pri!mates and humans indicate that medial temporal!lobestructures are crucially involved in episodic memory ð04\69Ł[ Whereas research has traditionally focused on con!tributions of the hippocampus\ results from recent studiesin non!human primates with isolated medial temporal!lobe lesions outside the hippocampus suggest that theparahippocampal gyrus\ including perirhinal andentorhinal cortex\ may also play an important role inmemory ð42\ 61Ł[ This notion is supported by functionalbrain imaging research in healthy humans in whichinvolvement of the hippocampus and the par!ahippocampal gyrus in episodic memory has been foundð08\ 43\ 44\ 50\ 62\ 67Ł[ Although there is evidence tosuggest memory contributions of both structures\ itremains to be determined whether the hippocampus andthe parahippocampal gyrus play a similar role in memoryor whether they support distinct processes ð66Ł[

Both the hippocampus and the parahippocampal gyrusare among the primary regions targeted by AD ð3\ 09\14\ 20\ 21Ł[ Recent advances in quantitative analysis ofmagnetic resonance "MR# imaging have made it possibleto quantify atrophy "i[e[ remaining tissue volume# of thehippocampus and of the parahippocampal gyrus inde!pendently with a high degree of reliability ð15\ 47\ 70\76Ł[ MR!volumetric estimates of atrophy can be used asindices of hippocampal and parahippocampal func!tioning in AD and can be correlated with behavioraldata to obtain insight into the functional role of thesestructures in memory[ Previous MR volumetry studies inAD have demonstrated that atrophy in the hippocampusbut not in structures outside the medial temporal!loberegion\ namely lateral temporal cortex and the caudatenuclei\ correlates with anterograde memory impairmentsð02\ 05\ 73Ł[ Within the medial temporal lobes\ hip!pocampal but not parahippocampal atrophy has beenfound to be associated with verbal anterograde memoryimpairments ð73Ł[ In line with _ndings in other organicmemory disorders and with lesion research in non!humanspecies ð31\ 46\ 56\ 74\ 75\ 78\ 89Ł\ this association hasbeen shown to be delay!dependent in AD in that it wasobserved for recall after a delay but not immediately afterlearning ð73Ł[

For the most part\ previous research using MR vol!umetry in AD has concentrated on verbal memory ð02\73Ł[ It has yet to be examined whether the reported pat!tern of relationships between memory de_cits and atro!phy in distinct medial temporal!lobe structuresgeneralizes to recall of non!verbal information[ Fur!thermore\ previous studies have failed to compare thebrain!behavior relationships observed in AD with thoseobserved in healthy control participants[ Thus\ it is

unclear whether the reported association between MRvolumes and memory performance is speci_c to AD orwhether it can also be observed when brain pathologyand behavioral de_cits are absent\ as the results of someð10\ 11Ł but not all studies ð48Ł that have focused onhealthy individuals alone\ suggest[

The goal of the present MR volumetry study was toinvestigate the relationship between atrophy in distinctmedial temporal!lobe structures and anterograde epi!sodic memory de_cits in a sample of AD patients and tocompare it with the relationship between volumes of thesestructures and memory performance in a sample of heal!thy normal control participants "NCs#[ We obtained MR!derived estimates of hippocampal and parahippocampalgyrus volumes in both groups and examined them inrelation to behavioral performance on immediate anddelayed recall trials of a verbal list!learning task and anon!verbal visual memory task[ To address thebehavioral speci_city of possible associations betweenMR volumes and memory performance\ we also obtainedan estimate of the overall level of cognitive functioningin each participant as a control measure[

Methods

Participants

Twenty!seven community!dwelling patients who met theNINCDS!ADRDA criteria ð34Ł for probable AD participatedin the study[ Their demographic characteristics are described inTable 0[ The severity of their disease as indicated by their Mini!Mental State Scores "MMS^ ð06Ł# ranged from mild to severe[Thirteen patients were mildly demented "MMS 10Ð16#\ 01 weremoderately demented "MMS 00Ð19# and two were severelydemented "MMS¾ 09#[ The control group was comprised of15 elderly community!dwelling individuals of comparable ageand educational background "Table 0# with no presence orhistory of neurological and psychiatric impairments "as deter!mined by a detailed health questionnaire#[ Inclusion in thiscontrol group was contingent on the absence of signs ofdementia and age!associated memory impairment "AAMI^ ð8Ł#on the Mattis Dementia Rating Scale "inclusion with TotalScore × 012# and the California Verbal Learning Test"inclusion with Immediate Recall age! and sex!adjusted T!Score × 39#\ respectively[ The average time that elapsedbetween neuropsychological testing and MR imaging was 2[9months in the AD sample and 2[8 months in NCs[ All par!

Table 0[ Demographic and clinical description of AD patientsand NCs

Variable AD patients NCs P

Number of participants 16 15 *Sex "male:female# 02:03 01:03 *Age 69[0 "7[4# 69[7 "5[2# nsYears of education 01[6 "1[6# 02[1 "1[7# nsMini mental state score 08[4 "3[6# 17[4 "0[2# ���

Note] ��� P ³ 9[990^ n[s[ P × 9[09[Standard deviations are shown in parentheses[

S[ Ko�hler et al[:Medial temporal lobes and memory in AD 892

ticipants "or their caregivers# gave written informed consent[Ethical approval was obtained from the Research Ethics Boardat Sunnybrook Health Sciences Centre[

Neuropsychological testing

Participants underwent individual testing on a large batteryof neuropsychological tests as part of their clinical work!up[Performance data for the Mattis Dementia Rating Scale "DRS^ð39Ł\ the California Verbal Learning Test "CVLT\ ð01Ł and theVisual Reproduction Test of the WMS!R "VRT\ ð71Ł were ana!lysed for the purpose of the present study[� The DRS is acognitive screening procedure that provides a score of overallcognitive functioning[ The CVLT is a neuropsychological testthat measures immediate and delayed recall for auditorily!pre!sented verbal material[ By contrast\ the VRT measures immedi!ate and delayed recall for visually!presented non!verbalmaterial[ The three test procedures were administered in thestandardized manner[ Administration of the CVLT\ however\did not include all subsections of the test[

Participants were asked to learn and recall List A of theCVLT in _ve learning and free recall trials "immediate recall#[This list is composed of 05 words from four di}erent semanticcategories[ Subsequently\ an interference list of 05 words "ListB# was presented for learning and free recall in a single trial[Upon completion of this trial\ memory for the words of the _rstlist "List A# was tested again in a free!recall trial "delayed recall#[The delay between the last learning trial and the delayed recalltrial was approximately 4 min[ In our analyses of the CVLTdata\ we focused on two measures[ To obtain a single score ofimmediate recall performance\ we computed the sum of cor!rectly recalled items from List A on Trials 0Ð4 "maximum of 79items#[ To quantify delayed recall performance\ we determinedthe number of correctly recalled items from List A after the 4min delay "maximum of 05 items#[

Administration of the VRT involved the visual presentationof four abstract _gures for 09 s each[ Participants were askedto memorize the _rst _gure[ Then\ immediately after learning\they were asked to draw this _gure from memory[ The sub!sequent _gures were presented in the same manner[ Memoryfor the designs was tested again in a delayed recall trial afterapproximately 29 min _lled with neuropsychological testing inunrelated domains[ Scores for the immediate and delayed recalltrials "each with a maximum of 30 points# were determinedusing the detailed scoring criteria of the WMS!R manual[ TheVRT was administered to only 07 of the 15 AD patients[ Dueto time constraints\ the clinical protocol did not include a non!verbal memory test initially[ Through the course of completionof the clinical investigation\ however\ the protocol was extendedto include the VRT[

MR scanning

Brain images were acquired on a 0[4!T Signa system with astandard head coil "General Electric Medical Systems\ Milwau!kee\ WI\ U[S[A[#[ A sagittally!acquired 2D T0!weighted SPGRsequence was used for determination of hippocampal and par!ahippocampal gyrus volumes "TR:TE of 24:4 ms^ 0 NEX^ ~ipangle of 24>^ _eld of view 11 cm^ matrix size 145×145^ imagingtime 03[3 min#[ This sequence produced 013 contiguous slicesof 0[2 mm thickness\ which covered the whole brain[ Images

� The CVLT and the VRT were the only neuropsychologicaltests of memory that were included in the larger battery of testsadministered for the clinical work!up[

from a second sequence were used to allow for correction ofvariations in overall brain size[ These additional images wereobtained from an axial two!spin!echo sequence "i[e[ protondensity and T1!weighted images^ half!Fourier sampling^ 081phase!encoding steps^ TR:TE of 2999:29\ 79 ms^ 9[4 NEX^ _eldof view 11 cm^ matrix size 145×145^ imaging time 00[5 min#that produced 47 contiguous and interleaved slices of 2 mmthickness\ which also covered the whole brain[

Volumes of interest were determined using ANALYZE soft!ware "Mayo Foundation\ Rochester\ MN\ U[S[A[# on a SUNworkstation "SUN Microsystems\ Mountain View\ CA\ U[S[A[#for display\ reformatting of images\ demarcation of structures\and volume calculations[ Hippocampal measurements wereobtained by adapting the protocol described by Jack et al[ð15\ 16Ł[ According to this protocol\ the hippocampus includesAmmon|s horn "CA0ÐCA3#\ dentate gyrus\ alveus\ _mbria andparts of the subiculum[ We modi_ed this protocol with the goalof excluding the subiculum\ which is transitional cortex thatconnects Ammon|s horn with the parahippocampal gyrus\ fromthe hippocampal measurements[ A protocol comparable to theone described by Yoneda et al[ ð76Ł was used to measure theparahippocampal gyrus and to distinguish it from the hip!pocampus[ This measurement included the parahippocampalgyrus proper "excluding portions inferior to the collateralsulcus#\ caudal aspects of entorhinal cortex\ underlying whitematter and medial aspects of the subiculum[ In addition\ itincluded caudal aspects of perirhinal cortex\ which lines thesuperior bank of the collateral sulcus before it becomes theparahippocampal gyrus proper ð2Ł[ Care was taken not toinclude parts of the amygdala in either of the medial temporal!lobe measurements[ Reference was made to a detailed atlas ofthe human hippocampus throughout the course of the measure!ments ð03Ł[

The measurements of the hippocampus and of intracranialcapacity were made by either of two raters[ The para!hippocampal measurements were obtained by a single rater[Raters were blind to the cognitive performance data[ To estab!lish that the volumetric measurements were su.ciently reliableintra!rater agreement "indexed by the intraclass coe.cient^ ICCð54Ł was determined for the measurements of the hippocampusand the parahippocampal gyrus[ In addition\ inter!rater agree!ment "also indexed by the ICC# was determined for the hippo!campal measurement[ All reliability coe.cients were found tobe above 9[79[ The intra!rater reliability for the hippocampalvolumes was estimated based on 25 hemispheres and was 9[80[The corresponding inter!rater reliability based on 19 hemi!spheres was 9[71[ The intra!rater reliability for the parahippo!campal volumes based on 19 hemispheres was 9[78[

The protocol for the volumetric measurements included thefollowing steps[ The T0!weighted images were _rst displayed inthe sagittal plane to determine the longitudinal axis of thehippocampus and its anterior and posterior boundaries[ Sub!sequently\ they were reformatted into a series of approximately44Ð59 contiguous slices of 9[748 mm thickness that were ori!ented perpendicular to this axis "oblique coronal orientation\see Fig[ 0# and covered the hippocampus in its complete rostral!caudal extent[ The anterior endpoint of the hippocampus wasdetermined on the oblique coronal slices as previously describedby Jack et al[ ð16Ł[ Speci_cally\ the most rostral slice was chosenon which the alveus "a white!matter marker with high signalintensity# forms the border between the hippocampus and theoverlying amygdala[ The posterior endpoint was determined bylocating the most rostral oblique coronal slice on which thetemporal horns separated from the main body of the lateralventricles ð4Ł[ The location of this bi!furcation relative to otheranatomical structures is presumably una}ected by ventricularenlargement or cortical atrophy[ The parahippocampal gyruswas also measured along the rostral!caudal extent of the hip!pocampal axis\ starting anteriorly on the _rst slice on which the

S[ Ko�hler et al[:Medial temporal lobes and memory in AD893

hippocampus was visible[ It was followed posteriorly up to thepoint where the anterior calcarine sulcus\ which separates theparahippocampal sulcus from the isthmus of the posteriorcingulate gyrus\ becomes visible[ In most cases\ this endpointwas found anterior to the most posterior slice on which thehippocampus was measured[ The hippocampal and par!ahippocampal regions were delineated manually between pos!terior and anterior endpoints in each hemisphere on every thirdslice of the reformatted images "Fig[ 1#[ Manual tracing "plan!imetry# was used to delineate the hippocampus[ To delineatethe parahippocampal gyrus\ we used stereology "point countingwith grid!size � 2×2×2\ shape coe.cient � 2[80# because itprovides a more e.cient method to determine volumes whenstructures are su.ciently large ð32\ 53Ł[ The demarcated areaswere multiplied by slice thickness\ interpolated across slices onwhich no measurements were taken and summed to providethe hippocampal and parahippocampal gyrus volume in eachhemisphere[ Because left and right volumes were found to behighly correlated "r � 9[83 for the hippocampus^ r � 9[69 forthe parahippocampal gyrus across participants of both groups#they were summed for the group comparison and most of theanalyses of brain!behavior correlations to provide single mea!sures of total hippocampal and parahippocampal volumes[ Dueto a technical problem with one of the scans\ measurements forthe parahippocampal gyrus were available in 15 of the 16 ADpatients only[

To correct the hippocampal and parahippocampal measure!ments for variations in total intracranial capacity\ whole brainvolume was determined\ using the T1!weighted images ð21Ł[Images were _rst {{edited|| using a semi!automated procedurewith intensity thresholds to separate brain tissue\ includingCSF\ from non!brain tissue[ Subsequently\ each slice was visu!ally inspected for the presence of remaining non!brain tissue[ Ifpresent\ this tissue was removed using manually!placed tracinglimit lines[ Finally\ the whole brain volume\ including cortex\cerebellum\ brainstem and surrounding CSF was calculatedfrom the edited images as an index of intracranial capacity[Following Jack et al[ ð16Ł\ this measure was included as a covari!ate in the multiple regression analyses on brain!behaviorrelationships[

Fig[ 0[ Sagittal slice showing hippocampal axis and orientation of slices used for the MR volumetric measurements[ The obliquecoronal slices on which measurements for the hippocampus and the parahippocampal gyrus were obtained were oriented perpen!

dicular to the longitudinal axis of the hippocampus[ The white arrow points to the hippocampus[

Statistical analysis

Two!sided t!tests were performed to compare AD patientswith NCs on the demographic\ neuropsychological\ and MR!volumetric measures[ The relationship between medial tem!poral!lobe volumes and neuropsychological test measures wasdetermined in each group separately using a three!stepprocedure[ First\ we computed the matrix of Pearson product!moment correlations between MR volumes and neuro!psychological test scores in each group and performed an omni!bus test on each matrix to determine whether any of thecorrelation coe.cients signi_cantly departed from zero[ Per!forming an omnibus test before examining individualcoe.cients is important in situations in which large numbers ofcoe.cients are tested because it guards against misinterpretingspuriously signi_cant individual coe.cients ð7Ł[ If the omnibustest showed a positive result we tested the signi_cance of eachindividual coe.cient in the matrix in a second step using t!tests[In a third step\ we further investigated those associations thatemerged as signi_cant in step two by applying a multipleregression approach[ For each neuropsychological measure thatcorrelated with MR volumes signi_cantly\ we tested a singleone!step regression model in which the test score was the depen!dent variable and the parahippocampal and hippocampal vol!umes were the predictors "i[e[ independent variables#[ Byincluding the volumes of both structures as predictors in thesame regression model\ we could directly determine their inde!pendent contributions to performance on each neuro!psychological measure examined "see ð05Ł\ for rationale#[Moreover\ we also controlled for possible confounding e}ectsof intracranial capacity\ age\ education and sex by includingthem as covariates "i[e[ additional independent variables# inthese regression models[

Results

A comparison of the group means for the MR!volu!metric measures and the neuropsychological measures is

S[Ko� hler

etal[:M

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andm

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inA

D894

Fig[ 1[ Samples of anterior "top# and posterior "bottom# oblique coronal slices perpendicular to the longitudinal hippocampal axis in an AD patient "left# and a NC "right#[ The topslices were taken at the level of the anterior commissure[ The bottom slices were taken approximately 4 mm anterior to the bi!furcation of the temporal horns of the lateral ventricles[For illustrative purposes\ the hippocampus is delineated in the left hemisphere "right!hand side# and the parahippocampal gyrus is delineated in the right hemisphere[ Note the atrophy

present in both medial temporal!lobe structures in the AD patient as compared to the NC[

S[ Ko�hler et al[:Medial temporal lobes and memory in AD895

Table 1[ Means and Ranges for MR!volumetric and Behavioral Measures of AD Patients and NCs

AD patients NCs

Variable Mean Range Mean Range P

MR volumetric measuresHippocampal volume "in mm2# 1954[1a 0119Ð2539 1502[6b 0406Ð2539 ��Parahippocampal volume "in mm2# 3231[0b 1459Ð4494 4109[9b 2459Ð6240 ���Intracranial capacity "in cm2# 0309[3a 0069Ð0571 0323[2b 0123Ð0692 n[s[

Dementia Rating ScaleOverall cognitive functioning score 097[1a 52Ð018 028[8b 018Ð033 ���

California Verbal Learning TestImmediate recall 06[7a 0Ð22 42[2b 28Ð61 ���Delayed recall 9[8a 9Ð5 00[0b 4Ð05 ���

WMS!R Visual ReproductionImmediate recall 00[8c 9Ð17 21[3b 6Ð30 ���Delayed recall 1[4c 9Ð02 15[4b 6Ð39 ���

Note] P values are for corresponding t!tests^ � P ³ 9[94^ �� P ³ 9[90^ ��� P ³ 9[990^ n[s[ P × 9[09^ a n � 16^ b n � 15^ c n � 07[

shown in Table 1[ The table also presents the results ofthe t!tests performed on these data[ Figure 2 dem!onstrates that the total "i[e[ sum of left! and right!sided#hippocampal volume "P³ 9[90# and the total par!ahippocampal volume "P³ 9[990# were smaller in ADpatients than in NCs[ This was also true when inter!individual di}erences in total intracranial capacity werepartialed out by means of analyses of covariance"F"0\49#�8[2\ P³ 9[90 for hippocampus^ F"0\38#�

Fig[ 2[ Scattergram for the observed MR!derived volumes ofthe hippocampus and the parahippocampal gyrus in AD pat!ients and NCs[ Mean volumes are indicated by horizontal lines[Both structures were smaller in AD patients than in NCs[ Thisdi}erence was also statistically signi_cant when interindividual

di}erences in total intracranial capacity were partialed out[

04[3\ P³ 9[990 for parahippocampal gyrus#[ Hippo!campal and parahippocampal volume were positivelycorrelated in AD patients "r�9[38\ P³ 9[94# but not inNCs "r�9[13^ P× 9[09#[

Behaviorally\ AD patients exhibited a signi_cantlylower level of overall cognitive functioning than NCs asindicated by their DRS scores "Table 1#[ When episodicmemory functioning was examined with the CVLT andthe VRT\ AD patients were found to be impaired on theimmediate and the delayed recall measure of both tests"Table 1#[ The di}erence between groups in delayed recallperformance persisted on both tests even when inter!individual di}erences in initial learning were partialedout by means of covariance analyses "F"0\49#�03[5\P³ 9[990 for the CVLT^ F"0\30#�6[5\ P³ 9[90 for theVRT#[ A subgroup of AD patients was unable to recallany items on the CVLT "05 patients# and the VRT "8patients# after the delay[

The omnibus test for the correlation matrix betweenneuropsychological test scores and volumetric measuresin AD patients revealed that some elements of the matrixwere signi_cantly di}erent from zero "x1"09#�18[9\P³ 9[994#[ When individual coe.cients were examined\we found that only delayed memory performance cor!related signi_cantly with hippocampal or parahippo!campal volume in AD[ Delayed recall on the CVLTcorrelated positively with hippocampal volume whereasdelayed recall on the VRT correlated positively with par!ahippocampal gyrus volume "Table 2#[ Importantly\ thedistribution of behavioral scores at ~oor also followedthis relationship\ i[e[ their occurrence was related to thedegree of hippocampal and parahippocampal gyrus atro!phy[ Those AD patients who obtained a delayed recallscore of zero on the VRT showed smaller par!ahippocampal gyrus volumes than those who obtained ascore above zero "P³ 9[90#[ Similarly\ those AD patients

S[ Ko�hler et al[:Medial temporal lobes and memory in AD 896

Table 2[ Pearson correlations between MR volumes andbehavioral measures in AD patients

MR!volumetric measures

ParahippocampalBehavioral variable gyrus Hippocampus

Dementia Rating ScaleOverall cognitive −9[93b −9[93a

functioning score

California Verbal Learning TestImmediate recall 9[04b 9[10a

Delayed recall 9[26b 9[50���a

WMS!R Visual ReproductionImmediate recall 9[21d −9[91c

Delayed recall 9[50��d 9[97c

Note] An omnibus test of the matrix showed an associationbetween MR!volumetric measures and behavioral variables"P ³ 9[994#[ Signi_cance levels are displayed for t!tests of indi!vidual coe.cients^ �P ³ 9[94^ �� P ³ 9[90^ ��� P ³ 9[990[a n � 16^ b n � 15^ c n � 07^ d n � 06[

who obtained a delayed!recall score of zero on the CVLTshowed a trend towards smaller hippocampal volumesthan those who obtained a score above zero "P³ 9[96#[To ascertain that recall scores of zero did not in~atethe observed correlations we also examined the brain!behavior correlations in only those individuals whoobtained recall scores above zero[ Both correlations\ i[e[the one between parahippocampal gyrus volume anddelayed VRT recall\ as well as the one between hippo!campal volume and delayed CVLT recall\ were numeri!cally larger in the subsamples of AD patients with recallscores above zero "VRT r�9[61^ P³ 9[94^ CVLTr�9[53^ P³ 9[94# than in the entire group[ Thus\ includ!ing AD patients with behavioral performance at ~oor inthe total sample led\ if at all\ to an under!estimationbut certainly not on over!estimation of the size of theseassociations[

In two subsequent multiple regression analyses\ inwhich CVLT delayed recall or VRT delayed recall servedas the dependent measure\ we determined the inde!pendent "i[e[ residualized for shared variance# con!tributions of hippocampal and parahippocampalvolumes to behavioral performance while controlling fore}ects of other confounding variables[ When the volumesof both structures together with intracranial capacity\sex\ age and education were used as predictors\ theregression analyses showed that the association betweenhippocampal volume and delayed CVLT performanceand the association between parahippocampal gyrus vol!ume and delayed VRT performance were robust anddissociable[ Both regression models reached statisticalsigni_cance "CVLT delayed recall R1 �9[38\ P³ 9[94^VRT delayed recall R1 �9[65\ P�9[90#[ In the modelfor the CVLT delayed recall score\ hippocampal volume

Table 3[ Independent contributions of hippocampal and par!ahippocampal gyrus volumes in multiple regression models fordelayed CVLT and delayed VRT performance in AD patients

Independent variable

ParahippocampalDependent variable gyrus Hippocampus

CVLT delayed recalla −9[96 9[67��WMS!R VRT 9[56� 9[00

delayed recallb

Note] Standardized regression coe.cients "b# are listed[ In bothmodels\ variations in intracranial capacity\ age\ sex\ and edu!cation were controlled for[ �P ³ 9[94^ ��P ³ 9[90^ all otherP × 9[09[ a n � 15^ b n � 06[

emerged as a signi_cant predictor but parahippocampalgyrus volume did not "Table 3#[ By contrast\ in the modelfor the VRT delayed recall score\ parahippocampal gyrusvolume emerged as a signi_cant predictor but hip!pocampal volume did not "Table 3#[ To ascertain thatthese results were not due to di}erences in sample size\we repeated the regression analysis for the delayed CVLTscore including only the subsample on which the delayedVRT regression analysis was based[ The pattern of resultsobserved with this subsample was identical to the patternobserved with the larger sample[

Because atrophy in medial temporal!lobe regions wasfound to be highly symmetrical in AD\ a}ecting left!and right!hemisphere structures to the same degree\ weconcentrated our analyses on total volumes of the hip!pocampus and the parahippocampal gyrus summedacross both hemispheres "as reported previously#[ Never!theless\ to address laterality e}ects in brain!behavior cor!relations\ we did perform additional regression analysesfor those behavioral measures which were found to berelated to hippocampal and parahippocampal volumes"CVLT delayed recall score\ VRT delayed recall score#[As in our previous analyses\ we included the behavioralmeasures as the dependent variable and the MR!volu!metric measures as the predictors while controlling fordi}erences in intracranial capacity\ age\ sex andeducation[ For each behavioral variable\ we examinedone model in which left!sided volumes served as pre!dictors and another model in which right!sided volumesserved as predictors[ For the CVLT delayed recall score\both the left!sided and right!sided models were signi_cant"R1 �9[37 and 9[38\ respectively^ P³ 9[94#[ In bothmodels\ hippocampal "left b�9[66\ P³ 9[94^ rightb�9[66^ P³ 9[94# but not parahippocampal gyrus vol!ume "left b�−9[96\ P× 9[09^ right b�−9[95^P× 9[09# emerged as a predictor for delayed CVLTrecall[ For the VRT delayed recall score\ the model inwhich right!sided volumes were included "R1 �9[67\P³ 9[90# showed that the volume of the par!ahippocampal gyrus "b�9[59\ P³ 9[94# but not of thehippocampus "b�9[27\ P× 9[09# was a signi_cant pre!

S[ Ko�hler et al[:Medial temporal lobes and memory in AD897

dictor of performance[ By contrast\ in the model withleft!sided volumes "R1 �9[54\ P�9[94#\ neither the vol!ume of the parahippocampal gyrus "b�9[36\ P× 9[09#nor of the hippocampus "b�−9[05\ P× 9[09# emergedas a signi_cant predictor of delayed VRT recall[ In sum\these analyses revealed laterality e}ects for delayed VRTrecall\ with the right but not the left parahippocampalgyrus emerging as a signi_cant predictor with a positiverelationship to behavioral performance[ By contrast\ nolaterality e}ects were found with respect to delayedCVLT performance\ presumably due to the higher cor!relation between left! and right!sided hippocampal vol!umes "r�9[86# as compared to parahippocampal gyrusvolumes "r�9[60# in our sample of AD patients[

The omnibus test for the matrix of brain!behavior cor!relations in NCs "Table 4# also revealed a signi_cantassociation between behavioral measures and MR vol!umes "x1"09#�07[8^ P³ 9[94#[ Inspection of individualcorrelation coe.cients showed signi_cant negativeassociations between hippocampal volume and immedi!ate CVLT recall and between hippocampal volume anddelayed CVLT recall[ These associations were furtherinvestigated in two multiple regression analyses in whichwe included the behavioral measures as the dependentvariable and the MR!volumetric measures as the pre!dictors while controlling for di}erences in intracranialcapacity\ age\ sex and education[ In the analysis for theCVLT immediate recall score\ neither the overallregression model "R1 �9[15\ P× 9[09# nor the stan!dardized regression coe.cients for the volume of thehippocampus "b�−9[17\ P× 9[09# and the par!ahippocampal gyrus "b�−9[09\ P× 9[09# reached stat!istical signi_cance[ By contrast\ the regression model forthe CVLT delayed recall score did reach statistical sig!

Table 4[ Pearson correlations between MR volumes andbehavioral measures in NCs

MR!volumetric measures

Behavioral variable Parahippocampal Hippocampusgyrus

Dementia Rating ScaleOverall cognitive −9[97 −9[21

functioning score

California Verbal Learning TestImmediate recall −9[07 −9[28�Delayed recall −9[19 −9[44��

WMS!R Visual ReproductionImmediate recall 9[93 −9[02Delayed recall −9[96 −9[00

Note] An omnibus test of the matrix showed an associationbetween MR!volumetric measures and behavioral variables"P ³ 9[94#[ Signi_cance levels are displayed for t!tests of indi!vidual coe.cients^ �P ³ 9[94^ �� P ³ 9[90^ ��� P ³ 9[990[n � 15 for all coe.cients[

ni_cance "R1 �9[42\ P³ 9[94#[ In this model\ the hip!pocampal volume showed a signi_cant trend as apredictor "b�−9[24\ P³ 9[98# whereas the volume ofthe parahippocampal gyrus failed to be of any predictivevalue "b�−9[05\ P× 9[09#[ The same pattern of resultswas obtained when we repeated these analyses with uni!lateral volumes as predictors\ using the strategy pre!viously employed for the analysis of AD data[ Regardlessof whether the regression model included left! or right!sided structures\ the hippocampal volume showed a trendas a negative predictor for CVLT delayed recall but notfor CVLT immediate recall[ By contrast\ left! and right!sided volumes of the parahippocampal gyrus showed nopredictive value in any of these models[

Discussion

The results of the present study demonstrate a distinctpattern of associations between episodic memory impair!ments and atrophy in medial temporal!lobe structures inAD[ MR!derived volumes of the hippocampus and theparahippocampal gyrus "with the latter including por!tions of perirhinal and entorhinal cortex# were foundto correlate with delayed memory recall but not withimmediate memory recall nor with an estimate of overallcognitive functioning[ Hippocampal volume correlatedpositively with delayed recall on a verbalÐauditory listlearning task "CVLT# but not on a non!verbal visualmemory task "VRT#[ In contrast\ parahippocampal gyrusvolume\ speci_cally in the right hemisphere\ was posi!tively associated with delayed recall on the non!verbalvisual memory task but not the verbalÐauditory list learn!ing task[ The pattern of brain!behavior correlationsobserved in AD contrasted with the pattern of cor!relations observed in NCs[ In the latter group\ a sig!ni_cant trend towards a ne`ative association betweenhippocampal volume and CVLT delayed recall emerged[Our _ndings have implications for an understanding ofmemory impairments in AD\ for theories concerning theneural organization of memory in the intact human brain\and for the functional interpretation of volumes of medialtemporal!lobe structures in healthy individuals[ Theseimplications will be discussed in the ensuing paragraphs[

The comparison of the group of AD patients with NCsshowed di}erences both on the structural and on thebehavioral level[ Our MR!volumetric results con_rm thatmedial temporal!lobe atrophy in AD a}ects the hip!pocampus as well as the surrounding parahippocampalgyrus ð15\ 20Ð22\ 52Ł[ Behaviorally\ AD patients showedanterograde episodic memory de_cits in verbal and non!verbal recall immediately after learning and after adelay[� Delayed recall of verbal material from the CVLT

� As previously noted\ our inclusion criteria for the group ofNCs took the CVLT immediate recall scores into consideration\biasing this comparison towards di}erences in memory per!formance between the two groups[ However\ comparable groupdi}erences have been documented in previous studies in whichparticipants were included as NCs irrespective of their per!formance level on memory tests "for review see ð13\ 49Ł#[

S[ Ko�hler et al[:Medial temporal lobes and memory in AD 898

and non!verbal material from the VRT was found to beimpaired even when di}erences in initial learning weretaken into account\ suggesting that the patients| memoryde_cits involve accelerated forgetting over short delaysand\ thus\ implicate abnormal storage ð12\ 26Ł[ Thatmemory de_cits were present even under immediate recallconditions is consistent with suggestions that the func!tional de_cits also pertain to processes at encoding ð25\28\ 36Ł[

Atrophy in the hippocampus and the parahippocampalgyrus was found to be related to delayed but not immedi!ate memory impairments in AD patients[ Delayed recallof the CVLT word list showed a positive association withremaining hippocampal volume whereas delayed recallof the VRT designs correlated positively with remainingparahippocampal gyrus volume[ We con_rmed this pat!tern of associations in multiple regression analyses thatallowed us to examine the independent contributions ofthese structures of interest while controlling for con!founding e}ects of age\ sex\ years of education and totalbrain capacity[ Hippocampal but not parahippocampalvolume emerged as a signi_cant predictor in theregression model for delayed CVLT performance[ Thereverse pattern was found in the model for delayed VRTperformance[ In both models\ the volume of the structurethat did not predict performance had regressioncoe.cients close to zero\ suggesting that the observeddissociations are not simply due to statistical thresholds[

Our results indicate that the relationship between atro!phy in medial temporal!lobe structures and episodicmemory impairments in AD is highly speci_c\ neu!roanatomically as well as behaviorally[ That atrophy inthe two structures of interest correlated with somebehavioral measures "i[e[ delayed recall on CVLT andVRT# but not others "i[e[ immediate recall on CVLT andVRT\ overall cognitive functioning on DRS# suggestsbehavioral speci_city^ that atrophy in the hippocampuswas associated with a di}erent type of delayed recalltask "CVLT# than atrophy in the parahippocampal gyrus"VRT# suggests neuroanatomical speci_city within themedial temporal lobes[ Taken together\ these data cannotbe accommodated by a hypothesis that assumes a unitarynon!speci_c factor\ such as disease severity or global cort!ical atrophy\ as the link between the various behavioralde_cits and structural abnormalities examined[ Aninterpretation that considers di}erences between memorytasks together with the notion of functional specializationwithin the medial temporal lobes\ as discussed later\ isclearly better suited to account for the observed patternof brain!behavior correlations in AD[

Our results con_rm those reported by Wilson et al[ð73Ł\ who also found a positive association between MR!derived volumes of the hippocampus but not the par!ahippocampal gyrus and delayed verbal recall of audi!torily!presented material in AD[ Our _ndings extendthese results by showing that the reverse pattern holdsfor delayed recall on a non!verbal visual memory task[Because no relationship was found between medial tem!

poral!lobe atrophy and immediate memory performance\our _ndings and those by Wilson et al[ hint that changesin structures outside the medial temporal lobes\ perhapsin dorsolateral prefrontal cortex ð5\ 00\ 29\ 40\ 45Ł\ under!lie the encoding de_cits observed in AD[ However\ giventhat the volumetric analysis in our study was focusedonly on the medial temporal!lobe region\ this relationshipcould not be addressed[

Impaired delayed recall over interference!_lled delaysis not only a core symptom of AD but has more generallybeen proposed to be the clinical hallmark of anterogradeamnesia caused by medial temporal!lobe damage ð37\ 58Ł[Several lesion studies in non!human species\ includingrhesus monkeys and rats\ have shown delay!dependentmemory impairments produced by hippocampal or com!bined hippocampal and parahippocampal gyrus damageð74\ 75\ 78\ 89Ł[ Studies in humans who underwent tem!poral lobectomy for the relief of intractable epilepsy havedemonstrated that the amount of medial temporal!lobetissue removed may crucially determine the degree ofdelayed!memory de_cits observed post!surgery whileleaving immediate memory performance una}ected ð46\56Ł[ Together with these _ndings\ the present results sup!port the hypothesis that the medial temporal lobes playa speci_c role in those memory processes that are involvedin storage and counteract forgetting during delays\ suchas consolidation or cohesion ð30\ 37\ 41\ 60Ł[ It should beemphasized\ however\ that the particular data presentedhere would not warrant the conclusion that the hip!pocampus and the parahippocampal gyrus are the onlybrain structures that support processes related to con!solidation and cohesion[ Other structures that were notexamined here may also contribute to these processes[They may include structures that have strong neu!roanatomical connections with the medial!temporal loberegion\ such as diencephalic nuclei or retrosplenial cortexð6\ 27\ 68Ł[

Although a small number of human cases with amnesiadue to isolated hippocampal lesions have been docu!mented ð59\ 79\ 77Ł in most reported cases of medialtemporal!lobe amnesia\ including those with amnesia dueto temporal lobectomy\ it is unclear whether the crucialdamage is localized in the hippocampus\ in surroundingparahippocampal gyrus\ or in both[ It has been suggestedthat medial temporal!lobe lesions in humans that includethe hippocampus and adjacent regions in par!ahippocampal gyrus produce more severe memoryimpairments than lesions restricted to the hippocampusð59Ł[ The present results suggest that this may be the casebecause damage "or atrophy# to each of these structuresa}ects di}erent aspects of memory functioning[ Thus\our _ndings in AD point to the exciting possibility thatthe memory contributions of the hippocampus and theparahippocampal gyrus can be double!dissociated ð08Ł[

Concerning the nature of these distinct contributions\it is important to note that the two memory tasks ofthe present study that were related to hippocampal andparahippocampal atrophy di}ered from each other in

S[ Ko�hler et al[:Medial temporal lobes and memory in AD809

several ways[ Di}erences in the type and amount of thematerial to be recalled\ the length of the delay\ themodality in which information was presented\ or a com!bination of these factors may have contributed to theobserved dissociation in brain!behavior correlations[ Itis unlikely that the di}erence in delay "4 min for CVLTversus 29 min for VRT# is a relevant factor because ano!ther MR volumetry study in AD found a positive cor!relation between hippocampal volume and CVLT recallafter a longer delay as well ð02Ł[ Instead\ it is more prob!able that the observed pattern of brain!behavior cor!relations re~ects modality speci_city or materialspeci_city in brain organization[

Our results are open to the interpretation that theparahippocampal gyrus plays a role in memory that isrestricted to visually!encoded information whereas thehippocampus plays this role for other sensory modalities[Our _nding that performance on a visual recall task ishighly sensitive to structural damage in the par!ahippocampal gyrus is in keeping with the evidence thatthis region has the strongest neuroanatomical con!nections with ventral occipito!temporal brain regionsinvolved in vision ð63Ł and that lesion studies with non!human primates ð19\ 35\ 42Ł and functional imaging stud!ies in humans ð0\ 08\ 24\ 44Ł have previously implicatedthe parahippocampal gyrus\ including entorhinal andperirhinal cortex\ in visual memory[ However\ visual rec!ognition memory impairments in non!human primateshave also been found after selective hippocampal lesionsð1Ł[ Moreover\ lesions to the parahippocampal gyrus canproduce memory impairments in modalities other thanthe visual one ð64Ł[ An interpretation of the correlationalpattern in terms of modality!speci_city is further com!plicated by the fact that both regions receive corticalinput from multiple sensory modalities with cortical pro!jections to the hippocampus being relayed in the par!ahippocampal gyrus ð2Ł[

While modality di}erences warrant further consider!ation\ it is also important to focus on the type of materialthat has to be recalled in each task[ The CVLT requiresthe recall of related words\ which can be grouped insemantic categories\ whereas the VRT requires the recallof unrelated meaningless shapes with distinct spatial con!_gurations[ Perhaps\ the human hippocampus plays arole in consolidation of verbal information encoded interms of its semantic context ð30Ł whereas the par!ahippocampal gyrus plays a role in consolidation of non!verbal patterns[ Future functional imaging studies inhealthy individuals can build upon these results in AD tocharacterize the distinct memory contributions of theparahippocampal gyrus and the hippocampus usingclosely matched verbal and non!verbal experimentaltasks[

Future functional imaging studies may also addressissues regarding the lateralization of hippocampal andparahippocampal gyrus contributions to memory[ Theanalysis of laterality e}ects in the present study revealedthat the volume of the right but not the left par!

ahippocampal gyrus is associated with delayed VRTrecall in AD\ con_rming earlier _ndings in patients withunilateral temporal lobectomy that showed lateralizationof non!verbal memory functions to the right hemisphereð18\ 23\ 46\ 55Ł[ By contrast\ our analyses revealed nolaterality e}ects with respect to the association betweenhippocampal volumes and delayed CVLT performancein AD\ presumably due to the high correlation betweenleft! and right!sided hippocampal atrophy that would notallow detection of any di}erences[ If the constraints ofsymmetrical hippocampal atrophy in AD are taken intoaccount our data are not inconsistent with the notionthat hippocampal contributions to delayed verbal recallare lateralized to the left hemisphere ð07\ 38Ł[

Although not the central focus of this article\ the pat!tern of brain!behavior correlations observed in the elderlyNCs also requires comment[ While there was no indi!cation for an association between parahippocampalgyrus volume and any of the memory measures in NCsthere was a statistical trend towards a ne`ative associationbetween hippocampal volume and delayed CVLT recallthat persisted even when possible confounding e}ectswere controlled for[ It is important to note that thisassociation replicates the speci_city of the relationshipbetween hippocampal volume and delayed memory per!formance observed in AD[ However\ the fact that theassociation is reversed in sign in NCs is intriguing andwe do not have a ready explanation for this[ It is unlikelythat di}erences in statistical power are responsible forthe observed di}erences in brain!behavior correlationsbetween the two groups because the volumetric measuresas well as the behavioral measures showed more varia!bility in the NC sample than the AD sample[

Previous research on the relationship between memoryperformance and MR!derived medial!temporal lobe vol!umes in NCs has provided inconsistent results[ Work byGolomb et al[ ð10\ 11Ł suggests a positive associationbetween hippocampal volumes and delayed memory per!formance in elderly NCs[ By contrast\ Raz et al[ ð48Ł havefound no association between hippocampal or par!ahippocampal gyrus volumes and performance on vari!ous delayed memory tasks in a large sample of NCs whoranged in age from 07Ð66 years[ Based on a comparisonof subgroups of their larger sample\ Raz et al[ arguedthat only samples with a large proportion of participantswith poor memory performance may reveal a positivelink between episodic memory and medial temporal!lobevolumes[ In line with this suggestion\ research in non!demented elderly individuals with age!associated mem!ory impairments "AAMI# has demonstrated a positiverelationship between memory performance and hippo!campal volumes "as indexed by left!right asymmetry\ð57Ł#[ In the present study\ unlike the one by Golomb etal[ ð10Ł\ elderly individuals with AAMI were speci_callyexcluded from the sample of NCs[ Eighty!eight percentof the NCs in our sample obtained performance scoresat or above the age! and sex!adjusted average ð01Ł onCVLT delayed recall\ the measure that showed the nega!

S[ Ko�hler et al[:Medial temporal lobes and memory in AD 800

tive association with hippocampal volumes[ It is likelythat the pathological mechanisms that cause hip!pocampal atrophy and link it to memory impairments inAD "and possibly AAMI# are absent in these highly!functioning healthy individuals[ Other "possibly revers!ible# neural mechanisms may determine the size of thehippocampus in this group and in~uence the relationshipto cognitive functioning ð33Ł[ It remains an importantgoal for future studies to specify these mechanisms inrelation to memory performance in AD and in healthyindividuals[

In conclusion\ the results of the present study showthat hippocampal and parahippocampal gyrus atrophyin AD are related to a speci_c subset of the patients|anterograde memory impairments[ We found an associ!ation between hippocampal tissue loss and delayed ver!balÐauditory memory de_cits and an association betweenparahippocampal gyrus loss and delayed non!verbal vis!ual memory de_cits in AD[ Although our use of a cor!relational design precludes establishing a causalrelationship between the structural and the behavioralchanges\ the present results are at least consistent withthe idea that delayed memory impairments in AD resultfrom atrophy in the hippocampus and the par!ahippocampal gyrus[ Our _ndings support theories whichposit that medial temporal!lobe structures play a crucialrole in episodic memory processes that occur after enco!ding such as consolidation or cohesion ð30\ 37\ 41\ 60Ł[Moreover\ they suggest that the hippocampus and theparahippocampal gyrus make separate contributions tothese processes[ On a more general level\ our study showshow investigating the relationship between structuralabnormalities in distinct brain structures and behavioralperformance in AD may not only further our under!standing of the cognitive impairments observed in ADbut may also advance our knowledge regarding theorganization of memory in the intact human brain[

Acknowled`ments*This research was funded by grants fromthe Ontario Mental Health Foundation and the MedicalResearch Council of Canada[ S[ K[ received a Woman|s Auxili!ary Alzheimer Research Fellowship from Baycrest Centre forGeriatric Care[ J[ K[ F[ is now at the Department of Psychology\University of Manchester\ Manchester\ U[K[\ and D[ K[ is atthe Chaim Sheba Medical Center\ Tel!Aviv\ Israel[ We wouldlike to thank A[ R[ McIntosh and M[ Binns for suggestionsregarding the statistical analysis\ as well as N[ Raz\ J[ Jenningsand B[ Buck for their helpful comments on earlier versions ofthis manuscript[

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