Estrogen modulates sexually dimorphic contextual fear conditioning and hippocampal long-term potentiation (LTP) in rats 1 1 Published on the World Wide Web on 1 December 2000

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The present study examined the role of ovarian steroids in contextual fear conditioning and hippocampal synaptic plasticity in female rats. In experiment 1, adult female rats were ovariectomized and submitted to contextual fear conditioning, a
  See discussions, stats, and author profiles for this publication at: Estrogen modulates sexually dimorphiccontextual fear conditioning and hippocampallong-term...  Article   in  Brain Research · February 2001 DOI: 10.1016/S0006-8993(00)03116-4 · Source: PubMed CITATIONS 130 READS 71 5 authors , including: Some of the authors of this publication are also working on these related projects: Neural circuits for fear renewal   View projectSrijan SenUniversity of Michigan 43   PUBLICATIONS   3,212   CITATIONS   SEE PROFILE Charles N RudickNorthwestern University 41   PUBLICATIONS   1,268   CITATIONS   SEE PROFILE Stephen MarenTexas A&M University 150   PUBLICATIONS   13,369   CITATIONS   SEE PROFILE All content following this page was uploaded by Stephen Maren on 30 November 2016. The user has requested enhancement of the downloaded file. All in-text references underlined in blueare linked to publications on ResearchGate, letting you access and read them immediately.  Brain Research 888 (2001) 356– Interactive report Estrogen modulates sexually dimorphic contextual fear conditioning 1 and hippocampal long-term potentiation (LTP) in rats a b a a,2 Rishi R. Gupta , Srijan Sen , Lisa L. Diepenhorst , Charles N. Rudick , a,b, *Stephen Maren a  Department of Psychology ,  University of Michigan , 525   E  .  University Ave .,  Ann Arbor  ,  MI   48109  - 1109,  USA b  Neuroscience Program ,  University of Michigan , 525   E  .  University Ave .,  Ann Arbor  ,  MI   48109  - 1109,  USA Accepted 30 October 2000 Abstract The present study examined the role of ovarian steroids in contextual fear conditioning and hippocampal synaptic plasticity in femalerats. In experiment 1, adult female rats were ovariectomized and submitted to contextual fear conditioning, a procedure in which ratsreceived unsignaled footshock in a novel observation chamber; freezing behavior served as the measure of conditional fear.Ovariectomized female rats froze at levels comparable to male rats, both of which froze significantly more than sham-operated femalerats. In experiment 2, estrogen replacement in ovariectomized female rats reduced fear conditioning to a level comparable to that of sham-operated females in experiment 1. In experiment 3, the influence of estrogen on the induction of long-term potentiation (LTP) atperforant path-dentate granule cell synapses in ovariectomized female rats was examined. Estrogen decreased both population spike LTPand EPSP-spike potentiation at perforant path synapses. Taken together, these experiments indicate that ovarian steroids regulate bothsexually dimorphic behavior and hippocampal plasticity in a fear-conditioning paradigm.  ©  2001 Elsevier Science B.V. All rightsreserved. Theme :   Neural basis of behavior Topic :   Learning and memory: systems and functions Keywords :   Estrogen; Fear conditioning; Hippocampus; Long-term potentiation; Rat 1. Introduction  ditioning in which an unconditional stimulus (US, afootshock) elicits freezing behavior (immobility except forFear is a psychological construct used to describe the breathing) in the context of US delivery. In this study,various behavioral and physiological changes that take male rats exhibited significantly higher levels of contextualplace when an organism is faced with a threatening freezing than female rats. Interestingly, we also discoveredsituation [16,23,35]. As with many behavioral systems a sex difference in perforant path-dentate granule cell[10,12,51], fear-related behavior exhibits a prominent long-term potentiation (LTP) [37]. LTP is an enduringsexual dimorphism in both rodents [7] and humans [31]. form of synaptic plasticity that has been posited to mediateRecently, we reported a reliable sex difference in con- various forms of learning [41,45], including contextual feartextual fear conditioning [43], a form of Pavlovian con- conditioning [25,39]. The positive correlation betweenhippocampal synaptic plasticity and contextual fear con-ditioning is consistent with the proposed role of the 1 Published on the World Wide Web on 1 December 2000.  hippocampus in contextual fear conditioning [5,40]. *Corresponding author. Tel.:  1 1-734-936-6532; fax:  1 1-734-763- An interesting question concerns the hormonal factors 7480. that regulate sexually dimorphic fear conditioning. Al-  E  - mail address : (S. Maren). 2  though the influence of gonadal steroids on several be- Present address: Northwestern University, Department of Neurobiol- havioral systems has been characterized [61], the in- ogy and Physiology, 2153 N Campus Dr., Evanston, IL 60208-0877,USA.  volvement of these hormones in fear conditioning is poorly 0006-8993/01/$ – see front matter  ©  2001 Elsevier Science B.V. All rights reserved.PII: S0006-8993(00)03116-4   R .  R .  Gupta et al .  /   Brain Research  888 (2001) 356  – 365   357 understood. Recently, Anagnostaras et al. found that effects of ovariectomy on fear conditioning. Specifically,castration in adult male rats did not affect the sex we predicted that estrogen administration prior to feardifference in either fear conditioning or hippocampal LTP conditioning would attenuate the acquisition of contextual[6]. In contrast, a role for ovarian steroids in fear con- fear.ditioning has been suggested by a study indicating that In experiment 3, we used electrophysiological tech-contextual fear conditioning in female rats varies across niques to characterize the influence of estrogen administra-the estrous cycle [44]. Specifically, female rats in proestrus tion on the induction of perforant path-dentate gyrus long-(when plasma levels of estrogen are high) exhibit reliably term potentiation (LTP) in ovariectomized female rats.lower levels of conditional freezing compared to females Perforant path LTP has been reported to be sexuallyin estrus (when plasma levels of estrogen are low). dimorphic [37,43] and it was therefore of interest toConsistent with these results, it has been reported that examine the modulation of this form of synaptic plasticitycycling female rats perform more poorly than ovariectom- by ovarian steroids.ized female rats in an aversively motivated avoidancelearning task [19,24]. Moreover, a preliminary report 2.2.  Subjects and surgery indicates that exogenous estradiol administration inovariectomized female rats reduces contextual fear con-Long–Evans rats were obtained from a commercialditioning [4]. These data suggest that ovarian steroids andsupplier (Harlan Sprague–Dawley, Indianapolis, IN). Theyestrogen, in particular, may be important in regulating fearwere individually housed in metal cages located in theconditioning in adult female rats.vivarium and maintained on a 14:10 h light/dark cycle.The possible role for ovarian steroids in contextual fearRats had unrestricted access to food and water and wereconditioning is interesting in the light of other work handled daily for 4 days prior to surgery to acclimate theimplicating estrogen in the regulation of hippocampalrats to the experimenter. Twenty-five female (150–174 g)neuronal morphology [63–66] and synaptic plasticityand 12 male (175–199 g) were used in experiment 1, 29[14,17,26] in female rats. In the present experiments wefemale (150–174 g) rats in experiment 2, and 33 femaleexamined the relationship between ovarian steroids andrats (150–174 g) in experiment 3.contextual fear conditioning. We conducted three experi-In experiments 1–3, rats were anesthetized with methox-ments to examine the hypothesis that estrogen exerts anyfluorane and were bilaterally ovariectomized; sham-oper-inhibitory influence on both contextual fear conditioningated males in experiment 1 underwent the same procedure,and perforant path-granule cell LTP in adult female rats.except that no tissue was removed. Three days afterOur results reveal an important role for estrogen inovariectomy, vaginal lavages were performed on themodulating contextual fear conditioning and hippocampaloperated females for 10 days to screen for improperlysynaptic plasticity in adult female rats.ovariectomized rats. The presence of cornified epithelialcells in the vaginal smears was evaluated, and a conserva-tive criterion was used for inclusion of animals in the 2. Materials and methods experiments. Sham-operated females and sham-operatedmales received mock swabs during the 10-day screening 2.1.  Experimental strategy period to equate for handling. Sham-operated (randomlycycling) females were used as a control in this experimentThree experiments were conducted. In experiment 1, theto replicate the sex difference in fear conditioning ob-effect of ovariectomy on contextual fear conditioning inserved by Maren et al., who also used randomly cyclingadult female rats was examined to test the hypothesis thatfemales and intact males [43].ovarian steroids in female rats play a role in sexuallydimorphic fear conditioning. We compared three groups of adult rats: (1) ovariectomized females, (2) sham-operated  2.3.  Hormone administration females, and (3) sham-operated males. The latter twogroups were included to replicate our previous report of In experiments 2 and 3, rats were administered twosex differences in contextual fear conditioning [43]. If successive hormone treatments 48 h and 4 h beforeovarian steroids influence sex differences in the formation conditioning (experiment 2) or electrophysiological testingof hippocampal-dependent memories then ovariectomized (experiment 3). This injection pattern was chosen to(OVX) rats should show similar contextual fear con- recapitulate the hormonal pattern of normally cycling rats.ditioning as the sham-operated male rats. The sham-oper- In experiment 2, the rats were divided into two treatmentated females should show lower levels of conditional fear groups: (1) rats that received peanut oil for both injectionsthan that in the other groups. (‘oil’ group) and (2) rats that received estradiol benzoateIn experiment 2, we examined the effects of estrogen for both injections (‘estrogen’ group). The estradiol ad-replacement on fear conditioning in OVX rats. We hypoth- ministration consisted of a 10- m g dose of estradiol ben-esized that estrogen replacement would counteract the zoate (Sigma Chemical, St. Louis, MO) dissolved in 0.1  358  R .  R .  Gupta et al .  /   Brain Research  888 (2001) 356  – 365  ml of peanut oil and injected subcutaneously in the nape of post-HFS recording period. All of the data were analyzedthe neck. using analysis of variance (ANOVA), and post-hoc com-parisons in the form of Fisher least significant difference 2.4.  Electrophysiological procedures  (LSD) tests were used for comparisons of means followinga significant omnibus  F  -test. All of the data are presentedFour hours after their final estrogen treatment, rats in as means 6 the standard errors of the means (S.E.M.s).experiment 3 were anesthetized with sodium pentobarbital(65 mg/kg, i.p.) and supplemented with pentobarbital  2.5.  Behavioral apparatus and procedure throughout the surgical procedure (0.05–0.15 ml) asneeded. After inducing surgical anesthesia, each rat was In experiments 1 and 2, eight identical observationmounted on a stereotaxic frame (David Kopf Instruments, chambers (28 3 21 3 22 cm; Lafayette Instrument Co.,Tujunga, CA). The scalp was exposed, the head was North Lafayette, IN) were used for both conditioning andleveled (bregma and lambda were oriented in the same contextual fear testing. The chambers consisted of horizontal plane), and small holes (1-mm diameter) were aluminum (side walls) and Plexiglas (rear wall, ceiling, anddrilled in the skull for placement of recording and stimula- hinged front door). The chambers were situated in cheststion electrodes and a stainless-steel ground screw. The located in a brightly lit and isolated room. The floor of recording electrodes consisted of an Epoxylite-coated each chamber consisted of 18 stainless steel rods (4 mmstainless steel insect pin (size 00) with a 50- m m uninsu- diameter) spaced 1.5 cm apart (center to center). The rodslated tip. The bipolar stimulating electrode consisted of were wired to a shock generator and scrambler (Lafayettetwo Epoxylite-coated insect pins with 500- m m tips sepa- Instrument Co.) for the delivery of the footshock USs.rated by 1 mm. The recording electrode was implanted in Each chamber rested on a load-cell platform and wasthe dentate gyrus (3.3 mm posterior to bregma and 2.4 mm connected to a computer that monitored each animal’slateral to bregma) and the stimulating electrode was motor activity. The chambers were cleaned with 5%implanted in the ipsilateral perforant path (8.1 mm poste- ammonium hydroxide solution, and stainless steel pansrior to bregma, 4.4 mm lateral to bregma). The position of containing a thin film of the same solution were placedthe recording electrode was optimized by using the laminar underneath the grid floors before the rats were placedprofile of the perforant path-evoked field potentials as a inside. Ventilation fans in each chest supplied backgroundguide. Body temperature was maintained at 37 8 C with a noise (70 dB, A scale).heating pad. On the conditioning day, the rats were transported to theExtracellular field potentials evoked by biphasic per- laboratory and placed in the conditioning chambers inforant path stimulation (100- m s/cycle) were amplified squads of eight rats; the chamber positions were counter-(gain 5 100), bandpass filtered (1 Hz–9 kHz), displayed on balanced for each squad and group. Three minutes afteran oscilloscope, digitized and written to a disk by a placement in the chambers, the rats received three foot-computer (DataWave Technologies, Longmont, CO). The shocks (2 s, 1.0 mA) with 60-s intershock intervals. Sixtyperforant path-evoked field potentials in the dentate gyrus seconds following the final shock, the rats were returned toconsisted of a characteristic gradual positive-going field their home cages.excitatory postsynaptic potential (EPSP) with a sharp Freezing was assessed on the conditioning day duringnegative population spike (PS) superimposed on the rising the 60-s periods following each of the three footshocksphase of the EPSP (see Fig. 3A). The population EPSP (i.e. immediate postshock freezing), and normalized byreflects synaptic currents at perforant path-dentate granule subtracting pre-shock freezing [6]. The extinction test wascell synapses in  stratum moleculare , whereas the PS repeated for 4 consecutive days. In all cases, freezing wasreflects the synchronous action potential discharge of assessed using a computerized system as described bygranule cell bodies in  stratum granulosum . Maren [38]. The freezing data were transformed to aAfter a stable hilar field potential was maintained for 10 percentage of total observations, a probability estimate thatmin, perforant path-evoked field potentials (stimulation allows for analysis with parametric statistics. These prob-current, 140–625  m A, adjusted to elicit a 1-mV population ability estimates of freezing were analyzed using ANOVA.spike) were sampled every 20 s during a 10-min period If a significant omnibus  F  -ratio was found, then post-hocbefore and a 60-min period after perforant path high- comparisons in the form of Fisher LSD tests were per-frequency stimulation (HFS). HFS consisted of 10 pairs of formed. All data are presented as means 6 S.E.M.s.400-Hz bursts (burst duration 5 25 ms, 10 pulses per burst,200 ms interburst interval) delivered at the same currentused for baseline recording. Each pair of bursts was  3. Results separated by 10 s. Averages of the perforant path-evokedfield potentials were computed for each 10-min block of   3.1.  Experiment   1:   ovariectomy eliminates the sexual the 70-min recording period. The percentage of change in  dimorphism in contextual fear conditioning the EPSP slope and the PS amplitude was measured fromthe pre-HFS baseline and the last 10-min block of the One rat died during surgery and five rats were excluded   R .  R .  Gupta et al .  /   Brain Research  888 (2001) 356  – 365   359 due to incomplete ovariectomy, leaving the following main effect of day ( F  (3,84) 5 37.3,  P , 0.0001) and agroup memberships: sham-males ( n 5 12), sham-females significant group 3 day interaction ( F  (6,84) 5 3.0,  P , 0.05).( n 5 11), and ovariectomized females ( n 5 8). Freezing on Ovariectomized female rats extinguished at the same ratethe conditioning day was averaged across the three post- as males, and both of these groups extinguished moreshock periods and is shown in Fig. 1A. All groups slowly than sham-operated females.exhibited immediate postshock freezing and the level of These data suggest that sham-operated female ratspostshock freezing was not different between the groups. exhibit a selective deficit in long-term memory for con-This observation was confirmed in an ANOVA by a non- textual fear conditioning, insofar as their short-term mem-significant main effect of group ( F  (2,28) , 1,  P 5 0.55). ory for contextual fear was intact on the conditioning day.Thus, ovariectomy did not affect either the performance of Alternatively, it is possible that intact females exhibit morethe freezing response or the short-term memory for con- rapid extinction of an equally strong long-term memory.textual fear conditioning. As discussed below, we favor the former interpretation inFreezing to the context of the conditioning chamber the light of our previous results [6,43].We did not examineduring the 8-min extinction tests conducted on 4 consecu- the relationship between estrous cyclicity in the intacttive test days is shown in Fig. 1B. For clarity, the 8-min females and contextual fear conditioning [44]. However, asmeans for each of the three groups are shown. All of the we have previously reported [43], intact female rats exhibitgroups exhibited robust conditional freezing on the first contextual freezing deficits (relative to males) when fearday of extinction testing. However, the sham-operated conditioning and testing occur randomly with respect tofemale group extinguished their fear more rapidly than the the estrous cycle.sham-operated male and ovariectomized females. This The present data replicate and extend those of Maren eteffect is apparent on days 3 and 4. A two-way ANOVA al., who showed that male rats exhibit higher levels of with factors of test day and group revealed a significant freezing than randomly cycling female rats after contextualfear conditioning [43]. In Maren et al., however, the sexdifference in conditional freezing was detected during thefirst 8-min extinction test [6,43], whereas it was notapparent until the third extinction test in the present study.It is likely that a ceiling effect contributed to this pattern of results, insofar as conditional freezing during the first twoextinction tests was near asymptote. In addition to replicat-ing previous studies [6,43], these data demonstrate thatcirculating ovarian steroids regulate fear conditioning inadult female rats. Indeed, ovarian steroids appear to exertan inhibitory influence on contextual fear conditioning,insofar as ovariectomy increased conditional freezingduring extinction testing. These data are consistent withthose from Markus and Zecevic, who showed that con-textual fear conditioning in females varied across theestrous cycle [44]. In this study, fear conditioning wasmaximal during estrus, when estrogen levels are relativelylow. Therefore, it seems likely that ovariectomy facilitatedcontextual fear conditioning in our experiments because iteliminated the inhibitory effect of estrogen on this form of learning. This hypothesis was tested in experiment 2. 3.2.  Experiment   2:   estrogen treatment attenuatescontextual fear conditioning in ovariectomized rats Two rats died during surgery and 15 rats were excludeddue to incomplete ovariectomies leaving the followinggroup memberships: oil ( n 5 6) and estrogen ( n 5 6). Freez- Fig. 1. (A) Mean ( 6 S.E.M.) percentage of freezing on the conditioning  ing on the conditioning day is shown in Fig. 2A. Both day in female rats that received sham surgery (filled), female rats that groups of rats exhibited robust immediate postshock  were ovariectomized (open), and male rats (hatched). The values were freezing, and the level of freezing did not differ between normalized to pre-shock freezing and averages across the three 1-min the groups. This observation was confirmed in an ANOVA post-shock periods. (B) Mean ( 1 S.E.M.) percentage of freezing collapsed by a non-significant main effect of group ( F  (1,10) , 1, across the 8-min of the context extinction test over the 4 consecutive daysof testing for the groups described in (A).  P 5 0.96). Thus, estrogen treatment did not affect either the
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