421Acta Physiologica Sinica, August 25, 2005, 57 (4): 421-432http://www.actaps.com.cn

Research Paper

Received 2005-03-03 Accepted 2005-05-09This work was supported by the EJLB-CIHR Michael Smith Chair in Neurosciences and Mental Health, Canadian Research Chair, and

NIH NINDS NS42722. *Corresponding author. Tel: +1-416-9784018; E-mail: min.zhuo@utoronto.ca **Contributed equally to this work.

Transcription factor Egr-1 is required for long-term fear memory and anxiety

Shanelle W. Ko1,**, AO Hu-Shan1,**, Amelia Gallitano-Mendel2, QIU Chang-Shen1, WEI Feng1, JeffreyMilbrandt2, ZHUO Min1,*

1Department of Physiology, University of Toronto, Faculty of Medicine, University of Toronto Centre for the Study of Pain,Medical Science Building, 1 King’s College Circle, Toronto, Ontario, M5S 1A8, Canada; 2Washington University School ofMedicine, Departments of Pathology and Psychiatry, St. Louis, MO 63110, USA

Abstract: The zinc finger transcription factor Egr-1 is critical for coupling extracellular signals to changes in cellular gene expression.In the hippocampus and amygdala, two major central regions for memory formation and storage, Egr-1 is up-regulated by long-termpotentiation (LTP) and learning paradigms. Using Egr-1 knockout mice, we showed that Egr-1 was selectively required for lateauditory fear memory while short term, trace and contextual memory were not affected. Additionally, synaptic potentiation induced bytheta burst stimulation in the amygdala and auditory cortex was significantly reduced or blocked in Egr-1 knockout mice. Our studysuggests that the transcription factor Egr-1 plays a selective role in late auditory fear memory.

Key words: Egr-1; long-term potentiation; fear memory; amygdala; auditory cortex

转录因子 Egr-1参与长期性恐惧记忆和焦虑

Shanelle W. Ko1,**，敖虎山1,**，Amelia Gallitano-Mendel2，邱长申1，魏 峰1，Jeffrey Milbrandt2，

卓 敏 1,*

1多伦多大学医学院生理系，多伦多大学痛觉研究中心，多伦多，安大略省 M5S1A8，加拿大；2华盛顿大学医学院病

理和精神病系，圣路易丝，密苏里州 63110，美国

摘 要：锌指转录因子 Egr-1在将细胞外信号和胞内基因表达的变化相耦联过程中发挥重要的作用。海马和杏仁体是记忆形成和储存的两个主要的脑区。在海马和杏仁体中，Egr-1可被长时程增强(long-term potentiation, LTP)和学习过程上调。在 Egr-1敲除小鼠上观察到晚时相声音恐惧记忆受损，而短时的痕迹和场景记忆却不受影响；另外，在 Egr-1敲除小鼠上，用 thetaburst刺激杏仁体和听觉皮层所引起的突触增强被明显减弱或完全阻断。因此，我们的研究表明，转录因子 Egr-1选择性地在晚时相听觉恐惧记忆中发挥作用。

关键词：E g r - 1；长时程增强；恐惧记忆；杏仁体；听觉皮层中图分类号：Q426; Q427; R338.64

Emotional learning and its expression in mammals dependson activity-dependent plasticity in higher brain structuresincluding the amygdala, hippocampus and related corticalareas[1-5]. Long-term changes in synaptic transmission, alsocalled long-term potentiation (LTP), have been predomi-nantly studied in brain slice preparations and are thought to

be required for the establishment and consolidation of fearmemory[3,6]. Two different temporal phases of synapticLTP have been reported in the hippocampus and amygdala:short-term potentiation requires rapid signaling in syn-apses[7-9] while late phases of LTP require gene activationand new protein synthesis[10,11]. Supporting the role of the

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late phase of LTP in long-term memory, inhibition of pro-tein synthesis has been shown to affect long-term memory[12-15].

The cyclic AMP-response element binding protein (CREB)is a major transcription factor associated with long-termmemory[16-21]. CREB in hippocampal neurons can be acti-vated by physiological learning and artificial high-frequencytetanic stimulation[16]. Activation of NMDA receptors and/or L-type voltage-gated calcium channels (VDCCs) leadsto activation of CREB in hippocampal neurons[10,22]. Nu-merous studies elucidate CREB’s role in memory. Inhibi-tion of CREB activity by blockade of its upstream signal-ing pathways, inactivation by antisense oligonucleotides,or genetic deletion, reduces or blocks late-phase LTP andproduces deficits in long term memory[16,20,21,23]. Other stud-ies show that CREB expression is important for memoryof fear associations and taste aversion[17,18]. Similarly, theoverexpression of CREB was reported to facilitate long-term fear memory[24,25]. Recent studies show that a signal-ing pathway consisting of mitogen activated protein kinase(MAPK), CREB, and the transcription factor Egr-1 maybe important for long term memory and associated synap-tic plasticity[20].

The zinc finger transcription factor Egr-1 (also calledNGFI-A, Krox24, or zif/268) is critical for coupling extra-cellular signals to changes in cellular gene expression[26-28].EGR-1 mRNA and protein are expressed in the neocortex,hippocampus, entorhinal cortex, amygdala, striatum andcerebellum[29-31]. The upstream promoter region of Egr-1contains binding sites for cyclic AMP-response elements(CRE), suggesting that Egr-1 may act downstream fromthe CREB pathway[20,32]. In the hippocampus, Egr-1 is up-regulated by tetanic stimulation[33-36] and during learning ormemory retrieval[19,37-40]. Deletion of Egr-1 leads to a re-duction or blockade of hippocampal LTP in the CA1 re-gion and dendate gyrus[41,42], as well as an impairment inlong term spatial memory[42]. A role for Egr-1 in the amygdalahas been reported in the acquisition[43] or recall[19,44] of con-textual fear memory. Long-term fear memory induced byfear conditioning triggered the NMDA receptor-dependentactivation of Egr-1 in neurons found in the amygdala[43,45,46].Two recent reports highlight the importance of Egr-1 inremote memory and the reconsolidation fear memory[44,47].An impairment in the reconsolidation of contextual fearmemory was reported when antisense oligodeoxynucleotides for Egr-1 were infused into the hippocampus ofrats[47]. In another study, Egr-1 expression was increasedin the anterior cingulate cortex of mice upon re-exposureto the chamber where they had received footshocks 36 d

prior[44]. While both studies reinforce the idea the Egr-1 isan important player in the retention of contextual fearmemory, the role of Egr-1 in auditory fear memory hasnot been investigated. In addition, no study has reportedthe role of Egr-1 in contextual and auditory fear memoryusing Egr-1 knockout mice.

In the present study, we use Egr-1 knockout mice toinvestigate the contribution of Egr-1 to different types offear memory. First, we studied two forms of associativefear memory: contextual and auditory fear conditioning.Contextual fear conditioning is thought to be hippocam-pus-dependent, while auditory fear conditioning isamygdala-dependent[48]; but see reference[49]. We then in-vestigated the role of Egr-1 in trace memory[50] as well asin the extinction of fear memory. Although there were nodifferences in response to contextual and trace memory orin the extinction of fear memory, there was a significantdecrease in fear responses during late auditory fear memoryin Egr-1 knockout versus wild-type mice. Our results sup-port a selective role for Egr-1 in the processing of long-term auditory fear memory.

1 MATERIALS AND METHODS

1.1 Animals and treatmentAdult male mice (wild-type and mutant Egr-1 mice gener-ated by Dr. J. Milbrandt) were used. Wild-type and ho-mozygous mutant Egr-1 mice were obtained by crossingheterozygous mutant mice bearing a targeted mutation ofthe Egr-1 gene. Genotypes were determined by PCR analy-sis[51] of genomic DNA extracted from mouse ear tissue.Mice were maintained in a C57BL/6 strain background andwere age-matched in each experiment. Wild-type and mu-tant mice were well groomed and showed no signs of ab-normality or any obvious motor defects. No indication oftremor, seizure or ataxia was observed. As it was impos-sible to visually distinguish mutant mice from wild-typemice, experimenters were blind to the genotype. The Ani-mal Care and Use Committees at Washington Universityand the University of Toronto approved the experimentalprotocols.1.2 Fear conditioningThe following experiments were performed in a condi-tioning shock chamber (30.5 cm× 24.1 cm× 21.0 cm)(Med Associates, Georgia, Vermont). Mice were allowedto habituate to the chamber for 2 min before fearconditioning. The conditioned stimulus (CS) used was an85 dB tone at 2 800 Hz for 30 s, and the unconditioned

423Shanelle Ko et al: Defects in Late Auditory Fear Memory and Anxiety in Egr-1 Knockout Mice

stimulus (US) was a continuous scrambled foot shock at0.75 mA for 2 s. During training, mice were presented witha 30 s tone (CS) and a shock (US) starting 28 s after theonset of the CS. Three CS/US pairings were delivered dur-ing multi-shock conditioning, while only one was used forsingle shock experiments. After the CS/US pairing, micewere allowed to stay in the chamber for an additional 30 sfor the measurement of immediate freezing. Freezing wasscored manually every 10 s. For contextual memory, eachmouse was placed back into the shock chamber and thefreezing response was recorded for 3 min. For auditoryfear memory, the mice were put into a novel chamber(different floor, ceiling and walls) and monitored for 3 minbefore the onset of a tone identical to the CS, which wasdelivered for 3 min, and freezing responses were recorded.Contextual and auditory fear memory was measured 1 h,1, 3, 7, and 14 d after training for all animals. To measuretrace memory, we used a trace fear conditioning paradigmas described[50]. For this paradigm, the US was delivered30 s after the end of the CS (trace) and mice were sub-jected to three training trails. To test the extinction of fearmemory, mice were trained with three shock-tone pair-ings and fear responses were measured during five trails at1 h intervals in both the context where they had receivedthe shock-tone pairing and in a novel chamber before andafter the onset of the tone (CS). The percentage change infear memory was normalized to control responses.1.3 Elevated plus mazeThe elevated plus maze (Med Associates, Georgia, Vermont)consists of two open arms and two closed arms situatedopposite each other and separated by a 6cm square centerplatform. Each runway is 6 cm wide and 35 cm long. Theopen arms have lips that are 0.5 cm high and the closedarms are surrounded on three sides by 20 cm walls. Thefloors and walls are black polypropylene and the floors are75 cm from the ground. For each test, the animal is placedin the center square and allowed to move freely for 5 min.The number of entries and time spent in each arm isrecorded.1.4 Open field activityTo record horizontal locomotor activity we used the Activ-ity Monitor system from Med Associates (43.2 cm× 43.2cm× 30.5 cm) (Med Associates, St. Albans, VT). Briefly,this system uses paired sets of photo beams to detectmovement in the open field and movement is recorded asbeam breaks. The open field is placed inside an isolationchamber with dim illumination and a fan. Each subject wasplaced in the center of the open field and activity was mea-

sured for 30 min.1.5 Slice electrophysiologyMice were anesthetized with halothane and transverse slicesof amygdala and cortex were rapidly prepared and main-tained in an interface chamber at 30°C, where they weresubfused with artificial cerebrospinal fluid (ACSF) con-sisting of (mmol/L) NaCl 124, CaCl2 4.4, MgSO4 2.0,NaHCO3 25, Na2HPO4 1.0, glucose 10, and bubbled with95% O2 and 5% CO2. In all experiments, slices recoveredin the chamber for at least 2 h before recording. In amygdalaslices, a bipolar tungsten stimulating electrode was placedin the ventral striatum, and an extracellular recording elec-trode (3~12 MΩ filled with ACSF) was placed in the lat-eral amygdala. In cortical slices, a bipolar tungsten stimu-lating electrode was placed in layer Ⅴ, and extracellularfield potentials were recorded using a glass microelectrodeplaced in layer Ⅱ /Ⅲ. Synaptic responses were elicited at0.02 Hz. For inducing LTP, we used five trains of theta-burst stimulation (TBS) at the same intensity of testingstimulation (each train contains four pulses at 100 Hz; de-livered at 200 ms interval). We found that this protocolinduced reliable LTP in the auditory cortex and amygdala(see Results).1.6 ImmunocytochemistryMice were deeply anesthetized with sodium pentobarbital(50 mg/kg) and transcardially perfused with heparinizedsaline (100 000 IU/L heparin, 0.1 mol/L PBS; 0.9% NaCl)followed by 4% paraformaldehyde in 0.1 mol/L PBS, pH7.4. Brains were removed and stored in the same fixativeovernight at 4°C, then cryopreserved in 30% sucrose inPBS buffer. Slices (14 µm) from frozen sections of theentire brain were cut. The primary antibodies used in thisstudy were directed against the following antigens (usingthe stated dilutions): astrocytes [glial fibrillary acidic pro-tein (GFAP) rabbit polyclonal, 1:4; Incstar, Stillwater,Minn.]; neurons [neuronal nuclear antigen (NeuN) mIgG1,1:500]; Species-specific secondary antibodies (1:200dilution) were conjugated to Cy3, fluorescein isothiocyanate(Jackson Immunoresearch, West Grove, Penn.) or Alexa488 (1:200 dilution; Molecular Probes). The samples re-ceiving Hematoxylin-eosin were embedded in paraffin, cutin sections 4 µm thick and stained.1.7 Data analysisResults were expressed as means ± SEM. Statistical com-parisons were made with one- or two-way analysis of vari-ance (ANOVA) with the Student-Newmann-Keuls test usedfor post hoc comparisons. In all cases, P<0.05 was con-sidered statistically significant.

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2 RESULTS

2.1 Anatomy of memory-related central regions inEgr-1 knockout miceIn general, Egr-1 knockout mice are visually indistinguish-able from wild-type littermates. To determine whether Egr-1knockout mice have neuroanatomic abnormalities in cen-tral regions related to sensory transmission and fearmemory, we carried out histochemical experiments in sev-eral brain areas. Analysis of serial coronal sections, exam-

ined by light microscopy, showed no detectable morpho-logical differences in the auditory cortex, amygdala andhippocampus. Higher magnification of the stained sectionsfurther demonstrated no apparent differences in the num-ber and distribution of cells in these areas ( Fig.1). A recentstudy in the hippocampus showed that both neuronal andglial cell populations were not affected in Egr-1 knockoutmice[42]. We wanted to confirm this finding in other centralareas such as the amygdala. As shown in Fig.2, we foundno difference in neuronal population and distribution be-

Fig. 2. Neuronal and glial staining of wild-type and Egr-1 knockout mice. Representative high-power sections of Anti-GFAP and Anti-NeuNepifluorescence from Egr-1 knockout and wild-type mice showed no detectable differences in the hippocampus, amygdala and auditorycortex. Scale bar, 20 µm.

Fig. 1. Brain morphology of wild-type and Egr-1 knockout mice. Representative coronal sections of brain and spinal cord from Egr-1 knockoutand wild-type mice show no detectable morphological differences in the hippocampus, amygdala and auditory cortex. Scale bar, 250 µm.

425Shanelle Ko et al: Defects in Late Auditory Fear Memory and Anxiety in Egr-1 Knockout Mice

tween Egr-1 knockout and wild-type mice. We also usedGFAP as a marker of glial cells. GFAP staining demon-strated that glial cells were similar between Egr-1 knock-out and wild-type mice.2.2 Contextual and auditory fear memoryPrevious studies show that fear conditioning activates Egr-1in the amygdala, a structure critical for fear memory.However, no study has reported a change in short or longterm fear memory in mice lacking Egr-1. We assessedtwo forms of associative emotional memory in wild-typeand Egr-1 knockout mice: contextual and auditory fearconditioning. Preliminary studies in wild-type mice showthat three shock-tone pairings produced long-term fearmemory that lasted for at least 2 weeks after conditioning(Fig.3, n=6). Pairing the tone with a single shock resultedin fear memory that lasted for about 1~3 d after training(Fig.4, n=6). We next measured fear responses from Egr-1knockout mice during contextual and auditory condition-ing 1 h, 1 d, 1 and 2 weeks after receiving multiple shock-tone pairings (Fig.3). As shown in Fig.3A, there was asignificant reduction in the freezing response during audi-tory conditioning in Egr-1 knockout mice as comparedto wild-type mice [F(1, 50)=19.5, P<0.001] (n=6 for

Fig. 3. Egr-1 required for late auditory fear memory induced by multiple shocks. A, B: Auditory and contexual fear memory induced by three-foot shock conditionings 1 h, 1 d, 3 d, 1 week and 2 weeks after training (wild-type, n=6; Egr-1 knockout, n=6). *P <0.05.

Fig. 4. Egr-1 is not required for fear memory induced by a single shock. Contexual and auditory fear memory induced by a single foot shockconditioning 1 h, 1 d and 7 d after training (wild-type, n=5; Egr-1 knockout, n=5).

each group). Post hoc analysis revealed that the reductionwas selective for late responses (P<0.01 for day 3, P<0.005 for 1 week and P<0.01 for 2 weeks), while earlyresponses (P=0.6 for 1 h and P=0.4 for 1 d) were notsignificantly different between wild-type and mutant mice.Unlike auditory fear memory, contextual memory was notsignificantly different between genotypes [F(1,50)=3.5,P=0.07](Fig.3B). This finding indicates that Egr-1 prefer-entially contributes to late auditory fear memory.

Since the defect in fear memory was not apparent untillater time points after training with multiple shocks, wewanted to test if fear memory induced by a single shock-tone pairing would also be altered. Neither contextual norauditory fear memory was significantly different in Egr-1knockout mice when compared to wild-type mice [F(1,24)=0.3, P=0.6, for context; F(1,24)=0.8, P=0.4, forauditory] (n=5, Fig.4). Both knockout and wild-type miceshowed a significant reduction in the freezing response by7 d after training (P<0.05 for both, 1 h vs 7 d, Fig.4B).This indicates that Egr-1 may not contribute to fear memoryinduced by a single shock-tone pairing.2.3 Trace fear memoryTo investigate if Egr-1 is truly selective for amygdalar (vs

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hippocampal) fear memory, we tested Egr-1 knockout andwild-type mice in the “trace” memory paradigm. Tracememory has recently been shown to be hippocampal-dependent, NMDA receptors in the hippocampus wereshown to be important for the formation of memories thatassociate events across time[50]. Previous studies showedthat Egr-1 contributes to NMDA receptor dependent hip-pocampal synaptic LTP in the CA1 and dentate gyrus re-gions[41,42]. Therefore, Egr-1 may contribute to fear memoryinduced by the hippocampal-dependent, trace fear-condi-tioning paradigm. We found significant fear memory in-duced by this ‘trace’ training paradigm in wild-type mice[compare (3.3±1.6) % freezing before training to (71.7±6.9)%freezing 1 d after training, P<0.001, n=5, Fig.5]. Threedays after training, the freezing responses of mice trainedunder the trace paradigm were comparable to those whohad received multiple paired training [compare (59.3±8.4)%freezing with (58.9+11.8)% freezing after trace condition-ing on day 3]. We found no significant difference in con-textual trace fear memory between wild-type and Egr-1knockout mice (n=7) [F(1,50)=1.5, P=0.2] 1, 3 d, 1 and2 weeks after training. Two weeks after training, bothgroups retained good trace fear memory [compare (52.8±5.6)% vs (54.8±6.2)% freezing, wild-type vs knockouton day 14]. This finding clearly indicates that Egr-1 is notrequired for trace fear memory.

2.4 Fear extinctionFear extinction is an active learning process that occurs

when a conditioned stimulus is repeatedly nonreinforced[52].To determine if Egr-1 knockout mice display significantchanges in the extinction of contextual and auditory fearmemory, we measured fear responses for five trails at 1 hintervals. Mice were trained with three shock-tone pair-ings as described in Fig.3. We normalized the percentagechange in fear memory to control responses and found nosignificant difference between Egr-1 knockout (n=5) andwild-type mice (n=6) in the extinction of either contextualor auditory fear memory [F(1,45)=1.4, P=0.2 for context,F(1,45), P=2.0 for auditory] (Fig.6). These results sug-gest that the deficit in long-term auditory fear memory isnot due to extinction of the fear response but due to re-peated testing.

2.5 Elevated plus maze and open field activityTo determine if anxiety-related behaviors were altered inEgr-1 knockout mice, we measured their performance inthe elevated-plus maze. The number of entrances and totaltime spent in the open arms were used as indicators ofanxiety-like behavior. As shown in Fig.7A, Egr-1 knock-out mice showed a significant increase in the total numberof entrances into the open arms (n=6) as compared withwild-type mice [n=6, t(11)=1.82, P<0.05]. Additionally,

Fig. 5. Egr-1 is not required for trace fear memory. Auditory (A) andcontexual (B) trace fear memory measured 1 h, 1 d, 3 d, 1 week and 2weeks after training showed no detectable difference between wild-type (n=5) and Egr-1 knockout mice (n=7).

Fig. 6. Deletion of Egr-1 did not affect fear extinction. Wild-typeand Egr-1 knockout mice showed similar rates of extinction for bothcontextual and auditory fear memory when tested repeatedly acrossfive trails (wild-type, n=6; Egr-1 knockout, n=5).

427Shanelle Ko et al: Defects in Late Auditory Fear Memory and Anxiety in Egr-1 Knockout Mice

the total time spent in the open arms was significantly in-creased in Egr-1 knockout mice, as compared with wild-type mice [t (11) = 2.68, P<0.05, Fig.7B]. However, thetotal entrances into the open arms and closed arms werenot significantly different between wild-type and Egr-1knockout mice, indicating that this finding is not simply aresult of general hyperactivity. We also evaluated the gen-eral locomotor behavior of both wild-type and Egr-1 knock-out mice in a novel open field (n=6 for each group). Therewas no difference between Egr-1 knockout and wild-typemice in the total distance traveled and the mean movingvelocity, indicating that the deletion of Egr-1 did not affectgeneral locomotor behavior (Fig.7C, D). However, the timespent ambulatory and ambulatory counts in Egr-1 knock-out mice were significantly smaller than that of wild-type

mice [t (10) = 1.87, P<0.05; t (10) = 1.81, P<0.05, Fig.7E].2.6 Fear memory-related synaptic plasticitySynaptic plasticity, such as long-term potentiation (LTP),is thought to be important for learning and memory. Herewe wanted to examine synaptic potentiation in theamygdala, a structure known to play an important role infear memory[53,54]. We examined synaptic potentiation at‘thalamic’ input synapses to the lateral amygdala by plac-ing a stimulating electrode in the ventral striatum[55]. Basedon our previous studies[56], we used five trains of thetaburst stimulation (TBS). In slices of wild-type mice, TBSinduced significant synaptic potentiation [(n=9 slices/8mice; (169.4 ± 8.0)%; Fig.8B]. However, synaptic poten-tiation in slices of Egr-1 knockout mice was significantlyreduced or blocked [n=6 slices/6 mice; (124.1±14.1)%,

Fig. 7. Elevated plus maze and open field locomotor activity for Egr-1 knockout and wild-type mice. A, B: Percent entries and percent of timespent in the open arms of the elevated plus maze (Egr-1 knockout, n=6; wild-type, n=6). C: Distance traveled in the open field over 30 minpresented in 5 min blocks (wild-type, n=6; Egr-1 knockout, n=6 mice). D: Total distance traveled in 30 min. E: Average moving velocity over30 min, total time spent ambulatory, and the total number of beam breaks while ambulatory. *P<0.05 vs wild-type group.

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t(13)=2.4, P<0.05 compared with slices of wild-type mice].We also examined synaptic potentiation in the auditorycortex. In slices of both wild-type and Egr-1 knockoutmice, similar baseline field EPSPs were induced by localstimulation (see samples in Fig.8). In the auditory cortex,TBS induced a long-lasting enhancement of synaptic re-sponses persisting for at least 40 min after induction [n=7slices/6 mice, (150.2±12.6)%]. In Egr-1 knockout mice,however, synaptic potentiation was abolished [n=8slices/8 mice, (81.9±10.0)%; P<0.05 compared with po-tentiation in wild-type mice] (Fig.8D). These results pro-vide strong evidence that Egr-1 contributes to synapticpotentiation in these cortical areas, and that Egr-1 contrib-utes to plasticity in both the amygdala as well as the audi-tory cortex.

3 DISCUSSION

Our results provide evidence for a role of Egr-1 in anxiety-related behavior and long-term auditory fear memory. Thesefindings are in good accord with previous reports that showEgr-1 was activated by associative fear conditioning[57].More interestingly, Egr-1 plays a selective role in late, butnot early, behavioral responses to auditory fear memory.Early auditory fear memory, measured 1 h or 1 d afterconditioning, was not affected in Egr-1 knockout micewhile fear responses were significantly decreased starting3 d after conditioning (Fig.3). Fear memory generated bythe single shock conditioning paradigm and contextualmemory were similar between knockout and wild-type mice(Fig.3 and 4). Additionally, Egr-1 knockout mice did not

Fig. 8. Requirement of Egr-1 for amygdala and cortical synaptic potentiation. A: Diagram depicting the location of stimulating and recordingelectrodes in the amygdala. B: TBS (indicated by the arrow) induced synaptic potentiation in the amygdala in wild-type (n=11 slices/9 mice)but not Egr-1 knockout mice (n=6 slices/6 mice). Insets: representative records of the EPSP before (Pre) and 40 min after (Post) TBS in a wild-type and Egr-1 knockout slices. C: Diagram depicting the location of stimulating and recording electrodes in the auditory cortex. D: Egr-1 isalso required for potentiation in the auditory cortex by TBS (wild-type, n=7 slices/5 mice; Egr-1 knockout mice, n=6 slices/6 mice). Insets:representative records of the EPSP before (Pre) and 40 min after (Post) TBS in a wild-type and Egr-1 knockout slices.

429Shanelle Ko et al: Defects in Late Auditory Fear Memory and Anxiety in Egr-1 Knockout Mice

differ from wild-type mice in fear memory using the “trace”memory paradigm[50](Fig.5). Egr-1 knockout mice showedno significant defects in several sensory and motor tests;such as acute pain threshold measurements[58] and open fieldmotor activity (Fig.7), therefore we argue that changes infear memory are unlikely to be due to general developmen-tal defects. Due to the use of Egr-1 knockout mice with-out regional selectivity, it is not possible for us to dissectthe role of Egr-1 in particular brain regions. Different partsof the amygdala have been shown to contribute to behav-ioral fear responses (e.g., lateral vs basal amygdala)[3,59,60].

In the hippocampus, Egr-1 is up-regulated by tetanicstimulation[33-36], behavioral learning, or memory re-trieval[37-39]. Deletion of Egr-1 leads to a reduction or block-ade of hippocampal LTP in the CA1 region and dendategyrus[41,42], as well as an impairment of spatial memory[42].Based on these results, the genetic deletion of Egr-1 shouldat least reduce hippocampus-dependent memory tasks suchas trace fear memory and contextual fear memory. In thepresent study, however, we found that contextual fearmemory and trace fear memory are intact in Egr-1 knock-out mice, although late auditory fear memories were sig-nificantly reduced. There are at least a few possible expla-nations for our findings. First, the functional role of Egr-1in hippocampus-dependent memory may be compensatedby alternative signaling pathways. Future studies using in-ducible knockout mice for Egr-1 may help to test thispossibility. Second, it is possible that hippocampal-depen-dent functions may be compensated by neuronal interac-tions between the hippocampus and its related central struc-tures or simply by other central nuclei in Egr-1 knockoutmice.

Electrophysiological results from in vitro brain slicesshowed that synaptic potentiation induced by multiple thetaburst stimulation was significantly reduced in Egr-1 knock-out mice, suggesting the possible contribution of Egr-1 tomemory related synaptic potentiation[41]. Since it has beennoted that significant and measurable levels of Egr-1 aredetected after LTP-inducing stimulation, this suggests thatEgr-1 may contribute, either directly or indirectly, to sig-naling pathways involved in synaptic potentiation. Althoughthere was no difference in early synaptic potentiation inthe hippocampus[41], Egr-1 deletion affected early synapticpotentiation induced by theta burst stimulation in theamygdala. Future studies are clearly needed to investigatethe synaptic mechanism for Egr-1 in early LTP (45~60min).

Many studies focus on the interaction between early syn-

aptic potentiation and long-term fear memory. These pro-cesses are obviously mediated by different time courses.In the case of synaptic plasticity in brain slices, most LTPlasts from several minutes to hours. For fear memory,behavioral responses usually last from days to weeks. It islikely that synaptic LTP in the amygdala and/or hippocam-pus contributes to the formation or induction of fear memoryduring early time periods, while late-phase plastic changes,including possible changes in cortical areas, may contrib-ute to long-term storage of memory. Based on our presentfindings related to synaptic potentiation in the amygdala,we propose that defects in early potentiation may contrib-ute to the early formation of long-term fear memory.Changes in potentiation in cortical areas, including the ACC,may contribute to long-term fear memory. We believe thatfuture studies are needed to investigate detailed synapticmechanisms for the loss of LTP in Egr-1 knockout mice.Since both NMDA receptors and L-type calcium channelsare reported to contribute to synaptic plasticity[61], it is ofobvious importance for future studies to determine if Egr-1may regulate the activity of NMDA receptors and L-typecalcium channels in central neurons.

It has been widely reported that many physiological andpathological stimuli are able to activate immediate earlygenes in central nuclei. While the exact physiological func-tions of these immediate early genes remain to be investigated,activation of immediate early genes has proven to be use-ful for studying activity-triggered plasticity in the centralnervous system. Since selective pharmacological antago-nists for immediate early genes are not available, geneti-cally manipulated mice become a good tool to investigatetheir physiological roles. CREB is probably the most thor-oughly studied immediate early gene. The role of CREB insynaptic plasticity and its related physiological and patho-logical changes have been reported[16]. In the present study,we found that Egr-1 plays a selective role in the late-formof auditory fear memory. Furthermore, we show that Egr-1knockout mice have reduced anxiety-like behavior in theelevated plus maze paradigm. This suggests that Egr-1 isan essential component in the pathway from sensory inputand interpretation of a fear-producing situation, to the mani-festation of an emotional response.

Our findings that Egr-1 knockout mice have intact shortterm fear memory agrees with numerous studies that re-port a selective role for both Egr-1 and CREB in the latephase of LTP or learning and memory[16,20,21,42]. A recentstudy using an Egr-1 antisense oligonucleotide to selec-tively knockout down expression in the hippocampus of

Acta Physiologica Sinica, August 25, 2005, 57 (4): 421-432430

rats reported that while Egr-1 was required for thereconsolidation of contextual memory, knockdown of Egr-1before conditioning had no affect on behavioral responses24 h later[47]. Additionally, levels of Egr-1 expression in theanterior cingulate cortex were increased after exposure tothe context 36 d, but not 1 d, after receiving multiplefootshocks[44]. Results from these and the present studysuggest a role for Egr-1 in the reconsolidation of long-term memory.

In the present series of experiments, we employed theuse of background contextual conditioning, i.e. the tone(conditioned stimulus) was the primary cue while the con-text remained static, as opposed to foreground condition-ing which occurs in the absence of a conditioned stimulusso that the static contextual cues are brought into the fore-ground by becoming associated with the shock(unconditioned stiumulus)[62]. Studies have shown that fore-ground contextual conditioning can activate Egr-1 expres-sion in the amygdala[43,57]. Additionally, lesion studies showthat background contextual conditioning is hippocampusdependent[62]. Since only background contextual condition-ing was performed here, future studies should evaluateforeground conditioning in Egr-1 knockout mice.

Compared to traditional fear memory, the molecularmechanisms for trace memory are less investigated. A re-cent report used a selective hippocampal deletion of NMDAreceptors to show that hippocampal structures are likelyrequired for the formation of trace memory in mice[50].This study shows that trace memory is not affected by thedeletion of Egr-1, while amygdala-dependent auditory fearmemory was significantly reduced. Our results thus pro-vide evidence that different signaling molecules from dif-ferent regions of the central nervous system may contrib-ute to trace vs classical fear memory as proposed previ-ously[50]. In summary, the present study provides evidencethat the immediate early gene Egr-1, which is activated bya fearful conditioning shock, contributes to late auditoryfear memory. These results show that Egr-1, in additionto CREB, may act as an important immediate early gene inemotional fear memory.

* * *ACKNOWLEDGEMENTS: We would like to thank IvanWine for assisting in the design and layout of Fig. 1 and 2.

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