转录因子Egr-1参与长期性恐惧记忆和焦虑

1、Research PaperReceived 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.zhuoutoronto.ca *Contributed equally to this wo

2、rk.Transcription factor Egr-1 is required for long-term fear memory and anxietyShanelle W. Ko 1,*, AO Hu-Shan 1,*, Amelia Gallitano-Mendel 2, QIU Chang-Shen 1, WEI Feng 1, Jeffrey Milbrandt 2, ZHUO Min 1,*1Department of Physiology, University of Toronto, Faculty of Medicine, University of Toronto Ce

3、ntre for the Study of Pain,Medical Science Building, 1 Kings College Circle, Toronto, Ontario, M5S 1A8, Canada; 2Washington University School of Medicine, Departments of Pathology and Psychiatry, St. Louis, MO 63110, USAAbstract: The zinc finger transcription factor Egr-1 is critical for coupling ex

4、tracellular 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-term potentiation (LTP and learning paradigms. Using Egr-1 knockout mice, we showed that Egr-1 was selectively require

5、d for late auditory fear memory while short term, trace and contextual memory were not affected. Additionally, synaptic potentiation induced by theta burst stimulation in the amygdala and auditory cortex was significantly reduced or blocked in Egr-1 knockout mice. Our study suggests that the transcr

6、iption 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. Ko 1,*,敖虎山1,*,Amelia Gallitano-Mendel 2,邱长申1,魏 峰1,Jeffrey Milbrandt 2,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 M5S1A

7、8,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theta burst 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

8、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; R338.64Emotional learning and its expression in mammals depends on activity-dependent plasticity in higher brain structures including the amygdala, hippocampus and related cortical areas 1-5. Long-term changes in synaptic transmission, also ca

9、lled long-term potentiation (LTP, have been predomi-nantly studied in brain slice preparations and are thought tobe required for the establishment and consolidation of fear memory 3,6. Two different temporal phases of synaptic L TP have been reported in the hippocampus and amygdala:short-term potent

10、iation requires rapid signaling in syn-apses 7-9 while late phases of LTP require gene activation and new protein synthesis 10,11. Supporting the role of thelate phase of LTP in long-term memory, inhibition of pro-tein synthesis has been shown to affect long-term memory12-15. The cyclic AMP-response

11、 element binding protein (CREB is a major transcription factor associated with long-term memory16-21. CREB in hippocampal neurons can be acti-vated by physiological learning and artificial high-frequency tetanic stimulation16. Activation of NMDA receptors and/ or L-type voltage-gated calcium channel

12、s (VDCCs leads to activation of CREB in hippocampal neurons10,22. Nu-merous studies elucidate CREBs 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 and prod

13、uces deficits in long term memory16,20,21,23. Other stud-ies show that CREB expression is important for memory of fear associations and taste aversion17,18. Similarly, the overexpression of CREB was reported to facilitate long-term fear memory24,25. Recent studies show that a signal-ing pathway cons

14、isting of mitogen activated protein kinase (MAPK, CREB, and the transcription factor Egr-1 may be important for long term memory and associated synap-tic plasticity20.The zinc finger transcription factor Egr-1 (also called NGFI-A, Krox24, or zif/268 is critical for coupling extra-cellular signals to

15、 changes in cellular gene expression26-28. EGR-1 mRNA and protein are expressed in the neocortex, hippocampus, entorhinal cortex, amygdala, striatum and cerebellum29-31. The upstream promoter region of Egr-1 contains binding sites for cyclic AMP-response elements (CRE, suggesting that Egr-1 may act

16、downstream from the CREB pathway20,32. In the hippocampus, Egr-1 is up-regulated by tetanic stimulation33-36 and during learning or memory retrieval19,37-40. Deletion of Egr-1 leads to a re-duction or blockade of hippocampal LTP in the CA1 re-gion and dendate gyrus41,42, as well as an impairment in

17、long term spatial memory42. A role for Egr-1 in the amygdala has been reported in the acquisition43 or recall19,44 of con-textual fear memory. Long-term fear memory induced by fear conditioning triggered the NMDA receptor-dependent activation of Egr-1 in neurons found in the amygdala43,45,46. Two re

18、cent reports highlight the importance of Egr-1 in remote memory and the reconsolidation fear memory44,47. An impairment in the reconsolidation of contextual fear memory was reported when antisense oligodeoxynucle otides for Egr-1 were infused into the hippocampus of rats47. In another study, Egr-1 e

19、xpression was increased in the anterior cingulate cortex of mice upon re-exposure to the chamber where they had received footshocks 36 d prior44. While both studies reinforce the idea the Egr-1 is an important player in the retention of contextual fear memory, the role of Egr-1 in auditory fear memo

20、ry has not been investigated. In addition, no study has reported the role of Egr-1 in contextual and auditory fear memory using Egr-1 knockout mice.In the present study, we use Egr-1 knockout mice to investigate the contribution of Egr-1 to different types of fear memory. First, we studied two forms

21、 of associative fear memory: contextual and auditory fear conditioning. Contextual fear conditioning is thought to be hippocam-pus-dependent, while auditory fear conditioning is amygdala-dependent48; but see reference49. We then in-vestigated the role of Egr-1 in trace memory50 as well as in the ext

22、inction of fear memory. Although there were no differences in response to contextual and trace memory or in the extinction of fear memory, there was a significant decrease in fear responses during late auditory fear memory in Egr-1 knockout versus wild-type mice. Our results sup-port a selective rol

23、e for Egr-1 in the processing of long-term auditory fear memory.1 MATERIALS AND METHODS1.1 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 crossing heterozygous mutant mice be

24、aring a targeted mutation of the Egr-1 gene. Genotypes were determined by PCR analy-sis51 of genomic DNA extracted from mouse ear tissue. Mice were maintained in a C57BL/6 strain background and were age-matched in each experiment. Wild-type and mu-tant mice were well groomed and showed no signs of a

25、b-normality or any obvious motor defects. No indication of tremor, seizure or ataxia was observed. As it was impos-sible to visually distinguish mutant mice from wild-type mice, experimenters were blind to the genotype. The Ani-mal Care and Use Committees at Washington University and the University

26、of Toronto approved the experimental protocols.1.2Fear conditioningThe following experiments were performed in a condi-tioning shock chamber (30.5 cm×24.1 cm×21.0 cm (Med Associates, Georgia, V ermont. Mice were allowed to habituate to the chamber for 2 min before fear conditioning. The co

27、nditioned stimulus (CS used was an 85 dB tone at 2 800 Hz for 30 s, and the unconditioned423 Shanelle Ko et al: Defects in Late Auditory Fear Memory and Anxiety in Egr-1 Knockout Micestimulus (US was a continuous scrambled foot shock at0.75 mA for 2 s. During training, mice were presented witha 30 s

28、 tone (CS and a shock (US starting 28 s after the onset of the CS. Three CS/US pairings were delivered dur-ing multi-shock conditioning, while only one was used for single shock experiments. After the CS/US pairing, mice were allowed to stay in the chamber for an additional 30 s for the measurement

29、of immediate freezing. Freezing was scored manually every 10 s. For contextual memory, each mouse was placed back into the shock chamber and the freezing response was recorded for 3 min. For auditory fear memory, the mice were put into a novel chamber (different floor, ceiling and walls and monitore

30、d for 3 min before the onset of a tone identical to the CS, which was delivered 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 measure trace memory, we used a trace fear conditioning paradigm

31、 as described50. For this paradigm, the US was delivered 30 s after the end of the CS (trace and mice were sub-jected to three training trails. To test the extinction of fear memory, mice were trained with three shock-tone pair-ings and fear responses were measured during five trails at 1 h interval

32、s in both the context where they had received the shock-tone pairing and in a novel chamber before and after the onset of the tone (CS. The percentage change in fear memory was normalized to control responses.1.3 Elevated plus mazeThe elevated plus maze (Med Associates, Georgia, V ermont consists of

33、 two open arms and two closed arms situated opposite each other and separated by a 6cm square center platform. Each runway is 6 cm wide and 35 cm long. The open arms have lips that are 0.5 cm high and the closed arms are surrounded on three sides by 20 cm walls. The floors and walls are black polypr

34、opylene and the floors are 75 cm from the ground. For each test, the animal is placed in the center square and allowed to move freely for 5 min. The number of entries and time spent in each arm is recorded.1.4Open field activityTo record horizontal locomotor activity we used the Activ-ity Monitor sy

35、stem from Med Associates (43.2 cm×43.2 cm×30.5 cm (Med Associates, St. Albans,VT. Briefly, this system uses paired sets of photo beams to detect movement in the open field and movement is recorded as beam breaks. The open field is placed inside an isolation chamber with dim illumination an

36、d a fan. Each subject was placed 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 slices of amygdala and cortex were rapidly prepared and main-tained in an interface chamber at 30°C, where they

37、were subfused with artificial cerebrospinal fluid (ACSF con-sisting of (mmol/L NaCl 124, CaCl24.4, MgSO42.0,NaHCO325, Na2HPO41.0, glucose 10, and bubbled with95% O2and 5% CO2. In all experiments, slices recovered in the chamber for at least 2 h before recording. In amygdala slices, a bipolar tungste

38、n stimulating electrode was placed in the ventral striatum, and an extracellular recording elec-trode (312 M filled with ACSF was placed in the lat-eral amygdala. In cortical slices, a bipolar tungsten stimu-lating electrode was placed in layer , and extracellular field potentials were recorded usin

39、g a glass microelectrode placed in layer /. Synaptic responses were elicited at 0.02 Hz. For inducing LTP, we used five trains of theta-burst stimulation (TBS at the same intensity of testing stimulation (each train contains four pulses at 100 Hz; de-livered at 200 ms interval. We found that this pr

40、otocol induced 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 heparinized saline (100 000 IU/L heparin, 0.1 mol/L PBS; 0.9% NaCl followed by 4% paraformaldehyde in

41、 0.1 mol/L PBS, pH 7.4. Brains were removed and stored in the same fixative overnight at 4°C, then cryopreserved in 30% sucrose in PBS buffer. Slices (14 µm from frozen sections of the entire brain were cut. The primary antibodies used in this study were directed against the following anti

42、gens (using the 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 (1:200 dilution were conjugated to Cy3, fluorescein isothiocyanate (Jacks

43、on Immunoresearch, West Grove, Penn. or Alexa 488 (1:200 dilution; Molecular Probes. The samples re-ceiving Hematoxylin-eosin were embedded in paraffin, cut in sections 4 µm thick and stained.1.7 Data analysisResults were expressed as means ± SEM. Statistical com-parisons were made with on

44、e- or two-way analysis of vari-ance (ANOV A with the Student-Newmann-Keuls test used for post hoc comparisons. In all cases, P<0.05 was con-sidered statistically significant.Acta Physiologica Sinica , August 25, 2005, 57 (4: 421-4324242 RESUL TS2.1 Anatomy of memory-related central regions in Egr

45、-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 fear memory, we carried out histochemical experiments in sev-er

46、al brain areas. Analysis of serial coronal sections, exam-ined by light microscopy, showed no detectable morpho-logical differences in the auditory cortex, amygdala and hippocampus. Higher magnification of the stained sections further demonstrated no apparent differences in the num-ber and distribut

47、ion of cells in these areas ( Fig.1. A recent study in the hippocampus showed that both neuronal and glial cell populations were not affected in Egr-1 knockout mice 42. We wanted to confirm this finding in other central areas such as the amygdala. As shown in Fig.2, we found no difference in neurona

48、l population and distribution be-Fig. 2. Neuronal and glial staining of wild-type and Egr-1 knockout mice. Representative high-power sections of Anti-GF AP and Anti-NeuN epifluorescence from Egr-1 knockout and wild-type mice showed no detectable differences in the hippocampus, amygdala and auditory

49、cortex. 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 knockout and wild-type mice show no detectable morphological differences in the hippocampus, amygdala and auditory cortex. Scale bar, 250 &

50、#181;m.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 used GFAP 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 Contex

51、tual 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 long term fear memory in mice lacking Egr-1. We assessed two forms of associative emotional memory in wil

52、d-type and Egr-1 knockout mice: contextual and auditory fear conditioning. Preliminary studies in wild-type mice show that three shock-tone pairings produced long-term fear memory that lasted for at least 2 weeks after conditioning (Fig.3, n =6. Pairing the tone with a single shock resulted in fear

53、memory that lasted for about 13 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 a significant reduction in t

54、he freezing response during audi-tory conditioning in Egr-1 knockout mice as compared to wild-type mice F(1, 50=19.5, P <0.001 (n =6 forFig. 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 condition

55、ings 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 shock conditioning 1 h, 1 d and 7 d after training (wild-type

56、, n =5; Egr-1 knockout, n =5.each group. Post hoc analysis revealed that the reduction was selective for late responses (P <0.01 for day 3, P <0.005 for 1 week and P <0.01 for 2 weeks, while early responses (P =0.6 for 1 h and P =0.4 for 1 d were not significantly different between wild-typ

57、e and mutant mice.Unlike auditory fear memory, contextual memory was not significantly 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 until later time

58、points after training with multiple shocks, we wanted to test if fear memory induced by a single shock-tone pairing would also be altered. Neither contextual nor auditory 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(

59、1,24=0.8, P =0.4, for auditory (n =5, Fig.4. Both knockout and wild-type mice showed a significant reduction in the freezing response by 7 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 memory induced by a single shock-tone pairing.2.3 Trace fear memoryTo investigate if Egr-1 is truly selective for amygdalar (vshippocampal fear memory, we tested Egr-1 knockout and wild-type