Effects of Estrogen on Activity andFear

M. A. MLaboratorNew Yor

Received 5, 2001

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Hormones and Behavior 40, 472482 (2001)doi:10.1006/hbeh.2001.1716, available online at http://www.idealibrary.com on

472ffects of estrogen on nonreproductive behav-h as activity; emotionality; and, more recently,

and memory, have been studied in both hu-d rodents. Reduced estrogen levels in humans

treatment in OVX rats has resulted in reduced activity(Palermo-Neto and Dorce, 1990), an indicator of in-creased anxiety. On measures of activity, EB treatmenthas also produced mixed results. On tests of sponta-neous motor activity, EB treatment in OVX rats re-sulted in increased activity (Diaz-Veliz, Urresta, Dus-saubat, and Mora, 1991) or no change in activity (Diaz-Veliz, Soto, Dussaubat, and Mora, 1989), dependingon the EB dose and duration of treatment. In the RW,

hom correspondence and reprint requests should be ad-ax: (212) 327-8664. E-mail: pfaff@rockvax.rockefeller.edu.ords: estrogen; fear; activity; mice. 1996) and to disrupt the anxiolytic action of diazepam(Nomikos and Spyraki, 1988). In the OF, estrogen-Related Behaviors in Mice

organ and D. W. Pfaff1

y of Neurobiology and Behavior, The Rockefeller University,k, New York 10021

October 25, 2000, revised March 17, 2001, accepted May 2

n has been shown to affect nonreproductive be-in humans and rodents, including anxiety, fear,ivity levels. Rat studies have shown increasesreases in these behaviors. Inconsistencies mayo differences in testing conditions and the extent

each test measures anxiety, fear, or activity.use studies have been performed. The presentas conducted to address these issues by exam-

effect of estradiol benzoate (EB) in ovariecto-OVX), C57BL/6 mice on a range of behavioral

s measuring anxiety [open field (OF), darklightn (DLT), elevated plus maze (EP)], activity [run-eel (RW)], and conditioned fear learning (FCon).hicle (Veh) animals spent more time in the cen-EB-treated animals and were more active over-LT, Veh animals were more active than EB-

animals in both the dark and light compartmentse more transitions between the two. In EP, Vehentered a greater number of arms. During FCon,als froze more than Veh to the conditioned stim-ontrast, in the home cage RW, EB animals were

tive than Veh. Factor analysis was used to char-intertask correlations of females behavior andre the possibility that estrogen may have an

on a general arousal factor. In sum, estrogennt heightened fear responses in a range of feariety-provoking situations (OF, DLT, EP, and

while increasing activity in the safer RW. Wethat EB treatment may result in a generally moreanimal. 2001 Elsevier Science

habativhethatefee(Hcrehysuhaberes

eleacloothanGotrepeveSpfouanen shown to lead to depression, sleep distur-rritability, anxiety, panic disorders, and cogni-function (Arpels, 1996; Campbell and White-977; Sherwin, 1998). Estrogen replacementin postmenopausal women has been associ-

th improved mood, increased energy levels,of general well being, and improved learning

ich, 1997; Sherwin, 1998). Anxiety and in-nervous energy have been linked to hypo- andtrogenism and the preovulatory estrogenrpels, 1996). In rodents, nonreproductive be-

related to anxiety, fear, and activity have alsofected by changes in estrogen level, but there controversial.easures of anxiety in the open field (OF) andplus maze (EP), estrogen has been found to

oth an anxiogenic and an anxiolytic. Studiesat open arm exploration in the EP have foundling female rats were more active and less

than males (Leret, Molina-Holgado, andz, 1994) and that estradiol benzoate- (EB)

ovariectomized (OVX) females spent a greaterage of the time in the open arms than did their

(Veh) treated counterparts (Nomikos and, 1988). However, other studies using EP haveB to act as an anxiogenic to progesteronesic actions (Mora, Dussaubat, and Diaz-Veliz,0018-506X/01 $35.00 2001 Elsevier Science

All rights reserved.

activity has consistently been shown to increase in thepresencand CoGillette,Thus, oactivity,across t

Severestrogenstudieslearningstrong e1999; Mretention1971; Ntreatmen(Vazqueand Schtion ofproestrutreated Obenzodipassiveance behdiol benone-waycating aactive aof avoidVaronosafter lonMillard,ment reance beh(Diaz-Vduced ahancedmice onin OVXVeh conruptedScherrermale rateyeblink(Shors, Lin contrcontextulus (CSto males1997). Tlearningdisrupt

These

ing effect on activity in the RW, while a less consistenttternct onotiondue

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473Estrogen, Activity, and Fear-Related Behaviorse of estrogen in rats (Ruiz de Elvira, Persaud,en, 1992; Thomas, Storlien, Bellingham, and1986; Wade and Zucker, 1970; Wang, 1923).

n measures of anxiety and different types ofthe effects of estrogen are varied within and

asks.al studies have also looked at the influence of

on fear learning tasks. While the results of thesehave primarily been interpreted in terms of thecomponent of the task, fear learning also has a

motional component (e.g., Altemus and Arleo,ora et al., 1996), with increased acquisition or

as a positive indicator of fear (e.g., Banerjee,elson and Young, 1998). In male rats, EBt facilitated retention of passive avoidance

z-Pereyra, Rivas-Arancibia, Loaeza-Del Castillo,neider-Rivas, 1995), while in female rats, reten-passive avoidance was inhibited both durings (when estrogen levels are highest) and in EB-VX animals (Mora et al., 1996). In female rats, a

azepine (an anxiolytic) disrupted retention ofavoidance and EB treatment restored the avoid-avior (Gibbs, Burke, and Johnson, 1998). Estra-

zoate treatment has also delayed extinction of aavoidance task (Telegdy and Stark, 1973), indi-prolongation of fear responding. On two-way

voidance, rats in proestrus exhibited facilitationance acquisition (Sfikakis, Spyraki, Sitaras, and, 1978), as did EB-treated OVX rats, particularlyg-term, chronic EB treatment (Singh, Meyer,and Simpkins, 1994). In contrast, acute EB treat-

sulted in reduced acquisition of two-way avoid-avior relative to OVX rats without replacement

eliz et al., 1989); similarly, low doses of EB re-voidance acquisition, while a higher dose en-acquisition (Diaz-Veliz et al., 1991). In a study ofa T-maze shock avoidance task, EB replacementfemales had no effect on acquisition relative totrols, but restored responding that was dis-

by progesterone administration (Farr, Flood,, Kaiser, Taylor, and Morley, 1995). Finally, fe-s in proestrus acquired a classically conditioned

response faster than females in other stagesewczyk, Pacynski, Mathew, and Pickett, 1998);

ast, female rats in proestrus froze less to theand no differently to the tone-conditioned stim-) in a fear conditioning paradigm as comparedand to females in estrous (Markus and Zecevic,

hus, on measures of fear, as indicated by feartasks, EB has been found to both facilitate and

learning.studies indicate that estrogen has a stimulat-

papaembeanwhleathinge

trefeatassigEBussovawaDepracstrlevexonaninhabasanbyonpogawe

M

An

ontim12anarrtreIDMof results emerges in terms of estrogens im-other activities, particularly those with an

al component. Part of this inconsistency mayto differences in testing conditions, method,

ration of EB administration and the extent toach test measures activity, anxiety, fear, orcapacity. Further, few studies have looked at

act of estrogen on nonreproductive behaviorsa species that must be characterized for futurestudies.following study, we examined the effect of EB

nt in OVX female mice on a battery of anxiety,d activity tests. All animals were run on eacha rigidly controlled protocol, a necessary de-

ponent for seeing any differential impact ofe same animals across several task types. We

e C57BL/6 mouse strain for the following rea-) the dose response for this strain in the acti-f sexual behaviors has been established andd to guide the current experiments (Morgan,de, and Pfaff, 2000); (b) genetic knock-out miced in this strain are available for subsequentand anxiety studies; and (c) the C57BL/6

as been characterized as having intermediatef anxiety (van Gaalen and Steckler, 2000). Wed the effect of three different estrogen doses

t of paradigms measuring anxiety (OF, DLT,, activity (RW), and learned fear. Due to thet complexity, reflected in the literature (seeof fear, anxiety, and activity in the various

data were looked at additionally using factorto help define variables potentially influencedgen. This technique has been used previouslyilar set of behavioral paradigms to explore the

ity of a general arousal system (Frohlich, Mor-awa, Burton, and Pfaff, 2001), a system whichothesize may be affected by estrogen.

ODS

and Procedures

le C57BL/6 mice, OVX by the supplier (Tac-ermantown, NY), were 89 weeks old at thearrival. They were maintained on a reversedlight:dark cycle (lights off at 8 AM), and foodter were available ad libitum. Seven days afterall animals were surgically implanted with ant capsule (Silastic tubing dimensions 1.57 mm

mm OD 3 2 cm in length; Dow Corning Co.,, MI) containing one of three doses of EB (25,

50, or 75 mg EB in 0.03 ml sesame oil) or 0.03 ml sesameoil vehifluranenary, Insubcutathree dreasonssuccessfmouse sbut prelinvertedpromptthat connot avadifficultthe 50-m2000) pconsiste(Sfikakicentratilastic cadifferenas the m(a) minsubjectisomethidaily inand (c)range tplant suthe remdivided(n 5 17(n 5 15

Ten dcommenof thedesign wan ordeous teston thewith eac5 min u5 min, E(OF2) fconditiotook plarandomfrom eaThe nuwas recdays fowere gi

were weighed; and their uteri were removed, trimmedfat, aer an

es.

perim

All bk pla

om. Oom, wges, ajacend tesall teuallytted tllowirdedth soldingls, at

om. Ds blin

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474 Morgan and Pfaffcle. Surgery was performed under methyoxy-anesthesia (Metofane; Mallinckrodt Veteri-c., Mundeline, IL), with the capsule locatedneously just below the nape of the neck. Theseoses of EB were selected for the following: the 50-mg EB in a Silastic capsule was usedully for eliciting sex behavior in the sametrain in a previous study (Morgan et al., 2000),iminary data in our lab were suggestive of an-U doseresponse curve on locomotor assays,

ing us to explore different EB doses aroundcentration. While estrogen plasma levels wereilable in the present study due to technicalies with the RIA kit, our previous study usingg dose in OVX C57BL/6 mice (Morgan et al.,

roduced estrogen levels of 163 6 41 pg/ml,nt with that found for female rats in proestruss et al., 1977). We thus achieved estrogen con-ons high enough that small differences in Si-psule performance would not lead to largeces in results. Treatment capsules were usedethod of hormone administration to allow for

imal handling and stressing of animals whenng them to sensitive anxiety and fear tasks,ng that is unavoidable in studies requiringjections; (b) steady levels of EB across tests;doses of EB within the high physiological

o avoid accidental variability. Following im-rgery, animals were housed individually forainder of the experiment. Animals were thusinto the following treatment groups: EB25), EB50 (n 5 17), EB75 (n 5 16), and Veh).ays following implantation, behavioral testingced. All tests occurred during the dark phase

cycle and began 23 h after lights out. Theas chosen such that tests were conducted in

r which would minimize the impact of previ-s on subsequent tests. All animals were testedfollowing paradigms in the following order,h test separated by 24 h: OF activity (OF1) fornder red light, darklight transition (DLT) forP for 5 min under red light, and OF activity

or 5 min under red light. Four days later,ned fear learning (FCon) training and testingce over a 2-day period. The following day asubset of animals (for a total of 12 animals

ch group) were placed in identical RW cages.mber of revolutions accrued per 72-h periodorded for three consecutive periods. Threellowing the test of RW activity, all animalsven an overdose of Nembutal (0.1 ml); they

ofothlin

Ex

toororocaAdateonvismiFocowihomarowa

plaabpla40eqthcuma5anMwiity

saacrigallThsamewa40suopwedaMnd weighed. All acquisition, handling, andimal procedures were within the NIH guide-

ental Design

ehavioral testing except running wheel (RW)ce in a two-room suite outside of the colonyne room was used as a darkened holdinghere all animals were placed, in their homet least 20 min prior to testing on each day.t to this room was a sound- and light-attenu-ting room. Animals were tested individuallysts except for FCon, where the presence of twoand auditorily isolated testing chambers per-

he training and testing of two mice at a time.ng each test, number of fecal boluses was re-and the test apparatus was thoroughly cleanedap and water. The animal was returned to theroom until testing was completed for all ani-which time they were returned to the colonyuring all phases of testing, the experimenterd to the animals treatment group.Field Activity (OF1 and -2). Testing took

nder a 50-W diffuse red light placed 40 cmhe floor of the chamber. Each subject wasn the center of a clear acrylic chamber (40.5 30 cm; Accuscan Instruments, Columbus, OH),d with 16 infrared sensors along each side formatic recording of all horizontal activity (Ac-

Instruments, Digiscan Model RXYZCM). Ani-re permitted to ambulate freely over the nextVariables recorded included overall activitye in the center of the field versus the margin.activity was defined as all behavior occurringinch of the chamber wall, while center activ-

ded all other behavior.light transition. Testing took place in theparatus as that used for OF. An enclosed blackox (40 3 20.5 3 20.5 cm) was inserted into the

lf of the chamber, with an opening (13 3 5 cm)g for passage between the two compartments.ck box had small holes along its base for pas-the infrared beams, thus permitting assess-activity in the dark. The open compartment

ectly illuminated by a 40-W white light placedove the floor of the compartment and directedt a crisp darklight line existed exactly at thebetween the two compartments. Animals

ntly inserted into the dark compartment, andllection commenced immediately for 5 min.es of particular interest were latency to emerge

into the light compartment, number of transitions be-tween tfined asactivity

Eleva50-W remaze wsisted oextendetwo oppopen (RYork, Narms hsubjectopen areach arm

Fear cover 2 csory-isolight plamentsChambe28 cm)shock flments,shockermal ShodelivereTX) locaconditiogrid floopersonatrainingmouse w210 s latpresenteshock dsame. TCSUS,ing chamto the h

Twenditioninresponsassociatof condBlanchaSakagucusing a1997; Loing theconditiodark co

anol rather than soap). The mouse was judged asezingior ton. Thntextntamiezing

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itter Atic swy ofd, an

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Analynductbehantlyus, dllapses maests oree anstatistry inreaseOVA

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mparBehavnt restor a

an 1l. 8).imalns ofns am

475Estrogen, Activity, and Fear-Related Behaviorshe two compartments (with a transition de-all four feet crossing the darklight line), andin the light compartment.ted plus maze. Testing took place under ad light suspended 180 cm above the maze. Theas elevated 40 cm above the floor and con-f a 5 3 5 cm center platform from whichd four acrylic arms in a cross formation, withosing arms enclosed by side walls and two leftockefeller University Instrument Shop, NewY). Arms were 30 3 5 cm, with the enclosed

aving 15-cm-high opaque acrylic walls. Thewas placed in the center of the maze, facing anm. Number of entries into and time spent in

were recorded over the next 5 min.onditioning. Training and testing took placeonsecutive days in one of two identical, sen-lated chambers illuminated by a 40-W whiteced 40 cm above the floor of the two compart-(Rockefeller University Instrument Shop).rs consisted of clear acrylic walls (29 3 26 3with a removable ceiling and a mouse gridoor (model E10-18 MF; Coulbourn Instru-

Lehigh Valley, PA) connected to an animal(SanDiego Instruments Programmable Ani-cker, San Diego, CA). The auditory CS wasd through a buzzer (Radio Shack, Fort Worth,ted on the ceiling of each chamber. The un-ned stimulus (US) was delivered through ther. Delivery of CS and US were controlled by a

l computer. Day 1 of the experiment was aday and consisted of two CSUS pairings. Theas placed in the chamber and approximately

er the CS (30-s, 4.5 6 0.5-kHz, 85-dB tone) wasd and coterminated with the US (2-s, 0.35-mA

elivered through the grid floor). Trial 2 was thehirty seconds after the offset of the secondthe mouse was removed from the condition-ber, placed in its home cage, and transferred

olding room.ty-four hours later mice were tested for con-g to the CS in a novel context. The freezinge (a lack of all visible movement except thoseed with respiration) was used as the measureitioned emotional responding (Blanchard andrd, 1969; Bouton and Bolles, 1980; LeDoux,hi, and Reis, 1984). Conditioning was assessedtime sampling method (Stiedl and Spiess,

gue, Paylor, and Wehner, 1997), and observ-animals behavior in a novel context (originalning box lined on the floor and all sides with

rrugated cardboard and cleaned with 70% eth-

freprtiococofresasuthcoex

thmobemasteMnedariosewa

Da

coallicaThcowat tthinenincAN72sorolusco

cafacthVoantertioor not once every 10 s during the 180-s periodthe CS and during a 180-s CStone presenta-is produced measures of fear to the noveland to the explicit CS (without contextualnation). Five hours later, mice were tested forto the original, training context using the time

g method as outlined above. Behavioral mea-ere calculated as the percentage of intervalsse was judged to be freezing during the novelexposure, the CS, and the training contexte.ing wheel activity. Activity was measured inny room in plastic-tub home cages (Nalgene

660-1284; Mini Mitter, Bend, OR) containing, food, water, and a running wheel. The ani-10 days of unlimited access to the stainless

ning wheel (24 3 8 cm; model 640-0701, Minictivity Wheel), to which was attached a mag-itch and counter (model 130-0023). The first

RW access was treated as an acclimation pe-d data were not included in statistical analy-mber of revolutions accrued per 72-h periodn recorded for three consecutive periods.

alysis

sis of variance (ANOVA) tests were initiallyed comparing the three EB-treated groups onvioral tests. No one EB group differed signif-from the other two on any test except RW.ata from the three EB-treated groups wered into a single group and a direct comparisonde between EB- and Veh-treated animals usingn all behavioral measures except RW. In EP,imals (two Veh and one EB) were not includedtical analyses, as they froze extensively uponto an open arm, giving a false impression ofd boldness. For RW, a repeated-measures, comparing all four groups across the three

riods, was conducted, and post hoc compari-pairs of means were made using the Bonfer-rection. For physiological measures (fecal bo-d uterine weight), ANOVA with post hoc

isons were conducted.ioral variables producing statistically signifi-ults in the above analyses were included in analysis, and factors with eigenvalues greaterwere treated with a varimax rotation (SPSS,Factor analysis requires the testing of each

on a battery of tests and involves using pat-cross-mouse comparisons to discern the rela-ong behavioral variables (e.g., see Ramos and

Mormedmax) ofof totalthe origwere thon the cdifferen

RESU

Open F

Estradful andthe firsttime in[t(63) 5cantly lthan themeasurebeam brrearing,during

DarkLight Transition Test

Estradl andres ofrtmeng. 2)ger t

ht coer s

rtmenent le

. 2. Dger totment

e EB-trht com-treated

partm50. D

FIG. 1. Ocenter ofgroup wa(*P , 0.0SEMs.

476 Morgan and Pfaffe, 1998). An orthogonal rotation (e.g., vari-the factors accounting for a significant portion

variability allows for a clearer interpretation ofinal variables. Two further factor analyses

en conducted, one on the Veh group and oneombined EB group, to determine whether EBtially affects various behavioral factors.

LTS

ield Tests

iol benzoate-treated animals were more fear-less active than Veh-treated animals duringOF test (Fig. 1). They spent significantly lessthe center of the OF than the Veh group3.33; P , 0.005]. They were also signifi-

ess active on the measure of overall activityVeh group (t 5 2.11; P , 0.05). This latter

, automatically calculated as the sum of alleaks, included such behaviors as ambulating,and grooming. These effects were not present

the second OF exposure 3 days later.

fusupa(Filonligfewpasp

FIGlonparThligEBcomn 5

pen field test. (A) The Veh group spent more time in thethe field than did the EB group (*P , 0.005). (B) The Vehs more active in the field overall than was the EB group5). Veh, n 5 15; EB, n 5 50. Data represent means 6iol benzoate-treated animals were more fear-less active than Veh-treated animals on mea-movement between the dark and light com-ts and activity levels in the two compartments

. Estradiol-treated animals took significantlyo emerge from the dark compartment into thempartment (t 5 2.29; P , 0.05) and madeubsequent transitions between the two com-ts (t 5 3.84; P , 0.001). The EB group alsoss time moving in the light (t 5 2.24; P ,

arklight transition test. (A) The EB-treated animals tookemerge from the dark compartment into the light com-for the first time than did Veh animals (*P , 0.05). (B)eated group made fewer transitions between the dark andpartments than did the Veh group (*P , 0.001). (C) The

animals spent less time engaging in activity in the lightent than did Veh animals (*P , 0.05). Veh, n 5 15; EB,ata represent means 6 SEM.

0.05) an0.005) c

Elevate

Vehicanimalsarms thprimarianalysethe opebetweenopen ar

Conditi

In feacontextand towere exfroze siganimalsfreezingnot diff

Runnin

RunnThe firswas notin RW aANOVA(RW1, Rmain efP , 0.0and a s

01),ch othst hore ac

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aine

ctor

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FIG. 3.into the c0.01). Ve

. 4. Ce freez, 0.0

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thirdpared

h perioiod; R

h, n 5resent

477Estrogen, Activity, and Fear-Related Behaviorsd less time moving in the dark (t 5 3.24; P ,ompartments than the Veh group.

d Plus Maze

le animals were more active in the EP than EB. Vehicle animals entered a greater number ofan EB animals [t(60) 5 2.60; P , 0.05],

ly closed arms (t 5 2.72; P , 0.01; Fig. 3). Thes of entries into open arms and time spent inn arms did not produce significant differences

the two groups; both groups entered thems a mean of 1.2 times (P . 0.80).

oned Fear Learning

r conditioning, levels of freezing to the novel(in which the CS test took place), to the CS,the context in which conditioning took placeamined. Estradiol benzoate-treated animalsnificantly more to the CS than did Veh-treated[t(63) 5 2.22; P , 0.05; Fig. 4). Levels ofto the conditioning and novel contexts did

er significantly between the two groups.

g Wheel

ing wheel activity was examined over 10 days.t day was treated as an acclimation period and

included in analysis. In order to see changesctivity over time, a 4 3 3 repeated-measuresof treatment group for the three 72 h periods

W2, and RW3) was conducted. There werefects of treatment group [F(3, 44) 5 5.801;05) and period [F(1, 44) 5 66.80; P , 0.001]ignificant interaction [F(3, 44) 5 7.93; P ,

0.0eaPomothressucoacrem

Fa

th

Elevated plus maze. The EB animals made fewer entrieslosed arms of the maze than did the Veh group (*P ,h, n 5 15; EB, n 5 49. Data represent means 6 SEM.

FIGtim(*PSE

FIGofandcom72-perVerepindicating that treatment groups differed fromer, depending on the time period (see Fig. 5).c analyses showed EB25 to be significantlytive than Veh and EB75 on the second and-h periods (vs Veh P , 0.005 and P , 0.001,vely; vs EB75 P , 0.05 for both periods). Ing. 5 shows that group EB25 was hyperactiveed to Veh and EB75, and while this increasedwas enhanced over time for EB25, Veh activityd constant.

Analysis

riables with statistically significant results inve analyses were included in factor analysis.

onditioned fear test. The EB-treated animals spent moreing to the CS in a novel context than did Veh animals5). Veh, n 5 15; EB, n 5 50. Data represent means 6

unning wheel activity. Group EB25 ran a greater numberions than did the Veh and EB75 groups during the second72-h periods (*P , 0.005 compared to Veh and P , 0.05to EB75). RW1, number of revolutions during the first

d; RW2, number of revolutions during the second 72-hW3, number of revolutions during the third 72-h period.

12; EB25, n 5 12; EB50, n 5 12; EB75, n 5 12. Datameans 6 SEM.

An initiconductunifyingvariabilcountednents wretainedfactorsplainedvariableEP and2 was asFactorsDLT vacontaineand secActivity

TwoVeh growas donpact ofone-fact46% ofwith inithem toaccounttor 1 cociated wtivity wcontaineproach/1998). Fthe Dar

ain coTim

For El aro

hen reatern, fouriancector 1d a nd 4 wntaineth ex

TABLE 1

Factor An than 1

ctor 2ary Ac

EEDOR 0.96R 0.94R 0.94DDFDO% 24.4

Note. F

BLE 2

478 Morgan and Pfaffal analysis combining Veh and EB animals wased. In order to determine the extent to which a

factor of general arousal accounted for totality, a one-factor solution was applied and ac-

for 34% of the total variance. When compo-ith initial eigenvalues greater than 1 wereand subjected to a varimax rotation, four

resulted, accounting for 78% of variance ex-(see Table 1). Factor 1 contained loadings fors primarily associated with exploration in theOF, and with DLT Activity in the Dark. Factorsociated solely with the RW activity variables.3 contained loadings for the remainder of theriables and for freezing to the CS. Factor 4d a primary loading with OF Time in Center

ondary but sizeable loadings from OF Overalland DLT Activity in the Light.

further analyses were conducted, one for theup, and one for the EB groups combined. Thise to determine if there was a differential im-

EB on fear and activity. For Veh animals, theor solution (general arousal) accounted fortotal variance. When retaining components

tial eigenvalues greater than 1 and subjectinga varimax rotation, four factors resulted and

ed for 87% of the variances (see Table 2). Fac-ntained loadings for variables primarily asso-ith locomotion/exploration, and here, RW ac-as strongly included in this factor. Factor 2d only DLT variables, associated with ap-avoidance/anxiety (Ramos and Mormede,

actor 3 combined loadings for DLT Activity ink negatively with freezing to the CS. Factor 4

agOF

eraWgrtiovaFahaancowi

alysis with Varimax Rotation of Factors with Eigenvalues Greater

VariablesFactor 1

ExplorationFa

Volunt

P Closed Entries 0.93P Arm Entries 0.92LT Activity in Dark 0.67F Overall Activity 0.55W RW2W RW3W RW1LT Time to EmergeLT TransitionsCon CSLT Activity in LightF Time in Centerof Total Variance 24.6

or clarity, factor loadings less than 0.5 are not shown.

TA

FacEig

RWRWRWOFEPEPDLDLDLDLFCOF%ntained a primary, now solitary, loading withe in Center.B-treated animals, the one-factor solution (gen-usal) accounted for 36% of total variance.

etaining components with initial eigenvaluesthan 1 and subjecting them to a varimax rota-r factors resulted, accounting for 81% of the(see Table 3). Here too, RW activity loaded on

. Notably, this locomotor activity factor nowegative loading of freezing to the CS. Factors 2ere the same as Veh Factors 2 and 4. Factor 3d loadings for variables primarily associatedploration, but rather than being associated

tivityFactor 3Fear

Factor 4Timidity

0.53

20.680.65

20.640.62 0.57

0.8115.4 13.2

alysis with Varimax Rotation of Factors withes Greater than 1

iables Factor 1 Factor 2 Factor 3 Factor 4

1.00.90.9

ll Activity 0.82ntries 0.82Entries 0.81

sitions 0.95to Emerge 20.90

vity in Light 0.82vity in Dark 20.87

0.77in Center 0.84l Variance 39.8 22.3 13.9 11.1

or clarity, factor loadings less than 0.5 are not shown.tor Anenvalu

Var

RW1RW2RW3

OveraArm EClosedT TranT TimeT ActiT Action CSTime

of Tota

Note. F

with RWDark w

Fecal B

Estradluses ththe sumRW, whEB grou[F(3, 61showed0.001),

Uterine

As exine sizeP , 0.0animalsother granimalsEB75 anand EB7

DISCU

This smice inactivity)in the lein all th

active and spent less time in the center of the OF, theyteredergee incklerasure

ouldatme

ree tain th

On thnificaer traincres ouorts

y (Pand t

th inkakislly co

. 6.ced mo

P ,uses frri thann EB5nifican01 com50, n 5

TABLE 3

Factor AnEigenvalu

Va

RW RW2RW RW1RW RW3FCon CSDLT TranDLT ActiDLT TimeEP ClosedEP Arm EDLT ActiOF OveraOF Time% of Tota

Note. F

479Estrogen, Activity, and Fear-Related Behaviors, as was the case for Veh, DLT Activity in theas included in this group.

oluses

iol benzoate animals produced more fecal bo-an Veh animals on most tasks. An analysis ofof fecal boluses from all tasks (not including

ere boluses could not be counted) showed thatps produced more boluses than Veh animals) 5 11.30; P , 0.001]. Post hoc analysesthis to be so for EB25 (P , 0.05), EB50 (P ,

and EB75 (P , 0.001) (see Fig. 6A).

Weight

pected, the difference between groups for uter-was EB-dose-dependent [F(3, 61) 5 58.57;01; Fig. 6B]. Post hoc analyses showed Vehto have significantly smaller uteri than all

oups (P , 0.001 for all groups). EB25-treatedhad significantly smaller uteri than EB50 andimals (P , 0.001 for both groups), and EB505 did not differ significantly from each other.

SSION

tudy demonstrates that EB treatment in OVXcreases fear and anxiety (and thus reducesin fearful situations, while increasing activity

ss stressful home cage RW. This was apparentree tests of anxiety: EB-treated mice were less

enemtivStemeshtrethis,

sigaftofingreporfouboSfica

FIGduandbolutethasig0.0EB

alysis with Varimax Rotation of Factors withes Greater than 1 for EB-Treated Animals

riables Factor 1 Factor 2 Factor 3 Factor 4

0.90.90.9

20.57sitions 0.89

vity in Light 0.89to Emerge 20.78Entries 0.85

ntries 0.83vity in Dark 0.82ll Activity 0.63 0.55in Center 0.92l Variance 24.8 22.7 22.5 10.7

or clarity, factor loadings less than 0.5 are not shown.fewer arms in the EP, and they took longer tointo the light compartment and were less ac-the light in the DLT test. Van Gaalen and(2000) have suggested that different tasksdifferent aspects of anxiety and that animals

be tested on a battery of tasks. We found EBnt to have a modest but significant effect on allsks, suggesting that the effect of EB on anxietyat sense, robust.e test of learned fear, EB-treated mice frozently more to the conditioned toneCS 24 hining than did Veh-treated mice. This findingased fear learning is consistent with the find-tlined above of increased anxiety and withthat estrogen may enhance learning and mem-ckard, 1998; Sherwin, 1998). Others have alsohat estrogen leads to increased fear learning,avoidance tasks (e.g., Diaz-Veliz et al., 1991;et al., 1978; Singh et al., 1994), and in a classi-

nditioned eyeblink response task (Shors et al.,

Physiological measures. (A) The three EB groups pro-re fecal boluses than the Veh group (*P , 0.05 for EB250.001 for EB50 and EB75). Displayed is the sum of fecalom all tasks except RW. (B) The Veh group had smallerall other groups, and the EB25 group had smaller uteri

0 and EB75. The EB50 and EB75 groups did not differtly from each other (*P , 0.001 compared to Veh; **P ,

pared to Veh and EB25). Veh, n 5 15; EB25, n 5 17;17; EB75, n 5 16. Data represent means 6 SEM.

1998). In contrast, Markus and Zecevic (1997) foundthat femand noing paraestrous.ences, ifemalestest (ouoverallcreasedother finFor exarats wermeasureRasia-FiCarobrenone ofwhich cother emice mishow athreatenand Blaence becreasedEB-treatcal bolustandarRamosnone ofeffectivetial diffanimals

Our fifear andgen ongreatlyestrogenrats (RuOur datmice, wthis stugan, Freven a 1elevatin

Previtask inteelementdency fobe constasks mRamossuccess

ables. For EP, time spent in open arms has been de-ibedtriesre ofaouloth feave inriableolvine freere ofutonsed tg whe of

luntaical

98). Oed a

Lookied, F

n, cosederall

ciatedT vant (C, 198taryctor 3ime td alsontainsActiv

ps bea loc

the a3.

Regard EBe twotivity.gelyxioustriesme c

andth opm otimalsegatials arFig. 5

480 Morgan and Pfaffale rats in proestrus froze less to the contextdifferently to the toneCS in a fear condition-digm as compared to males and to females inSeveral factors may account for these differ-

ncluding species tested (mice vs rats), cyclingvs OVX with EB replacement, and the time ofr 24 h posttraining vs their 2 weeks). Ourfindings of elevated estrogen resulting in in-anxiety and fear in mice also contrast withdings in rats, as reviewed in the introduction.

mple, several studies have found that femalee less anxious than males in the EP, a popular

of anxiety (Lucion, Charchat, Pereira, andlho, 1996; Imhof, Coelho, Schmitt, Morato, andz, 1993; Johnston and File, 1991). However,these studies controlled for estrous cycle stage,ould have a large impact on the results. An-xplanation for differences between rats andght come from findings that mice, unlike rats,

strong initial risk assessment tendency ining situations (Blanchard, Griebel, Henrie,nchard, 1997), a tendency which could influ-havior in the EP. Finally, our findings of in-fear were further supported by the fact thated animals produced a greater number of fe-ses on several tasks than did Veh animals, a

d indicator of fear (Hall, 1934; Lister, 1990;and Mormede, 1998). Among these measures,the three EB doses stood out as being morethan the others, despite resulting in substan-

erences in uterine weight; thus, all EB-treatedwere considered as one group on these tasks.ndings of reduced activity in tasks measuringanxiety cannot be due to an impact of estro-

general motor function, as RW activity wasenhanced in these same animals. The effect of

on RW activity has been found previously iniz de Elvira et al., 1992; Thomas et al., 1986).a confirm that this is also true for EB-treatedith EB25 indicated as the optimal EB dose indy. Another study from our lab (Garey, Mor-ohlich, McEwen, and Pfaff, 2001) found that03 smaller dose of EB had a similar effect ong RW activity in mice.ous reports have pointed out the complexity ofrpretation. Tasks such as EP and OF combine

s of fear of novel places with a natural ten-r exploration. Further, emotionality itself may

idered a multidimensional trait, and differentay measure different forms of anxiety (seeand Mormede, 1998, for review). However,has been had in characterizing several vari-

scrensuChwiativarevThsuBoponintypvotyp19bin

bintioClOvsoDLmeleyunFa(Tancoallhailyortor

anthaclaranEnhoRWwifroana nimatas a measure of anxiety, while the number ofinto closed arms has been considered a mea-

activity (Ramos, Mellerin, Mormede, andff, 1998). The OF test also combines activityr, and Center Time may be considered a neg-dicator of anxiety (Crawley, 1999). The DLTs have been described as measures of fear,g around an approach/avoidance conflict.zing response has long been used as a mea-learned fear (Blanchard and Blanchard, 1969;and Bolles, 1980; LeDoux et al., 1984) as op-

o the innate fear of the other measures. Run-eel activity has been described as a differentmotor activity from the above tasks, being ary activity rather than the locomotion moreof exploratory activity (Ramos and Mormede,ur variables can be distinguished and com-

long similar lines.ng at the factor analysis for all groups com-actor 1, which we speculatively call Explora-ontains loadings for EP Arm Entries andEntries, DLT Activity in the Dark, and OFActivity. These are all variables primarily as-with locomotion or exploration, though the

riable has been considered to have a fear ele-haouloff, Durand, and Mormede, 1997; Craw-1; Ramos and Mormede, 1998). Factor 2, Vol-Activity, contains only the RW variables., Fear, contains the remaining DLT variableso Emerge, Transitions, and Activity in Light)

FCon freezing to the CS. Factor 4, Timidity,OF Time in Center and secondarily OF Over-ity and DLT Activity in Light and may per-distinguished from Factor 3 by being primar-omotor fear measure rather than the freezing

pproach/avoidance more characteristic of Fac-

ding the separate factor analyses for the Vehconditions, the primary point of interest is how

hormonal states differentially grouped RWFor Veh animals, the Exploration variables

grouped with RW, suggesting that for lessanimals, OF Overall Activity and EP Arm

are perceived as similar to activity in the saferage RW. For EB-treated animals, in contrast,

CS freezing loaded together negatively (i.e.,posite signs), while maintaining a distinctionher motor functions. This suggests that, for EB, Voluntary Activity in particular may requireve fear component such that more fearful an-e reliably less active in the RW. Also, looking, it can be seen that group EB25 had a pro-

gressive increase in activity over time while Veh per-formancthis incpotentiaindepenpairinguntreateVeh- anOur resaffects t

Anothgen inhypothawell estenviron(Carey,1995; Hand Meshow grcosteronbol, Ber1991). FACTHcage forEstradioresponsAbitbolmales,levels athe AC(Viau avided areducedupon a(Calvo,Siriczmport thsensitivbehaviomiliar pstudy.

In sumEB treatmice toactivityity in ththe studlized thFrohlichThe preanimal:and rep

safe environments, but also (b) engenders increasedels o

FER

emus,eprodun Moodngton,pels, J.rom thnd revz, M.,

nvolve0, 795erjee,ones

90.nchardf fear.nchard1997).ects onevs. 21

uton, Mreezingearn B

lvo, N.etyra

hemicampbell

enoparey, M

loet, Eamic-p44, 311aouloffctivityat lighwley,nockoealth,

ests. Bwley,nimalhem. Bz-Veliffects

ioned0, 61z-Velince ofpon thhysiol.r, S. Aorley

nce le15723hlich,tatisticale m

481Estrogen, Activity, and Fear-Related Behaviorse remained relatively flat; one explanation forrease may be a progressive decrease in anyl fear associated with the RW. In two otherdent studies from our lab, this factor analyticof RW with CS freezing was also found ford OVX animals (Frohlich et al., 2001) and ford EB-treated OVX animals (Garey et al., 2001).ults suggest that EB treatment qualitativelyhe RW locomotor experience.er line of evidence supporting a role of estro-fear and activity comes from work on thelamic-pituitary-adrenal (HPA) axis. It is fairly

ablished that the HPA axis is most sensitive tomental stressors in the presence of estrogenDeterd, de Koning, Helmerhorst, and de Kloet,anda, Burgess, Kerr, and OKeefe, 1994; Viauaney, 1991). In response to stress, female ratseater adrenocorticotrophin (ACTH) and corti-e (cort) responses than males (Le Mevel, Abit-aud, and Maniey, 1979; Viau and Meaney,or example, one study found that levels of

are much greater in the OF versus the homefemales relative to males (Handa et al., 1994).l-treated OVX females show a greater HPA

e than untreated OVX females (Le Mevel,, Beraud, and Maniey, 1978). In cycling fe-it is during early proestrus, when estrogenre high but progesterone levels are low, thatTH and cort responses to stress are greatestnd Meaney, 1991). Further studies have pro-direct link between stress, levels of cort, andactivity in EP, an effect which is reversed

dministration of a cort synthesis inhibitorMartijena, Molina, and Volosin, 1998; Baez,

an, and Volosin, 1996). These results all sup-e idea that females with estrogen are moree to environmental stressors and may translaterally into an increased fear/reduced (nonfa-lace) activity response as seen in the present

, our data on this range of assays show thatment has at least a dual effect on OVX femaleincrease various aspects of fear (thus reducinglevels in fear situations) and to increase activ-e safer home cage RW. A recent approach toy of hormone effects in female mice has uti-e concept of arousal (Frohlich et al., 2001;, Ogawa, Morgan, Burton, and Pfaff, 1999).

sence of estrogen may produce a more arousedThis drives (a) elevated levels of RW activityroductive behavior sequences in familiar and

lev

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METHODSFIG. 1

RESULTSFIG. 2FIG. 3FIG. 4FIG. 5TABLE 1TABLE 2TABLE 3

DISCUSSIONFIG. 6

REFERENCES