Cued and spatial learning in the water maze: Equivalent learning in male and female mice

Cued and spatial learning in the water maze: Equivalent learning in male and female mice
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  Neuroscience Letters 483 (2010) 148–151 Contents lists available atScienceDirect NeuroscienceLetters  journal Cued and spatial learning in the water maze:Equivalent learning in male and female mice Lissandra C. Baldan Ramsey a , Christopher Pittenger a , b , ∗ a Department of Psychiatry, Yale University, United States b Interdepartmental Neuroscience Program, Yale University, United States a r t i c l e i n f o  Article history: Received 24 May 2010Received in revised form 27 July 2010Accepted 29 July 2010 Keywords: Spatial learningCued learningWater mazeSex differencesMice a b s t r a c t Mammals navigate a complex environment using a variety of strategies, which can operate in parallelandevencompetewithoneanother.Wehaverecentlydescribedavariantwatermazetaskinwhichtwoofthesestrategies,hippocampus-dependentspatiallearningandstriatum-dependentcuedlearning,canbe dissociated. Male rodents perform better at some spatial learning tasks, while female rodents morereadily learn certain striatum-dependent behavioral strategies. We therefore predicted that sex woulddifferentially influence spatial and cued learning in the water maze. We trained adult male and femaleC57Bl/6 mice for 7 days in the two-cue variant of the water maze, with probe trials on days 5 and 7. Intwo independent experiments, males and females performed similarly, with both groups showing goodspatial learning after 5 and 7 days of training, and both groups showing trend-level cued learning after 5days and robust learning after 7. Therefore, contrary to our hypothesis, sex does not significantly affectcued or spatial learning in this task. © 2010 Elsevier Ireland Ltd. All rights reserved. Navigating a complex environment requires flexible use of learn-ing strategies that can accommodate both reliable regularities andunpredictabledeviationsfromtheexpected[4].Themultiplemem- orysystemshypothesisproposesthatdifferentbraincircuits,whichcan be dissociated by experimental manipulations, employ differ-entlogicsoflearningandareinvokedunderdifferentcircumstances[26].We have recently described a water maze learning task inmice that permits assessment of cued or spatial learning in twootherwise identical task variants[13].We found spatial learning to depend on the hippocampus, while cued learning dependedon the striatum, consistent with earlier literature[16,17,15].We found that the two systems compete with one another duringlearning[21],suchthatdisruptionsofstriatalfunctioncanactually enhance spatial learning, while hippocampal disruptions enhancecued learning[13].Male and female rodents have been shown to learn differen-tiallyundersomeconditions.Maleratsshowconsistentlysuperiorperformance in hippocampus-dependent spatial reference andworking memory tasks, in both the Morris water maze and theradial maze[11].Male mice have also been reported to show bet- ter spatial memory in a radial maze[9,12],though findings in the water maze have been more equivocal[11,8,6]. Interestingly, ∗ Corresponding author at: 34 Park Street, W315, New Haven, CT 06508, UnitedStates. Tel.: +1 203 974 7675; fax: +1 203 974 7662. E-mail address: Pittenger). ‘sex-reversed’ female mice (which carry the male Y chromosome)perform better in the Morris water maze than normal females,suggesting an important role for male genotype (as opposed tohormonalcomplement)onhippocampus-dependentspatiallearn-ing[25].Several explanations for this sexual dimorphism have been proposed, including evolved, adaptive differences in brainwiringbetweenthetwosexes(e.g.[24]),differentialglucocorticoid responses to new tasks[1],differential responsivity to appetitive motivation[14],and differential response to non-spatial pretrain- ing[19].In contrast, limited data suggest that female mice more rapidlyacquire certain striatum-dependent learning tasks. In an instru-mental habit task, which is sensitive to striatal disruptions in bothrats[27]andmice(J.J.Quinn,C.Pittenger,etal,unpublishedobser- vations), genotypically female mice show more rapid acquisitionof habitual responding than males or ‘sex-reversed’ females[22].In navigation tasks, it has been suggested that females rely moreon landmark cues rather than on spatial cognition[11,23].Earlier studiesinrats[17,18],ourresultsinacuedwatermazetaskinmice [13],and neuroimaging studies in humans (e.g.[10])suggest that such cue-based navigation depends on the striatum.We therefore speculated that our variant water maze task,which tests both spatial and cued learning and can detect com-petition between them[13],would reveal predictable sexual dimorphismsinbehavior.Specifically,wepredictedthatmalemicewouldperformbetterthanfemalesinthespatialtask,whilefemalemicewouldshowsuperiorperformanceinthecuedtask.Wetestedthis hypothesis in adult C57Bl/6 mice. 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.07.082  L.C. Baldan Ramsey, C. Pittenger / Neuroscience Letters 483 (2010) 148–151 149 All experiments were conducted under the supervision of theYale University Institutional Animal Care and Use Committee(AnimalWelfareAssuranceNumberA3230-1).Food(standardlab-oratorychow)andwaterwereavailable adlibitum .Allexperimentsused adult male and female C57Bl/6J mice, aged 2.5–4 months;male and female mice were of the same age in each experiment.Animalsweretrainedoneitherthecuedorspatialvariantofthewater maze, using a slight variation on the protocol described indetail by Lee et al.[13].The apparatus consisted of a large circular pool (172cm diameter) filled with opaque water, which is main-tained at 25 ◦ C. A 12cm circular Atlantis platform (Med Associates,Vermont), 1.5cm below the surface of the water, permits escapefrom the water. The platform is marked with a visible cue, con-sistingofaplasticcylinder(2.5cmdiameter × 11cmhigh),paintedeitherauniformgrayorwithhigh-contrast1-cmhorizontalorver-ticalstripes,asdetailedbelow.Duringtwo-cuetraining(seebelow),a second cue was held on a stand elsewhere in the pool, such thatit was not possible to discern from above the water which cue wasassociated with the platform and thus the possibility of escape.All training days consisted of four trials with a 20-min inter-trial interval. The first 5 days consisted of shaping to the task. Onday 1, the gray cue was used and the animal was placed on theplatform for 30s for each trial. On days 2–5 the gray cue was againused; animals were placed in the water at the edge of the pool andallowedtosearchfortheescapeplatformforupto120s.Anyanimalunable to find the platform in this time period was guided to it bytheexperimenter.After15sontheplatform,animalswerereturnedtothehomecage.Followingshaping,animalswerereturnedtothevivarium and left undisturbed for 1–3 days.Shapingwasfollowedby7daysofcuedorspatial2-cuetraining.Vertically and horizontally striped cues were used for this phase;ineachtrial,oneofthesecuesmarkedthelocationoftheplatform,whiletheotherwasplacedelsewhereinthepoolanddidnotpermitescape. Each animal was assigned to either cued or spatial learn-ing.Inthecuedtask,theplatformwasconsistentlyassociatedwithoneofthetwocues(verticalorhorizontalstripes,counterbalancedacross animals), but varied in location among the four quadrantsof the pool. In the spatial task, the platform was consistently in thesame place (counterbalanced across animals), but the cue markingit varied pseudo-randomly. In both tasks, the other cue (termedthe lure cue) was placed on the stand, not permitting escape, in anadjacent quadrant. On each training trial, the animal was placedin the pool against the edge, opposite both cues, and permitted tosearch for 120s for the escape platform. After finding the escapeplatform, the animal was permitted to rest there for 15s beforebeing removed to its home cage.Probe trials were performed on the fourth trial of days 5 and 7of 2-cue training. On a probe trial, the Atlantis platform was low-eredsothatitdidnotpermitescape.Searchwasmonitoredfor60susinganoverheaddigitalcameraandautomatedtrackingsoftware(Any-Maze:Stoelting),afterwhichtheplatformwasraisedandtheanimal permitted to escape to it. Any animal failing to escape after60s (or a total of 120s of search) was guided to the platform. Sys-tematicbiastowardsthegoalcueintheprobetrialwasinterpretedasevidenceoflearning,ineitherthecuedorthespatialtask.Probetrial performance was quantified as either quadrant occupancy oroccupancy in a circular zone, 25cm in diameter, centered on thegoal or lure cue. For probe trials, the time of each animal’s firsttouch of the goal cue was used as a latency measure in the latencyanalysis (Fig. 1).Analysis of latency and probe trial data was by RM-ANOVA;effects within individual tasks were probed using lower-orderANOVAs. Analysis was performed using SPSS.61 adult C57Bl/6J mice were trained in the cued and spatialwater maze tasks[13]in two independent balanced experi-ments ( n =16 male/spatial, 16 male/cued, 15 female/spatial, 14 Fig. 1. Escape latencies during shaping and training. (A) Male and female miceshowed similar escape latencies during 1-cue training during the shaping phase.‘Cued’ and ‘Spatial’ mice received identical training during this phase but are sep-arated out for illustrative purposes. All values are mean ± SEM. (B) In the 2-cuetask,animalslearningthespatialtaskshowedfasterescapethanthoselearningthecued task, but there was no difference between male and female mice. See text forstatistical analysis. female/cued). Similar results were found in the initial experi-ment and the replication, and data were pooled for analysis, withexperimentnumberasanindependentvariableinallprimaryanal-yses.Latency to find the escape platform improved, as expected,across training trials. There was no difference between sexesin escape latencies in the one-cue shaping task (Fig. 1A). In the two-cue task, latencies improved with training for both thecued and spatial task (Fig. 1BRM-ANOVA: day, F  [6,318]=22.6,  p <0.0001; trial, F  [3,159]=3.53, p <0.02; day × trial interaction, F  [18,954]=2.17, p <0.005). Latencies were shorter in the spatialtask than in the cued task ( F  [1,53]=34.7; p<0.0001), consistentwithwhatwehaveobservedpreviously[13].Therewasnoeffectof  sexonlatencyineithertask,andnointeractions(all  p >0.05).Simi-larly,therewasnoeffectofexperimentnumberandnointeractions(all p >0.05).Probe trials were substituted for normal training trials on thefourth trial of days 5 and 7. Analysis of all probe trial data (RM-ANOVAofquadrantoccupancydatawithsex,task,andexperimentas between-subjects factors and with probe trial day and quad-rant as within-subject factors) showed a clear bias towards thegoal quadrant (main effect of quadrant: F  [1,53]=68.36, p <0.0001)thatdifferedbetweenspatialandcuedtasks(quadrant × taskinter-action: F  [1,53]=33.8, p <0.0001) but was not influenced by sex(quadrant × sex: F  [1,53]=0.036, p >0.1; interactions of sex withtask and trial were likewise non-significant).In the spatial task (Fig. 2A), significant bias towards the goal quadrant was apparent across both probe trials (RM-ANOVArestrictedtothespatialtask:maineffectofquadrant,F[1,27]=84.1,  p <0.0001) that increased from days 5 to 7 (quadrant × trial inter-action: F  [1,27]=9.013, p =0.006). However, there was no effect of sexonquadrantbias(quadrant × sex: F  [1,27]=0.03,  p >0.5),oranyinteractionsinvolvingsex.Similarly,significantgoal-quadrantbiasbut no effects of sex were found in ANOVA analysis restricted today 5 or to day 7.Less dramatic learning was apparent in the cued task (Fig. 2B), consistent with our previous findings[13].Bias towards the goal quadrant was at trend level across the experiment (RM-ANOVA  150 L.C. Baldan Ramsey, C. Pittenger / Neuroscience Letters 483 (2010) 148–151 Fig. 2. Probe trial performance. (A) In the spatial task, both males and femalesshowed robust learning on days 5 and 7, illustrated here by a significantly greatersearch time in the target quadrant relative to the lure quadrant. There was no sig-nificant effect of sex in this or any other measure of probe trial performance. (B)Learning was less robust in the cued task, but both groups showed preference forthe goal quadrant after 7 days of training. There was again no effect of sex on learn-ing or probe trial performance. See text for details and statistical analysis. *  p <0.05;all values are mean ± SEM. restrictedtothecuedtask:maineffectofquadrant: F  [1,26]=3.732,  p =0.064), emerging as significant on day 7 (day 5, main effectof quadrant: F  [1,26]=0.82, p >0.3; day 7: F  [1,26]=4.85, p <0.04).Thequadrant × sexinteractionwasnon-significantwhenanalyzedeither across the whole experiment ( F  [1,26]=0.01, p >0.5) or inde-pendently on days 5 and 7.Werepeatedtheseanalysesusingothermeasuresofprobetrialperformance, including occupancy in a circular target zone aroundthe goal and lure cues and mean distance from the goal and lurecues during search[13].The same effects were seen in these other analyses;nosignificanteffectofsexorinteractionwasfoundusingthese alternative measures.We have described a variant water maze task that permitsassessment of cued and spatial learning in mice; we have previ-ouslyshown[13]thatthespatialtaskdependsonthehippocampus, whilethecuedtaskdependsonthedorsalstriatum,consistentwitha large body of previous work[26,16,17,27].Strikingly, we found that these two memory systems can destructively interfere withoneanother,orcompete,duringlearning[13,20].Wehypothesized thatthistaskwouldrevealsexualdimorphisminlearningstrategy,such that male mice would exhibit superior performance in thespatial task while female mice would be superior in the cued task.However, in two independent experiments with a large number of animals, we did not find any differences between adult male andfemalemiceineithertask.Therefore,atleastasassayedbythistask,adultmaleandfemaleC57Bl/6micedonotmarkedlydifferintheirability to use hippocampus-dependent and striatum-dependentstrategies during learning.Superiorhippocampus-dependentspatiallearninginmaleshasbeen demonstrated in rats and mice in a variety of tasks[11].For example, in a water escape-motivated radial arm maze, maleC57Bl/6 mice have been reported to exhibit better spatial refer-ence and working memory than females[9].However, results in both radial maze and more standard water maze tasks have var-ied,withmanystudiesshowingnocleardifferencebetweensexes,especially in mice[11].Indeed one study actually showed female superiority in spatial learning in aged mice[6],and another recent study found object location memory, which also depends on hip-pocampalfunction,tobesuperiorinfemales[24].Therefore,while the absence of a clear male advantage in spatial learning in ourmodified water maze task is contrary to our initial hypothesis, it isconsistent with a significant body of the literature.Itremainspossiblethatdetailsofourtrainingprotocolservedtomitigateadifferencebetweenmaleandfemaleanimalsthatmightbe revealed by a different protocol. Although non-spatial shapingdid not interfere with our ability to show distinct effects of tar-getedbrainlesionsonspatialandcuedlearninginoursrcinalstudy[13],similarpretraininghasbeenshowntomitigatesexdifferencesin spatial learning in rats[1,19].This effect was found to corre- latewithagreatercorticosteroneresponsetotraininginthewaterin females than in males; pretraining reduced both the increasedcorticosterone level and the relative deficit in spatial learning[1].Other studies have similarly found that sex differences in spatialandnon-spatiallearningwereeliminatedoverthecourseofexten-sive training[11].Our paradigm includes substantial pretraining in a one-cue water escape task, which may have eliminated a sexdifference that would otherwise have been seen in two-cue spa-tial training. However, it might have been expected that such aneffect would manifest as a sex difference in latencies during theearly phase of one-cue pretraining; such an effect is not apparentin our data (Fig. 1A). It is possible that male and female mice are achieving similarperformance in the spatial task by applying different strategies.Consistent with this possibility, a recent study showed C57Bl/6malemicetohavesuperiorspatialmemory(asassayedbytheclas-sic Morris water maze task), while female mice exhibited superiorobjectrecognition[3];thisreproducedasimilarearlierstudyinrats [24].Earlier work in rats has similarly suggested that females aremorereliantonindividual‘landmark’cuesforefficientspatialnavi-gation[11,23].Ourspatialtaskisactuallyahybrid,inwhichoptimal performance requires using spatial information to pick which of two prominent cues (i.e. landmarks) to swim to; it may be that astrategy of being guided by individual landmarks is better able tocompensate for a subtle deficit in spatial learning in such a taskthan in the classic Morris water maze.Finally, it may be that a deficit in spatial learning in femalemice,relativetomales,wasmaskedbyvariabilityintroducedbythefemales’ hormonal cycling. However, several observations argueagainstthispossibility.First,traininginthespatialtaskoccursover7days(nearlytwotypicalestruscycles),soanysuchsourceofvari-ability would be expected to average out over training. Effects of estrus cycling have been observed in spatial water maze tasks inmiceonlyusingprotocolsinwhichalltrainingoccursduringasin-gle day (e.g.[5]).Second, this explanation would predict a larger variance in probe trial performance in females than in males; butvarianceinourexperimentwasactuallysmallerinfemalesthaninmales on day 5—an effect that approached statistical significance(Levene’stestforequalityofvariances,day5goalquadoccupancy,  p =0.05).Therewasnodifferencebetweensexesinvarianceonday7. These data argue against the commonly held view that estruscycling adds variance and thus complicates analysis of behavioral  L.C. Baldan Ramsey, C. Pittenger / Neuroscience Letters 483 (2010) 148–151 151 tasks in females. Finally, while an effect of estrus stage on Morriswater maze performance has been suggested in rats even whentrainingextendsacrossseveraldays,theeffecthasbeensubtleandlimited to early in training in some studies (when learning is likelyto be non-spatial; e.g.[7]),and absent in others (e.g.[2]). We tested striatum-dependent cued learning andhippocampus-dependent spatial learning in parallel. This strategyhas proven valuable for dissociating the anatomical substrates of different forms of learning and for examining their interactionsduringtraining[13,17,18].Femaleratsandmicehaveshownbetter acquisition of striatum-dependent tasks in some contexts[22].In addition, as mentioned above, female rats are thought to dependmore on discrete cues during navigation than males[11,23].We therefore predicted that females would be superior to males inour striatum-dependent cued task; we further anticipated that,since spatial and cued strategies compete with one another duringlearning in this task[13],the predicted sexual dimorphisms in cued and spatial learning should amplify one another. However,no such female superiority in the cued task was seen.It is likely that different forms of striatum-dependent-learningwill be differently affected by sexual dimorphism both inmnemonic capacity and in other parameters that affect perfor-mance, such as attention and motivation. In this water maze task,however,nosignificantsexualdimorphismineithercuedorspatiallearning is apparent.  Acknowledgments This work was supported by NIH grants T32MH014175 (LBR)and K08MH081190 (CP) and by a NARSAD Young InvestigatorAward (CP). References [1] J. Beiko, R. Lander, E. Hampson, F. Boon, D.P. Cain, Behav. Brain Res. 151 (2004)239–253.[2] B. Berry, R. McMahan, M. Gallagher, Behav. Neurosci. 111 (1997) 267–274.[3] T.J. Bettis, Jacobs LF, Behav. Process. 82 (2009) 249–255.[4] N.D. Daw, Y. Niv, P. Dayan, Nat. Neurosci. 8 (2005) 1704–1711.[5] K.M. Frick, J. Berger-Sweeney, Behav. Neurosci. 115 (2001) 229–237.[6] K.M. Frick, L.A. Burlingame, J.A. Arters, J. Berger-Sweeney, Neuroscience 95(2000) 293–307.[7] C.A. Frye, Physiol. Behav. 57 (1995) 5–14.[8] L.A. Galea, M. Kavaliers, K.P. Ossenkopp, D. Innes, E.L. Hargreaves, Brain Res.635 (1994) 18–26.[9] J.E. Gresack, K.M. Frick, Brain Res. 982 (2003) 98–107.[10] T. Hartley, E.A. Maguire, H.J. Spiers, N. 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