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Prenatal Ethanol Exposure Enhances NMDAR-Dependent Long-Term Potentiation in the Adolescent Female Dentate Gyrus Andrea K. Titterness 1,2,3 and Brian R. Christie 1,2,3,4 * ABSTRACT: The dentate gyrus (DG) is a region of the hippocampus intimately involved with learning and memory. Prenatal exposure to ei- ther stress or ethanol can reduce long-term potentiation (LTP) in the male hippocampus but there is little information on how these prenatal events affect LTP in the adolescent female hippocampus. Previous stud- ies suggest that deleterious effects of PNEE can, in part, be mediated by corticosterone, suggesting that prenatal stress might further enhance any alterations to LTP induced PNEE. When animals were exposed to a combination of prenatal stress and PNEE distinct sex differences emerged. Exposure to ethanol throughout gestation significantly reduced DG LTP in adolescent males but enhanced LTP in adolescent females. Combined exposure to stress and ethanol in utero reduced the ethanol- induced enhancement of LTP in females. On the other hand, exposure to stress and ethanol in utero did not alter the ethanol-induced reduc- tion of LTP in males. These results indicate that prenatal ethanol and prenatal stress produce sex-specific alterations in synaptic plasticity in the adolescent hippocampus. V V C 2010 Wiley-Liss, Inc. KEY WORDS: sex differences; prenatal ethanol; adolescent INTRODUCTION Changes to the in utero [A1]environment can have a profound impact on the developing fetus. The consumption of alcohol during pregnancy, in particular, can lead to a number of abnormalities that include central nervous system (CNS) dysfunction, impaired cognition, and reduced physical growth. Collectively these deficits are grouped under the term fetal alcohol spectrum disorder or FASD (Hoyme et al., 2005). Maternal stress can also negatively impact the development and maturation of the CNS; however, it is not clear if combined exposure to stress and ethanol in utero produces similar, distinct, or even synergistic effects in the brains of the offspring, particularly during adolescence when brain structure and function is further altered by sexual development. Hippocampal-dependent spatial learning and memory is impaired in male offspring following exposure to ethanol in utero in both humans and animals (Reyes et al., 1989; Uecker and Nadel, 1996; Uecker and Nadel, 1998; Richardson et al., 2002; Wilcoxon et al., 2005; Ryan et al., 2008). Consistent with this impairment, long term potentiation (LTP) of synaptic efficacy, a putative model of learning and memory (Bliss and Colling- ridge, 1993), is reduced in males following prenatal ethanol exposure (PNEE) across gestation (Sutherland et al., 1997; Swartzwelder et al., 1988; Richardson et al., 2002; Christie et al., 2005). While these observa- tions are consistent in males, very few studies have investigated the effects of PNEE on synaptic plasticity in females. Gestational ethanol exposure can reduce CA1 LTP and impair spatial memory in adult male and female guinea pigs (Richardson et al., 2002), but spatial memory impairments in adolescent female rats are equivocal (Zimmerberg and Weston, 2002; O’Leary- Moore et al., 2006). It also remains to be determined whether PNEE alters synaptic plasticity in the adoles- cent female rat hippocampus, an animal model that is distinctly different than guinea pigs because significant brain hippocampal growth occurs postnatal. Similar to PNEE, prenatal stress (PS) can also impair spatial learning and memory (Son et al., 2006; Yang et al., 2006; Hosseini-Sharifabad and Hadinedoushan, 2007; Yang et al., 2007; Yaka et al., 2007) and reduce the capacity for LTP in the CA1 region of the hippo- campus (Son et al., 2006; Yang et al., 2006; Yaka et al., 2007). Stress activates the hypothalamic-pituitary-adrenal [hypothalamo-pituitary-adrenal (HPA)] axis, and, during gestation, increased secretion of adrenal hormones in the mother can produce lasting impairments in spatial learn- ing and memory in male offspring (Zagron and Wein- stock, 2006). Secretion of the stress hormone corticoster- one (CORT: rodents) or cortisol (humans) by the adre- nal glands has been directly implicated in the long- lasting harmful effects of prenatal stress (Barbazanges et al., 1996). For example, CORT has been shown to directly mediate the growth deficits that result from PNEE (Tritt et al., 1993), implicating HPA activity in ethanol-induced pathology. In addition, ethanol con- sumption directly stimulates HPA activity (Pohorecky, 1990; Nash and Maickel, 1988) and ethanol-consuming dams exhibit a heightened CORT response to an acute stressor (Weinberg and Gallo, 1982). Therefore, it is not unreasonable to hypothesize that combined exposure to stress and ethanol in utero might act synergistically, exac- erbating any deleterious effects normally produced by stress or ethanol alone. The goals of the current study were to investigate (1) whether PNEE alters synaptic plasticity in the dentate gyurs of adolescent males and females and (2) whether 1 Graduate Program in Neuroscience, University of British Columbia, Vancouver, BC, Canada; 2 Department of Cellular and Physiological Sci- ences, University of British Columbia, Vancouver, BC, Canada; 3 Divi- sion of Medical Sciences, University of Victoria, Victoria, BC, Canada; 4 Department of Biology, University of Victoria, Victoria, BC, Canada Grant sponsors: PEO, ABMRF, CFI, CIHR, MSFHR, and NSERC, MSFHR. *Correspondence to: Brian R. Christie, Division of Medical Sciences, University of Victoria, 3800 Finnerty Road, Victoria, BC, Canada, V8P 5C2. E-mail: [email protected] Accepted for publication 10 June 2010 DOI 10.1002/hipo.20849 Published online in Wiley Online Library (wileyonlinelibrary.com). HIPPOCAMPUS 00:000–000 (2010) V V C 2010 WILEY-LISS, INC.

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Page 1: Prenatal Ethanol Exposure Enhances NMDAR-Dependent ......Prenatal Ethanol Exposure Enhances NMDAR-Dependent Long-Term Potentiation in the Adolescent Female Dentate Gyrus Andrea K

Prenatal Ethanol Exposure Enhances NMDAR-DependentLong-Term Potentiation in the Adolescent Female Dentate Gyrus

Andrea K. Titterness1,2,3 and Brian R. Christie1,2,3,4*

ABSTRACT: The dentate gyrus (DG) is a region of the hippocampusintimately involved with learning and memory. Prenatal exposure to ei-ther stress or ethanol can reduce long-term potentiation (LTP) in themale hippocampus but there is little information on how these prenatalevents affect LTP in the adolescent female hippocampus. Previous stud-ies suggest that deleterious effects of PNEE can, in part, be mediated bycorticosterone, suggesting that prenatal stress might further enhanceany alterations to LTP induced PNEE. When animals were exposed toa combination of prenatal stress and PNEE distinct sex differencesemerged. Exposure to ethanol throughout gestation significantly reducedDG LTP in adolescent males but enhanced LTP in adolescent females.Combined exposure to stress and ethanol in utero reduced the ethanol-induced enhancement of LTP in females. On the other hand, exposureto stress and ethanol in utero did not alter the ethanol-induced reduc-tion of LTP in males. These results indicate that prenatal ethanol andprenatal stress produce sex-specific alterations in synaptic plasticity inthe adolescent hippocampus. VVC 2010 Wiley-Liss, Inc.

KEY WORDS: sex differences; prenatal ethanol; adolescent

INTRODUCTION

Changes to the in utero [A1]environment can have a profoundimpact on the developing fetus. The consumption of alcohol duringpregnancy, in particular, can lead to a number of abnormalities thatinclude central nervous system (CNS) dysfunction, impaired cognition,and reduced physical growth. Collectively these deficits are groupedunder the term fetal alcohol spectrum disorder or FASD (Hoyme et al.,2005). Maternal stress can also negatively impact the development andmaturation of the CNS; however, it is not clear if combined exposure tostress and ethanol in utero produces similar, distinct, or even synergisticeffects in the brains of the offspring, particularly during adolescencewhen brain structure and function is further altered by sexualdevelopment.

Hippocampal-dependent spatial learning and memory is impaired inmale offspring following exposure to ethanol in utero in both humansand animals (Reyes et al., 1989; Uecker and Nadel, 1996; Uecker andNadel, 1998; Richardson et al., 2002; Wilcoxon et al., 2005; Ryan

et al., 2008). Consistent with this impairment, longterm potentiation (LTP) of synaptic efficacy, a putativemodel of learning and memory (Bliss and Colling-ridge, 1993), is reduced in males following prenatalethanol exposure (PNEE) across gestation (Sutherlandet al., 1997; Swartzwelder et al., 1988; Richardsonet al., 2002; Christie et al., 2005). While these observa-tions are consistent in males, very few studies haveinvestigated the effects of PNEE on synaptic plasticityin females. Gestational ethanol exposure can reduceCA1 LTP and impair spatial memory in adult male andfemale guinea pigs (Richardson et al., 2002), but spatialmemory impairments in adolescent female rats areequivocal (Zimmerberg and Weston, 2002; O’Leary-Moore et al., 2006). It also remains to be determinedwhether PNEE alters synaptic plasticity in the adoles-cent female rat hippocampus, an animal model that isdistinctly different than guinea pigs because significantbrain hippocampal growth occurs postnatal.

Similar to PNEE, prenatal stress (PS) can also impairspatial learning and memory (Son et al., 2006; Yanget al., 2006; Hosseini-Sharifabad and Hadinedoushan,2007; Yang et al., 2007; Yaka et al., 2007) and reducethe capacity for LTP in the CA1 region of the hippo-campus (Son et al., 2006; Yang et al., 2006; Yaka et al.,2007). Stress activates the hypothalamic-pituitary-adrenal[hypothalamo-pituitary-adrenal (HPA)] axis, and, duringgestation, increased secretion of adrenal hormones in themother can produce lasting impairments in spatial learn-ing and memory in male offspring (Zagron and Wein-stock, 2006). Secretion of the stress hormone corticoster-one (CORT: rodents) or cortisol (humans) by the adre-nal glands has been directly implicated in the long-lasting harmful effects of prenatal stress (Barbazangeset al., 1996). For example, CORT has been shown todirectly mediate the growth deficits that result fromPNEE (Tritt et al., 1993), implicating HPA activity inethanol-induced pathology. In addition, ethanol con-sumption directly stimulates HPA activity (Pohorecky,1990; Nash and Maickel, 1988) and ethanol-consumingdams exhibit a heightened CORT response to an acutestressor (Weinberg and Gallo, 1982). Therefore, it is notunreasonable to hypothesize that combined exposure tostress and ethanol in utero might act synergistically, exac-erbating any deleterious effects normally produced bystress or ethanol alone.

The goals of the current study were to investigate (1)whether PNEE alters synaptic plasticity in the dentategyurs of adolescent males and females and (2) whether

1Graduate Program in Neuroscience, University of British Columbia,Vancouver, BC, Canada; 2Department of Cellular and Physiological Sci-ences, University of British Columbia, Vancouver, BC, Canada; 3Divi-sion of Medical Sciences, University of Victoria, Victoria, BC, Canada;4Department of Biology, University of Victoria, Victoria, BC, Canada

Grant sponsors: PEO, ABMRF, CFI, CIHR, MSFHR, and NSERC, MSFHR.*Correspondence to: Brian R. Christie, Division of Medical Sciences,University of Victoria, 3800 Finnerty Road, Victoria, BC, Canada, V8P5C2. E-mail: [email protected] for publication 10 June 2010DOI 10.1002/hipo.20849Published online in Wiley Online Library (wileyonlinelibrary.com).

HIPPOCAMPUS 00:000–000 (2010)

VVC 2010 WILEY-LISS, INC.

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combined exposure to stress and ethanol in utero exacerbates anyalterations that arise from exposure to ethanol alone.

METHODS

Animals

Male (275–300 g) and virgin female (250–275 g) Sprague-Dawley rats were obtained from Charles River Laboratories (St.Constant, PQ, Canada) and housed in a colony room that wasmaintained on a 12 h light: dark cycle (lights on at 0600 h) withconstant humidity and temperature (228C). Unless specified, allrats had ad libitum access to standard rat chow (Lab Diets, 5,001)and water. All animal procedures were performed in accordancewith the University of Victoria, University of British Columbia,and the Canadian Council for Animal Care policies.

Breeding and Diets

An experimental timeline is shown in Figure 1. Following anacclimation period of at least one week, individual femaleswere paired with a single male. Vaginal smears were taken dailyat 09:00 h and checked for the presence of sperm, indicatinggestation Day 1 (GD1), at which point females were individu-ally housed and randomly assigned to one of three feedingconditions: (1) ethanol (E): ad libitum access to a liquid dietcontaining 35.5% ethanol-derived calories; (2) pairfed (PF):liquid diet with maltose-dextrin isocalorically substituted forethanol derived calories. PF dams received the same amount offood in g/kg/day as the matched E dam; (3) Ad Libitum (AL):ad libitum access to standard rat chow. All dams had unre-stricted access to water. E dams were gradually introduced tothe liquid diet across the first three days of gestation by com-bining 1/3 ethanol liquid diet with 2/3 pairfed diet on GD1,2/3 ethanol diet with 1/3 pairfed diet on GD2, and 3/3 etha-nol diet on GD3. Freshly prepared liquid diets were given 2 hprior to lights off to prevent a shift in the cort circadianrhythm (Weinberg and Gallo, 1982). At this point bottles fromthe previous day were weighed to determine the amount offood consumed. Liquid diets were obtained from Dyets (Beth-lehem, PA) and are sold as Weinberg/Keiver high protein liquid

diet-control (no. 710109) for the pairfed diet and Weinberg/Keiver high protein liquid diet-experimental (no. 710324) forthe ethanol diet. Both liquid diets are nutritionally fortified toprovide adequate nutrition for pregnant rodents (Weinberg,1985). Liquid diets were replaced with ad libitum access tostandard rat chow on GD22, which helps reduce any furtherdeficits of ethanol exposure on offspring (Weinberg, 1989).

Females were weighed on GD1, 7, 14, and 21 duringroutine cage changing to minimally disturb the animals. The dayon which females gave birth indicated postnatal day 1 (PND1)and litters were then culled to 10 pups (5 male/5 female whenpossible) on PND2. After birth, cages were changed twice weeklyand dams and offspring were weighed on PND 2, 8, 15, and22. Offspring were weaned on PND 22 and group housed (2–3 per cage) by sex and electrophysiological experiments were per-formed on adolescent offspring between PND30–35. To reducelitter effects (Zorrilla, 1997), only two male and female offspringfrom each dam were used for electrophysiology recordings inthese experiments.

Prenatal Stress

AL, PF, and E dams were randomly assigned to one of twohousing conditions: (1) stress (S): consisting of three, 45-minrestraint sessions (09:00 h, 12:00 h, and 15:00 h) during gesta-tion days 12–21 or (2) nonstress (NS): the dams remainedundisturbed in their home cage. Pregnant females were individ-ually placed in a clear plastic tube (diameter 5 7 cm; length 519 cm) under bright light (Zuena et al., 2008). Tubes con-tained holes at either end for airflow. The last restraint sessionoccurred at 15:00 h to ensure that the timing of liquid dietadministration was consistent across stress/nonstress conditions.

Ethanol and Corticosterone Assays

Blood ethanol concentration assay

To determine blood ethanol concentrations (BEC), bloodsamples were taken from three randomly chosen stress andnonstress ethanol dams on GD15. Approximately 2 h afterlights out, tail vein blood was collected in a microcentrifugetube and allowed to clot overnight at 48C. The following day,

FIGURE 1. Timeline of experiments. The first day on whichsperm was present in the vaginal smear indicated gestation day 1(GD1). On GD1, females were individually housed and assignedto one of three diets (E, PF, AL). Stress dams were exposed torestraint stress during GD12–21; non-stress dams were left undis-

turbed in their home cage. Liquid diets were replaced with stand-ard rat chow on GD22, litters were culled to 5 males and 5females on postnatal day 2 (PND2) and electrophysiology experi-ments were performed on offspring between PND30 and PND35.

2 TITTERNESS AND CHRISTIE

Hippocampus

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samples were centrifuged at 3,000g and the supernatant wascollected and stored at 2208C until assayed. BECs were deter-mined using the Analox Alcohol Analyzer (Model AMI; AnaloxInstruments, Lunenberg, MA).

Corticosterone assay

To determine the CORT levels following restraint stress, tailvein blood samples were collected from restraint dams immedi-ately following cessation of the first restraint (�9:45 am) onGD 12, 17, and 21. Blood samples were also taken from non-stress AL, PF, and E dams at 9:45 am on GD12 to assess basalCORT levels; offspring from these nonstress dams were notused in this study. Blood samples were collected from nonstressdams within 2 min of touching the cage in order to reduce theinfluence of HPA activation on the basal blood sample(Davidson et al., 1968). Once collected, all blood samples wereallowed to clot for �30 min at room temperature then centri-fuged at 3,000g for 15 min; supernatant was collected andstored at 2208C until assayed. CORT levels were assayed viaenzyme-linked immunoassay (ELISA, Assay Designs; AnnArbor, MI; catalog #900–097) according to manufacturer’sinstructions. The minimum detection of the kit is 26.99 pg/mL and has the cross reactivity is 100% for cort, 28.6% fordeoxycorticosterone, 1.7% for progesterone and less than 0.3%for testosterone, aldosterone, and cortisol.

Electrophysiology

Animals were anesthetized with urethane (1.5 g/kg, i.p.) andplaced in a Kopf stereotaxic apparatus. Rectal temperature wasmonitored and maintained at 37 6 18C with a grounded homeo-thermic temperature control unit (Harvard Instruments, MA).One electrode was inserted into the skull anterior to bregma anda second posterior to lambda to serve as a reference and groundfor the recording and stimulating electrodes, respectively. A 125lm diameter stainless steel recording electrode was directedthrough a trephine hole to the dorsal dentate gyrus (3.5 mmposterior, 2.0 mm lateral to bregma). A monopolar stimulatingelectrode (125 lm diameter) was directed through a trephinehole to the ipsilateral perforant path (7.4 mm posterior, 3 mmlateral to bregma). Once both stimulating and recording electro-des were lowered to elicit a maximal response, and the minimalstimulation required to elicit a 1–2 mV population spike deter-mined. A single pulse (0.12 ms in width) was delivered at 0.067Hz for a minimum of 15 min to assess the baseline excitatorypostsynaptic potential (EPSP). Once a stable baseline was ob-tained, long-term potentiation (LTP) was induced by administer-ing a u-burst protocol consisting of four trains of 10 bursts offive pulses at 400 Hz with a 200-ms interburst interval. The pulsewidth was changed to 0.24 ms during u-burst stimulation (TBS)and there was a 15-s delay between trains. Following TBS, base-line stimulation resumed for 60 min, after which the animal waseuthanized by an overdose of urethane to the brain. Electrical sig-nals were acquired using custom written software (Lee Campbell;Getting Instruments) and National Instruments data acquisitionhardware. Signals were amplified and filtered at 1 Hz and 3 Hzusing a differential amplifier (Getting Instruments, San Diego,

CA) and digitized at 5 kHz before being stored on a PC. All elec-trophysiology data are presented as the percent change from base-line (mean 6 SE) of the initial phase of the EPSP slope (10–80%). The following is a summary of the number of offspring ineach group: ad libitum, nonstress male (n 5 10); ad libitum,nonstress female (n 5 12); ad libitum, stress male (n 5 11); adlibitum, stress female (n 5 11); pairfed, nonstress male (n 5 10);pairfed, nonstress female (n 5 14); pairfed, stress male (n 5 11);pairfed, stress female (n 5 11); ethanol, nonstress male (n 5 10);ethanol, nonstress female (n 5 13); ethanol, stress male (n 5 8);ethanol, stress female (n 5 14).

Drug

The competitive antagonist against N-methyl-D-aspartatereceptors (NMDARs) (6)-3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) was obtained from Sigma and dis-solved in 0.9% saline. Drug was administered intraperitoneal90 min prior to application of TBS at a dose of 10 mg/kg(Farmer et al., 2004).

Data and Statistical Analyses

All data are presented as mean 6 standard error of the mean(S.E.M.). A 2-way analysis of variance (ANOVA) for prenataldiet (AL, PF, E) and stress condition (NS, S) was conducted ondam weight gain across gestation. Pup weight was analyzed usinga two-way ANOVA for sex (male, female) 3 diet (AL, PF, E) onPND2 because PNEE has been shown to reduce birth weight(Christie et al., 2005; Weinberg and Gallo, 1982); offspringweight for PND 8–35 were analyzed repeated measuresANOVA. LTP data were analyzed by assessing the initial phaseof the EPSP slope (10–80%) at 55–60 min post-TBS. A three-way ANOVA of prenatal diet (AL, PF, E) X prenatal stress (NS,S) and sex (male, female) was performed on LTP data. For allANOVAs, Newman-Keuls post hoc tests were performed whereappropriate. Based on the a priori hypothesis that PS wouldreduce LTP in ad libitum male offspring, we also performed astudent’s t-test between male ad libitum stress and nonstressoffspring and between female ad libitum stress and nonstressoffspring. We also hypothesized that application of CPP willblock LTP and thus performed t-tests to determine if the percentchange in EPSP slope was (1) significantly different from baselineand (2) significantly reduced compared with saline counterparts.To control for multiple comparisons, Bonferroni corrections wereapplied when analyzing the data and statistical significance wasset at P < 0.016 for CPP data. Statistical analyses were per-formed using Statistica software (Statsoft, Tulsa, OK) with statis-tical significance set at a P < 0.05 unless otherwise stated.

RESULTS

Developmental Data

Exposure to prenatal stress did not increase ethanolconsumption in the pregnant females, and the blood ethanol

SEX-SPECIFIC EFFECTS OF PRENATAL ETHANOL ON DG LTP 3

Hippocampus

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concentration for nonstress ethanol dams (87 6 15 mg/dL)was not significantly different from stress dams (114 6 27 mg/dL; t(11) 5 0.91, P > 0.05). However, maternal weight gainwas affected by prenatal diet (F(2,74) 5 8.38, P < 0.05) withethanol dams (27 6 2%) showing significantly less gain com-pared to ad libitum (38 6 3%, P < 0.05) and pairfed (33 62%, P < 0.05) dams. Prenatal stress also attenuated weightgain in all females regardless of prenatal diet (F(1,74) 5 15.40,P < 0.05). The altered weight gain in dams was not due toreduced litter size since neither prenatal stress (F(1,70) 5 0.49,P > 0.05) nor prenatal diet (F(2,70) 5 0.65, P > 0.05)decreased litter size. A significant interaction between prenataldiet and stress was observed for the ratio of male/female pupsborn (F(2,70) 5 3.28, P < 0.05), however post hoc analysesfailed to reveal any significant differences in the sex ratio for thedifferent treatment groups. Finally, gestation length was also notaffected by either prenatal diet (F(2,47) 5 0.15, P > 0.05) orprenatal stress (F(1,47) 5 0.07, P > 0.05). The gestationaldevelopmental data for the dams are presented in Table 1.

Offspring weight was analyzed across the postnatal period todetermine if prenatal treatments altered offspring weight gain.When assessed on PND2, a significant main effect of prenataldiet (F(2,140) 5 6.38, P < 0.05) indicated that birth weight ofethanol-exposed pups was significantly reduced compared to adlibitum (P < 0.05) and pairfed (P < 0.05) offspring. OnPND2, there was also trend toward a main effect of both sex(F(1,140) 5 3.62, P 5 0.06) and stress (F(1,140) 5 3.51, P 50.06) with males weighing more than females (P < 0.05) andstress reducing birth weight (P < 0.05). Across PND 8–35, alloffspring gained weight across the postnatal period as indicatedby a significant main effect of postnatal day (F(3,321) 51,923.32, P < 0.05). A significant main effect of prenatal diet(F(2,107) 5 3.58, P < 0.05) revealed that ethanol-exposed off-spring weighed less than ad libitum offspring (P < 0.05) witha trend toward reduced weight compared to pairfed offspring(p 5 0.06). A significant main effect of sex (F(1,107) 5 24.05,P < 0.05) revealed that males weighed more than femaleswhen they reached the experimental ages of PND30–35

TABLE 1.

Gestation Outcome Measures for Ad Libitum, Pairfed and Ethanol Dams

Ad libitum Pairfed Ethanoala

Gestation outcomes measures Non-stress Stressb Non-stress Stressb Non-stress Stressb

Number of dams 10 10 18 9 18 15

Gestation length (days) 22 6 0.15 21.8 6 0.20 21.89 6 0.20 20.78 6 1.30 21.92 6 0.14 22 6 0.00

Dam weight gain (% change from GDI) 42.62 6 4.97 34.05 6 3.64 35.29 6 2.22 28.13 6 2.14 32.63 6 2.56 19.69 6 2.13

Number or live pups 15.42 6 0.47 14.37 6 0.92 14.46 6 1.14 13.83 6 1.76 15.05 6 0.63 15.23 6 0.50

Pup sex ratio (male/female) 1.11 6 0.18 1.61 6 0.33 1.51 6 0.19 1.06 6 0.13 1.28 6 0.15 0.95 6 0.12

aEthanol diet significantly reduced weight gain across gestation.bPrenatal stress significantly reduced weight gain across gestation.

TABLE 2.

Offspring Developmental Data

Ad libitum Pairfed Ethanol

Offspring weight (g) Non-stress Stress Non-stress Stress Non-stress Stress

Male

PND 2 7.90 6 0.39 7.59 6 0.28 7.37 6 0.36 7.30 6 0.33 7.30 6 0.20a 6.669 6 0.14a

PND 8 19.77 6 0.95 34.96 6 16.43 19.77 6 0.79 18.29 6 0.93 18.04 6 0.81 16.97 6 0.38

PND 15 36.87 6 1.23 35.66 6 0.56 36.29 6 0.91 38.20 6 1.84 37.08 6 0.84 35.21 6 1.32

PND 22 59.13 6 1.92 59.21 6 1.67 58.12 6 1.64 60.07 6 3.31 58.68 6 2.26 55.28 61.38

PND 30–35 135.46 6 6.31 127.14 6 7.18 134.82 6 4.83 125.67 6 9.55 124.36 6 6.20 121.69 6 5.86

Female

PND 2 7.30 6 0.31 7.21 6 0.27 7.23 6 0.39 6.90 6 0.10 7.01 6 0.19a 6.56 6 0.18a

PND 8 17.88 6 0.52 18.86 6 1.04 19.28 6 0.79 17.40 6 0.49 18.17 6 0.38 16.01 6 0.45

PND 15 34.25 6 0.93 34.71 6 0.69 33.55 6 2.12 36.45 6 0.89 36.57 6 0.58 32.70 6 1.12

PND 22 55.68 6 1.59 56.88 6 1.56 53.34 6 3.31 58.06 6 1.82 58.49 6 1.69 50.59 6 1.67

PND 30–35b 119.00 6 4.11 112.71 6 5.36 116.45 6 6.16 105.17 6 5.90 116.75 6 6.38 97.77 6 2.99

aE < AL, PF.bFemale < Males.

4 TITTERNESS AND CHRISTIE

Hippocampus

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(F(3,321) 5 12.53, P < 0.05).There was also a trend of prenatalstress on offspring weight (F(1,107) 5 2.76, P 5 0.09) and asignificant interaction between postnatal day and prenatal stress(F(3,321) 5 6.13, P < 0.05) revealed that the reduction inweight did not occur until PND30-35 (P < 0.05). The devel-opmental data for all offspring are presented in Table 2.

Ethanol Consumption During PregnancyDoes Not Exacerbate the CORT Responseto Restraint Stress in Female Rats

To determine the impact of restraint stress on CORT levels,we compared the blood samples taken on GD12 for nonstressand stress dams in each feeding condition. As shown in Figure2A, CORT was significantly increased in all dams, irrespectiveof their experimental grouping, immediately following the ces-sation of the first restraint session on GD12 (F(1,42) 5 31.41,P < 0.05). Although there was a trend of prenatal diet onCORT levels for GD12 (F(2,42) 5 2.46, P 5 0.09), post hocanalyses did not reveal significant differences in CORT levelsacross the prenatal diets. CORT levels were then assayed inblood samples taken on GD12, 17, and 21 immediatelyfollowing the first restraint session to determine if CORT levelsattenuated across gestation, but arepeated measures ANOVAdid not reveal a main effect of gestation day (F(2,48) 5 0.36,P > 0.05) or diet (F(2,48) 5 0.79, P > 0.05; Fig. 2B).

Prenatal Ethanol Exposure Reduces LTPin Adolescent Males but Enhances LTPin Adolescent Females

We sought to determine how prenatal events (i.e., PNEEand prenatal stress) alter LTP in the DG of adolescent maleand female rodents. A three-way factorial ANOVA for sex(male, female) 3 prenatal diet (ad libitum, pairfed, ethanol)and prenatal stress (nonstress, stress) revealed a significant maineffect of stress (F(1,123) 5 9.05, P < 0.05) and sex (F(1,123) 512.06, P < 0.05), as well as significant interactions between pre-natal diet and sex (F(2,123) 5 5.45, P < 0.05) and prenatal dietx prenatal stress 3 sex (F(2,123) 5 5.91, P < 0.05). The posthoc analyses for this three-way interaction are presented below.

In the absence of prenatal stress, LTP was significantly reducedin ethanol-exposed males (27 6 2%) compared with ad libitummales (39 6 1%, P < 0.05; Fig. 3A). LTP in pairfed males(32 6 2%) was not significantly different from either ad libitum(P > 0.05) or ethanol (P > 0.05) males. In contrast, LTP inethanol-exposed females (34 6 3%) was significantly greaterthan ad libitum females (21 6 2%, P < 0.05; Fig. 3B). LTP inpairfed females (30 6 2%) was not significantly different fromad libitum (P > 0.05) or ethanol (P > 0.05) females.

As shown in Figure 3C, ad libitum females had significantlyless LTP than ad libitum males (P < 0.05), supporting previ-ous findings of basic sex differences in LTP capacity duringearly adolescence (Maren et al., 1994). This basic sex differencein LTP was abolished following either prenatal food deprivation(P > 0.05) or prenatal ethanol exposure (P > 0.05). Interest-ingly, LTP in ethanol-exposed females was equivalent to that

observed in ad libitum males (P > 0.05), while the LTP inethanol-exposed males was now equivalent to that observed inad libitum females (P > 0.05). That is, prenatal ethanol expo-sure increased LTP in females to a point normally seen incontrol males, but reduced LTP in males to a level normallyobserved in control females. These findings are graphicallydepicted in Figure 3D.

Prenatal Stress Reduced LTP in EthanolExposed Adolescent Females but not Males

A test of our a priori prediction confirmed that prenatal

stress significantly reduced LTP in ad libitum males (31 6 3%)

compared with nonstress ad libitum males (39 6 1%; t(19) 52.18, P < 0.05; Fig. 4A). Conversely, prenatal stress did not

reduce LTP in pairfed (28 6 4%, P > 0.05; Fig. 4B) or etha-

FIGURE 2. Restraint stress increased serum corticosteronelevels equally across prenatal diet. A. CORT levels were signifi-cantly increased following restraint stress on GD12 in ad libitum,pairfed and ethanol dams. B. When assessed immediately followingthe first restraint session, CORT levels were not significantly differ-ent between ad libitum, pairfed or ethanol dams on gestation days12, 17, and 21. There was not a significant difference in cort levelsacross gestation days following restraint stress. *P < 0.05.

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nol (28 6 1%, P > 0.05; Fig. 4C) males compared to non-

stress pairfed (32 6 2%) or ethanol (27 6 2%) counterparts.

Results of prenatal stress on DG LTP in adolescent males are

summarized in Figure 4D.Post hoc analyses also revealed that prenatal stress did not

reduce LTP in ad libitum females (24 6 2%; P > 0.05) com-pared with nonstress ad libitum females (21 6 2%), which wasfurther supported by a student’s t-test of our a priori prediction(t(23) 5 20.09, P > 0.05; Fig. 5A). Prenatal stress also did notreduce LTP in pairfed females (23 6 3%, P > 0.05; Fig. 5B)compared with nonstress pairfed females (30 6 2%). However,combined exposure to stress and ethanol in utero significantlyreduced LTP (21 6 3%, P < 0.05; Fig. 5C) compared with

nonstress ethanol females (34 6 3%). The effect of prenatal stresson DG LTP in adolescent females are summarized in Figure 5D.

Prenatal Stress Alters NMDAR Contributionto DG LTP in Adolescent Females

In order to determine if PNEE altered NMDAR contri-bution to LTP in the DG, CPP, a competitive NMDARantagonist, was administered 90 min prior to the application ofu-patterned stimulation. The change in EPSP slope was signifi-cantly different from baseline in ad libitum (t(17) 5 23.67,P < 0.016) and pairfed males (t(12) 5 23.33, P < 0.016) butnot in ethanol males (t(11) 5 21.84, P > 0.016) following

FIGURE 3. Prenatal ethanol exposure produced sex-specificeffects on DG LTP. A. LTP was reduced in ethanol-exposed malescompared to ad libitum males. B. LTP was enhanced in ethanol-exposed females compared to ad libitum females. C. Ad libitumfemales and ethanol males had significantly less LTP compared toad libitum males. Ethanol females had significantly more LTPthan ad libitum females. D. Representative traces from ad libitum,

pairfed and ethanol males (D1–3) and females (D4–6). Darkertraces were taken immediately prior to HFS and lighter traceswere taken 55–60 min post-HFS. *P < 0.05; a: significantly lessthan ad libitum male (P < 0.05); b: significantly greater than adlibitum female (P < 0.05). Scale bar: vertical: 4 mV, horizontal20 ms.

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application of CPP as shown in Figure 6A. LTP was signifi-cantly reduced in all male offspring compared to saline coun-terparts (all P-values <0.016). In females, the EPSP slope wassignificantly different from baseline in ad libitum females (t(19)5 27.68, P < 0.016) but not ethanol (t(6) 5 21.77, P >0.016) and pairfed females (t(6) 5 20.68, P > 0.016; Fig. 6B).The EPSP slope was significantly reduced in ethanol (t(15) 54.17, P < 0.016) and pairfed females (t(16) 5 4.36, P < 0.016)and there was a trend toward reduced LTP in ad libitum femalesfollowing CPP administration (t(21) 5 2.59, P 5 0.017).

We next investigated whether prenatal stress altered NMDARcontribution to LTP. LTP was significantly reduced by CPP inethanol (t(16) 5 4.05, P < 0.016) and pairfed (t(14) 5 4.26, P <

0.016) males but not ad libitum males (t(14) 5 2.16, P > 0.016;Fig. 7A). The change in EPSP slope was not significantly differentfrom baseline in ad libitum males (t(8) 5 22.96, P > 0.016),pairfed (t(18) 5 20.82, P > 0.016) or ethanol males (t(14) 521.92, P > 0.016) following CPP administration. In females, thepercent change in EPSP slope following CPP administration wasnot significantly different in ad libitum (t(13) 5 0.11 P > 0.016)or ethanol females (t(17) 5 2.00, P > 0.016) compared with salinecounterparts; the EPSP slope was reduced by CPP in pairfedfemales (t(28) 5 6.70, P < 0.016; Fig. 7B). Furthermore, theEPSP slope was not significantly different from baseline in ad libi-tum (t(6) 5 23.00, P > 0.016), pairfed (t(14) 5 1.56, P > 0.016)or ethanol (t(8) 5 22.75, P > 0.016) females.

FIGURE 4. Prenatal stress reduced LTP in ad libitum males.A. Prenatal stress significantly reduced LTP in ad libitum males. B.LTP was not reduced by prenatal stress in pairfed males. C. LTPin ethanol-exposed males was not further reduced by prenatalstress. D. Summary LTP illustrating that prenatal stress reducedLTP in ad libitum males. Representative traces from non-stress

(NS) and stress (S) offspring are included within ad libitum,pairfed and ethanol LTP graphs. Darker traces were taken immedi-ately prior to HFS and lighter traces were taken between 55 and60 min post-HFS. *P < 0.05. Scale bar: vertical: 4 mV, horizontal20 ms.

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DISCUSSION

These experiments revealed that PNEE reduced LTP in theDG of adolescent males but, conversely, enhanced LTP in theDG of adolescent females. For the male subjects, these resultsare consistent with previous research in the adult male hippo-campus (Sutherland et al., 1997; Swartzwelder et al., 1988;Richardson et al., 2002; Christie et al., 2005). This is the firststudy to show that PNEE produces disparate effects on synapticplasticity in the adolescent male and female hippocampus. Inaddition, these experiments revealed that prenatal stress alsoproduces sex specific reductions in synaptic plasticity. That is,prenatal stress reduced LTP in the DG of adolescent control

males, but did not produce a similar deficit in adolescentcontrol females. Interestingly, prenatal stress did reduce theenhanced LTP observed in adolescent females following PNEE,but did not produce any further alterations in synaptic plastic-ity in the adolescent male DG.

It has previously been suggested that the HPA axis contrib-utes to the deleterious effects of PNEE on offspring. Ethanolcan stimulate maternal HPA activity (Wilkins and Gorelick,1986; Spencer and McEwen, 1990; Wand and Dobs, 1991;Rivier, 1996) and maternal adrenalectomy can rescue some ofthe behavioral deficits induced by PNEE (Slone and Redei,2002; Taylor et al., 2002; Wilcoxon et al., 2003). Indeed,CORT can directly mediate the deleterious effects of maternalethanol consumption on offspring growth (Tritt et al., 1993).

FIGURE 5. Prenatal stress reduced LTP in ethanol females. A.Prenatal stress did not reduce LTP in ad libitum females. B. LTPwas not reduced by prenatal stress in pairfed offspring. C. Prenatalstress significantly reduced LTP in ethanol-exposed females. D.Summary graph indicating that prenatal stress reduced LTP only

in ethanol females. Representative traces from non-stress (NS) andstress (S) offspring are included within ad libitum, pairfed andethanol LTP graph. Darker traces were taken immediately prior toHFS, and lighter traces were taken between 55 and 60 min post-HFS. **P < 0.01. Scale bar: vertical: 4 mV, horizontal 10 ms.

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Although basal CORT levels were not significantly different inethanol consuming dams compared with ad libitum dams onthe morning of GD12 (Fig. 2A), it is possible that maternalHPA activity could be significantly enhanced above controlsimmediately following the consumption of the ethanol liquiddiet at the beginning of the dark cycle. Some authors haveshown that exposure to a stressor during gestation can alsoenhance CORT levels above controls in ethanol consumingdams (Weinberg and Gallo, 1982), however, we report herethat CORT levels in our animals were not significantly elevatedabove controls in ethanol consuming dams exposed to arepeated restraint stress. Although many of the procedures usedin these experiments are identical to that of Weinberg andGallo (1982), the discrepancy in these findings might be dueto the type of stressor used in this study.

PNEE increases fetal levels of glutamate (Karl et al., 1995)and administration of an NMDAR antagonist following PNEE

can rescue ethanol-induced deficits in offspring spatial memory(Thomas et al., 2004). These studies suggest that heightenedNMDAR activity due to ethanol-induced increases in gluta-mate might also contribute to ethanol toxicity. Interestingly,CORT-induced release of glutamate within the hippocampus ofnear-term fetal guinea pigs is enhanced by PNEE (Iqbal et al.,2006) suggesting that glutamate-induced damage caused byPNEE might be exaggerated by exposure to stress in utero.Our results indicate that combined exposure to stress and etha-nol in utero produce distinct effects on synaptic plasticity inthe adolescent female dentate gyrus. Specifically, more LTP wasobserved in females following PNEE, than in ad libitumfemales, yet this ethanol-induced enhancement was abolishedby prenatal stress. On the other hand, LTP in adolescent malesfollowing PNEE was not significantly different from males

FIGURE 6. CPP blocked LTP in male and female offspringfollowing prenatal ethanol exposure. A. CPP reduced LTP in adlibitum and pairfed males but blocked LTP in ethanol males. B.LTP was neither blocked nor reduced by CPP in ad libitumfemales. CPP blocked LTP in pairfed and ethanol females. *Signifi-cantly different from saline, P < 0.016; ^significantly differentfrom baseline, P < 0.016.

FIGURE 7. Sex-specific effects of CPP on LTP following pre-natal stress. A. Although CPP neither blocked nor reduced LTP inad libitum males exposed to prenatal restraint stress, LTP wasblocked by CPP in pairfed and ethanol males exposed to prenatalrestraint stress. B. LTP was neither reduced nor blocked by CPPin ad libitum or ethanol females exposed to prenatal restraintstress. The change in EPSP slope was reduced but not significantlydifferent from baseline following application of CPP in pairfedfemales exposed to stress in utero. *significantly different fromsaline (P < 0.016); ^significantly different from baseline (P <0.016).

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exposed to stress and ethanol in utero. These findings suggestthat in utero exposure to stress and ethanol produce specificeffects in males and females but the mechanism behind thesedifferences remains to be determined.

The contribution of NMDARs to synaptic plasticity follow-ing prenatal stress and PNEE was also investigated and a com-plex relationship between sex and prenatal condition wasfound. NMDARs can be recruited for the induction of LTP bythe repeated bursts of depolarization that are characteristic ofu-burst stimulation (Larson and Lynch, 1988; Capocchi et al.,1992; Mott and Lewis, 1992; Farmer et al., 2004). Applicationof the competitive NMDAR antagonist CPP, however, onlyreduced, but did not block, LTP in the DG of adolescent malesand neither blocked nor reduced in the female DG. Shankaret al. (1998) found that hippocampal LTP is more reliant uponNMDARs in the adult vs. aged male rat (Shankar et al., 1998)and an NMDAR antagonist blocks LTP in adult but not ado-lescent rats (de Marchena et al., 2008). We have also previouslydemonstrated that CPP completely blocks DG LTP in adultmales (Farmer et al., 2004), indicating that there is likely adevelopmentally regulated change in the contribution ofNMDARs to synaptic plasticity. There is also a greaterNMDA-mediated component during u-burst stimulation inadolescent males than females (Maren, 1995) suggesting a sex-specific contribution of NMDARS to LTP following u-burststimulation. CPP application completely blocked LTP induc-tion following PNEE, suggesting that prenatal ethanol exposurecauses LTP to become solely reliant upon NMDARs. This issurprising because PNEE reduces [3H]MK-801 binding, NMDAR-mediated calcium entry, and expression of NR2A and NR2BNMDAR subunits in males (Lee et al., 1994; Diaz-Granadoset al., 1997; Spuhler-Phillips et al., 1997) and activation ofintracellular pathways that contribute to LTP (Samudio-Ruizet al., 2009). Prenatal stress can also reduce NMDAR subunitexpression in the hippocampus (Son et al., 2006; Yaka et al.,2007) indicating that altered expression and/or function ofNMDARs following prenatal ethanol or prenatal stress mightaffect NMDAR contribution to synaptic plasticity in the ado-lescent hippocampus. It is unknown how PNEE or prenatalstress affects the kinetics of CPP (e.g., half-life, absorption, dis-tribution, etc.) and we therefore cannot exclude the possibilitythat alterations in these parameters contributed to the resultsobtained in this study.

There is a dearth of evidence as to how PNEE affects hippo-campal synaptic plasticity in the female offspring. It has previ-ously been shown that LTP in the CA1 region of adult maleand female guinea pigs is reduced following PNEE (Richardsonet al., 2002) indicating that females are equally sensitive to thedeleterious effects of PNEE in this animal model. We have pre-viously shown that PNEE selectively abolished CA1 LTD inadolescent females (Titterness and Christie, 2008) and thereforesought to investigate how PNEE alters LTP in the adolescentfemale DG. In contrast to the reduced LTP observed in males,PNEE enhanced LTP in adolescent females. These findingssuggest that the threshold for bidirectional synaptic plasticitymight be shifted in favor of LTP in females following PNEE.

A mechanism for this shift may involve PNEE-induced altera-tions in the expression of gonadal hormones. In contrast toadults (Warren et al., 1995; Good et al., 1999), estradiolreduces LTP in the adolescent hippocampus (Ito et al., 1999).Pubertal estradiol is closely related to vaginal opening (Ojedaet al., 1976; Germain et al., 1978) and since vaginal opening isdelayed in females following PNEE (McGivern et al., 1992;McGivern and Yellon, 1992; Sliwowska et al., 2008), pubertalestradiol levels might be reduced in females following PNEE.We were unable to determine estradiol levels in the currentstudy and thus cannot confirm this hypothesis. However, thisintriguing possibility could account for the enhanced LTPobserved in the adolescent ethanol-exposed female if thedepressing actions of estradiol on synaptic plasticity are indeedreduced in these animals. Future experiments will explore thispossibility.

The behavioral ramifications of these alterations in thecapacity for LTP following PNEE have yet to be fully explored.It is tempting to infer that reduced hippocampal synaptic plas-ticity will result in impaired spatial learning because previousstudies have shown that both hippocampal LTP and spatiallearning are impaired in adult males following PNEE (Blan-chard et al., 1987; Kim et al., 1997; Matthews and Simson,1998; Cronise et al., 2001; Christie et al., 2005). Yet spatiallearning and memory can be impaired in adolescent femalesfollowing PNEE (Zimmerberg and Weston, 2002) and we haveshown that PNEE enhances DG LTP in adolescent females.This suggests that the enhanced LTP in adolescent females maynot be advantageous for hippocampal-dependent learning andmemory. Indeed, impaired spatial performance can be accom-panied by enhanced LTP (Vaillend et al., 2004) and the magni-tude of LTP can be negatively correlated with spatial perform-ance (Jeffery, 1995). Consistent with previous studies (Marenet al., 1994), adolescent ad libitum females exhibited less LTPthan ad libitum males, which might contribute to impairedspatial performance in adolescent females compared with males(Mendez-Lopez et al., 2009). In this particular study, however,sex-differences in spatial performance did not persist with age(Mendez-Lopez et al., 2009) although sex-differences in synap-tic plasticity are still apparent in adults (Maren et al., 1994;Maren, 1995). Taken together, these studies indicate a complexrelationship between synaptic plasticity and behavior. Thecurrent study did not directly investigate the relationshipbetween LTP and behavior in adolescent offspring followingPNEE but this work highlights an area that should be furtherinvestigated.

It is important to consider the influence of food deprivationon the results observed in this study. The diets employedwithin the current study were designed to impart proper nutri-tion to dams regardless of the amount of diet consumed (Wein-berg, 1985) yet ethanol dams tend to consume less of the liq-uid diet, and therefore, fewer calories than control dams (Wein-berg, 1985). As a result, the pairfed dams receive a limitedration of food. Although not meant as a stressor, the behaviorof pairfed dams suggests that an element of stress results fromthis feeding regime. Specifically, pairfed dams are sensitive to

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cues associated with feeding and rush to the front of the cagewhen the food is presented. Pairfed dams also immediately startto consume the liquid diet upon presentation and typicallyconsume all of the diet that is presented. CORT levels in stresspairfed dams were not significantly different from ethanol orcontrol dams indicating that all of the dams responded equallyto the restraint stress. LTP in pairfed offspring was not signifi-cantly different from either control or ethanol offspring sug-gesting that the reduced food intact of ethanol dams mightcontribute to LTP changes observed in ethanol-exposed off-spring. LTP in ethanol-exposed animals was significantly differ-ent from controls, as opposed to pairfed offspring, so therewere specific alterations that resulted from the ethanol diet.Therefore, it is possible that prenatal food restriction may par-tially mitigate or augment the LTP in ethanol exposed malesand females, respectively. However, the LTP in ethanol-exposedoffspring was significantly different from controls suggestingdistinct effects of the ethanol and pairfed diets on LTP.

SUMMARY

Prenatal ethanol exposure produced sex-specific alterations toNMDAR-dependent DG LTP in early adolescent offspring. Asthis is the first study to investigate how PNEE alters LTP inadolescent females, it remains to be determined if the enhancedLTP is correlated with improved performance on hippocampal-dependent learning and memory tasks. We also found thatsynaptic plasticity in ethanol-exposed offspring is differentiallyaffected by prenatal stress. These data highlight a complex rela-tionship between sex, prenatal stress, and prenatal ethanol ex-posure on hippocampal function and future research should beaimed at elucidating the mechanisms behind these changes andbehavioral implications of these interactions.

Acknowledgments

The authors would like to acknowledge B. Eadie for helpfuldiscussions as well as G. Keyes and A. Kwasnica for technicalassistance.

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