See discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/12609956Effects of prenatal alcohol exposure on thehippocampus: Spatial behavior,electrophysiology, and neuroanatomyARTICLEinHIPPOCAMPUS JANUARY 2000Impact Factor: 4.3 DOI: 10.1002/(SICI)1098-1063(2000)10:13.0.CO;2-T Source: PubMedCITATIONS190DOWNLOADS722VIEWS1222 AUTHORS:Robert F BermanUniversity of California, Davis157 PUBLICATIONS 3,840 CITATIONS SEE PROFILEJohn H HanniganWayne State University120 PUBLICATIONS 2,574 CITATIONS SEE PROFILEAvailable from: John H HanniganRetrieved on: 12 July 2015Effects of Prenatal Alcohol Exposure on theHippocampus: Spatial Behavior, Electrophysiology,and NeuroanatomyRobert F. Berman1* and John H. Hannigan21Department of Neurological Surgery, Center forNeuroscience, University of California at Davis,Davis, California2Departments of Obstetrics and Gynecology,and Psychology, C.S. Mott Center for Human Growthand Development, Wayne State University School ofMedicine, Detroit, MichiganABSTRACT: Prenatal exposuretoalcohol canresult infetal alcoholsyndrome (FAS), characterized by growth retardation, facial dysmorpholo-gies, and a host of neurobehavioral impairments. Neurobehavioral effectsin FAS, and in alcohol-related neurodevelopmental disorder, include poorlearning and memory, attentional decits, and motor dysfunction. Many ofthese behavioral decits can be modeled in rodents. This paper reviewsthe literature suggesting that many fetal alcohol effects result, at least inpart, fromteratogenic effects of alcohol on the hippocampus. Neurobehav-ioral studies show that animals exposed prenatally to alcohol are impairedinmany of the same spatial learning andmemory tasks sensitive tohippocampal damage, including T-mazes, the Morris water maze, and theradial armmaze. Directevidenceforhippocampal involvementispro-vided by neuroanatomical studies of the hippocampus documentingreduced numbers of neurons, lower dendritic spine density on pyramidalneurons, and decreased morphological plasticity after environmentalenrichment inrats exposedprenatallytoalcohol. Electrophysiologicalstudies also demonstrate changes in synaptic activity in in vitro hippocam-pal brain slices isolated from prenatal alcohol-exposed animals. Consid-ered together, these observations demonstrate that prenatal exposure toalcohol can result in abnormal hippocampal development and function.Such studies provide a better understanding of neurological decitsassociated with FAS in humans, and may also contribute to the develop-ment of strategies to ameliorate the effects of prenatal alcohol exposure onbehavior. Hippocampus 2000;10:94110. 2000 Wiley-Liss, Inc.KEY WORDS: fetal alcohol syndrome; radial maze; Morris maze;environmental enrichment; neural plasticityINTRODUCTIONFetal alcohol syndrome (FAS) was rst dened in the early 1970s as apattern of growth retardation, facial anomalies, and mental retardation ininfants bornto alcoholic women(Jones andSmith,1973). Similar patterns of development hadbeenre-ported earlier but were not widely recognized (Lemoineet al., 1968). Children with FAS showdistinctive,persistent, and subtle patterns of cognitive dysfunctionthat becomeincreasinglyevident withmoresophisti-cated psychological assessments (e.g., Mattson and Riley,1998;Jacobson,1998;Roebucketal.,1999).Prenatalalcohol-exposed children with alcohol-related neurode-velopmental disorders (ARNDs) show similar cognitiveeffects (Stratton et al., 1996; Tanaka, 1998), but withoutthe characteristic facial features of FAS(Aase, 1994;Mattsonetal.,1998).Althoughthediagnosticcriteriafor FAS and other fetal alcohol effects continue to evolve(Sokol and Clarren, 1989; Stratton et al., 1996), early onit was proposed that changes in central nervous system(CNS) functionmay be the most sensitive signs ofprenatal alcohol exposure (Clarren, 1982). Considerableresearch continues to investigate the CNS abnormalitiesand dysfunction responsible for the cognitive and behav-ioral fetal alcohol effects, inbothhumanandanimalmodels.Even prior to the denition of FAS, it was known thatplacentallytransferredalcohol accumulatesinthefetalhippocampus of rodents and primates (Ho et al., 1972).It is now evident that hippocampal anatomy andelectrophysiology are altered by prenatal or early postna-tal alcohol exposure. One recent review of clinical andexperimental outcomesconcludedthat damagetothevulnerable hippocampus may be responsible for many ofthe behavioral sequelae (Tanaka, 1998). Riley et al.(1986) argued that the results of many behavioral studiesup to that time, mostly in rats, supported the hypothesisof enduringpostnatal hippocampal dysfunction. Thiswas based upon the similarity of effects on behavior afterprenatal alcohol exposureanddirect hippocampal le-sions. That analysis focused on response inhibitionGrant sponsor: National Institute on Alcohol Abuse and Alcoholism; Grantnumber: P50-AA-07606.*Correspondence to: Robert F. Berman, Ph.D., Department of Neuro-logical Surgery, University of California at Davis, Medical NeurosciencesBuilding, Room502B, 1515NewtonCourt, Davis, CA95616. E-mail:rfberman@ucdavis.eduAccepted for publication 19 October 1999HIPPOCAMPUS10:94110(2000)2000WILEY-LISS,INC.decits in various avoidance paradigms (Riley et al., 1986). Morerecent studies have characterized the involvement of the hippocam-pusinneurobehavioraldysfunctionafterprenatalalcoholexpo-surebyusingtasksassessingspatial learningandmemory(e.g.,Blanchard et al., 1987; Hall and Berman, 1995). In this paper wereview evidence that the hippocampus is profoundly susceptible totheteratogeniceffects of prenatal alcohol exposure. Findings frombehavioral, neuroanatomical, andneurophysiological experiments fromour laboratories and others, including newresearch, are presented.ALCOHOL EXPOSURE PROTOCOLSIN ANIMALSAnimal models, particularly those using rodents, are powerfultools for determiningthe mechanisms andoutcomes of earlyalcohol exposure because the physiological responses to alcohol indevelopment are similar to those in humans (Hannigan and Abel,1996). The neurobehavioral sequelae of prenatal alcohol exposurein animals can also be remarkably consonant with the clinical andbehavioral outcomes in humans (Driscoll et al., 1990; Hannigan,1996). However, rats do not normally voluntarily consumeenough alcohol to maintain chronically high blood alcoholconcentrations (BACs) during pregnancy. As a result, severalprocedures have beendevelopedfor alcohol administrationtopregnant dams (HanniganandAbel, 1996). Oneof themostwidely used procedures limits food and water consumption to analcohol-containing liquid diet (Sherwin et al., 1979), often madefrom commercially available nutritional formulations (e.g., Liqui-diet, Bio-Serv, Frenchtown, NJ or Sustacal, Mead Johnson andCo., Evansville, IN). Sufficient alcohol is added to this diet (about12g/kg/day, andupto 18g/kg/dayinsomestudies)sothat18% (low dose) or 35% (high dose) of total daily calories areprovided by the alcohol (e.g., 35% ethanol-derived calories (35%EDC)). Control diets canbe made isocaloric tothe alcohol-containing diets by the substitution of another carbohydrate foralcohol. A second procedure involves direct intragastric gavage orintubation of dams with alcohol solutions, producing dosestypically between 26 g/kg/day. A third technique involvesarticial rearing, theso-calledpup-in-the-cup technique, de-signedtoexposeneonatal ratpupstoalcohol duringadenedperiod within the rst 10 postnatal days (e.g., Kelly et al., 1988).This earlyneonatal periodof development inrodents shares anumberof functional andmaturational similarities withthird-trimester gestational development in humans (Dobbing andSands, 1979), and neonatal alcohol exposure in the rat is used tomodel third-trimester alcohol exposure in human FAS. In arti-cial rearing, rat pups are surgically implanted on postnatal day 4(PN4) withachronicallyindwellingintragastrictubethroughwhich an alcohol-containing liquid diet is delivered periodicallyover37days. Thisprotocolrequirescontrolsfortheeffectsofseparating the pups from their dams and litters. Finally, a methodfor direct intragastric administration of alcohol to neonates,neonatal gavage (Sonderegger et al., 1982), has been revived andrevised to allow the pups to remain with either biological or fosterdams throughout the period of early neonatal alcohol administra-tion (Light et al., 1989; Goodlett and Johnson, 1997; Goodlett etal., 1998). Thegreat majorityof all publishedstudies of fetalalcohol effects in animals have relied on these procedures,although direct intraperitoneal injections of alcohol solutions andinhalation of vaporized ethanol in special exposure chambers havealso been used (e.g., Pal and Alkana, 1997; Bellinger et al., 1999).Each procedure has advantages and disadvantages in the experimen-tal study of prenatal alcohol exposure (Hannigan and Abel, 1996).The interpretation of results among studies can be inuenced byprocedural differences.SPATIAL LEARNING AND MEMORYIN RODENTSThe general ndings for most of the studies reviewed here arethat earlyalcohol exposureimpairs spatial response-dependentlearning, that decits are greater upon reversal training, and aremost evident when tested in younger animals.T-MazesDecits in acquisition and retention of spontaneous andconditional alternation in T-mazes, and in spatial discriminationreversal learninginanimals exposedprenatallytoalcohol, areconsistent withthe behavioral effects of hippocampal damage(OKeefe and Nadel, 1978). Lochry and Riley (1980) rstreportedthat rats exposedtoalcohol viamaternal liquiddiets(17.5%EDCand35%EDC)haddose-dependentincreasesinerrors on a T-maze during both acquisition and retention testing,with or without reversal training. Abel (1982) and Zimmerberg etal. (1989) later founddecits inspontaneous alternationinaT-mazeinratsafterprenatal exposuretoliquiddietsproviding35%EDC. Wainwright et al. (1990) reported that prenatalalcohol-exposed B6D2F2 mice that had learned to escape a simplewater T-mazebyalwaysturninginonedirection(rightorleft)showed decits in learning the reverse. Decits in acquisition andreversal of aspatial alternationwere alsoreportedafter eitherprenatal alcohol exposure (Zimmerberg et al., 1991) or neonatal,binge-like alcohol exposure (Thomas et al., 1996, 1997). InZimmerberg et al. (1991), rats were tested in a T-maze with twochoice points. The rst choice point tested spatial referencememorybecausetheanimalalwayshadtogototheright. Thecorrect choice (left or right) at the second choice point alternatedwithprevious choiceat that point, providingatest of spatialworkingmemory. Offspringexposedprenatallytoalcohol wereimpaired on both the spatial reference memory (males andfemales) andthe spatial workingmemorycomponents (malesonly).Morris MazeInthe basic Morris maze task(Morris, 1981), rodents arerequired to swim to a hidden escape platform submerged in a large___________________________________________HIPPOCAMPAL AND SPATIAL DYSFUNCTION IN FAS 95tank of opaque water. The task appears to require spatialmapping baseduponlocalizationofextra-mazecues(Morris,1981). Certainprobe measures takenwiththe platformre-moved (e.g., percent time spent near the platform location, angleof rst approach) help discriminate spatial learning and memoryperformance (i.e., knowingwhere the platformis) fromsomemotor responses (e.g., learning a swimpatternthat leads toescape). Hippocampal damage substantially impairs rodentsabilitytolearnplatformlocation(Morrisetal., 1982). Studiesidentifying spatial learning decits in a Morris maze by young rats(PN20PN30) after perinatal (i.e., prenatal or neonatal) alcoholexposure, and thereby suggesting hippocampal dysfunction, wererst published in1987 (Blanchard et al., 1987; Goodlett et al.,1987). Inonestudy, youngLong-Evansratsborntodamsfed35%EDCliquiddiets tooklonger paths toreachtheescapeplatform in the Morris maze than controls. The prenatal alcohol-exposed females tended to perform more poorly than the malesover the 4 days of acquisitiontraining. Probe trials revealedspecic spatial rather thangeneral response decits, andheremales tended to be more affected on spatial learning than females(Blanchard et al., 1987).Goodlett et al. (1987) tested juvenile Sprague-Dawley rats afterearlyneonatal alcohol exposureonpostnatal days410(PN410). The pup-in-the-cup articial rearing procedure was used togenerate high peak BACs (i.e., binge exposures) during the periodofpostnatalbraingrowthspurt.Inadetailedanalysisofspatiallearningandmemoryover 12days of testing, bothmaleandfemale alcohol-exposed pups had signicantly longer escapelatencies and traveled more circuitous paths in the Morris mazethan controls. Analyses of probe trials without a platformconrmedspecic decits inspatial learning(Goodlett et al.,1987). Similar spatial decits in juvenile rats were reportedfollowing direct intubations of alcohol to neonates from PN79,without the complications of the articial rearing method (Goodlettand Johnson, 1997). Matthews and Simon (1998) modied theMorris task by increasing the delay between training and testingfrom 1 to 3 days. They found that the offspring of Long-Evans ratsintubated with 3 g/kg/day ethanol during gestation showedsignicantly increased escape latencies as adults. Kim et al. (1997)compared the effects of prenatal alcohol exposure via a 36% EDCdietonnonspatialandspatiallearninginmaleSprague-Dawleyrats. There were no decits on a nonspatial delayed nonmatching-to-sample object recognition task. However, the same rats showedincreased latencies in Morris maze acquisition, demonstrating thespecicity of spatial decits.Radial Arm MazeSpatial learning decits can also be demonstrated in Olton-typeradial armmazes. Reyes et al. (1989) weretherst toreportdecits in eight-arm radial maze acquisition in surrogate-fostered,food-restricted, prenatal alcohol-exposed, maleandfemalerats(17% EDC or 35% EDC) tested on PN60. Only half of thealcohol-exposed offspring were able to reach criterion, and thosethat didrequiredsignicantly more training trials relative topair-fed controls. Pick et al. (1993) reported similar effects in miceinjected with ethanol (3 g/kg or 5 g/kg; sc) on PN214. At PN50,these mice took twice as many trials as controls to reach criterion,and continued to require signicantly more trials than controls toenter all eight arms (Pick et al., 1993). Hall et al. (1994) showedthat midgestational alcohol exposure (i.e., gestational days (GD)713), via 35% EDC liquid diets, was sufficient to impair radialmaze acquisition when rats were tested as either juveniles(PN26)oradults(PN80). Incontrast, Opitzetal. (1997)showednodifferences inanunrewardedcompact versionof aradial maze testing offspring of mice intubated with 3.19 g/kg/dayof alcohol from GD1418, even though litter mates of these micehad signicant decits in acquiring a conditioned taste aversion.Stone et al. (1996) alsoreporteddecits inradial armmazeperformance in prenatal alcohol-exposed rats. The same rats werenot impaired in passive avoidance, suggesting that spatial perfor-mancemaybeamoresensitivebehavioral measureof alcoholteratogenicity in the hippocampus than response inhibition (Rileyet al., 1986; Greene et al., 1992).To elaborate the role of learning and attention in spatialbehavior after prenatal alcohol, we adapted a radial arm maze toassess attentional processes by requiring animals to use visual orspatial cues in a food-search task. In the visual cue task, rats weretrained to nd food in three black arms in an eight-arm maze withwhite, black, or gray arms. The position of the colored arms variedfrom day to day so that spatial position was irrelevant. Animalshad to attend to the color of the arms to nd food in this cuedcondition. After 3 weeks, the food was switched from the black tothewhitearmsfor3moreweeksoftraining. Next, aspatialcontingency was used where the location of three goal arms wasreinforcedandthecolor cues becameirrelevant. As showninFigure 1, under the spatial cue condition, rats exposed prenatallytoeither4g/kg/dayor6g/kg/dayshowedsignicantlylongerlatencies to locate food in the three reinforced arms compared toalcohol-naivecontrols. Prenatal alcohol-exposedrats alsomademore errors than controls when the contingency was shifted fromthecued tothespatial condition(datanot shown), againsuggestingaspatial learningdecitinprenatal alcohol-exposedanimals.Spatial Decits and Age of TestingGrowth retardation and neurobehavioral decits after prenatalalcohol exposure may reect delayedmaturationof the CNS(Sherwin et al., 1979; Abel, 1982; Pettigrew, 1986; Rintelmann etal., 1994). This argument is based in part on experimental studiesthat show a catching up of alcohol-exposed animals to alcohol-naiveanimalsinsometasks(Meyeretal., 1990), aswell asinhumans where the most severe decits are often observed in theadolescent(Sampsonetal., 1994).Thespatial decitsrevealedabove inT-mazes, Morris mazes, and radial armmazes alsoillustrate the variable age-dependency of fetal alcohol effects.While the effects of fetal alcohol exposure in children and rodentscan be long-lasting (Riley, 1990; West et al., 1990), there are alsoreports that fetal alcohol effects are sometimes transient oroutgrown (e.g., Meyer and Riley, 1986). Such reports support96 BERMAN AND HANNIGANthe possibility that some fetal alcohol effects may modiable withage, and indeed may respond to treatment.Spatial and temporal serial pattern learning and memory in ratsare disrupted by prenatal alcohol (e.g., Riley et al., 1993; LaFietteet al., 1994). Working memory and spatial learning decits werereported in both younger and older rats, but older rats with priorexperience in the maze apparently remembered, showing nodecitsinrelearning(Halletal.,1994). Theeffectsofprenatalalcohol exposure on these learning tasks may also indicateattentionproblems, althoughsuchattentiondecits have notbeen identied reliably in rats (e.g., Hayne et al., 1992). However,even a single in utero episode of high peak BACs in mice was ableto produce a profound decit in memory retrieval in 2-year-oldmice that was not evident in3-month-oldmice (Dumas andRabe, 1994).We have examined the role of age at testing, using a radial armmaze task that appears to be solved by young (PN21) and adult(PN90) rats using fundamentally different arm selection strate-gies. At least two general strategies can be used by rats to solve theeight-armradial maze. Animals can select a highly variablepatternsofarmseachday, asshowninFigure2A, ortheycanselect adjacent arms, as indicated in Figure 2B. The adoption ofhighlyvariable,andseeminglyrandom,armselectionsbyadultrats has beenarguedtoindicatetheuseof aspatial mappingstrategy that is highly dependent on hippocampal function(OKeefe and Nadel, 1978). Adult animals typically use therandom choice of arms whensolving the radial maze, andyounger rats (e.g., tested beginning on PN21) tend to adopt theadjacent arm choice strategy. Data from Hall and Berman (1995)supporting this conclusion are shown in Figure 2C. These resultsFIGURE 1. Mean total latency (sec SEM) to locate foodreinforcement in 3 of 8 arms of a radial arm maze by either visualcues or by spatial location. In the cued condition, food was foundin three arms of the same color (e.g., black), while under the spatialcondition, foodwas associatedwiththree specic armlocationsregardless of armcolor. Animals exposed prenatally to alcoholperformedaswell ascontrolsunderthevisual cuecondition, butwere impaired in learning spatial locations of food reward asindicated by increased latencies to complete the maze. *P F.05 vs.none and zero.___________________________________________HIPPOCAMPAL AND SPATIAL DYSFUNCTION IN FAS 97are similar to those reported earlier by Einon (1980). Therefore,we hypothesized that if prenatal alcohol delayed development ofthe CNS, then adult rats exposed prenatally to alcohol might usethe adjacent arm strategy.This hypothesis was tested in offspring of Sprague-Dawley ratsgiven 35% EDC liquid diet from GD8 through parturition. Ratswere cross-fostered to alcohol-naive dams and weaned on PN21.OnPN90, radial maze training began. Briey, animals wereplacedonarestrictedfeedingscheduleandhabituatedtotheeight-arm maze for 5 min per day for 5 days without food reward.Thearmsofthemazewerethenbaited,andadultrats(PN90)weretrainedtoentereacharmasingletimewithin5mintoretrieve a food reward. The pattern of arm choices was analyzedover 12 days of training.FIGURE 2. Examples of two general search strategies for locat-ing food reward in the radial arm maze. A: Highly variable daily armselection pattern typical of adult rats. B: An adjacent arm selectionstrategy observed in juvenile rats. C: Juvenile rats (26 days of age) usean adjacent armselection strategy, while adult rats (90 days of age) donot (adaptedfromHall andBerman, 1995). D: Animalsexposedprenatally to 35%EDCtend to use an adjacent armstrategycompared to alcohol-naive controls (P F0.05).98 BERMAN AND HANNIGANThe results are shown in Figure 2D. Rats exposed prenatally toalcohol showed a pattern of arm choices that indicated adoptionof an adjacent arm strategy, similar to the maze strategy observedin younger animals. Although the use of the adjacent arms choicestrategy was not as robust as in juvenile animals, the difference inaverage adjacent arms selected between animals exposed prenatallyto alcohol and alcohol-naive controls was statistically signicant(P 0.05). As suggested by Hall and Berman (1995), onepossible explanation for the shift in strategies from young to adultrats could be maturation of a hippocampus capable of supportinga spatial strategy for solving the maze. This explanation would beconsistent withthe hypothesis that prenatal alcohol exposuredelays development of the hippocampus.Persistence of Fetal Alcohol EffectsSpatial learning decits canbe long-lasting. Nagahara andHanda (1997) assessed delayed spatial alternation in a T-maze forfood reward in male offspring of Long-Evans dams fed 35% EDCliquid diets during pregnancy. At three separate ages tested,PN45, PN85, or PN175, there were signicant decreases inpercent correct alternations at delays greater than30sec. Theyoungest animals were also impaired at the 10-sec delay, and noanimals were impaired in the no-delay condition (Nagahara andHanda, 1997). Stone et al. (1996) reported similar prenatalalcohol-induced decits in spontaneous alternation in a T-maze inrats tested as old as 18 months of age (i.e., PN540).Gianoulakis(1990)reportedspatialnavigationdecitsintheMorris watermazeafterprenatal alcohol exposure(35%EDCliquiddiet). Alcohol-exposedSprague-Dawleyrats showedin-creased escape latencies at 40, 60, and 90 days of age, as well asevidence of poor spatial memory during probe trials without theescape platform. Gianoulakis (1990) also controlled for maternalinuences by cross-fostering and found that being raised by a damthat had been exposed to alcohol did not contribute to the spatialperformance decits. Westergrenet al. (1996) testedSprague-Dawleyratsat6monthsofageintheMorriswatermazeandreported marginally signicant impairments in acquisition. Suther-land et al. (2000) reported small but signicant increases in escapelatencies in alcohol-exposed adult (58 months of age) males, in astandard Morris maze with a xed platform position. In contrast,when the platformwas moved fromday to day, prenatalalcohol-exposedratshadshorterlatenciesthancontrolsontherst trial. This was interpreted as evidence for poor memory of theprevious days position compared to alcohol-naive controls (e.g.,less interference, or for the use of a nonspatial strategy; Sutherlandet al., 2000).Dose, Gender Effects, and Critical Periodsof ExposureGoodlett and Johnson (1999) recently reviewed the literaturedemonstrating the importance of dose, gender, age of testing, andespecially periodof alcohol exposure, tobehavioral effects ingeneral. Each of these factors can inuence how prenatal alcoholexposure affects spatial learning. Dose effects were evident inseveral of thestudies reviewedsofar. Tomlinsonet al. (1998)reported that Morris maze escape latency was increased in62-day-old Wistar rat offspring exposed to 6 g/kg of alcohol onPN5, butnottolowerdoses(2g/kgor4g/kg). However, themagnitude of prenatal alcohol effects is not always related to dosein any simple way. For example, Clausing et al. (1995) assessedspatial learning in a seven choice-point complex maze that thirstyratscompletedforawaterreward.Offspringofratsexposedto18%EDCor 36%EDCinliquiddiets weretestedbetweenPN6980.Amongthemultiplemeasuresofperformance,threereectedspatial learning. Ineachof three measures of spatiallearning, the18%EDCgroup, but not thehigher-dose36%EDCgroup, was signicantly impaired relative to controls.Clausing et al. (1995) noted that the high alcohol dose group, butnot the low-dose group, evidenced behavioral signs of increasedstress/anxiety (i.e., freezing) that could have masked spatiallearning decits.Prenatal alcohol effects can also be inuenced signicantly bygender, although the critical factors inuencing gender differencesinresponse toalcohol are unclear at this time. For example,Blanchard et al. (1997) found that male rats exposed prenatally toalcohol (i.e., 35%EDCthroughout gestation) weremoreim-paired then females in spatial memory tested during probe trials inthe Morris water maze. Both were impaired in water mazeacquisition. Incontrast, Minetti et al. (1996), using a singleprenatal alcohol exposure(i.e., 5.8mg/kg, ip)onGD8, foundgreaterdecitsinfemalesthanmalesduringprobetrialsintheMorris water maze. Initial acquisition did not differ between sexes.Goodlettetal. (1987)reportedthatbothmaleandfemaleratsshowed Morris maze acquisition decits following alcohol expo-sure on postnatal days 410. However, females were reported toshow greater decits when high, repeated, binge-like, peak BACsweregeneratedduringthissamepostnatal period(Kellyet al.,1988). The timing anddurationof exposure may alsoaffectgenderdifferencesinthedevelopmentaleffectsofalcoholexpo-sure. Goodlett and Peterson (1995) reported spatial learningdecitsintheMorriswatermazeinbothmaleandfemaleratsexposed to ethanol on postnatal days 410. However, the durationof exposure was important, in that males exposed to alcohol over 2postnatal days (i.e., PN46 or PN79) also showed spatiallearningdecits,whereasthefullexposureperiod(i.e.,PN49)was required to reveal decits in females. Even a single neonatalday of alcohol exposure on either PN5 or PN10 may be sufficientto impair Morris maze performance in male rats tested between4154 days of age (Pauli et al., 1995).Behavioral SummaryConsidered together, the pattern of effects on spatial learningand memory indicates consistent decits in animals exposedprenatally to ethanol. Such decits have been observed inspontaneous and conditional alternation, the Morris water maze,andradialarmmazetasks.Despitethefactthatthedetailscandiffer amongvarious experiments, signicant decits inspatiallearning and/or memory are consistently seen across alcoholexposure procedures, alcohol dose, age of testing, and gender. Ingeneral, decits tend consistently to be greater in younger animals,___________________________________________HIPPOCAMPAL AND SPATIAL DYSFUNCTION IN FAS 99followinglongerdelaysbetweentrainingandtesting, andafterhigher doses of alcohol (i.e., higher peak BACs). These results alsoindicate that the pattern of spatial learning and memory decits issimilar to that observed after direct hippocampal damage, and isconsistent withthe hypothesis that prenatal alcohol exposureinterferes withthe normal development and functionof thehippocampus (Riley et al., 1986). Of course, the effects of prenatalalcohol exposure are not limited to the hippocampus, and growthretardation throughout the CNS is a hallmark of FAS.NEUROANATOMICAL FINDINGSCell LossBarnes and Walker (1981) reported a signicant 20% reductioninthe numbers of dorsal hippocampal CA1regionpyramidalneurons, whereas there was only a nonsignicant 3% decrease ingranule cells in the dentate gyrus in young adult (PN60) rats afterprenatal alcohol exposure via liquiddiet. Perez et al. (1991)reportedsignicant 10%reductions incell numbers inallhippocampalCAeldsonPN36in Wistarrats,usingasimplecell-counting procedure in Luxol fast-blue-stained tissue. OnPN65 these reductions ranged from 31% in CA3 to 46% in CA1(Perez et al., 1991). These relatively large effects were remarkablein that the 12 g/kg alcohol dose was administered in fourintraperitoneal injectionsoverasingleday, GD7, adatewhichanticipates the period of initial hippocampal pyramidal cellgenerationbyabout 45days (Perezet al., 1991). Signicant3240%reductionsinthenumbersofCA3pyramidal neuronswere also reported by Ba et al. (1996) following alcohol exposureduring development. However, the attribution of these effects to aparticular period of perinatal exposure is impossible becausealcohol (i.e., 12% solution) was administered as the sole drinkinguid to dams for 2 months before pregnancy, throughoutpregnancy and lactation, and directly to offspring until PN45 (Baet al., 1996).Similar to the effects of high BAC binge exposures on behavior,BACinuences neuroanatomical effects inthe hippocampus.Greeneetal.(1992)reportedsignicantreductionsintheCA1area and pyramidal cell counts on both PN21 (10% decrease)and PN60 (20% decrease) after neonatal binge alcohol exposureconcentratedintofour of 24daily administrations. However,whenthe same total daily dose was distributedacross all 24administrations each day, there were no signicant effects in thehippocampus(Greeneetal., 1992). Also, usinganuncorrectedsampling procedure to count cells, Wigal and Amsel (1990)reported that prenatal alcohol exposure via 35% EDC liquid diet,but not neonatal exposure via articial rearing methods, signi-cantly reduced the estimated numbers of CA1 pyramidal cells andmature granule cells on PN21. Combining prenatal plus neonatalexposures in the same animals did not further reduce the numbersof either CA1 pyramidal cells or granule cells (Wigal and Amsel,1990). Inalaterstudyfromthissamelaboratory, theoppositetemporal pattern was found, with higher peak BACs than thoseused in Wigal and Amsel (1990). Specically, early neonatal butnot prenatal alcohol exposure reduced CA1 pyramidal cell num-bersandtheCA1areainweanlingSprague-Dawleyrats(Diaz-Granados et al., 1993). In addition, the density of mature granulecells was signicantly reduced relative to untreated offspring aftercombinedprenatal plus neonatal alcohol exposure. Prenatal orneonatal exposure alone also reduced granule cell density, but didnot do so signicantly (Diaz-Granados et al., 1993).Changes in cell counts can vary as the animals age. Lobaugh etal. (1991) tested offspring of Sprague-Dawley dams weaned ontoprogressively greater alcohol concentrations in liquid diet proce-dure beginning on GD1, up to 35% EDC. At PN21 or PN180,following nonspatial behavioral testing, the number of pyramidalcells and cell density were measured in the midtemporal hippocam-pal CA1. In contrast to the previous studies, there were nosignicant differences incell counts amongprenatal treatmentgroups at either age, although there was a signicant decrease incell density in all groups with age.Recent morphological studies using unbiased stereologicalprocedures indicate that reducedneuronal populations inthehippocampusfollowingprenatal alcohol exposureareregionallyselective and are due to decreases inneuronal generationorproliferation, rather than to cell death or changes in density due tochangesinareaorvolume.Miller(1995)assessedhippocampaldevelopment inLong-Evans rats exposedprenatallytoalcoholfromGD621, or postnatally fromPN412. Injections of[3H]-thymidinewereusedtoassessthebirthdays ofneuronalpopulations in the hippocampus. Stereological cell-countingprocedures were used to correct for potential overestimationcaused by cell fragments, or by underestimating mean celldiameter. A signicant 17% reduction was found in the numberof pyramidal neurons in the hippocampal CA1region afterprenatal exposure. Similar reductions were seeninCA1afterneonatal alcohol exposure to high BACs (339 17 mg/dl).Lower BACs (i.e., 132 14 mg/dl to 222 24 mg/dl) did notaffect pyramidal cell number. In contrast, prenatal alcohol expo-suredidnotaffectthenumbersof granulecellsinthedentategyrus, while postnatal alcohol exposure affected granule-cellnumbers as a biphasic function of postnatal BAC. At moderateBACs, dentategyrus-cell numbers weresignicantlyhigher; atmoderatelyhigh BACs, therewas noeffect; andonlyat thehighest BACs were cell numbers signicantly reduced. Based onananalysisof[3H]-thymidineuptake, Miller(1995)concludedthat alcohol exposure during hippocampal development delayedthe generation of neurons in both the CA1 and dentate gyrus.Neuronal Branching and SpinesWest et al. (1981) rst reportedworkwithaTimms stainshowing that prenatal alcohol exposure via 35% EDC in a liquiddiet causedabnormal branchingof mossybers intheventralhippocampus. Mossybers invadedtheCA3infrahippocampalregion, where they are not normally present (West and Hodges-Savola, 1983). This was conrmed with a horseradish peroxidase(HRP)stain(WestandPierce,1984). WigalandAmsel(1990)later found that neonatal alcohol exposure, whether or not100 BERMAN AND HANNIGANcombined with prenatal exposure, but not prenatal exposurealone, increased the area of CA4 region. Hoff et al. (1984) foundno evidence of signicant change in the dimensions of thehippocampus or dentate gyrus on PN10 or PN20 in offspring ofSprague-Dawleyrats feda35%EDCliquiddiet. Davies andSmith (1981) studied dendritic arbors in hippocampal pyramidalneuronsin14-day-oldmiceandreporteda20%stunting intotalbasilardendriticlength.Dendriticarborsweresimplerinconguration following maternal feeding with a 25% EDC diet(5.5g/kg/day)duringgestationandearlylactation, althoughcomplexity was not quantied.Abel et al. (1983)counteddendriticspinesonPN90andfound signicant 27% and 31% decreases in numbers of spines onapical and basilar tertiary dendritic branches, respectively, in theCA1hippocampal region after prenatal alcohol exposure viaintragastricintubation(6g/kg/day) throughout gestation. Inabrief report byPerez et al. (1991), usinga single-daydosingregime in Wistar rats, there were signicant 2747% decreases inapical and basilar dendritic spine densities, which appeared to besummedacross pyramidal cells inall of hippocampus proper(CA1, CA2, CA3, and CA4). Ferrer et al. (1988) reported 50%reductioninapical andbasilar secondary, rather thantertiary,dendriticspinedensityinCA1onPN15,followingupto25%ethanol in drinking water for 4 weeks before, and a 35% EDC dietduring gestation. However, in contrast to Abel et al. (1983) andPerez et al. (1991), there were no signicant differences indendriticspinedensitiesonPN90inWistarrats(Ferreretal.,1988). Ferreretal. (1988)interpretedtheirndingstosuggestthat there was recovery from the teratogenic effects of alcohol onthehippocampusduringpostnatal maturationbetweenPN1590, although other reports suggest that effects on spine density inCA1 are persistent.UltrastructurePrenatal alcohol-induced changes in the hippocampus are alsoevident at the ultrastructural level. SmithandDavies (1990)assessed the ultrastructure of hippocampal CA1 pyramidal cells inC57B1/6J mice after alcohol exposure extending from GD12 toPN9. Pyramidal neurons were more densely packedinadultalcohol-exposedrats withless elaborate dendritic arbors inamanner typical of younger animals, suggesting developmentaldelay. There were also fewer dendritic spines, with no changes inspine length or numbers of microtubules. Morphological changeswere also found in mitochondria and in the distribution ofendoplasmic reticulum in CA1 (Smith and Davies, 1990). Tanakaet al. (1983) had previously reported dilated, rough endoplasmicreticulum in the hippocampus of offspring of Wistar dams given10% ethanol in drinking water. They also found decreased densityof synapses quantied under electron microscopy in the hippocam-pal CA3 eld (Tanaka et al., 1991).Anatomical PlasticityChanges in numbers of dendritic spines, synapses, or complex-ity of dendritic elds with development or environmental stimula-tion reect neuronal plasticity within the hippocampus. Ferrer etal. (1988) suggested that the absence of the 50% reduction insecondarydendriticspinedensityinCA1onPN90meantthatthere was recovery between PN1590, perhaps mediated by somekindof residual neuronal plasticity. Ontheotherhand, inanelectron microscopy study, Hoff (1988) argued that a signicantdecrease in synapse turnover, measured as changes in the propor-tion of synapses of different shapes, indicated that synapticplasticity in the hippocampus was impaired after prenatal alcoholexposure. Dewey and West (1985b,c) demonstrated that althoughprenatal alcohol exposure itself did not inuence the developmentof hippocampal commisural or perforant path projections to thedentate gyrus, subsequent neuronal plasticity within these projec-tionswasimpaired. Thiswasevidentasadecreaseincollateralsprouting of hippocampal bers in response to unilateral entorhi-nal lesions and a decrease in neurite outgrowth in prenatalalcohol-exposed rats (Dewey and West, 1984, 1985a; West et al.,1984).HIPPOCAMPAL ELECTROPHYSIOLOGYAFTER PRENATAL ALCOHOLEXPOSUREThe effects of prenatal alcohol exposure on hippocampalelectrophysiology have been studied at several levels, ranging fromEEGmeasurements tostudies of invitrohippocampal slices.Evidence exists at each level for abnormal neural electrical activityconsistent with the behavioral and neuroanatomical studiesdemonstratingthesensitivity ofthehippocampustotheterato-genic effects of alcohol.In Vitro Hippocampal Slice ElectrophysiologyTheinvitroratbrainslicepreparationhasbeenusedexten-sivelytocharacterizetheelectrophysiologyofthehippocampusisolatedfromrats exposedprenatallytoalcohol (BermanandKrahl, 1996). Stimulation-evokedextracellular eldpotentialshave been characterized in brain slices fromthese animals,includingmeasures of synapticstrength(i.e., input/output re-sponses), synapticinhibitorycircuitry(i.e., paired-pulseinhibi-tion), and synaptic plasticity (i.e., long-term potentiation or LTP).The initial report of abnormal hippocampal electrophysiologyafter prenatal alcohol exposurewas byHablitz(1986). Inthisstudy, hippocampal subregion CA1 was examined at PN4060 inrats that had been exposed prenatally to a 35% EDC liquid dietfromGD321. Paired-pulse response inhibitiontypically ob-servedat short interpulseintervals (50msec) inslices fromcontrolswasabsentinslicesfromtheprenatal alcohol-exposedanimals. Inaddition, paired-pulsepotentiationseenat paired-pulse intervals longer than 100 msec was greatly enhanced. Therewerenosignicant effects of prenatal alcohol oninput/outputcurves, although hippocampal slices isolated fromprenatal alcohol-exposed offspring were described as less responsive than those ofcontrols.__________________________________________HIPPOCAMPAL AND SPATIAL DYSFUNCTION IN FAS 101These results were essentially replicated by Tan et al. (1990). Inthis study, eld potentials evoked in CA1by stimulation ofSchaffer collateral axons were examinedinhippocampal slicesisolated from rats exposed prenatally to alcohol, using a liquid dietcontaining either 17.5% EDC or 35% EDC. Controls receivedeither an isocaloric alcohol-free liquid diet or were fed normal ratchowadlibitum. Adose-relatedreductioninbirthweightandretardedgrowthonPN21were observedinprenatal alcohol-exposedanimals(Tanetal.,1990).AsinHablitz(1986),therewas a loss of paired-pulse response inhibition at short intervals anda markedenhancement of paired-pulse potentiationat longerinter-pulse intervals (Tan et al., 1990).LTP, aneurophysiological correlateofsynapticplasticity, hasalso been evaluated in animals exposed prenatally to alcohol. InTan et al. (1990), the magnitude of LTP in hippocampalsubregion CA1 was lowest in the 35% EDC group, but did notdiffer signicantly among groups. Swartzwelder et al. (1988)examinedhippocampal LTPinanimalsexposedprenatallytoadietcontaining18.8%EDC. LTPwaselicitedinCA1usinga10-sec, 60-Hz stimulus train to the Schaffer collaterals at 20% ofthe maximum evoked population spike response. The magnitudeof LTPintheprenatal alcohol-exposedanimalsshowedonlya75% increase over baseline measured 30 min after tetanus. Thiswassignicantlylessthanthe140%increaseelicitedinCA1ofalcohol-naive animals fedrat chow, andless thanthe 260%increase in animals maintained on an alcohol-free liquid diet. Theapparent differenceinLTPmagnitudebetweencontrol groupswas not signicant. Savage et al. (1998) demonstrated that the rateof decay inLTPwas acceleratedinhippocampal slices fromprenatal alcohol-exposed rats.Inarecent studyfromour laboratory(Krahl et al., 1999),input/output responses, paired-pulseinhibition, andLTPwereexamined in region CA1 of in vitro hippocampal slices. Slices wereisolatedfromadolescent (PN2532) andyoungadult animals(PN6377) exposedprenatallyto0, 4, or 6g/kg/dayethanoladministered to the dams via intragastric gavage from GD821.Slices from the higher-dose, 6 g/kg/day prenatal alcohol-exposedanimals on PN30 showed signicantly reduced maximal evokedpopulation spike amplitudes compared to animals receiving loweralcohol doses (i.e., 4 g/kg/day), or to age-matched alcohol-naivecontrols. In contrast, there were no differences in maximalpopulation spike amplitude in the hippocampus of prenatalalcohol-exposedanimals examinedas youngadults (PN70).There were also no differences at any age among groups ininput/output proles or paired-pulse responses at either short orlong interpulse intervals. The Kral et al. (1999) study supports theconclusionthat prenatal alcohol exposure results inabnormalhippocampal electrophysiological responses, and demonstratesthat the effects of prenatal alcohol exposure are both dose-relatedand age-dependent.Therouteandpatternof prenatal alcohol exposuremaybeimportant variables contributing to the electrophysiological out-comesofprenatal alcohol exposure. Theliquiddietproceduresused by Hablitz (1986), Tan et al. (1990), and Swartzwelder et al.(1993)allproducedabnormalpaired-pulseresponsesandinter-fered with the generation of LTP in CA1. These evoked responseswere intact in CA1 in the study by Krahl et al. (1999) that used anintragastricgavageexposuretechnique. Thismaybeimportant,because liquid diet exposure procedures typically result in lowermeanpeakandlongersustainedBACsthanthoseachievedbyintragastric intubation. Similarly, Sutherland et al. (1997) assessedeldexcitatorypostsynaptic potentials (EPSPs) inthe dentategyrusofratsexposedprenatallytoaliquiddietcontaining5%ethanol. This regimeproducedmoderate BACs inthedams(83mg/dl). AtPN120150, theseratshadnodifferencesininput-output curves but had smaller LTPs than controls. Recently,Bellinger et al. (1999) reported that hippocampal slices preparedat PN4560 from rats that had been exposed to ethanol vaporsfrom PN49, yielding BACs of 350 mg/dl, had similar reductionsininput/outputcharacteristicswithoutchangesinLTPorpair-pulse potentiation. Liquid diet procedures may be more represen-tativeof steadyalcohol consumptioninhumans, whilegavageprocedures andtheir concomitant higher BACs maybe moresimilar to binge-drinking patterns. Each pattern of alcoholconsumption, therefore, could result in different levels and peaksof prenatal alcohol exposurethat leadtodifferent neurobehav-ioral, anatomical, and electrophysiological effects. Understandingthe inuence of patterns of drinking and alcohol exposure to thefetus is clearlyimportant, andstudies addressingthis questionneed to be carried out in this area.EEG StudiesTwo EEGstudies in animals demonstrated fetal alcoholexposure effects on the hippocampus. Kaneko et al. (1993)studiedauditoryevent-relatedpotentials(ERPS)inoffspringofrats fed an alcohol-containing liquid diet during gestation.Electrophysiological data revealed that the prenatal alcohol-exposed animals had signicantly longer latencies of the P1 andN1 ERP components recorded in the hippocampus, againsuggestingthat thehippocampusisalteredbyprenatal alcoholexposure. Cortese et al. (1997) reportedthat prenatal alcoholexposure altered hippocampal theta activity, a slow rhythmic EEGactivity in the 412-Hz frequency range that is characteristic ofthehippocampus(Bland, 1986). Ratswereeitheruntreatedorexposed prenatally to either 4 or 6 g/kg/day ethanol via anintragastric gavage procedure. Theta activity was measured whileanimals were moving or still, and the ratio of the relative power oftheta during these two periods (i.e., ratio of movement theta tostill theta) was analyzed. A signicantly higher ratio of moving-to-still theta was observed in the 6 g/kg/day male animalscompared to controls, again demonstrating changes in a hippocam-pal electrophysiological responsethat has beenassociatedwithprocessingspatial information(OKeefeandNadel,1978)afterprenatalalcoholexposure.However,thisstudycouldnotdeter-mine whether the change in ratio was the result of an increase intype I (i.e., movement-related) or a decrease in type II(still-related) theta, or both.Insummary, invivoandinvitrondingsfromavarietyofanimal studies clearly demonstrate abnormal hippocampal electro-physiological activityresultingfromprenatal alcohol exposure.These electrophysiological changes are consistent with neuroana-102 BERMAN AND HANNIGANtomical studies demonstratingloss of principal neurons inthehippocampus as well as aberrant mossy ber projections, asdescribed above. These changes are also consistent with behavioralstudies demonstrating decits in spatial learning tasks, tasksknowntobeimpairedafter direct hippocampal damage(e.g.,lesions). Considered together, the weight of the evidence leads totheconclusionthatthehippocampusisaprimetargetforfetalalcohol effects, andfurthersupportsthepossibilitythatatleastsomeof thecognitivedecits seenwithFASmayresult fromhippocampal damage. Additional evidence for compromisedplasticity and growth in the hippocampus after prenatal alcoholexposure comes fromour recent studies on the effects ofenvironmental enrichment onhippocampal spinedevelopment(Hannigan et al., 1993; Berman et al., 1996).ENVIRONMENTAL ENRICHMENTAMELIORATION OF FETALALCOHOL EFFECTSRearing animals inenrichedor complex environments canreliably stimulate CNS development, facilitate recovery of func-tion, andenhancebehavioral performance(Weileretal., 1995;Rosenzweig, 1996; Kolbet al., 1998).Therefore, wepredictedthat environmental enrichment might ameliorate some fetalalcohol effects in our rodent models. We have been systemicallyexaminingtheeffects of postnatal environment enrichment onboth brain development and behavior in rats exposed prenatally toalcohol. These studies are designed to assess the inuence of thepostnatal environment on the expression of fetal alcohol effects onhippocampal function, and to evaluate the potential of environ-mental enrichment to ameliorate some of the damaging effects ofalcohol exposureonbraingrowthandbehavioral development(Hannigan et al., 1993; Berman et al., 1996).Morris Maze Learning After EnvironmentalEnrichmentWe and others have documented that rearing rats in anenriched environment after prenatal alcohol exposure can signi-cantly improve behavioral performance, including learning,memory, and motor performance (Hannigan et al., 1993; Wain-wright et al., 1993; Berman et al., 1996; Klintsova et al., 1998;but see Opitz et al., 1997). In our rst studies, Long-Evans ratswere exposedprenatallytoeither 6kg/dayalcohol or sucrosevehiclefromGD819, usinganintragastricgavagetechnique.There were two separate equal intubations of 3 g/kg delivered 2 hrapart tooptimize boththe concentrationandvolume of thealcohol solution. This method of alcohol administration results inBACs between 155220 mg/dl measured 30 min after the seconddaily intubation (e.g., Church et al., 1990). Animals in theuntreated control group were not intubated.At weaning on PN21, male and female pups from each prenataltreatment group were assigned to one of two postnatal environmen-tal rearing conditions: Isolated or Enriched. In the Isolated controlcondition, pups were housed individually in small hangingsteel-wirecagesandnotdisturbed. RatsrearedintheEnrichedconditionwerehousedinsame-sexgroupsof 1012, withratsfrom each of the three prenatal treatment groups housed together.Theenrichedenvironment arenas werelargeNalgene(NalgeNunc International, Rochester, NY) tubs (75 75 60 cmhigh) with hardwood chip bedding, feed, and water sources, andwere suppliedwithtoys (e.g., dowels, plastic pipe, ladders)whichtheanimalscouldmanipulate, chew, andclimbon. Thetoys were changed every 34 days.After the enrichment period, at about PN6372, rats weretrained in the Morris water maze. Animals were given four trialsper day over 4 consecutive days. The platform was moved to a newlocationonthe fth training day, and learning of this newposition was evaluated (Fig. 3). Although there was no effect ofprenatal alcohol exposure on Morris water maze acquisition in thisstudy, there was an effect in learning the new platform position ontest day 5. Environmental enrichment improved water mazeperformanceinallgroups,regardlessofprenataltreatment,andthe behavioral impairment in prenatal alcohol-exposed animals onday 5 was reduced (Hannigan et al., 1993). Although these effectswere subtle, we recently replicated these ndings using a modiedMorris maze protocol with a longer intertrial interval (i.e.,Hanniganet al., 1999). BecauseMatthews andSimon(1998)reportedMorris mazeimpairments followinglongerdelays be-tweentrainingandtesting, weincreasedtheintertrial intervalfrom5 min to 60 min and reduced the number of trials per dayfromvetothree. Animalswereagainrequiredtolearnanewplatformpositiononthefthday. Despitethesechanges, themagnitude of fetal alcohol effects was unchanged. When the goalplatformwasmovedtoanewpositionwithinthemazeafter4training days (i.e., a fth reversal day), prenatal alcohol-exposedrats reared in the Isolated condition took signicantly longer thancontrols to nd the platform in its new location. However, afterenrichment there were no signicant differences due to prenatalalcohol exposure. These results with Sprague-Dawley rats onreversal trainingreplicateandextendtoourearlierndingsofamelioration of fetal alcohol effects in Long-Evans rats (Hanniganet al., 1993).Effects of Enrichment on Hippocampal DendriticSpine DensityAs described above, 6 weeks of an enriched environment canimprovelearning, memory, andmotorperformanceinprenatalalcohol-exposed rats (Hannigan et al., 1993). However, we havealso found that the ability of environmental enrichment toincreasedendriticspinedensityonCA1andCA3hippocampalpyramidal cells is severely compromised by prenatal alcoholexposure (Berman et al., 1996).Inthese studies, spine densities onhippocampal pyramidalneurons in CA1 and CA3 were quantied. Briey, slices from thedorsal hippocampal CA1 region were processed with a modiedversion of the rapid Golgi method. Single apical shaft pyramidalcells were selected that were well-impregnated, contained minimaltruncation, andwerenotobscuredbysurroundingcells, blood__________________________________________HIPPOCAMPAL AND SPATIAL DYSFUNCTION IN FAS 103vessels, or artifact. Tertiary apical or basilar terminal (nonbranch-ing) dendrites (one per cell) were drawn. Ten cells per rat (ve forapical and ve for basilar dendrites) were analyzed. The numbersofspineswerequantiedusingadigital morphometricanalysissystem, and unbiased correction procedures were used to estimatethree-dimensional spine density distributed about a dendrite shaft(Feldman and Peters, 1979). Data were expressed as the estimatednumber of spines per 10 m tertiary dendritic segment.Mean spine densities in CA1 are depicted in Figure 4 for malesandfemalesdemonstratingtheeffectsof postweaningenviron-ment and prenatal alcohol. There were no signicant differencesinapical spine density among the prenatal treatment groupsreared in the Isolated condition. Postweaning environmentalenrichment signicantly increased apical or basilar dendritic spinedensityinthetwocontrol groups, but didnot increasespinedensities in CA1 in animals exposed prenatally to alcohol (Bermanet al., 1996). Among Isolated groups, the fact that prenatalexposure to alcohol did not affect dendritic spine density in CA1may reect in part the negative impact of the Isolated conditionitself on controls.The refractory hippocampal response to neuroanatomical signsof environmentally induced plasticity may reect a developmentaldelay. Waters et al. (1997) demonstrated that the maturation of aspatial behavior (spontaneous alternationina Y-maze) wasassociated with the developmental emergence of a novelty-induced elevation in FOS-like immunoreactivity (FL-IR) in thehippocampus. Normal rats younger than PN30 do not alternateanddonot increaseFL-IR. Perhaps adelayinthis or similarneuroplastic responses to the environment can account for poorspine growth after prenatal alcohol exposure, but a delay does notaccountforthegeneral improvementinspatial behaviorintheMorris maze after enrichment.We recently extended our analyses of the ameliorative effects ofpostnatal environmental enrichment on hippocampal neuro-anatomy to CA3. Damage to this region has been implicated in thememory impairments seen after prenatal alcohol exposure (Tanaka,FIGURE 3. Morris water-maze performance after environmentalenrichment. Rats reared in an enriched environment (dashed lines)showed shorter escape latencies (mean sec) than animals reared underisolatedconditions (solidlines). This was true for bothprenatalalcohol-exposed rats and alcohol naive rats. During reversaltraining (inset bar graph), isolated rats exposed prenatally to alcoholtooksignicantlylonger tondthe newlocationof the escapeplatformthanisolateduntreatedcontrols(P F0.05). Incontrast,environmentally enriched rats exposed prenatally to alcohol did notdiffersignicantlyfromenrichedalcohol-naiverats(dataadaptedfrom Hannigan et al., 1993).104 BERMAN AND HANNIGAN1998),andCA3alsoappearstoberesponsivetoenvironmentalmanipulations. Thesamebrains describedabovefromanimalsexposed prenatally to 6 g/kg/day of alcohol were assessed in thisnewanalysis. Therewerenomaineffectsorinteractionsduetogender. The results are depicted in Figure 4. Unlike in CA1, therewas a signicant main effect of prenatal treatment (F(2, 40) 4.50,P 0.02), whichplannedcomparisons indicatedwas due tosignicantly reduced spine density in the prenatal alcohol grouprelativetoboththeuntreated(F(1, 40) 4.52, P 0.04)andintubated control groups (F(1, 40) 7.74, P 0.01). Thecontrol groups did not differ from each other (P 0.65). Therewere no signicant effects or interactions due to postnatalenvironmentalenrichmentinCA3(P 0.11and0.70).Inourmodel, hippocampal pyramidal cell spine density in CA3 may bemore sensitive to prenatal alcohol exposure than in CA1, but notto enrichment. Other studies have also reported teratogenic effectsin CA3 anatomy, as noted above (e.g., Perez et al., 1991; Tanaka etal., 1991). There may be enduring, possibly life-long fetal alcoholeffects on brain growth and neuronal plasticity in the hippocam-pus, even in the presence of postnatal stimulation that is able toimprove performance and eliminate decits on learning andmotor tasks (Hanniganet al., 1993). Our results suggest thatprenatal alcohol exposure compromises the ability of the CNS toincrease the complexity of its neuropil in response to environmen-tal stimulation. Because environmental enrichment can improvespatial learning and memory and motor performance in prenatalalcohol-exposedrats, these ndings suggest that the benecialeffects of environmental enrichment onbehavior maynot bedirectlymediatedbyanyhippocampal anatomical responsetoenrichment.CLINICAL INDICATIONS OFHIPPOCAMPAL DYSFUNCTION IN FASTherehavebeenrelativelyfewstudies of spatial learninginchildren or adolescents with FAS of ARNDs. A recent review ofneuropsychological ndingsinchildrenwithFASandARNDsincluded reports of visuospatial decits (Roebuck et al., 1999).Sampsonetal. (1987)proposed, onthebasisofapartial leastsquares multiple covariate analysis using several overlappingmeasures of alcohol consumption, that there are latent indica-tions of poor spatial organization. However, it is difficult todiscern the nature of such decits with this type of analysis. Huntet al. (1995) measured44214-year-oldchildrenwithknownlevels of prenatal alcohol exposure ona computerizedtest ofspatial visualization involving recall of shapes. Performance corre-lated withtwo measures of maternal self-report of drinking,including anindex of binge drinking (number of drinks peroccasioninthe monthbefore the pregnancy). The more themothers drank, the faster andless accurately the adolescentsresponded. Children exposed prenatally to high levels ofalcohol, with or without diagnoses of FAS, were impaired on testsof visuomotor integration (Mattson and Riley, 1998) or global-local visuospatial abilities (Mattson et al., 1998). Steinhausen etal.(1982)reportedsignicantdecitsinspatialrelationshipsinsevere FAScomparedtomildFAS. Olsonet al. (1998) alsoreporteddecitsinvisual-spatial skillsin9nonretardedadoles-centswithFAS. Inanotherstudy, 4boysdiagnosedwithfetalalcohol effects built only nonstacking asymmetrical designs withblocks. After the boys were shownavideomodel of stacked,symmetric designs, none imitated, suggesting not only an inabilitytomodel, but alsoadecit inspatial perceptual tasks (Meyer,1998).It is unclear what the relationships are between these kinds oftestsand thespatialdecitsseeninanimalmodels.Uecker andNadel (1996, 1998)arguedthatdecitsincertainspatial taskscould be indicative of hippocampal damage after prenatal alcoholexposure, andthathippocampal-associateddecitsidentiedinrats should be explored in children. They also argued that manyspatial tasks have no clear association with the hippocampusFIGURE 4. Analysis of dendritic spine densities on CA1 and CA3hippocampal pyramidal neurons after prenatal alcohol exposure and6weeks of differential rearing ineither anisolatedor enrichedenvironment. Data are presented separately for males and females. InCA1, prenatal alcohol exposure didnot signicantly affect spinedensity for males and females. However, only alcohol-naive rats (i.e.,none, zero) showed signicant increases in spine density afterenvironmental enrichment (data adapted from Berman et al., 1996).In CA3, male and female rats exposed prenatally to alcohol showedsignicantly reduced dendritic spine densities compared to controls,regardless of rearing conditions. The effect of environmental enrich-ment on spine densities for control groups (i.e., none and zero) wasnot statistically signicant.__________________________________________HIPPOCAMPAL AND SPATIAL DYSFUNCTION IN FAS 105(Uecker and Nadel, 1996). Decits on some tasks (e.g., Milnersstepping-stone maze) are apparently not indicative of exclusivehippocampal involvement (Milner, 1965). Uecker and Nadel(1996) administeredthe memoryfor 16objects taskto15children with FAS and 15 matched controls (4 girls/11 boys; 10yearsold). Therewerenodecitsinimmediaterecall,althoughupondelayed(24hr) recall, there were signicant decits inmemory for what the 16 objects were (e.g., screwdriver, whistle,toy car). FAS children recalled 17%fewer objects than didcontrols after a24-hr delay. However, thelarger effect was inremembering where the objects were located in the 60-cm2eld.Spatial recall was measured as the mean difference in distance (cm)from the original positions of the objects and where the objectswereplaceduponrecall. Regardlessof delay, FASchildrenhaddistance differences that were42%larger that controls. Ananalysis of the object displacement patterns, relative to the originalpositions in a fairly uniform dispersal of objects across the eld,indicated that most FAS children tended to line up objects left torightacrosstheeld.Incontrast,thefewcontrolchildrenwhohadpoorerspatial recall tendedtodisplaceobjectsaroundtheperiphery.Uecker and Nadel (1998) recently elaborated their analyses ofhippocampal damage in FAS children using an Ellis-type spatialmemorytask(cf. Ellis et al., 1989). PerformanceontheEllisspatial memorytaskcandissociateobjectmemoryfromspatialmemory. Children with mental retardation and Downs syndromeperformpoorlyonbothobjectandspatialmemoryinthistask(Ellis et al., 1989). Although FAS children took longer to recallobjects seen in a picture book, there were no signicantdifferencesineitherimmediateordelayedrecall of what thoseobjects were. In contrast, the FAS children recalled signicantlyfewer correct locations than did controls at both immediate recall(49%vs.73%)anddelayedrecall (38%vs.49%)(UeckerandNadel, 1998). Uecker and Nadel (1998) argued that the pattern ofspatial decits in this task for FAS is consistent with thehypothesis that thesechildrenhavehippocampal damage. Theresultsonboththememoryfor16objects andtheEllis-typespatial memory task in these FAS children are very similar to thedissociation between object memory and spatial memory decitsreportedinrats (e.g., Kimet al., 1997). Overall, theauthorsconcluded that these results indicate at least hippocampaldamage (Uecker and Nadel, 1996).Werecognizetwoimportantlimitationstointerpretationsofbehavior, in children and animals, as an index of localized alcoholteratogeniceffectswithintheCNS. Similarityofoutcomedoesnotimplycausality: spatial decitsinthemselvesdonotprovehippocampal involvement in teratogenic outcome. In fact, Ueckerand Nadel (1996) also identied spatial decits in FAS childrenon other tests that were indicative of involvement of the prefrontalcortex. Indeed, prenatal alcohol exposure has profound neuroana-tomical and neurophysiological effects on many other brain areasin FAS children, including the neocortex and cerebellum (Bermanand Krahl, 1996; Roebuck et al., 1998, 1999). Damage to theseotherareascertainlyalsocontributestobehavioral dysfunction,including spatial learning and memory decits.A few autopsy studies of fetal or infant brain list damage to thehippocampus as part of the gross alterations (cited in Mattson andRiley, 1996), althoughstudiesassessingdendriticspinesinthehuman brain are restricted to the neocortex (Ferrer et al., 1988).Todate,therehavebeennoreportsfromimagingstudies(e.g.,MRI or CAT) identifyinghippocampal damage(MattsonandRiley, 1996; Roebuck et al., 1998; Moore et al., 1998). Informa-tion about abnormal hippocampal electrophysiology after prena-tal alcohol exposure has all been derived from research in animalmodels of fetal alcohol effects (BermanandKrahl, 1996). Nohuman studies to date have directly examined hippocampalelectrophysiologyinFAS. However, cortical EEGandevokedpotential studies inhumans clearlydocument abnormal brainelectrical activityassociatedwithprenatal alcohol exposure(seeBerman and Krahl, 1996).CONCLUSIONSCognitivedecits,includingimpaired learning,memory,andattentional problems, are hallmarks of fetal alcohol effects on theCNS. Thestudiessummarizedinthisreviewstronglyimplicatedamage to the hippocampus as an important mediator of some ofthebehavioral andcognitivedecitsassociatedwithFAS.Thisconclusionis basedonthe weight of evidence demonstratingspatial learning and memory decits in animals exposed prenatallyand/or neonatally to alcohol in the same behavioral tasks that areimpaired, sometimesselectively, bydirecthippocampal damage(e.g., lesions, pharmacological manipulations). This behavioralevidence is supported by neuroanatomical studies demonstratingabnormal axonal projections inthe hippocampus of prenatalalcohol-exposedrodents, as well as direct loss of hippocampalpyramidal neurons. Electrophysiological studies also show abnor-mal electrical activityinthehippocampus, bothinvivo(EEG,ERPs) and in vitro using hippocampal brain slices. The variabilityin many of these outcomes in hippocampal function illustrates theimportanceof thedetailsof alcohol administration(e.g., dose,route, diet), patterns of exposure (e.g., bingeing), critical periods(e.g., prenatal vs. neonatal), and individual differences (e.g.,gender, genetics (strain)). Finally, the ability of postnatal environ-mental enrichment to stimulate increased dendritic spine densityinthe hippocampus is bluntedby prenatal alcohol exposure.Reduced neuronal plasticity long after removal of alcohol demon-stratesthattheeffectsof prenatal alcohol exposureextendwellbeyond the prenatal or early neonatal periods, when the CNS isundergoing rapid development (i.e., neuronal proliferation, neu-rite extension, and synaptogenesis) in the presence of alcohol.Several issues remain concerning the effects of prenatal alcoholon brain growth and development, and development and functionof the hippocampus in particular. First, it is now abundantly clearthat the hippocampus is not unique inits sensitivity to theteratogenic effects of prenatal alcohol. Other brain regions,includingthecerebellum(Klintsovaetal., 1997; GoodlettandJohnson, 1999)andfrontal cortex(Miller, 1986; Mooreetal.,106 BERMAN AND HANNIGAN1998; Hanniganet al., 1999), are also adversely affectedbyprenatal alcohol exposure and contribute to the motor andcognitive decits in FAS. Understanding how different regions ofthebrainareaffectedbyperinatal alcohol andbyenrichmentshouldimprovediagnosis of FASandprovideinsight intothenature of neurological decits. The development of more effectivetreatments for fetal alcohol effects might then be possible.Second, just as critical periods are being described for effects ofneonatal alcohol exposure(WestandGoodlett, 1990; Goodlettand Johnson, 1999), there are likely to be critical periods for theeffects of prenatal alcohol exposure on brain development. If so,critical periods may exist for the postnatal ameliorative effects ofenvironmental enrichment, and these should be identied.Third, more researchis neededtoprovide insight intothemolecular and biochemical mechanisms by which prenatal alcoholdamages the hippocampus (e.g., second messenger systems, geneexpression, trophic factors). Finally, more human studies ofhippocampal structure and function after prenatal alcohol expo-sure are needed. Relatively few human studies have attempted toexamine the effects of prenatal alcohol exposure in FAS, but thendings fromanimal studies clearlyindicate the potential forhippocampal damage in humans (e.g., Uecker and Nadel, 1996,1998). The technology for carrying out suchstudies may beavailable (i.e., MRI, fMRI).In conclusion, the behavioral signs of FAS or ARNDs,including spatial learning and memory decits, can have under-standable neurological bases. The effects of perinatal alcoholexposure canextendintime far beyondthe periodof actualexposure and inuence neural plasticity throughout life. Finally,and potentially of the most importance, basic research on animalmodels indicates that prenatal alcohol-damaged animals canbenet from being reared in an appropriately structured postnatalenvironment. Suchndingsprofferhopethatuseful behavioralstrategies for amelioration of fetal alcohol can be developed to takeadvantageof this remainingresponsiveness of thebrain. Suchremediation would improve the lives of the many children borneach year damaged by prenatal alcohol.AcknowledgmentsWeappreciatethesubstantiveandeditorial commentsonanearlier versionof this paper byB. Cortese, andthesecretarialassistance of A. English. The technical assistance of M. Sperry, T.Smith, C. Siebert, and L. DiCerbo is also appreciated.REFERENCESAase JM. 1994. Clinical recognition of FAS: difficulties of detection anddiagnosis. Alcohol Health Res World 18:59.Abel EL. 1982. Inuteroalcohol exposureanddevelopmental delayofresponse inhibition. Alcohol Clin Exp Res 6:369376.Abel EL, JacobsonS, SherwinBT. 1983. Inuteroalcohol exposure:functional and structural brain damage. Neurohbehav Toxicol Teratol5:363366.Ba A, Seri BV, Han SH. 1996. Thiamine administration during chronicalcohol intake in pregnant and lactating rats: effects on the offspringneurobehavioral development. Alcohol Alcohol 31:2740.Barnes DE, Walker DW. 1981. Prenatal ethanol exposure permanentlyreduces the number of pyramidal neurons in rat hippocampus. DevBrain Res 1:333340.Bellinger FP, Bedi KS, WilsonP, WilcePA. 1999. Ethanol exposureduring the third trimester equivalent results in long-lasting decreasedsynaptic efficacy but not plasticity inthe CA1regionof the rathippocampus. Synapse 31:5158.Berman RF, Krahl SE. 1996. Neurophysiological correlates of fetalalcohol syndrome. In: Abel EL, editor. Fetal alcohol syndrome: frommechanism to prevention. Boca Raton: CRC Press. p 6984.Berman RF, Hannigan JH, Sperry MA, Zajac CS. 1996. Environmentalenrichment and dendritic spine density in hippocampus after prenatalalcohol exposure in rats. Alcohol 13:209216.BlanchardBA, Riley EP, HanniganJH. 1987. Decits ona spatialnavigation task following prenatal exposure to ethanol. NeurotoxicolTeratol 9:253258.BlandBH. 1986. Thephysiologyandpharmacologyof hippocampalformation theta rhythms. Prog Neurobiol 26:1.Church MW, Abel EL, Dintcheff BA, Matyjasik C. 1990. Maternal ageandbloodalcoholconcentrationinthepregnantLong-Evansrat.JPharmacol Exp Ther 253:192199.Clarren SK. 1982. The diagnosis and treatment of fetal alcoholsyndrome. Compr Ther 8:4146.Clausing P, FergusonSA, HolsonRR, AllenRA, Paule MG. 1995.Prenatal ethanol exposureinrats: long-lastingeffects onlearning.Neurotoxicol Teratol 17:545552.Cortese BM, Krahl SE, BermanRF, HanniganJH. 1997. Effects ofprenatalethanolexposureonhippocampalthetaactivityintherat.Alcohol 14:231235.Davies DL, Smith DE. 1981. A Golgi study of mouse hippocampal CA1pyramidal neurons followingperinatal ethanol exposure. NeurosciLett 26:4954.Dewey SL, West JR. 1984. Evidence for altered lesion-induced sproutingin the dentate gyrus of adult rats exposed to ethanol in utero. Alcohol1:8188.Dewey SL, West JR. 1985a. Direct evidence for enhanced axon sproutingin adult rats exposed to ethanol in utero. Brain Res Bull 14:339348.Dewey SL, West JR. 1985b. Organization of the commissural projectiontothedentategyrusisunalteredbyheavyethanolexposureduringgestation. Alcohol 2:617622.Dewey SL, West JR. 1985c. Perforant pathway lamination in the dentategyrus is unaffected by prenatal ethanol exposure. Alcohol 2:221225.Diaz-Granados JL, Greene PL, Amsel A. 1993. Mitigatingeffects ofcombinedprenatal andpostnatal exposure toethanol onlearnedpersistence in the weanling rat: a replication under high-peakconditions. Behav Neurosci 107:10591066.Dobbing J, Sands J. 1979. Comparative aspects of the brain growth spurt.Early Hum Dev 3:7983.Driscoll CD, Streissguth AP, Riley EP. 1990. Prenatal alcohol exposure;comparability of effects on humans and animal models. NeurotoxicolTeratol 12:231238.Dumas RM, Rabe A. 1994. Augmented memory loss in aging mice afteroneembryonicexposuretoalcohol. Neurotoxicol Teratol 16:605612.Einon DF. 1980. Spatial memory and response strategies in rats: age, sexandrearingdifferencesinperformance.QJExpPsychol323:473489.Ellis NR, Woodley-Zamthos P, Dulaney CL. 1989. Memory for spatiallocationinchildren, adults, andmentallyretardedpersons. AmJMent Retard 93:521527.FeldmanML, Peters A. 1979. Atechniquefor estimatingtotal spinenumbers on Golgi-impregnated dendrites. J Comp Neurol 188:527542.FerrerI, GalofreE, Lopez-TejeroD, LloberaM. 1988. Morphological__________________________________________HIPPOCAMPAL AND SPATIAL DYSFUNCTION IN FAS 107recovery of hippocampal pyramidal neurons in the adult rat exposedin utero to ethanol. Toxicology 48:191197.Gianoulakis C. 1990. Rats exposed prenatally to alcohol exhibit impair-ment in spatial navigation test. Behav Brain Res 36:217228.Goodlett CR, Johnson TB. 1997. Neonatal binge ethanol exposure usingintubation:timinganddoseeffectsonplacelearning.NeurotoxicolTeratol 19:435446.Goodlett CR, JohnsonTB. 1999. Temporal windows of vulnerabilitywithin the third trimester equivalent: why knowing when matters.In: Hannigan JH, Goodlett CR, Spear LP, Spear NE, editors. Alcoholand alcoholism: effects on brain and development. Mahwah, NJ: L.Earlbaum Associates. p 5991.Goodlett CR, PetersonSD. 1995. Sexdifferences invulnerabilitytodevelopmental spatial learning decits induced by limited bingealcohol exposureinneonatal rats. Neurobiol LearnMem64:265275.Goodlett CR, Kelly SJ, West JR. 1987. Early postnatal alcohol exposurethat produces high blood alcohol levels impairs development of spatialnavigation learning. Psychobiology 15:6474.Goodlett CR, Pearlman AD, Lundahl KR. 1998. Binge neonatal alcoholintubations induce dose-dependent loss of Purkinje cells. Neurotoxi-col Teratol 20:285292.Greene PL, Diaz-Granados JL, Amsel A. 1992. Blood ethanol concentra-tion from early postnatal exposure: effects on memory-based learningandhippocampal neuroanatomy ininfant andadult rats. BehavNeurosci 106:5161.Hablitz JJ. 1986. Prenatal exposure to alcohol alters short-term plasticityin the hippocampus. Exp Neurol 93:423427.Hall JL, Berman RF. 1995. Juvenile experience alters strategies used tosolve the radial arm maze in rats. Psychobiology 23:195198.Hall JL, Church MW, Berman RF. 1994. Radial arm maze decits in ratsexposed to alcohol during midgestation. Psychobiology 22:181185.HanniganJH. 1996. What researchwithanimals is tellingus aboutalcohol-related neurodevelopmental disorder. Pharmacol BiochemBehav 55:489499.Hannigan JH, Abel EL. 1996. Animal models for the study ofalcohol-related birth defects. In: Spohr H-L, Steinhaussen H-C,editors. Alcohol pregnancy and child development. Cambridge:Cambridge University Press. p 77102.HanniganJH,BermanRF,ZajacC.1993.Environmentalenrichmentandthebehavioral effects of prenatal exposuretoalcohol inrats.Neurotoxicol Teratol 15:261266.Hannigan JH, Berman RF, Sperry MA, Steward TM, Cortese BM. 1999.Prenatal alcohol and enrichment-induced increases in dendritic spinesin rat prefrontal neocortex. Alcohol Clin Exp Res 23:29A.Hayne H, Hess M, Campbell BA. 1992. The effect of prenatal alcoholexposure on attention in the rat. Neurotoxicol Teratol 14:398.HoBT, Idanpaan-HeikkilaJE, McIssacWM. 1972. Placental transferandtissue distributionof ethanol-1-14C: a radiographic study inmonkeys and hamsters. Q J Stud Alcohol 33:485493.Hoff SF. 1988. Synaptogenesis in the hippocampal dentate gyrus: effectsof in utero ethanol exposure. Brain Res Bull 21:4754.Hoff SF, Laurie M, Perron J. 1984. Effects of prenatal ethanol exposureon the development of the dendritic elds of the hippocampalformation in rats. Res Commun Substance Abuse 5:253260.Holson RR, Ali SF, Scallet AC, Slikker W, Paule MG. 1989. Benzodiane-pine-like behavioral effects following withdrawal fromchronic delta-9-tetrahydrocannabinaol administrationinrats. Neurotoxicology10:605620.HuntE, StreissguthAP, KenB, Carmichael-OlsonH. 1995. Mothersalcohol consumption during pregnancy: effects on spatial-visualreasoning in 14-year-old children. Psychol Sci 6:339342.Jacobson SW. 1998. Specicity of neurobehavioral outcomes associatedwith prenatal alcohol exposure. Alcohol Clin Exp Res 22:313320.JonesKL,SmithDW.1973.Recognitionoffetalalcoholsyndromeinearly infancy. Lancet 2:9991001.Kaneko WM, Riley EP, Ehlers CL. 1993. Electrophysiological andbehavioral ndings inrats prenatallyexposedtoalcohol. Alcohol10:169178.Kelly SJ, Goodlett CR, Hulsether SA, West JR. 1988. Impaired spatialnavigation in adult female but not adult male rats exposed to alcoholduring the brain growth spurt. Behav Brain Res 27:247257.Kim CK, Kalynchuk LE, Kornecook TJ, Mumby DG, Dadgar NA, PinelJP, WeinbergJ. 1997. Object-recognitionandspatial learningandmemory in rats prenatally exposed to ethanol. Behav Neurosci5:985995.Klintsova AY, Matthews CR, Goodlett CR, Napper RM, Greenough WT.1997. Therapeutic motor training increases parallel ber synapsenumber per Purkinje neuron in cerebellar cortex of rats givenpostnatal bingealcohol exposure: preliminaryreport. Alcohol ClinExp Res 21:12571263.Klintsova AY, Cowell RM, SwainRA, Napper RMA, Goodlett CR,Greenough WT. 1998. Therapeutic effects of complex motor trainingon motor performance decits induced by neonatal binge-like alcoholexposure in ratsI. Behavioral results. Brain Res 800:4861.Kolb B, Forgie M, Gibb R, Gorny G, Rowntree S. 1998. Age, experienceand the changing brain. Neurosci Biobehav Rev. 22:143159.Krahl SE, Berman RF, Hannigan JH. 1999. Electrophysiology ofhippocampal CA1 neurons after prenatal ethanol exposure. Alcohol17:125131.LaFiette MH, Carlos R, Riley EP. 1994. Effects of prenatal alcoholexposure on serial pattern performance in the rat. NeurotoxicolTeratol 16:4146.Lemoine P, Harrousseau H, Borteyru JP, Menuet JC. 1968. Les infants deparents alcooliques: anomalies observees a`propos de 127 cas [Childrenof alcoholic parents: abnormalities observed in 127 cases]. Quest Med25:476482.LightKE, SerbusDC, SantiagoM. 1989. Exposureofratstoethanolfrom postnatal days 4 to 8: alterations of cholinergic neurochemistryin the hippocampus and cerebellum at day 20. Alcohol Clin Exp Res13:686692.LobaughNJ, Wigal T,GreenePL,Diaz-GranadosJL,AmselA.1991.Effects of prenatal ethanol exposure on learned persistence andhippocampal neuroanatomy in infant, weanling and adult rats. BehavBrain Res 44:8186.Lochry EA, Riley EP. 1980. Retention of passive avoidance and T-mazeretentioninratsexposedtoalcoholprenatally.Neurobehav Toxicol2:107115.MatthewsDB, SimonPE. 1998. Prenatalexposuretoethanoldisruptsspatialmemory:effectofthetraining-testingdelayinterval.PhysiolBehav 64:6367.Mattson SN, Riley EP. 1996. Brain anomalies in fetal alcohol syndrome.In: Abel EL, editor. Fetal alcohol syndrome: frommechanisms toprevention. Boca Raton: CRC Press. p 191213.Mattson SN, Riley EP. 1998. A review of the neurobehavioral decits inchildren with fetal alcohol syndrome or prenatal exposure to alcohol.Alcohol Clin Exp Res 22:313320.Mattson SN, Riley EP, Gramling L, Delis DC, Jones KL. 1998.Neuropsychological comparison of alcohol-exposed children with orwithout physical features of fetal alcohol syndrome. Neuropsychology12:146153.Meyer LS, Riley EP. 1986. Behavioral teratology of alcohol. In: Riley EP,Vorhees CV, editors. Handbook of behavioral teratology. New York:Plenum Press. p 101140.Meyer LS, KotchLE, Riley EP. 1990. Alterations ingait followingethanol exposure during the brain growth spurt in rats. Alcohol ClinExp Res 14:2327.Meyer MJ. 1998. Perceptual differences infetal alcohol effect boysperforming a modeling task. Percept Mot Skills 87:784786.Miller MW. 1986. Effects of alcohol on the generation and migration ofcerebral cortical neurons. Science 233:1308.Miller MW. 1995. Generation of neurons in the rat dentate gyrus andhippocampus: effects of prenatal and postnatal treatment withethanol. Alcohol Clin Exp Res 19:15001509.108 BERMAN AND HANNIGANMilner B. 1965. Visually guided maze-learning in man: effects of bilateralhippocampal, bilateral frontal, and unilateral cerebral lesions. Neuro-psychologia 3:317338.Minetti A, Arolfo MP, Virgolini MB, Brioni JD, Fulginiti S. 1996. Spatiallearning in rats exposed to acute ethanol intoxication on gestationalday 8. Pharmacol Biochem Behav 53:361367.Moore GJ, JacobsonSW, Rosenberg DR, Cortese BM, FaulkMW,Hannigan JH. 1998. Proton magnetic resonance spectroscopic imag-ing in children with fetal alcohol syndrome (FAS): ndings in caudatenucleus and corpus callosum. Alcohol Clin Exp Res 22:25.Morris RGM. 1981. Spatial localization does not require the presence oflocal cues. Learn Mem 12:239260.Morris RGM, Garrud P, Rawlins JNP, OKeefe J. 1982. Place navigationimpaired in rats with hippocampal lesions. Nature 297:681683.Nagahara AH, Handa RJ. 1997. Fetal alcohol exposure producesdelay-dependent memory decits in juvenile and adult rats. AlcoholClin Exp Res 21:710715.OKeefe J, Nadel L. 1978. The hippocampus as a cognitive map. Oxford:Oxford University Press. p 328.OlsonHC, FeldmanJJ, StreissguthAP, SampsonPD, BooksteinFL.1998. Neuropsychological decitsinadolescentswithfetal alcoholsyndrome: clinical ndings. Alcohol Clin Exp Res 22:19982012.Opitz B, Mothes HK, ClausingP. 1997. Effects of prenatal ethanolexposure and early experience on radial maze performance andconditioned taste aversion in mice. Neurotoxicol Teratol 19:185190.Pal N, Alkana RL. 1997. Use of inhalation to study the effect of ethanolandethanol dependenceonneonatal mousedevelopment withoutmaternal separation: a preliminary study. Life Sci 61:12691281.PauliJ,WilceP,BediKS.1995.Spatial abilityofratsfollowingacuteexposure to alcohol during early postnatal life. Physiol Behav58:10131020.Perez HD, Villanueva JE, Salas JM. 1991. Behavioral and hippocampalmorphological changes induced by ethanol administered to pregnantrats. Ann NY Acad Sci 625:300304.PettigrewAG. 1986. Prenatal alcohol exposure anddevelopment ofsensory systems. In: West JR, editor. Alcohol and brain development.New York: Oxford University Press. p 225274.PickCG, CoopermanM, TrombkaD, Rogel-FuchsY, Yanai J. 1993.Hippocampal cholinergicalterationsandrelatedbehavioral decitsafter early exposure to ethanol. Int J Dev Neurosci 11:379385.Reyes E, WolfeJ, SavageDD. 1989. Theeffects of prenatal alcoholexposure on radial arm maze performance in adult rats. Physiol Behav46:4548.RileyEP. 1990. The long-termbehavioral effects of prenatal alcoholexposure in rats. Alcohol Clin Exp Res 14:670673.RileyEP, BarronS, HanniganJH. 1986. Responseinhibitiondecitsfollowingprenatal alcohol exposure: acomparisontotheeffectsofhippocampallesionsinrats.In: WestJR,editor.Alcoholandbraindevelopment. New York: Oxford Press. p 71102.Riley EP, Barron S, Melcer T, Gonzalez D. 1993. Alterations in activityfollowing alcohol administration during the third trimester equivalentin P and NP rats. Alcohol Clin Exp Res 17:12401246.Rintelmann WR, Simpson TH, Church MW, Root LE. 1994. Effects ofmaternal alcohol and/or cocaine on neonatal ABRs. Presented at theAmerican Speech and Hearing Association, p 85.RoebuckTM, Mattson SN, Riley EP. 1998. A reviewof the neuroanatomi-cal ndings inchildrenwithfetal alcohol syndrome or prenatalexposure to alcohol. Alcohol Clin Exp Res 22:339344.Roebuck TM, Mattson SN, Riley EP. 1999. Prenatal exposure to alcohol:effects onbrainstructure andneurophysiological functioning. In:Hannigan JH, Goodlett CR, Spear LP, Spear NE, editors. Alcohol andalcoholism: effects on brain and development. Mahwah, NJ: L.Earlbaum Associates. p 116.Rosenzweig MR. 1996. Aspects of the search for neural mechanisms ofmemory. Annu Rev Psychol 47:132.Sampson PD, Streissguth AP, Barr HM, Bookstein FL. 1987. Neurobe-havioral effects of prenatal alcohol: part II. Partial least squaresanalysis. Neurotoxicol Teratol 11:477491.SampsonPD,BooksteinFL,BarrHM,StreissguthAP.1994.Prenatalalcohol exposure, birthweight, and measures of child size from birthto age 14 years. Am J Public Health 84:14211428.SavageDD, CruzLL, DuranLM, PaxtonLL. 1998. Prenatal ethanolexposurediminishesactivity-dependentpotentiationof aminoacidneurotransmitter release in adult rat offspring. Alcohol Clin Exp Res22:17711777.Sherwin BT, Jacobson S, Troxell SL, Rogers AE, Pelham RW. 1979. A ratmodel of the fetal alcohol syndrome. Curr Alcohol 7:1530.SmithDE, Davies DL. 1990. Effects of perinatal administrationofethanol on the CA1 pyramidal cell of the hippocampus and Purkinjecell of the cerebellum: an ultrastructural study. J Neurocytol 19:708717.Sokol RJ, Clarren SK. 1989. Guidelines for use of terminology describingthe impact of prenatal alcohol on the offspring. Alcohol Clin Exp Res13:597598.Sonderegger TB, Calmes H, Corbitt S, Zimmermann EG. 1982. Lack ofpersistent effects of low-dose ethanol administered postnatally in rats.Neurobehav Toxicol Teratol 4:463468.Steinhausen H-C, Nestler V, Spohr H-L. 1982. Development andpsychopathologyof childrenwiththefetal alcohol syndrome. DevBehav Pediatr 3:4954.Stone WS, Altman HJ, Hall J, Aranowsky Sandoval G, Parekh P, GoldPE. 1996. Prenatal exposuretoalcohol inadult rats: relationshipsbetween sleep and memory decits, and effects of glucose administra-tion on memory. Brain Res 742:98106.Stratton K, Howe C, Battaglia F, editors. 1996. Fetal alcohol syndrome:diagnosis, epidemiology, prevention, and treatment. Washington,DC: National Academy Press. p 1213.SutherlandRJ,McDonaldRJ,SavageDD.1997.Prenatalexposuretomoderate levels of ethanol can have long-lasting effects on hippocam-pal synaptic plasticity in adult offspring. Hippocampus 7:232238.SutherlandRJ,McDonaldRJ,SavageDD.2000.Prenatalexposuretomoderatelevelsofethanolcanhavelong-lastingeffectsonlearningand memory in adult offspring. Psychobiology, in press.Swartzwelder HS, Farr KL, WilsonWA, SavageDD. 1988. Prenatalexposure to ethanol decreases physiological plasticity in the hippocam-pus of the adult rat. Alcohol 5:121124.Tan SE, Berman RF, Abel EL, Zajac CS. 1990. Prenatal alcohol exposurealters hippocampal slice electrophysiology. Alcohol 7:507511.TanakaH. 1998. Fetal alcohol syndrome: aJapaneseperspective. AnnMed 30:2126.Tanaka H, Inomata K, Arima M. 1983. Zinc supplementation inethanol-treatedpregnantratsincreasesthemetabolicactivityinthefetal hippocampus. Brain Dev 5:549554.TanakaH, NasuF, InomataK. 1991. Fetal alcohol effects: decreasedsynaptic formations in the eld CA3 of fetal hippocampus. Int J DevNeurosci 9:509517.Thomas JD, Wasserman EA, West JR, Goodlett CR. 1996. Behavioraldecits inducedbybingelikeexposuretoalcohol inneonatal rats:importanceofdevelopmental timingandnumberofepisodes. DevPsychobiol 29:433452.Thomas JD, Weinert SP, Sharif S, Riley EP. 1997. MK-801 administra-tion during ethanol withdrawal in neonatal pups attenuates ethanol-induced behavioral decits. Alcohol Clin Exp Res 21:12181225.TomlinsonD, WilceP, Bedi KS. 1998. Spatial learningabilityofratsfollowing differing levels of exposure to alcohol during early postnatallife. Physiol Behav 63:205211.Uecker A, Nadel L. 1996. Spatial locations gone awry: object and spatialmemory decits in children with fetal alcohol syndrome. Neuropsy-chologia 34:209223.Uecker A, Nadel L. 1998. Spatial but not object memory impairments inchildren with fetal alcohol syndrome. Am J Ment Retard 103:1218.Wainwright PE, Ward GR, Wineld D, Huang YS, Mills DE, Ward RP,McCutcheon D. 1990. Effects of prenatal ethanol and long-chain n-3__________________________________________HIPPOCAMPAL AND SPATIAL DYSFUNCTION IN FAS 109fattyacidsupplementationondevelopment inmice. 1. Bodyandbrain growth, sensorimotor development, and water T-maze reversallearning. Alcohol Clin Exp Res 14:405412.Wainwright PE, Levesque S, Krempulec L, Bulman-Fleming B, McCutch-eon D. 1993. Effects of environmental enrichment on cortical depthand Morris-maze performance in B6D2F2 mice exposed prenatally toethanol. Neurotoxicol Teratol 15:1120.Waters NS, Klintsova AY, Foster TC. 1997. Insensitivity of the hippocam-pustoenvironmental stimulationduringpostnatal development. JNeurosci 17:79677973.Weiler IJ, Hawrylak N, Greenough WT. 1995. Morphogenesis inmemoryformation:synapticandcellularmechanisms.BehavBrainRes 66:16.West JR, Goodlett CR. 1990. Teratogeniceffects of alcohol onbraindevelopment. Ann Med 22:319325.West JR, Hodges-Savola CA. 1983. Permanent hippocampal mossy berhyperdevelopment following prenatal ethanol exposure. NeurobehavToxicol Teratol 5:139150.West JR, Pierce DR. 1984. The effects of in utero ethanol exposure onhippocampal mossy bers: an HRP study. Brain Res 317:257259.West JR, Hodges CA, Black AC. 1981. Prenatal exposure to ethanol altersorganization of hippocampal mossy bers in rats. Science 211:957959.West JR, Dewey SL, Cassell MD. 1984. Prenatal ethanol exposure altersthe post-lesion reorganization (sprouting) of acetylcholinesterasestaining in the dentate gyrus of adult rats. Brain Res 314:8395.West JR, Goodlett CR, Brandt JP. 1990. New approaches to research onthe long-term consequences of prenatal exposure to alcohol. AlcoholClin Exp Res 14:684689.WestergrenS, RydenhagB, BassenM, Archer T, Conradi NG. 1996.Effects of prenatal alcohol exposure on activity and learning inSprague-Dawley rats. Pharmacol Biochem Behav 55:515520.Wigal T, Amsel A. 1990. Behavioral andneuroanatomical effects ofprenatal, postnatal, or combined exposure to ethanol in weanling rats.Behav Neurosci 104:116126.ZimmerbergB, MattsonS, RileyEP. 1989. Impairedalternationtestperformance in adult rats following prenatal alcohol exposure.Pharmacol Biochem Behav 32:293299.Zimmerberg B, Sukel HL, Stekler JD. 1991. Spatial learning of adult ratswithfetalalcoholexposure: decitsaresex-dependent.Behav BrainRes 42:4956.110 BERMAN AND HANNIGAN