RAPID COMMUNICATION

Interfering With Perirhinal Brain-Derived Neurotrophic FactorExpression Impairs Recognition Memory in Rats

Ana Seoane, Chris J. Tinsley, and Malcolm W. Brown*

ABSTRACT: The role of brain-derived neurotrophic factor (BDNF) inrecognition memory was investigated by locally infusing oligodeoxynu-cleotides (ODNs) into perirhinal cortex, a region of the temporal lobeessential for familiarity discrimination. Antisense but not sense BDNFODN impaired consolidation of long-term (24h) but not shorter-term(20min) recognition memory. VVC 2010 Wiley-Liss, Inc.

KEY WORDS: familiarity discrimination; perirhinal cortex; neurotrophin;oligodeoxynucleotide; consolidation

INTRODUCTION

Brain-derived neurotrophic factor (BDNF) is a neurotrophin involvedin synaptic plasticity, including long-term potentiation and long-termdepression (Pang et al., 2004; Bramham and Messaoudi, 2005; Wooet al., 2005; Bekinschtein et al., 2008; Lu et al., 2008). As synaptic plas-ticity is believed to underlie memory processes, it is interesting to testlinks between BDNF and memory. Indeed, interfering with BDNFactions in hippocampus, amygdala, or parietal cortex impairs fear-condi-tioning, inhibitory avoidance learning, or spatial memory (Ma et al.1998; Mu et al., 1999; Mizuno et al., 2000, 2003; Alonso et al., 2002,2005; Lee et al., 2004; Rattiner et al., 2004, 2005; Bekinschtein et al.,2007; Heldt et al., 2007). Recently, an increase in BDNF mRNA hasbeen reported in perirhinal cortex 2 h after exposure to novel objects(Romero-Granados et al., 2009). However, the necessity for recogni-tion memory of BDNF activity within perirhinal cortex remains to betested.

The familiarity discrimination component of recognition memory forindividual items relies on the integrity of the perirhinal cortex of thetemporal lobe (Brown and Aggleton, 2001). We investigated the role ofBNDF in the acquisition, retrieval, and consolidation of recognitionmemory processes at long and short delays using a novel object prefer-ence task (Ennaceur and Delacour, 1988). Infusion of an antisense oli-godeoxynucleotide (ODN) into perirhinal cortex was used to interfere

with BDNF expression, as previously used in hippo-campus (Ma et al., 1998; Lee et al., 2004; Bekinsch-tein et al., 2007).

Two groups (n 5 9 and n 5 10) of adult male pig-mented Dark Agouti rats (220–250 g; Bantin andKingman, UK) were maintained on a 12-h light–darkcycle, with the dark phase during normal daylight. Allexperiments were performed in accordance with theUK Animals Scientific Procedures Act (1986) and hadthe approval of the University of Bristol EthicalReview Committee. ODNs were PAGE-purified phos-photioate end-capped 18-mer sequences (SigmaGenosys, Haverhill, UK): BDNF antisense ODN:50-TCTTCCCCTTTTAATGGT-30; BDNF senseODN (control sequence): 50-AGAAGGGGAAAATTACCA-30. Antisense or sense ODN was infusedlocally into the perirhinal cortex through bilaterallyimplanted cannulae at 1 nmol in 1-ll normal saline,the dose following that used by Lee et al. (2004).

Cannula implantation into perirhinal cortex wascarried out stereotaxically in rats deeply anesthetizedwith Isoflurane (Merial Animal Health, Harlow, UK).Details of procedures have been published previously(Warburton et al., 2003; Barker et al., 2006a,b,2008). Two stainless-steel guide cannulae (26 gauge,Plastics One, Roanoke, VA, via Semat in UK) wereimplanted through holes in the skull, at an angle of208 to the vertical (coordinates relative to bregma: AP25.6 mm, L 6 4.5 mm, and V 26.7 mm relative tothe skull surface) and anchored to the skull. Behav-ioral testing began >20 days later. Infusions through acannula in each hemisphere were made by inserting a33 gauge cannula (Plastics One), which protruded 1mm beyond the guide cannula tip and which wasconnected by PVC tubing (Barloworld Scientific,Berkshire, UK) to a Hamilton syringe (HamiltonBonaduz, Bonaduz, Switzerland). To minimize theamount of ODN used, the PVC tubing was prefilledwith a nonwater soluble solution of 0.3 mg/ml OilRed O (Sigma-Aldrich Chemie, Steinheim, Germany)in paraffin oil (Fluka, Steinheim, Germany). The sy-ringe was advanced with an infusion pump (HarvardBioscience, Holliston, MA) at a rate of 0.7 ll/min for1 min and 30 s; 5 min later the injection cannulaewere withdrawn. Cannula locations were checked, aspreviously (Warburton et al., 2003; Barker et al.,

Department of Anatomy, Medical Research Council, Centre for SynapticPlasticity, University of Bristol, Bristol, United KingdomGrant sponsor: Medical Research Council; Grant number: G9713086.*Correspondence to: Malcolm W. Brown, Department of Anatomy,University of Bristol, Bristol, BS8 1TD, United Kingdom.E-mail: M.W.Brown@bristol.ac.ukAccepted for publication 18 November 2009DOI 10.1002/hipo.20763Published online 19 January 2010 in Wiley Online Library(wileyonlinelibrary.com).

HIPPOCAMPUS 21:121–126 (2011)

VVC 2010 WILEY-LISS, INC.

2006a,b, 2008) using coronal (40 lm), cresyl violet sections.All reported data are from animals where the tips of the cannu-lae were within perirhinal cortex (areas 35 and 36) frombregma 25.5 to 24.5 (Paxinos and Watson, 1998; Shi andCassell, 1999; Fig. 1).

To visualize the extent of ODN diffusion within the brain, 1nmol of biotinylated antisense BDNF ODN (ODN sequencelabeled with biotin on 50 end) in 1 ll saline was infused intothe PRH via cannula 1 h (n 5 2) before being anesthetizedwith Euthatal. The brain was removed and fixed in 4% para-formaldehyde in 0.1 M phosphate buffer (pH 7.4) for at least6 days before being immersed in 30% sucrose in 0.1 M phos-phate buffer for at least 2 days. The right hemisphere of thebrain was marked with an incision before being sectioned (40lm) coronally on a cryostat. Floating sections were collectedand washed in 0.1 M phosphate buffer saline containing 0.2%Triton X-100 (PBST; pH 7.4), and processed with avidin-bio-tinylated horseradish peroxidase complex (Vectastain ABC Kit;Vector Laboratories) in PBST for 1 h at room temperature,and the reaction was visualized using 3,30-diaminobenzidine(Sigma FastTM, Sigma-Aldrich). As the brain needed to beimmersion-fixed, the sections also contain black blood cells,identifiable by their size and configuration. Some of the sec-tions were counter-stained with cresyl violet. Biotinylated anti-sense BDNF ODN labeling was chiefly restricted to the PRHand was clearly visible within neurons (see Fig. 2). Staining inthe entorhinal cortex or area Te2 was estimated to be <5% ofthese structures in each case. No stained neurons were found inthe hippocampus. The spread in the anterior–posterior axis was900 6 80 lm (mean 6 standard error of mean for four infu-sion sites) and estimates indicated that �31% of the total peri-rhinal cortex volume contained biotinylated antisense BDNFODN. In the central 500 lm �45% of the perirhinal cortexcontained such BDNF staining. It should be noted that asdeep cortical layers were chiefly involved in area 36 and super-ficial layers in area 35, more of perirhinal cortex’s processingcapacity was likely to have been affected by the ODN infusions

than the above-quoted percentage figures might imply. In previ-ous studies, biotinylated BDNF ODN spread was shown at 90min, 2 h, or 24 h after a local infusion in the hippocampususing doses of 1 or 2 nmol ODN in 1 ll of saline (Lee et al.,2004; Bekinschtein et al., 2007). A wide ODN spread wasfound at 90 min or 2 h, but by 24 h the presence of biotinyl-ated ODN in the hippocampus was greatly reduced. This sug-gests that at time points later than 24 h, such as 1 week,BDNF ODN may be expected to have disappeared.

Recognition memory was tested using the novel object pref-erence test as previously (Warbuton et al., 2003; Barker et al.,2006a,b, 2007). In brief, this task took place in an arena (903 100 3 50 cm) surrounded to a height of 1.5 m with blackcurtains and with sawdust on the floor. Rat behavior was moni-tored using a camera and a video recorder. The objects weremade of Duplo bricks (Lego Produktion A.G., Baar, Switzer-land) and varied in size (range: 10 3 5 3 15 cm to 20 3 203 30 cm), color and shape and were placed near the two cor-ners at either end of one side of the arena (15 cm from eachadjacent wall). Prior to the start of memory testing, each ratwas habituated to the empty arena for 5 min daily for 4 days.The novel object preference test comprised two phases, acquisi-tion and test, separated by a delay of 20 min or 24 h. In theacquisition phase, an animal was allowed to explore two identi-cal objects for 40 s of exploration or a maximum of 4 minspent in the arena. In the test phase, the rat was allowed toexplore an identical third copy of the previously explored objectand a novel object for 3 min from the start of the objects’ ex-ploration. In both acquisition and test phases, the time spentexploring each of the objects was recorded. Exploration wasscored only when the animal’s nose was directed toward theobject at a distance of less than 1 cm. If the time of explora-tion was less than 15 s in the acquisition phase or less than 10s in the test phase, the animal was discarded from the analysisof that experiment. The objects used as novel or familiar andtheir position in the arena were counterbalanced between ani-mals receiving the same treatment and between control anddrug-treated animals. Each experiment used a cross-over designwith half the animals receiving sense and half receiving anti-sense ODN infusions followed a week later by infusions of theopposite compounds. The scorer was blind as to the treatmentof each animal. The role for BDNF in recognition memorywas tested using experimental conditions differing both in thetime of infusion of antisense or sense ODN and in the delaybetween the acquisition and test phases (see Table 1).

There were no significant differences between antisense- andsense-treated rats in the time spent in exploration in either ac-quisition or test phases in any of the experiments (see Table 2).Thus, there was no evidence that the memory impairmentswere due to problems with general exploration. Moreover, inall the experiments, sense ODN treatment produced discrimi-nation and total exploration values similar to those of previousstudies for rats injected or infused with saline (Warburtonet al., 2003; Barker et al., 2006b). Accordingly, there was noevidence that sense ODN infusion affected familiaritydiscrimination.

FIGURE 1. Infusion sites. The location of cannula tips, asobserved in cresyl violet stained sections, for all implanted animalsused in object recognition test (n 5 9 and n 5 10) fell within thedark gray area, which was located within the perirhinal cortex(PRH) from bregma 25.5 to 24.5 (Paxinos and Watson, 1998;Shi and Cassell, 1999).

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The discrimination ratio (DR) was calculated as the time inthe test phase spent exploring the novel object minus that spentexploring the previously experienced object divided by the totalexploration time. Comparisons with other studies (Warburton

et al., 2003; Barker et al., 2006b) established that the perform-ance of rats under control conditions (sense ODN infusion)showed no evidence of residual effects from the antisenseODN infusion 1 week earlier. Comparisons between treatment

FIGURE 2. Visualization of biotinylated antisense BDNFODN (1 nmol in 1 ll saline) 1 h after infusion into the perirhinalcortex. Top, photomicrographs showing a section stained for bio-tinylated BDNF ODN (A) and counterstained with cresyl violet(B). The section is �26.0 relative to bregma (Paxinos and Wat-

son, 1998) and immediately posterior to the cannula track. Thegreat majority of biotinylated BDNF ODN labeling was restrictedto perirhinal cortex. Bottom, a magnification of the same sectionshowing incorporation of biotinylated BDNF ODN into neurons(C and D). Scale bars: A, B 5 1 mm; C, D 5 200 lm.

TABLE 1.

Infusion Times Relative to and Delays Between Acquisition and Test Phases for Each Condition

Condition abbreviation Infusion time Delay between acquisition and test

Acquisition 20 min 1 h before acquisition 20 min

Acquisition 24 h 1 h before acquisition 24 h

Consolidation 24 h 2 min after acquisition 24 h

Retrieval 24 h 1 h before test 24 h

6 h postacquisition 24 h infusion 6 h after acquisition 30 h (test done 24 h after infusion)

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conditions were analyzed by within-subject analyses ofvariances (ANOVAs). One-sample t-tests were used to deter-mine whether the DR for each condition was significantlydifferent from zero (no discrimination between novel andfamiliar objects). All tests used a significance level of P 5

0.05 and were two-tailed.The effects of BDNF ODNs were determined on the differ-

ence in exploration of a novel rather than a familiar object(DR values). In three experiments with a 24-h memory delayperiod (n 5 9), sense or antisense ODN was administered ei-ther 1 h before the acquisition phase, so that it was active dur-ing and after acquisition, 1 h before the test phase, so that itwas active during retrieval, or within 2 min after the acquisi-tion phase, so that it was active at the start of consolidation.That antisense ODN impaired early consolidation was indi-cated by the following findings. The effect of treatment (senseor antisense ODN) was significant [ANOVA F(1,7) 5 23.94;P 5 0.002], but the interaction between treatment and experi-ment (time of infusion) was also significant [ANOVA F(2,14)5 3.92; P < 0.05]. Further analysis of the data in the twoexperiments for infusions given before or immediately after ac-quisition indicated that the effect of treatment was highly sig-nificant [F(1,7) 5 15.14; P 5 0.006] and that there was nosignificant difference between the effects of treatment for thetwo infusion times [interaction between treatment and experi-ment: ANOVA F(1,7) 5 3.63; P 5 0.1]. With infusion beforeacquisition, rats given the antisense ODN failed to discriminatethe familiarity of objects, whereas when given the sense ODN(controls), they did discriminate (DR � 0: antisense, t(8) < 1,P > 0.1; sense, t(8) 5 4.36, P 5 0.002; Fig. 3). Similarly,with infusion immediately after acquisition, rats treated withantisense ODN did not show significant discrimination,whereas when treated with sense ODN they did (DR � 0:antisense, t(8) < 1, P > 0.1; sense, t(8) 5 6.95, P < 0.0001;Fig. 3). As there was impairment when the antisense-ODN wasnot present at acquisition and might be expected to be no lon-ger active at test at the 24-h delay, the impairment could not

be ascribed to a difference in the presence against absence ofthe compound at acquisition and retrieval (i.e., impairmentwas not due to state-dependency). Neither sense nor antisenseBDNF ODN produced an impairment in recognition memorywhen infused before retrieval [ANOVA F(1,8) < 1; P > 0.1]:rats discriminated in both conditions [DR > 0: antisense: t(8)5 3.15, P 5 0.01; sense: t(8) 5 3.38, P 5 0.01; Fig. 3].

In contrast, when the memory delay was 20 min rather than24 h (n 5 9, same animals as used in the previous experi-ments), antisense BDNF ODN administered 1 h beforeacquisition did not cause an impairment in familiarity discrimi-nation; rats when given sense or antisense ODN spent a signifi-cantly greater proportion of time exploring the novel ratherthan the familiar object [DR > 0: antisense: t(8) 5 5.681,P < 0.0001; sense: t(8) 5 2.31, P < 0.01; Fig. 3].

To determine whether the familiarity discrimination impair-ment found at the 24-h delay was because of an effect on earlybut not late memory consolidation processes, sense or antisenseODN was administered to an additional group of rats (n 5

10) 6 h after the acquisition phase, with the test phase 24 h af-ter the infusion. Rats preferred the novel to the familiar objectfor both the sense and antisense-treated conditions (antisense:mean DR 5 0.36 6 0.1; DR > 0, t(9) 5 3.46, P < 0.01;sense: mean DR 5 0.30 6 0.09; DR > 0, t(9) 5 3.22, P 5

0.01), and their mean DRs did not differ [t(18) < 1, P >0.1]. Thus, antisense ODN infusion did not impair long-termfamiliarity discrimination when the antisense ODN was admin-istered 6 h after acquisition. This lack of impairment addition-ally indicates that under these circumstances long-term recogni-tion memory is normal 24 h after infusion of the antisenseODN; this lack of impairment excludes a long-term nonspe-cific action of the ODN as an explanation for the difference inits effects on memory at 20 min and 24-h delays.

The results establish a role for BDNF in long-term recogni-tion memory: familiarity discrimination was impaired whenantisense BDNF ODN was locally infused into perirhinal cor-tex either before or immediately after acquisition (but not 6 hafter acquisition) and memory measured after a 24-h delay.Additionally, antisense BDNF ODN did not affect retrieval. Bycontrast, memory was not impaired at a 20-min delay. Theantisense, but not the sense, BDNF ODN will bind to andhence prevent translation of BDNF mRNA, so preventingBDNF production. Thus, these findings suggest a role forBDNF in early (<7 h) consolidation processes underlying peri-rhinal familiarity discrimination.

The BDNF impairment of long-term but not shorter-termrecognition memory found here is in accord with the impair-ment in fear conditioning following antisense BDNF ODN oranti-BDNF antibody infusions into the hippocampus (Alonsoet al., 2002; Lee et al., 2004) or parietal cortex (Alonso et al.,2005); these impairments were also interpreted as arising frominterference with consolidation. For example, infusion of anti-BDNF antibody into the hippocampus 15 min prior or 1 or 4h after inhibitory avoidance training caused memory impair-ment in one-trial performance carried out at 24-h delay(Alonso et al., 2002). Impairment of long-term but not

TABLE 2.

Exploration Time (Mean 6 SEM; in s) in Acquisition and Test Phases

Acquisition 20 min Acquisition 24 h

Sense Antisense Sense Antisense

Acquisition 32.4 6 2.1 32.5 6 1.5 22.5 6 1.4 25.5 6 2.0

Test 27.5 6 2.6 28.7 6 4.4 18.9 6 2.4 19.6 6 2.3

Retrieval 24 h Consolidation 24 h

Sense Antisense Sense Antisense

Acquisition 28.4 6 3.0 23.6 6 2.1 28.6 6 1.4 28.1 6 2.9

Test 33.4 6 4.8 30.1 6 2.7 38.5 6 3.9 36.3 6 1.5

6 h Postacquisition 24 h

Sense Antisense

Acquisition 31.5 6 2.7 30.8 6 2.0

Test 29.7 6 2.0 24.7 6 2.1

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shorter-term perirhinal recognition memory has been foundpreviously for antagonism of either L-type voltage-dependentcalcium channels (Seoane and Brown, 2006), NMDA (Barkeret al., 2006a), or metabotropic (Barker et al., 2006b) glutamatereceptors in PRH. By contrast, perirhinal kainate receptorantagonism resulted in the opposite pattern of memory loss(amnesia after a 20 min but not a 24-h delay), indicating thatthere must be more than one underlying mechanism that sup-ports perirhinal familiarity discrimination (Barker et al.,2006a).

BDNF receptor TrkB colocalizes with proteins of theNMDA-receptor complex (Aoki et al., 2000; Yoshii and Con-stantine-Paton, 2007; Lu et al., 2008). Moreover, BDNF canfacilitate glutamate release and increase phosphorylation ofNR1 and NR2B subunits of the NMDA-receptor complex(Suen et al., 1997; Levine et al., 1998; Lin et al., 1998; Car-valho et al., 2008). Glutamate receptor transmission and plas-ticity are essential for perirhinal recognition memory (Masseyet al., 2004; Winters and Bussey, 2005; Barker et al., 2006a,b;Griffiths et al., 2008). However, it seems unlikely that theimpairments reported here arise from a direct effect on gluta-matergic transmission, because memory was normal when anti-sense BDNF ODN was infused before retrieval or memory wasmeasured at a 20-min delay.

One possible route through which BDNF might influenceconsolidation of perirhinal recognition memory is via activation

of the transcription factor cAMP responsive element-bindingprotein (CREB). Phosphorylation of CREB within perirhinalcortex is essential for long-term plasticity and recognitionmemory mechanisms (Warburton et al., 2005). Exposure ofneurons to BDNF can lead to the activation of CREB (Fink-beiner et al., 1997; Alonso et al., 2005; Bekinschtein et al.,2008); and the activation of CREB transcriptional processeshas been linked with learning and memory and synaptic plas-ticity (Bourtchuladze et al., 1994; Deisseroth et al., 1996; Silvaet al., 1998; Genoux et al., 2002). Moreover, CREB phospho-rylation can lead to Fos production (Ahn et al., 1998; Silvaet al., 1998) and oligonucleotide antagonism of Fos productionin perirhinal cortex impairs familiarity discrimination (Seoaneand Brown, 2007).

In conclusion, the results indicate that BDNF plays a role inthe early (<7 h) consolidation of long-term (24 h) perirhinalrecognition memory.

Acknowledgments

We are grateful to the Medical Research Council for finan-cial support, and to J. Robbins and K. Narduzzo for technicalassistance.

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FIGURE 3. Effects of anti-BDNF ODN on familiarity discrim-ination. With a 24-h memory delay period, antisense ODNimpaired familiarity discrimination when it was administeredbefore or just after acquisition but not if the infusion was beforethe test (retrieval) phase or if administration was before acquisitionbut the memory delay period was 20 min. DR, discriminationratio (difference in time exploring the novel and familiar objectsdivided by the total exploration time in the test phase). Withinsubject ANOVA treatment difference: ***P < 0.001. One-sample t-test of DR value against zero: OP < 0.05.

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