The beneficial effects of berries on cognition, motor behaviour and neuronal function in ageing

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1 British Journl of Nutrition (215), 114, doi:1.117/s Cmridge University Press 215. This is work of the U.S. Government nd is not suject to copyright protection in the United Sttes. The eneficil effects of erries on cognition, motor ehviour nd neuronl function in geing Brr Shukitt-Hle*, Donn F. Bielinski, Frncis C. Lu, Luren M. Willis, Amnd N. Crey nd Jmes A. Joseph USDA-ARS, Humn Nutrition Reserch Center on Aging, Tufts University, Boston, MA 2111, USA (Sumitted 18 Mrch 215 Finl revision received 17 July 215 Accepted 1 August 215 First pulished online 22 Septemer 215) Astrct Previously, it hs een shown tht strwerry (SB) or lueerry (BB) supplementtions, when fed to rts from 19 to 21 months of ge, reverse ge-relted decrements in motor nd cognitive performnce. We hve postulted tht these effects my e the result of numer of positive enefits of the erry polyphenols, including decresed stress signlling, incresed neurogenesis, nd incresed signls involved in lerning nd memory. Thus, the present study ws crried out to exmine these mechnisms in ged nimls y dministering control, 2 % SB- or 2 % BB-supplemented diet to ged Fischer 344 rts for 8 weeks to scertin their effectiveness in reversing ge-relted deficits in ehviourl nd neuronl function. The results showed tht rts consuming the erry diets exhiited enhnced motor performnce nd improved cognition, specificlly working memory. In ddition, the rts supplemented with BB nd SB diets showed incresed hippocmpl neurogenesis nd expression of insulin-like growth fctor 1, lthough the improvements in working memory performnce could not solely e explined y these increses. The diverse polyphenolics in these erry fruits my hve dditionl mechnisms of ction tht could ccount for their reltive differences in efficcy. Key words: Strwerries: Blueerries: Sptil memory: Lerning: Stress signlling: Neurogenesis Although the mechnisms involved in motor (1,2) nd cognitive (3) ehviourl deficits during geing remin to e discerned, it is cler tht oxidtive stress (OS) (3) nd inflmmtion (INF) (4,5) re involved. Incresed susceptiility to the long-term effects of OS nd inflmmtory insults re thought to e contriuting fctors to the decrements in cognitive nd/or motor performnce seen in geing nd other neurodegenertive diseses. Deficits in rin functions due to OS my e the result of declines in endogenous ntioxidnt defence mechnisms (6 11) nd increses in the vulnerility of the rin to the deleterious effects of oxidtive dmge (12). Reserch lso indictes tht not only is the CNS prticulrly vulnerle to OS ut tht this vulnerility increses during geing (see Joseph et l. (13,14) for review) nd my lso enhnce centrl vulnerility to INF (13,15).Withge,therere increses in inflmmtory meditors (e.g. cytokines) (16 18), s well s incresed moilistion nd infiltrtion of peripherl inflmmtory cells, which hve een shown to produce deficits in ehviour similr to those oserved during geing (4). Furthermore, ge-relted chnges in rin vulnerility to OS nd INF my e the result of memrne chnges nd differentil receptor sensitivity (19). The question then ecomes how to reduce OS/INF vulnerility to these ge-relted chnges nd reverse the forementioned deficits in motor nd cognitive function. In this respect, studies hve shown tht plnts, including fruit- or vegetle-ering plnts, synthesise vst rry of chemicl compounds tht re not necessrily involved in the plnt s metolism. These secondry compounds insted serve vriety of functions tht enhnce the plnt s survivility, including comting OS nd INF. Reserch suggests tht polyphenolic compounds contined in colourful fruits nd vegetles exhiit potent ntioxidnt nd ntiinflmmtory ctivities tht cn reduce the ge-relted sensitivity to OS or INF (2). In previous studies, we found tht crude lueerry (BB) or strwerry (SB) extrcts significntly ttenuted ge-relted motor nd cognitive deficits in senescent rodents (21,22), s well s rdition-induced cognitive ehviourl decrements in young rts (23). Specificlly, young rts given n AIN-93 diet supplemented with SB extrct or spinch extrct (1 2 % of the diet) for 8 months did not exhiit ge-relted decrements in cognitive performnce or neuronl function tht were seen in the unsupplemented controls (22). In susequent experiment, Arevitions: BB, lueerry; BrdU, romodeoxyuridine; IGF-1, insulin-like growth fctor 1; INF, inflmmtion; MWM, Morris wter mze; OS, oxidtive stress; SB, strwerry; TBST, Tris-uffered sline with Tween. * Corresponding uthor: B. Shukitt-Hle, fx , emil rr.shukitthle@rs.usd.gov In memory of Jmes A. Joseph, our vlued collegue nd friend, who pssed wy while this pper ws eing written.

2 Beneficil effects of erries 1543 BB or SB supplementtion for 8 weeks reversed ge-relted deficits in neuronl nd ehviourl (motor nd cognitive) function in ged (19 months) Fischer 344 (F344) rts (21). The rodents in ll diet groups, ut not the control group, showed improved working memory (short-term memory) performnce in the Morris wter mze (MWM) test (21). Lter studies suggested tht, in ddition to MWM performnce, BB supplementtion ws lso effective in reversing cognitive declines in oject recognition (24,25). Susequent reserch hs suggested tht erry fruit polyphenols my possess multiplicity of ctions side from ntioxidnt ctivity. Other possile mechnisms for the erry fruit s positive effects include direct effects on signlling to enhnce neuronl communiction (26 31), the ility to uffer ginst excess C (32), incresed neurogenesis (27), enhncement of neuroprotective stress shock proteins (33), ltertion of inflmmtory gene expression nd protection ginst neurodegenertion following excitotoxic stress (34,35), nd reduction of stress signls such s NFκB (24,28). In ddition, the nthocynins contined in BB hve een shown to enter the rin, nd their concentrtions were correlted with cognitive performnce (36). The present study ws crried out to further ssess the ehviourl enefits of BB or SB in ged rts supplemented with different diets for 8 weeks. In ddition, we lso ssessed insulin-like growth fctor 1 (IGF-1) in the hippocmpus, neuronl signl tht hs een shown to e ssocited with lerning nd memory, in the supplemented nd control groups. Finlly, mesurements were mde of dentte neurogenesis in control nd supplemented nimls. Methods Animls In ll, forty-two 19-month-old mle Fischer 344 rts, otined from the NIA colony (Hrln Sprgue Dwley), were individully housed in stinless steel mesh suspended cges, provided food nd wter d liitum, nd mintined on 12 h light 12 h drk cycle. Following 1-week cclimtistion period to the fcility, the rts were weight-mtched nd then rndomly ssigned to one of three diet groups until euthnised (n 14/group): control, 2 % SB or 2 % BB. Weights were recorded t severl time points throughout the study nd during ehviourl testing. A food intke ssessment (over 96-h period) ws mde during the 6th week of the experimentl feeding. All rts were oserved dily for clinicl signs of disese. During the course of the study, two rts in the control group, one in the BB group nd two in the SB group were euthnised ecuse of excessive weight loss. Animls were utilised in complince with ll pplicle lws nd regultions s well s with principles expressed in the Ntionl Institutes of Helth, USPHS, Guide for the Cre nd Use of Lortory Animls. This study ws pproved y the Animl Cre nd Use Committee of our Center. Diets The diets were prepred t Hrln Tekld y dding freezedried fruit powder to the control diet, which ws modifiction of the NIH-31 diet (2 g/kg diet, 2 % w/w). The mount of mize in the control diet ws djusted to compenste for the dded volume of the fruit powder. The control NIH-31 diet ws the sme s used in erlier studies in which other fruits, including BB, were found to e eneficil in mitigting rin geing (24,26,27,37,38). The BB powder ws prepred y homogenising BB, otined from US Highush Blueerry Council, with deionised wter (1:1, w/v) for 3 min nd then centrifuging the recovered homogente t 27 5 g for 15 min t 4 C. The superntnt ws then frozen, crushed nd lyophilised. The SB powder ws prepred y freeze-drying SB supplied y the Cliforni Strwerry Commission; this powder hs een previously descried (39). The mount of nthocynins in ech diet ws determined y liquid chromtogrphy-tndem MS ccording to the methods of Milury et l. (4) (see Tle 1). Behviourl tests Psychomotor testing. A ttery of ge- nd diet-sensitive tests of psychomotor ehviour (2,21,37,38,41,42) ws dministered in rndomised order to the nimls during the 8th week of tretment. Ech test ws performed once, seprted y rek etween tsks. Rts were tested in rndom order, with the restriction tht one rt from ech diet group e tested in succession. Briefly, the tests included the following: (1) rod wlking, which mesures psychomotor coordintion nd the integrity of the vestiulr system y requiring the niml to lnce on sttionry, horizontl rod; (2) wire suspension, which mesures muscle strength nd the prehensile reflex, n niml s ility to grsp horizontl wire with its forepws nd to remin suspended; (3) plnk wlking, which mesures lnce nd coordintion y exposing the rts to horizontl plnks of three different widths; (4) inclined screen, which mesures muscle tone, strength, stmin nd lnce y plcing the niml on wire mesh screen tht is tilted 6 to the horizontl plne of the floor; nd (5) ccelerting rotrod, which mesures fine motor coordintion, lnce nd resistnce to ftigue y ssessing the durtion tht rt cn remin stnding/wlking on Tle 1. Anthocynin content (ng/g) of the rt diets s mesured y liquid chromtogrphy-tndem MS (4) Anthocynin Control diet (ng/g) Blueerry diet (ng/g) Strwerry diet (ng/g) Cynidin-3-glctoside Cynidin-3-glucoside Cynidin-3-rinoside Delphinidin-3-glucoside* 35 5 Mlvidin-3-rinoside* Mlvidin-3-glctoside* Mlvidin-3-glucoside* Pelrgonidin-3-( cetoyl)glucoside* Pelrgonidin-3-glucoside* Peonidin-3-glctoside Peonidin-3-glucoside Petunidin-3-glucoside* * Becuse of the lck of nlyticl stndrds ville, nlytes were clculted using molr equivlency to cynidin-3-glucoside

3 1544 B. Shukitt-Hle et l. rotting, slowly ccelerting rod. (For more detiled description of the tests, see Shukitt-Hle et l. (42).) Cognitive testing. The MWM test, n ccepted method of testing sptil lerning nd memory, is n ge- (41 43) nd dietsensitive (21,37,44) lerning prdigm tht requires the rt to use sptil lerning to find hidden pltform (1 cm in dimeter) sumerged 2 cm elow the surfce of the wter in circulr pool of wter (134 cm in dimeter 5 cm in height), mintined t 23 C, nd to rememer its loction from the previous tril. Accurte nvigtion is rewrded with escpe from the wter onto the pltform, which the rt effectively loctes y using distl cues. The working memory version of the MWM (43,45) ws performed dily for 4 consecutive dys during the 9th week of tretment, with morning nd n fternoon session, two trils t ech session, with 1-min inter-tril intervl etween the two trils. Rts were tested in rndom mnner, with the restriction tht one rt from ech group e tested in succession. At the eginning of ech tril, the rt ws gently immersed in wter t one of four rndomised strt loctions. Ech rt ws llowed 12 s to escpe onto the pltform; if the rt filed to escpe within this time, it ws guided to the pltform. Once the rt reched the pltform, it remined there for 15 s (tril 1; reference memory or cquisition tril). The rt ws returned to its home cge etween trils (1 min). Tril 2 (the working memory or retrievl tril) used the sme pltform loction nd strt position s tril 1. Performnces were videotped nd nlysed with imge trcking softwre (HVS Imge), which llows mesurements of ltency to find the pltform (s), pth length (cm) nd swimming speed (cm/s; ltency/pth length). (For more detiled description of the mze nd the prdigm used, see Shukitt-Hle et l. (42).) Neurogenesis At lest 1 d following ehviourl testing, the rts were injected with romodeoxyuridine (BrdU, 5 mg/kg) for 3 d. On the 4th dy, one-hlf of the rts in ech diet group (n 6 7/group) were perfused with PBS nd their rins post-fixed in 4 % prformldehyde in order to study the prolifertion of newly divided cells vi immunohistochemistry. The remining rts (n 6/group) were perfused 28 d lter, s ove, to study cell survivl. For single immunostining of BrdU, 4-μm sections were cut with cryostt, nd every tenth section spnning the entire extent of the hippocmpus ws treted to denture DNA with 2 N-HCl nd DNse I. Sections (6 8/rt) were incuted with mouse monoclonl nti-brdu ntiody, followed y incution with rt-sored iotinylted nti-mouse secondry ntiody. Immunorectivities were visulised y the vidin iotin peroxidse procedure using 3,3'-diminoenzidine (DAB) s chromogen (Vectstin Elite ABC Kit; Vector). For doule stining of BrdU nd glil firillry cidic protein (GFAP), sections were treted nd stined for BrdU s descried ove, except tht Ni ws used in conjunction with DAB to produce lck colourtion. After BrdU stining, sections were locked with norml horse serum, incuted with polyclonl rit nti-gfap primry ntiody nd followed y incution with iotinylted nti-rit secondry ntiody. GFAP-positive cells were detected with the ABC Kit using DAB lone to otin rown-stined cells. BrdU-positive cells were counted y two independent rters. Estimtes were mde of the totl numer of positive cells locted throughout the entire extent of the grnulr cell lyer (GCL), including the sugrnulr zone (s defined y two-cellody-wide zone t the edge of the GCL) nd the hilus, s well s these regions seprtely, to exmine migrtion pttern chnges. The estimted totl numer of BrdU-positive cells for ech hippocmpus ws clculted y first tking the men count etween two rters for ech sectioned dentte gyrus re, then verging the mens of six sections counted per hippocmpus, nd multiplying this verge for 6 sections y 6 (6 eing the totl numer of sections cross the nlysed portion of hippocmpus for ech niml). Western immunolotting: insulin-like growth fctor 1 For nlysis of IGF-1 y western immunolotting, smple preprtion ws done ccording to slightly modified procedure of Willims et l. (3) ; specificlly, hippocmpl rin regions were homogenised on ice with Teflon-pestle motorised hndheld homogeniser in uffer contining Tris (5 mm), Triton X-1 ( 1 %), NCl (15 mm) nd ethylene glycol tetrcetic cid/edta (2 mm), ph 7 4, with mmmlin protese inhiitor cocktil (1:1 dilution), sodium pyrophosphte (1 mm), phenylmethnesulfonylfluoride (1 μg/ml), sodium vndte (1 mm) nd sodium fluoride (5 mm). Homogentes were left on ice for 45 min efore centrifugtion t 1 g for 5 min t 4 C to remove unroken cell deris nd nuclei. Protein concentrtion in the superntnts ws determined y the Dc Protein Assy (Bio-Rd). Gel smples were oiled for 5 min in Lemmli smple uffer with finl concentrtion of 2 5% 2- mercptoethnol. Smples were stored t 8 C until nlysis. Western immunolotting procedures were conducted s per Ferrer et l. (46) nd Mendelson et l. (47). Briefly, 2 μg of protein smples nd pooled protein stndrd were run on 12 5% SDS-PAGE gel. The gel ws soked in protein trnsfer uffer (2 mm-glycine, 25 mm-tris se, 2 % methnol) for 1 min, nd then proteins were electrophoreticlly trnsferred to nitrocellulose memrne. Blots were locked with TBST (Trisuffered sline with Tween; 14 mm-ncl, 2 mm-tris, 5 % Tween-2, ph 7 5) with 4 % non-ft dried milk nd incuted in the IGF-1 ntiody (Millipore) in TBST without milk, followed y wshing with TBST nd incution with the pproprite horserdish peroxidse-conjugted secondry ntiody in TBST without milk. After finl wshing in TBST, the signl ws detected using ECL Plus Regents. The immunorective nds, corresponding to the moleculr weight of the protein of interest, were visulised with digitl chrge-coupled device cmer (Hmmtsu Photonics) ttched to BioImging System (EC 3 Drkroom; UVP), nd the opticl densities were quntified with LWorks Imging Acquisition nd Anlysis softwre (version 4.5; UVP). The pooled protein stndrd ws used to normlise the intensities of the ntiody-specific nds, nd the signls were expressed s percentge of opticl density of the stndrd for comprison of signl cross the lots.

4 Beneficil effects of erries 1545 Sttisticl nlyses For ech mesure, etween-sujects ANOVA models compring the diet groups were performed using Systt (SPSS Inc.) to test for sttisticl significnce t the P < 5 level. Dys or trils, when pproprite, were included in the model s within-sujects vrile. Post hoc comprisons, to determine differences etween the diet groups, were performed using Fisher s lest significnt difference post hoc nlysis. To nlyse working memory, seprte t tests were conducted for ech group etween the tril 1 nd tril 2 ltencies. Correltions etween ehviour nd rin mesures were crried out using Person s r correltion. Results There were no differences in weight (P > 5; verge weight: control group = (SEM 1 27); BB group = (SEM 7 2); SB group = (SEM 7 43)) or food intke (P > 5; verge food intke: control group = (SEM 88); BB group = 22 2 (SEM 71); SB group = (SEM 1 16)) etween the diet groups during the study. Psychomotor tests Differences in psychomotor performnce were seen mong the diet groups (Fig. 1). Specificlly, ltency to fll in the rod wlking test (men vlues with their stndrd errors) ws higher in the BB group compred with the SB group (P < 5); however, neither diet group ws different from the control group (group effect: F 2,34 = 4 3; P < 5; Fig. 1()). Rotrod performnce ws improved y the BB diet compred with control (P < 5), wheres the SB group ltency ws not different from either the control or the BB group (group effect: F 2,34 = 2 35; P = 1; Fig. 1()). On the lrge plnk wlk test, the SB group hd longer ltency to fll compred with the BB group (P < 5), wheres neither diet group ws different from the control group (group effect: F 2,34 = 4 52; P < 5; Fig. 1(c)). However, the BB group lso mde significntly more 18 turns on the lrge plnk compred with the SB or control group (P < 5), which might hve contriuted to the shorter ltency to fll (group effect: F 2,34 = 3 31; P < 5; dt not shown). There were no dditionl differences etween the groups on ny of the other motor tests. Cognitive tests When exmining cognitive performnce, the erry fruit diet groups showed improved performnce compred with the control diet group (Fig. 2). There were no overll group differences in MWM ltency or distnce to the pltform in tril 1 or tril 2 performnce. However, seprte t tests were performed etween the two tril ltencies or distnces for ech group for dys 3 nd 4 (the dys when performnce relies more on memory thn on lerning) to determine whether the different diet groups significntly improved their performnce from tril 1 to tril 2, n indiction of working memory. We found tht rts in oth the BB (t(12) = 3 21; P = 7) nd SB (t(11) = 2 11; P = 5) groups showed significnt reduction in ltency to find the pltform etween tril 1 nd tril 2; tht is tril 2 ltencies were significntly less thn tril 1 ltencies, showing tht these rts demonstrted one-tril lerning, even with the 1-min retention intervl (Fig. 2()). This one-tril lerning ws not found in the control group (t(11) = 1 4; P = 32). In ddition, oth the BB (t(12) = 3 51; P = 4) nd SB (t(11) = 3 14; P = 9) diet groups showed significnt decreses in distnce to find the pltform etween tril 1 nd tril 2; tht is, tril 2 distnces were significntly less thn tril 1 distnces (Fig. 2()), wheres this difference ws not different in the control group (t(11) = 1 44; P = 178). Therefore, rts consuming the erry fruit diets showed reversl of the deleterious effects of geing on cognitive performnce, prticulrly on working memory. These differences were not due to swim speed s there were no differences etween the groups in this prmeter on dys 3 nd 4. (A) Ltency to fll (s) (B) Ltency to fll (s) (C) Ltency to fll (s) , Control Blueerry Strwerry Control, Control Rodwlking Rotrod Blueerry Lrge plnk Blueerry, Strwerry Strwerry Fig. 1. Ltency to fll in the rod wlking (A), rotrod (B) nd lrge plnk (C) tests for the control, 2 % lueerry nd 2 % strwerry diet groups. Vlues re mens, with their stndrd errors represented y verticl rs., Men vlues with unlike letters were significntly different (P < 5; Fisher s lest significnt difference).

5 1546 B. Shukitt-Hle et l. (A) 6 Ltency to pltform (s) Ltency to pltform (s) (B) Control Blueerry Strwerry Control Blueerry Strwerry Fig. 2. Morris wter mze performnce ssessed s ltency in seconds (A) nd distnce in metres (B) to find the hidden pltform over dys 3 nd 4 of testing for nimls in the control, 2 % lueerry nd 2 % strwerry diet groups. Vlues re mens, with their stndrd errors represented y verticl rs. Sttisticl difference in performnce (i.e. n improvement) etween,tril1 nd, tril 2 (* P < 5, ** P < 1), indicting improved working memory. ** ** * ** (A) 4 Numer of surviving cells (B) 4 Numer of proliferting cells 3 2 1, Control Blueerry Strwerry Control Blueerry Strwerry Fig. 3. The numer of surviving (A) nd proliferting (B) precursor cells (mesured s BrdU-positive cells) in the dentte gyrus of the hippocmpus of rts in the control, 2 % lueerry nd 2 % strwerry diet groups. Vlues re mens, with their stndrd errors represented y verticl rs., Men vlues with unlike letters were significntly different (P < 5; Fisher s lest significnt difference). Neurogenesis Differences were seen in cell survivl mong the diet groups (group effect: F 2,12 = 2 95; P = 9; Fig. 3()), with rts in the SB group showing significnt increse in the numer of cells surviving in the dentte gyrus of the hippocmpus compred with the control diet group (P < 5). However, even though rts in the SB group lso showed higher numers of proliferting precursor cells (Fig. 3()), these differences did not rech significnce (P > 5). In oth mesures, rts in the BB diet group showed numers tht fell etween those of the SB nd control rts, nd were not different from either group (Fig. 3() nd ()). The BrdU-positive cells did not co-loclise with the GFAP-positive cells (dt not shown). Correltions etween neurogenesis nd cognitive performnce reveled tht, s the numer of proliferting cells incresed, the men difference in ltency etween tril 1 nd tril 2 on dys 3 4 lso incresed (r 414; P = 87; Fig. 4). The difference score is mesure of working memory, or short-term memory, in tht the higher the difference score, the more improvement from tril 1 to tril 2, s the score implies tht the rts re le to find the pltform more quickly on tril 2 y rememering where it ws on tril 1, fter the 1 min dely. Interestingly, when exmined seprtely y diet group, only the BB group showed significnt correltion etween prolifertion nd working memory performnce (r 816; P < 5); neither the control group (r 29; P > 5) nor the SB group (r 4; P > 5) yielded significnce. In contrst, cognitive performnce did not significntly correlte with surviving cells, nd none of the motor tests showed positive correltion with neurogenesis. Western immunolotting IGF-1 levels were incresed y oth erry diets compred with control (P < 5), ut levels in the SB group were lso significntly higher (P < 5) thn those in the BB group (group effect: F 2,12 = 33 7; P < 5; Fig. 5). Correltions etween IGF-1 nd cognitive performnce reveled no overll reltionship etween IGF-1 levels nd the men difference in ltency etween tril 1 nd tril 2 on dys 3 4 (r 28; P > 5). However, when exmined seprtely y diet group, only the BB group showed positive correltion etween IGF-1 levels nd working memory performnce (mening tht, s IGF-1 levels incresed, the difference score for ltency from tril 1 to tril 2 incresed, showing improved lerning), lthough this vlue did not rech sttisticl significnce (r 772; P = 126). Correltion vlues for oth the control group (r 884; P < 5) nd the SB group (r 357; P > 5) were negtive, lthough only the vlue for the control group ws sttisticlly significnt, showing tht higher IGF-1 levels were ssocited with poorer working memory performnce in this group. Discussion Overll, results from this study showed tht consuming errysupplemented diets led to enhnced motor performnce, improved cognition, specificlly working memory, incresed

6 Beneficil effects of erries 1547 Numer of proliferting cells IGF- 1 (density units) r. 414, P = Diffrence score, T1 T2, dys 3 4 Fig. 4. Correltions etween neurogenesis (numer of proliferting cells) nd cognitive ehviour (difference score of tril 1 (T1) nd tril 2 (T2) in the Morris wter mze on dys 3 4) of rts in the control, 2 % lueerry (BB) nd 2 % strwerry (SB) diet groups., Control: r.29, P >.5;, BB: r.816, P <.5;, SB: r.4, P >.5. neurogenesis nd incresed IGF-1 signlling. However, these results lso indicte tht the diverse polyphenolics in the different erry fruits might e cting differentilly to produce their positive effects. For exmple, on the motor tests, the BB diet group ws etter on the rod wlk, which mesures psychomotor coordintion nd the integrity of the vestiulr system, wheres the SB group ws etter on the plnk wlk, which ssesses more generl lnce nd coordintion. Both supplemented groups showed positive effects on the rotrod test, which is mesure of fine coordintion, lnce nd resistnce to ftigue. Conversely, oth diets showed positive effects on the cognitive tests. However, the improvements in working memory performnce could not solely e explined y the increses in neurogenesis nd neuronl signlling. For exmple, only the ehviour of the BB group ws significntly correlted with the numer of proliferting cells in the dentte gyrus, even though overll the SB group hd the highest numer of proliferting cells nd significntly greter numer of surviving cells compred with ny other group. Similrly, only the ehviour in the BB group showed positive reltionship with IGF-1 levels, even though the SB-fed group hd the highest IGF-1 levels. Becuse oth erry fruit diets did not produce equivlent results, the types nd reltive mounts of polyphenols in the Control Blueerry Strwerry Fig. 5. Insulin-like growth fctor 1 (IGF-1) levels in the hippocmpus of rts in the control, 2 % lueerry nd 2 % strwerry diet groups. Quntittive mesurements were sed on the western lots nd represented s men nd density.,,c Men vlues with unlike letters were significntly different (P < 5; Fisher s lest significnt difference). c different erries could ccount for their reltive differences in efficcy. Therefore, we mesured the nthocynin content of the diets s we feel these polyphenols re the most relevnt nd the most distinct mong these two fruits. The control diet hd trce mounts of few nthocynins. However, the BB diet showed higher levels of four different nthocynins, specificlly cynidin, delphinidin, mlvidin nd peonidin, compred with the SB diet, which hd more pelrgonidin nd petunidin thn the BB diet. In fct, the only nthocynin tht the two erry diets hd in common ws cynidin-3-glucoside. Wheres the BB diet hd smller mounts of more nthocynins, the nthocynin content of the SB diet ws driven y the lrge quntity of pelrgonidin-3-glucoside. We hve shown previously tht nthocynins contined in BB, specificlly the four seen in the rt diet in this study, cross the lood rin rrier in glycosylted form nd loclise in the rin, nd the totl numer of nthocynin compounds in the cortex nd hippocmpus correltes with MWM performnce (36). Future studies should mesure polyphenols nd their metolites in the serum nd rin of rts, to determine which specific nthocynins contriute to the improvements in ehviour. These results gree with n erlier study in our lortory (27) tht found tht ged rts fed 2 % BB-supplemented diet hd significnt increses in hippocmpl prolifertion of neuronl precursor cells compred with non-supplemented ged rts, nd these increses correlted with reductions in the numer of memory errors in rdil rm wter mze. Furthermore, this study found tht BB-fed rts hd increses in the neuroprotective trophic fctor IGF-1 tht were ssocited with significnt decreses in memory errors (27). Another study found tht rts fed 2 % BB diet prior to centrl dministrtion of the neurotoxin kinic cid showed incresed hippocmpl mrna expression of IGF-1 compred with control-fed rts (34). In the present study, sptil working memory performnce in the BB group ws lso ssocited with increses in hippocmpl plsticity prmeters of neurogenesis nd IGF-1 levels. Interestingly, these ssocitions were not seen in the SB group. It remins to e determined whether chnges in other mrkers would correlte etter with the cognitive improvements in the SB group or whether nother mesure of cognition would correlte etter with these mrkers. Given the well-documented link etween hippocmpl neurogenesis, cognitive performnce nd ge, the fct tht n ssocition ws found etween the prolifertion of neuronl precursor cells nd sptil memory performnce in our nimls suggests tht erry-medited increses in neurogenesis my ply key role in the reported improvements in cognition in such ged nimls. However, despite this ssocition etween prolifertion nd cognition, neither erry diet group showed significntly higher numer of proliferting cells compred with the control group, possily ecuse of the smll numer of rts in ech group mesured for neurogenesis (6/group). It is lso surprising tht, even though the erry diets incresed the survivl of hippocmpl neurons, sptil working memory in the MWM did not correlte with this prmeter. Perhps prolifertion itself is more importnt in improving sptil memory nd lerning in the BB-supplemented nimls. Alterntively, it my e tht diet-induced chnges in survivl

7 1548 B. Shukitt-Hle et l. tke longer time to develop, nd the length of the feeding prior to ehviourl testing ws not long enough time to cpture this chnge. It is lso possile tht BB my e more effective t stimulting neuronl differentition nd in improving neuroplsticity compred with SB; future studies should exmine these mesures. Another niml study showed tht supplementtion with BB diet (2 % of the diet for 12 weeks) improved sptil working memory in ged rts in cross mze, nd tht, s in the present study, these performnce improvements were ssocited with ttenuted levels of vriety of signlling molecules involved in stress, survivl nd neurl plsticity in the hippocmpus (3). Memory performnce correlted with the ctivtion of cyclic AMP-response element-inding protein nd increses in rin-derived neurotrophic fctor in the hippocmpus, which were linked to increses in the phosphoryltion stte of extrcellulr signl-relted kinse nd chnges in the ctivtion stte of mmmlin trget of rpmycin. Interestingly, one key pthwy tht the IGF-1 receptor signls through is regulted y phosphtidylinositol-3 kinse nd its downstrem prtner mtor (48). The current study highlights the need to study the effects of erry fruits on humn helth nd, to dte, only few studies hve investigted chnges in cognition with ge. Most notly, in one study, older dults with mild cognitive impirment consuming BB juice (6 9 ml/kg per d) for 12 weeks showed improved word list recll nd incresed pired ssocite lerning, reltive to oth seline nd plceo controls (49). Recent clinicl reserch hs lso linked chnges in wlking speed nd git vriility to declines in cognitive ility during norml geing (5,51). On the sis of these findings nd existing evidence for the protective effects of erry fruits on motor ility in rodent models, from this nd previous studies, it cn e stted tht future clinicl reserch should incorporte mesures of cognition pired with mesures of motor control, lnce nd moility to determine whether erry supplementtion cn enhnce motor control in older, nonpthologicl, dults. Tken together, these preclinicl findings suggest tht dietry supplementtion with erry fruits hs the potentil for multiplicity of effects, in ddition to their ntioxidnt nd nti-inflmmtory properties, including the ctivtion of vriety of signlling pthwys tht result in neuroprotection, neurogenesis nd, ultimtely, spred cognitive nd motor ehviour. Individul polyphenols in the erry fruits might exert their effects through different nd/or independent mechnisms. Future studies should ssess which specific polyphenolic compounds nd/or metolites enter the serum, nd from there into which res of the rin in nimls, to scertin the extent to which regionl loclistion nd iovilility of the BB nd SB polyphenols contriute to their mechnism(s) of ction. Acknowledgements B. S.-H. thnks Json Wlsh, PhD, nd Oliver Chen, PhD, of the Antioxidnts Reserch L t the HNRCA t Tufts University for their help in identifying the nthocynins in the rt chow. This reserch ws supported y USDA intrmurl funds nd greements etween the USDA nd the Wild Blueerry Assocition of North Americ (WBANA), the US Highush Blueerry Council (USHBC) nd the Cliforni Strwerry Commission. A. N. C nd B. S.-H. rn the ehviourl tests nd nlysed the dt; D. F. B. rn the western lots; L. M. W. nd F. C. L. performed the neurogenesis ssys nd nlysed the results; J. A. J. nd B. S.-H. designed the experiments nd prepred the mnuscript; nd B. S.-H. hd primry responsiility for finl content. 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