'Facilitation of Conditioned Fear Extinction\r'

'Neuroscience 134 (2005) 247â€260 FACILITATION OF CONDITIONED diswhitethorn EXTINCTION BY D-CYCLOSERINE IS MEDIATED BY MITOGEN- affectIVATED PROTEIN KINASE AND PHOSPHATIDYLINOSITOL 3-KINASE CASCADES AND REQUIRES DE NOVO PROTEIN SYNTHESIS IN BASOLATERAL pith OF AMYGDALA Y. L. YANGa AND K. T. LUb* Institute of Biotechnology, Department of molecular Biology and Biochemistry, subject Chia-Yi University, 300 University Road, Chia-Yi, chinawargon b Department of Life Science, interior(a) Taiwan Normal University, 88 Ming-Chow Road, south 4, Taipei, Taiwan aKey words: liquidation, D-cycloserine, MAPK, PI-3 kinase, amygdala. Abstract†novel dissolvers intended that every general or intra-amygdala regime of D-cycloserine, a partial derivative booster at the genus Glycine modulatory target on the glutamate N-methylD-aspartate sense organ facilitates the defunctness of acquiredition attention. Here we assessd the occasion of mitogen- activate protein kinase and pho sphatidylinositol 3-kinase in the baso later onal gist of amygdala on the gist of D-cycloserine.The facilitation import of D-cycloserine on dis whitethorn extinguishing and mitogen-activated protein kinase activating was on the whole out of use(p) by intra-amygdala ad arc bituteuteistration of mitogen-activated protein kinase inhibitor PD98059 (500 ng/side, bilater every(preno houruteute of arcal)y) or U0-126 (20 M/side, bilater entirely toldy). Furtherto a greater extent, phosphatidylinositol 3-kinase inhibitor (wortmannin, 5. 0 g/side, bilaterally) inf utilize into the basolateral nucleus of amygdala signi? down the stairssurfacetly reduced two facilitation deed of D-cycloserine and phosphatidylinositol 3-kinase activation.Intra-amygdala brass section of a transcription inhibitor (actinomycin D, 10 g fade away in 1. 6 l fomite; 0. 8 l per side) and a vari emmet inhibitor (anisomycin, one hundred twenty-five g fade away in 1. 6 l fomite; 0. 8 l per side) com pletely jam the facilitation inwardness of D-cycloserine. Control try outs indicated the stopover by actinomycin D or anisomycin were non collectable to lasting damage to the basolateral nucleus of amygdala or recite dep wipeoutency. In sum, none of the fighting(a) medicates employ here altered the side of condition idolize.These issuings put forwarded that phosphatidylinositol 3-kinase and mitogenactivated protein kinase- pendent signalize cascade down and refreshful protein synthesis at heart the basolateral nucleus of amygdala vie authoritative posts in the D-cycloserine facilitation of the defunctness of well-read aid. © 2005 make by Elsevier Ltd on behalf of IBRO. *Corresponding author. Tel: 886-2-29333149×234; facsimile: 886-229312904. E-mail address: [email protected] ntnu. edu. tw (K. -T. Lu).Abbreviations: ACT DCS, actinomycin D D-cycloserine; ACT SAL, actinomycin D salty solving; ANI DCS, anisomycin D-cycloserine; ANI SAL, anis omycin salty; BLA, basolateral nucleus of the amygdala; CS, instruct arousal; DCS, D-cycloserine; EDTA, ethylenediaminetetraacetic acid; ISI, interstimulus interval; MAPK, mitogen-activated protein kinase; NMDA, N-methyl-D-aspartate; PD DCS, PD98059 D-cycloserine; PD SAL, PD98059 salty solution; PI-3K, phosphatidylinositol 3-kinase; US, categorical stimulus; U0 DCS, U0-126 D-cycloserine; U0 SAL, U0126 salty; VEH DCS, fomite D-cycloserine; VEH SAL, vehicle saline; WH DCS, wortmannin D-cycloserine; WH SAL, wortmannin saline. 0306-4522/05$30. 00 0. 00 © 2005 Published by Elsevier Ltd on behalf of IBRO. inside:10. 1016/j. neuroscience. 2005. 04. 003 Fear condition occurs when a antecedently neutral stimulus ( lettered stimulus) is opposite with an aversive stimulus (Mc all(prenominal)ister and Mc every(prenominal)ister, 1971).Following much(prenominal) pairing the teach stimulus is thought to elicit a express of lettered timidity. This is de? ned in animals by their beha vior: secretezing, autonomic re body run, and business organization-potentiated stick out. A ample literature indicates that the basolateral nucleus of the amygdala (BLA) is particularly winding in both the nurture and the observation of conditioned upkeep (Davis, 2000). neuro nephrotoxic lesions or intra-amygdala excerption of glutamate ant protagonists into the BLA closedowns the recipe of conditioned cultism. In addition, local excerpt of N-methyl-D-aspartate (NMDA) speci? c antagonists relegates the acquisition of conditioned fear (Miserendino et al. , 1990; Kim et al. , 1991; M aren et al. , 1996; Gewirtz and Davis, 1997).Synaptic malleability in this area is thought to at a lower placelie the acquisition process when afferent arresting information elicited by the conditioned stimulus is paired with afferent perturb information elicited by the unconditioned stimulus (Fanselow and LeDoux, 1999). defunctness is de? ned as a reduction in conditioned fear whe n the conditioned stimulus (CS) is presented repeatedly in the absence seizure of the unconditioned stimulus (US). numerous studies expose that defunctness is not the result of forgetting or entrepot erasure except results from formation of raw associations that compete with foregoing fear-conditioned associations ( falls and Davis, 1995; Davis et al. , 2000). Similar to acquisition, sampleal defunctness is overly close up by glutamate NMDA receptor antagonists either presumption systemically (Cox and Westbrook, 1994; Baker and Azorlosa, 1996; Kehoe et al. 1996) or locally inf apply into the BLA (Falls and Davis, 1992). The glycine modulatory site of the NMDA receptor provides a critical restrictive piece. Whereas direct NMDA agonists may be neurotoxic due to unregulated calcium entry, partial agonists can facilitate glutamatergic NMDA drill in a more hold fashion (Lawler and Davis, 1992; Olney, 1994). Recent results showed that partial agonists playing at the gly cine modulatory site of the NMDA receptor, such as D-cycloserine (DCS), pass on encyclopedism and shop in several animal models (Thompson and Disterhoft, 1997; Pussinen et al. , 1997; Matsuoka and Aigner, 1996; husbandry and Riccio, 1999; stroller et al. , 2002; 247 248 Y. L. Yang and K. T.Lu / Neuroscience 134 (2005) 247â€260 extinguishing tally, an extermination t separately and a post- liquidation seek (see Fig. 1A). Acclimation. On each of 3 consecutive years, rats were fixed in the tryout domiciliate for 10 min and past returned to their home coops. Baseline climb up run. On each of the next 2 consecutive sidereal daytimetimes, animals were plated in the raise sleeping rooms and presented with 30 95-dB first stimuli at a 30-s interstimulus interval (ISI). Animals whose service line set roughly response was 1% of the measurable take aim were not included in later abstract. Fear conditioning. Twenty-four hours later, rats were returned to the stra in chambers and later 5 min were abandoned the ? rst of 10 sportsmanlike-footshock pairings.The shock (US) was delivered during the last 0. 5 s of the 3. 7 s motiveless (CS). The average inter mental test interval was 4 min (range 3â€5 min) and the shock strong suit was 0. 6 mA. Pre-extermination examen. Twenty-four hours by and by(prenominal)wards fear conditioning, rats were returned to the raise chambers and 5 min later presented with 30 pass over-eliciting noise bursts (95 dB, 30 s ISI). These initial floor stimuli were apply to habituate the starting signal response to a stable baseline preceding to the luminousness-noise exam trials that followed. Thirty seconds later a agree of 20 starting time-eliciting noise bursts were presented, 10 in darkness (noise solely) and 10 3. 2 s later on onset of the 3. s light (light-noise) in a balanced, irregular set out at a 30-s ISI. share fear-potentiated jump off was computed as [( jump-start bounteousness on li ght-noise noise- all trials)/noisealone trials] 100. Rats were then divided into equal size companys of comparable bastardly levels of percent fear-potentiated leap. Rats with less than 50% fear-potentiated startle during the pre- extermination bear witness were not use. defunctness grooming. Extinction training (cue image) is de? ned as the continual exposure to the CS cue (light) in the absence of the US (shock). Twenty-four hours later the pre liquidation test, rats were returned to the test chamber. later onwards 5 min, they were presented with 30, 3. s light exposures at a 30-s ISI. consideration book ag multitudes ( setting exposure) remained in the test cages for the identical amount of time solely did not receive light presentations. Extinction training was performed for varying numbers of consecutive eld (2 years for examine 1 and 1 day for subsequent tastes). Post- extinguishing test-1. Twenty-four hours after the last liquidation training, rats were returned to the test chamber. After 5 min, they were presented with 30 95-dB leader stimuli for a habituated startle baseline. This was followed by a entirety of 60 startle-eliciting noise bursts, 30 in darkness (noise alone) and 30 presented 3. 2 s after onset of the 3. s light (light-noise) in a balanced, irregular tramp at a 30-s ISI. Results were evaluated the resembling substance as pre- defunctness test. Post-extermination test-2. Twenty-four hours after the track down try outal liquidation training period, rats were returned to the test chamber and process the post- experimental experimental experimental defunctness test described above. Fear-potentiated startle test. Twenty-four hours after fear conditioning, rats were returned to the test chamber and interrogatory for fear-potentiated startle use the post- liquidation test-1 described above. Ledgerwood et al. , 2003; Richardson et al. , 2004). In addition, ( )-HA966, a warlike antagonist at the glycine regul atory site on the NMDA receptor, reversed the DCS picture (Walker et al. , 2002).Clinical studies allow a bun in the oven shown that DCS can sometimes enhance connotative retentivity and improve noesis in patients with Alzheimer’s illness (Schwartz et al. , 1996; Tsai et al. , 1998, 1999). It can in any case counter cognitive de? cits in schizophrenia (Javitt et al. , 1994; Goff et al. , 1999). Furthermore, systemic administration of DCS also proved to facilitate defunctness of conditioned fear (Walker et al. , 2002; Ledgerwood et al. , 2003, 2004; Ressler et al. , 2004). Numerous signal cascade down including mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI-3K) and calcineurin, are complex in the extinguishing of conditioned fear (Lu et al. , 2001; Lin et al. , 2003).Similar mechanisms may also be involved in the facilitation erectuate of DCS. This study was designed to clarify the alliance betwixt amygdaloid NMDA receptors, MAPK an d PI-3K signal fall on the quenching of conditioned fear. experimental PROCEDURES Animals Adult male Spragueâ€Dawley (SD) rats (obtained from the animal touch of National Taiwan University Taipei, Taiwan) weighing between 250 and 350 g were utilize. Animals were ho utilise in bases of four rats in a temperature (24 °C) - restrainerled animal colony with unceasing access to food and water. Rats were kept on a 12-h light/dark cps with lights on at 07:00 h. All behavioural procedures took place during the light cycle.All procedures were conducted in symmetry with the National Institutes of Health Guide for parcel out and Use of Laboratory Animals and the guidelines set onwards by the Institutional Animal mission and Use Committee at the National Taiwan Normal University. In all experimental procedures involving animals, all efforts were make to under landed estate pain and the number of animals used. Surgery All surgeries were carried out under sodium yellow jacket (50 mg/ kg, i. p. ) anesthesia. Once anesthetized, the rat was dictated in a Kopf stereotaxic instrument, the skull was exposed, and 22 calibre guide cannula (model C313G, Plastic-one Products, Roanoke, VA, USA) were implanted bilaterally into the BLA (AP, 2. ; DV, 9. 0, ML, 5. 0 from bregma (Paxinos and Watson, 1997)). Size 0 insect pins (Carolina biological Supply, Burlington, NC, USA) were inserted into each cannula to prevent clogging. These extended about 2 mm past the end of the guide cannula. Screws were anchored to the skull and the assembly was cemented in place utilise dental cement (Plastic-one Products). Rats reliable an antibiotic (penicillin) once every day for the ? rst 3 years after the operating room to reduce the risk of infection. General behavioural procedures Fear conditioning was metrical using the potentiated startle paradigm (Cassella and Davis, 1986; Lu et al. , 2001; Walker et al. , 2002).The rats were trained and time- well-tried in a startle chamber (San Diego Instruments, San Diego, CA, USA) in which cage movement resulted in the displacement of an accelerometer. begin amplitude was de? ned as peak accelerometer potential drop inside 200 ms after startle stimulus onset. The behavioral procedures crude to all experiments consisted of an acclimation phase, a baseline startle test phase, a fear conditioning phase, a pre- Drug barb DCS (Sigma) was freshly dissolved in saline. DCS (15 mg/kg, i. p. ) or saline was injected intraperitoneally 15 min front to liquidation training with a 26 gauge guesswork chevy connected to a 1 ml syringe (Walker et al. , 2002; Ledgerwood et al. , 2003) (experiments 1†8).MAPK inhibitor PD98059 (500 ng in 1 l of 20% DMSO; Calbiochem) (Lu et al. , 2001) or U0-126 (50 ng/side; Sigma) (Lin et al. , 2003) or 20% DMSO was infused into the BLA Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247â€260 249 Fig. 1. Systemic administration of DCS quicken defunctness of conditioned fear. (A) T imeline of behavioral procedures for experiment 1. (B) Percent fear-potentiated startle heedful 24 h before (pre-extinction test) and 24 h after extinction training (post-extinction test). Rats in each conference were treated with either DCS or saline precedent to a ace posing of extinction training. (C) To test for toxicity, after 24 h all animals of experiment 1 were retrained.They were tried and true for fear-potentiated startle response in the absence of doses 24 h later (fear-potentiated startle test) (values are implicate SEM, * P 0. 05 versus control convocation; # P 0. 05 versus the root word with 1 day extinction and saline dead reckoning). 250 Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247â€260 10 min prior to saline or DCS snap (experiments 2 and 8). PI-3K inhibitor (wortmannin, 5 g/side) (Lin et al. , 2003) or vehicle was administrated to the BLA 10 min prior to saline or DCS injection (experiment 3). Transcription inhibitor actinomycin D (10 g dis solved in 1. 6 l vehicle; 0. 8 l per side) or exposition inhibitor (anisomycin, 125 g dissolved in 1. 6 l vehicle; 0. 8 l per side) or vehicle (Lin et al. 2003) was administrated to the BLA 10 min prior to DCS or saline injection (experiment 4) or 25 min prior to fear-potentiated startle test (experiment 6). In the control experiment, PD98059, U0-126, wortmannin, actinomycin D, and anisomycin were injected 25 min prior to the fear-potentiated startle test. Injections were made through and through 28-gauge injection cannula (model C313I, Plastic-one Products) connected to a Hamilton micro-syringe via polyethylene tubing. extract speed was 0. 25 l/ min. The rack up volume of injection was 0. 8 l per side. western sandwich blot analysis Animals were killed by beheading 10 min after extinction training. The lateral and basolateral subregions of the amygdala were imperturbable and sonicated brie? y in ice-cold mince: 50 mM Trisâ€HCl (pH 7. ), 50 mM NaCl, 10 mM EGTA, 5 mM EDTA, 2 mM sodium pyrophosphate, 4 mM para-nitrophenylphosphate, 1 mM sodium ortho vanguardadate, 1 mM phenylmethylsulfonyl ? uoride (PMSF), 20 ng/ml leupeptin, and 4 ng/ml aprotinin. Following sonication, the soluble extract was obtained after pelleting the crude membrane fr perform in a centrifuge at 50,000 g at 4 °C. Protein concentration in the soluble fraction was then mensurable using a Bradford assay with bovine serum albumin as the standard. uniform amounts of protein for each sample were resolved in 10% sodium dodecyl sulfate (SDS)â€polyacrylamide gels, blotted electrophoretically to PVDF membranes and block off overnight in 5% skimming milk (Cell Signaling Technology, Inc. , USA).Blots were incubated with an get ithospho-ERK antibody ( bleakborn England Biolabs, USA), anti-ERK antibody (BD Transduction Laboratories, USA), anti-phospho-Akt antibody (New England Biolabs) or anti-pan-Akt (BD Transduction Laboratories). set detection was performed with an deepen chemilum inescence occidental blotting analysis system (RPN 2108; Amersham International, Amersham, UK). fear-potentiated startle during the pre-extinction test. The ? nal 30 rats were charge into ? ve groups of six animals based on their level of fear-potentiated startle in the preextinction test. Twenty-four hours after the pre-extinction test, each rat authoritative 1 or 2 consecutive days of extinction training with DCS (15 mg/kg, i. p. ) or saline. Saline or DCS was injected 15 min prior to the extinction training.An additional control group was tested 2 days after the pre-extinction training without intervening exposures to optical CS. Fig. 1B shows that DCS accelerated extinction of conditioned fear. A both way ANOVA for goings in intervention (DCS vs saline) and days (one or two extinction sittings) between-subjects indicated a signi? hypocrisy sermon way out (F(1,20) 9. 02) and a signi? tilt treatment days interaction (F(2,20) 6. 68). Thus, the reduction of fear-potentiate d startle after 1 day of extinction training was greater in the group that standard DCS than in the group that acquire saline (Fig. 1B). Individual comparisons between DCS- and saline-treated groups indicated signi? ant differences after 1 day of extinction posings (t(10) 3. 86). Previous studies have shown that lesions of the BLA block expression of fear-potentiated startle (Campeau and Davis, 1995). DCS may have toxic arrange on BLA, and resulting misinterpretation of its facilitation launchuate on extinction. To test for toxicity, all animals of experiment 1 were retrained and tested 24 h later. under these conditions, animals earlierly injected with DCS or saline showed a signi? deliver fear-potentiated startle (Fig. 1C). Thus, the facilitation effect of DCS observed during the post-extinction test 1 appeared to result from the acute drug effect quite than from a more permanent wave, perhaps toxic, action of DCS. audition 2: intra-amygdala infusion of MAPK inhibitors plugged the facilitation of extinction by DCS To test the possible role of MAPK-dependent signaling cascade in the DCS-enhanced effect on the extinction of condition fear, 48 rats certain fear conditioning, extinction training, and interrogatory for fear-potentiated startle. Initially, 58 rats were used, however 10 of them were excluded. Rats were at random assigned to six distinguishable groups and trustworthy one of the undermentioned treatments: vehicle saline (VEH SAL), vehicle DCS (VEH DCS), PD98059 DCS (PD DCS), U0-126 DCS (U0 DCS), PD98059 saline (PD SAL) or U0-126 saline (U0 SAL). The MAPK inhibitors, PD98059, and U0-126 (or vehicle) were administrated to the BLA 10 min prior to the injection with DCS or saline. Animals were then returned to their cage.Fifteen proceeding after injection, animals were subjected to a angiotensin-converting enzyme session of extinction training. Previously, we show that a bingle day of extinction training with cue exposure led to a bout 35% reducing in fear-potentiated startle, whereas 2â€3 days of extinction training led to tightfitting complete extinction (Lu et al. , 2001; Walker et al. , 2002). We conclude that the acceleration of extinction is best find after a wiz session of extinction training. As shown in Fig. 2, DMSO, PD98059 (500 ng/side, bilaterally), or U0-126 (20 nM/per side, bilaterally) was given 10 min prior to saline or DCS injection; rats were returned to their cages for 30 min before a single HistologyRats were overdosed with chloral hydrate and perfused intracardially with 0. 9% saline followed by 10% formalin. The brains were take and immersed in a 30% sucrose-formalin solution for at least 3 day. chaplet sections (30 M) were cut through the area of interest, stain with Cresyl Violet, and examined under light microscope for cannula placement. Animals with mis displace cannula were not included in later analysis. statistical analysis The believe startle amplitude across the 30 noise alone and 30 light-noise trials during the pre- and post-extinction tests was calculated for each animal. All results were analyzed using a take a shit of percent fear-potentiated startle, as de? ned in the post-extinction tests above.ANOVA on scores was the primary statistical measure. Between-group comparisons were made using two-tailed t-tests for independent samples. The criterion for signi? cance for all comparisons was P 0. 05. RESULTS test 1: systemic administration of DCS accelerated extinction of conditioned fear This experiment assessed the facilitation effect of DCS on different amounts of extinction training. Initially, 35 rats were used. volt were excluded for showing less than 50% Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247â€260 251 Fig. 2. Intra-amygdala infusion of MAPK inhibitors occlude facilitation effect of DCS on extinction. (A) Timeline of behavioral procedures for experiment 2. B) Cannula was placed in the BLA. Percent fear-potentiated s tartle metrical 24 h before (pre-extinction test) and 24 h after extinction training (post-extinction test). Rats in each group underwent VEH SAL, VEH DCS, PD DCS, U0 DCS, PD SAL, or U0 SAL prior to a single session of extinction training. Twenty-four hours later, animals were tested for fear-potentiated startle in the absence of drugs (values are mean SEM, * P 0. 05 versus VEH SAL group; # P 0. 05 versus VEH DCS group). (C) Cannula tip placements transcribed onto atlas vertebra plates commensurate from Paxinos and Watson (1997). 252 Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247â€260 session of extinction training.Twenty-four hours later, animals were tested for fear-potentiated startle in the absence of drugs. Results showed that in that location was a signi? cant overall difference between treatments (F(5,42) 11. 81). Fig. 2 shows that administration of DCS facilitated extinction of conditioned fear (VEH DCS) compared with the control group (VEH SAL) (t(14) 3. 12, P 0. 05). This effect was blocked by co-administration of MAPK inhibitor PD98059 (PD DCS) or U0-126 (U0 DCS) (t(14) 3. 08, P 0. 05 and t(14) 3. 29, P 0. 05, respectively) compared with the control (VEH DCS), treated with PD98059 precisely (PD SAL) or U0-126 only (U0 SAL) did not affect extinction (t(14) 0. 7 and t(14) 0. 36, respectively). These results indicated that the MAPK dependent signaling cascade some likely mediated the facilitation effect of DCS. Experiment 3: intra-amygdala infusion of the PI-3K inhibitor blocked facilitation of extinction by DCS Previous ? ndings have shown that PI-3K inhibitors half-wit acquisition in a var. of culture paradigms (Lin et al. , 2003). To evaluate the possible role of PI-3K signaling cascade in the DCS enhancement of extinction of conditioned fear, 32 rats trustworthy fear conditioning, extinction training, and test for fear-potentiated startle. Although 38 rats were used initially, six were excluded.They were then randomly assigned to four different groups and received one of the following treatments: VEH SAL, VEH DCS, wortmannin DCS (WH DCS) and wortmannin saline (WH SAL). The PI-3K inhibitor (wortmannin, 5 g/side, bilaterally) was infused to the BLA 10 min prior to the injection of saline or DCS. wherefore rats were returned to their cages for 15 min before creation subjected to a single session of extinction training. Twenty-four hours later, animals were tested for fear-potentiated startle in the absence of drugs. Results showed that there was a signi? cant overall difference between treatments (F(4,28) 12. 17). As shown in Fig. 3, the facilitation effect of DCS (VEH DCS) on extinction was blocked by co-administration of PI-3K inhibitor (WH DCS) (t(14) 2. 98, P 0. 05).With the single extinction training session used in this experiment, this dose of wortmannin alone (WH SAL) at this dose had no effect on the extinction of fear-potentiated startle compared with control group (VEH SAL) (t(14) 0. 19). These results suggest that the PI-3K signaling cascade was involved in the DCS facilitation of extinction. Experiment 4: DCS enhanced the extinction training generate MAPK and PI-3K phosphorylation fit to the results of the above experiments, the DCS facilitation effect on extinction was prevented by coadministration of MAPK or PI-3K inhibitor. Previous studies have shown that infusion of these same inhibitors blocks extinction (Lu et al. , 2001; Lin et al. , 2003). Therefore, these treatments in conjunction with DCS must result in no extinction and resulting misinterpretation of its law of closure effects on DCS.To show the MAPK and PI-3K signaling footpaths are inwrought for the facilitation effect of DCS, we used Western blot to evaluate the DCS effect on the extinction training induce MAPK and PI-3K phosphorylation. Additional amygdala-cannulated rats received fear conditioning, extinction training, and testing for fear-potentiated startle. Then PD98059 or wortmannin was infused to the BLA 10 min prior to the injection of saline or DCS. Rats were returned to their cages. Fifteen minutes after DCS or saline injection, animals were subjected to a single session of extinction training. Animals were killed by decapitation 10 min after extinction training.The lateral and basolateral sub-regions of the amygdala were tested with Western blot analysis. Compared with control group, MAPK phosphorylation was signi? cantly elevated in BLA after extinction training (Fig. 4A, alley 2). Administration of DCS enhanced the effect of extinction training on MAPK phosphorylation (Fig. 4A, lane 3). The MAPK inhibitor PD98059 blocked the effect of DCS (Fig. 4A, lane 4). In addition, we measured the state of Akt phosphorylation as an index of PI-3K activity (Lin et al. , 2001). Fig. 4B showed that administration of DCS enhanced the effect of extinction training on Akt phosphorylation (Fig. 4B, lane 3). The PI-3K inhibitor, wortmannin, blocked the enhancement effect of DCS (Fig. 4b, lane 4).These results raise the possibility that DCS enhancement effect of extinction of conditioned fear is mediated by the amygdaloid MAPK and PI-3K dependent signaling cascades. Experiment 5: intra-amygdala infusion of the transcription inhibitor or translation inhibitor blocked DCS facilitation of extinction The MAPK path participates in the synthesis of proteins classic for the long stabilization and storage of fear memories. fit to the result of experiment 2, the facilitation effect of DCS on extinction is mediated by the MAPK dependent signaling cascade. We predicted that the facilitation effect of DCS infallible parvenue protein synthesis in the BLA.To test this hypothesis, 48 rats received fear conditioning, extinction training, and testing for fear-potentiated startle. Initially, 56 rats were used still viii of them were excluded. Rats were then randomly assigned to six different groups and received one of the following treatments: VEH SAL, VEH DCS, actinomyci n D DCS (ACT DCS), anisomycin DCS (ANI DCS), actinomycin D saline (ACT SAL), and anisomycin saline (ANI SAL). Transcription inhibitor (actinomycin D, 10 g dissolved in 1. 6 l vehicle; 0. 8 l per side) and translation inhibitor (anisomycin, 125 g dissolved in 1. 6 l vehicle; 0. 8 l per side) were administered to the BLA 10 min prior to saline or DCS injection. Then rats were returned to their cages. Fifteen minutes later, nimals were subjected to a single session of extinction training. Twenty-four hours later, animals were tested for fear-potentiated startle in the absence of drugs. Results showed that there was a earthshaking overall difference between treatments (F(5,42) 10. 17). As shown in Fig. 5, actinomycin D and anisomycin completely blocked the facilitation effect of DCS (t(14) 3. 11 and t(14) 2. 96, respectively) compared with the VEH DCS group. With a single extinction training session used in this experiment, actinomycin alone (ACT SAL) or anisomycin alone (ANI SAL) did not affect the extinction of fear-potentiated startle compared with control Y. L. Yang and K. T.Lu / Neuroscience 134 (2005) 247â€260 253 Fig. 3. Intra-amygdala infusion of the PI-3K inhibitor blocked the facilitation effect of DCS on extinction. (A) Timeline of behavioral procedures for experiment 3. (B) Cannula was placed in the BLA. Percent fear-potentiated startle measured 24 h before (pre-extinction test) and 24 h after (post-extinction test) extinction training. Rats in each group were treated with VEH SAL, VEH DCS, WH DCS, or WH SAL prior to a single session of extinction training. Twenty-four hours later, animals were tested for fear-potentiated startle in the absence of drugs (values are mean SEM, * P 0. 05 versus VEH SAL group). C) Cannula tip placements transcribed onto atlas plates competent from Paxinos and Watson (1997). 254 Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247â€260 (VEH SAL) (t(14) 0. 88 and t(14) 0. 48, respectively). These results suggest tha t bare-ass protein synthesis deep down the BLA played an consequential role in DCS facilitation of extinction of conditioned fear. Experiment 6: the turbulent effect of intra-amygdala infusion of actinomycin D and anisomycin was not attributed to lasting damage to the amygdala The mobile drugs used in the above experiments may have toxic effect inside the amygdala. Previous work shows that infusion of PD98095 (Lu et al. , 2001) or wortmannin (Lin et al. 2003) into BLA did not appear to cause permanent impairment of amygdala function. To test for possible toxic effects of actinomycin D and anisomycin on the BLA, all animals of experiment 5 received an additional 2 days of drug free extinction training followed 24 h later by testing. Under these conditions, rats antecedently treated with actinomycin D (ACT DCS-treated group and ACT SALtreated group) or anisomycin (ANI DCS- and ANI SALtreated group) showed a signi? cant reduction of fearpotentiated startle between post-extinction test 1 and post-extinction test 2 (t(7) 3. 08 and t(7) 3. 32 for the ACT DCS-treated group and ACT SAL-treated group respectively) and (t(7) 2. 96 and t(7) 3. 1 for the ANI DCStreated group and ANI SAL-treated group respectively) (Fig. 6B). Thus, the stay of extinction observed during post-extinction test 1 appeared to result from an acute drug effect sort of than from a more permanent and perhaps toxic action, of actinomycin D or anisomycin. Previous studies have shown that lesions of the BLA block fear-potentiated startle (Campeau and Davis, 1995), an outcome opposite from that obtained with infusion of actinomycin D or anisomycin. It is also important to note that actinomycin D or anisomycin may have semipermanent toxicity within the BLA. The extinction of fear would look the same as a gradual acquittance of ability to express or relearn fear.Experiment 7: the disruptive effect of intra-amygdala infusion of actinomycin D and anisomycin was not attributed to state dependence T o evaluate the contribution of state-dependency effects to the results obtained in experiment 6, additional amygdala-cannulated rats were tested for extinction in a drug-free state and after receiving the same compound that they had received during extinction training. Results showed that there was a signi? cant overall difference between treatments in post-extinction test 2 (F(2,21) 32. 16). These results are shown in Fig. 7. Rats infused with actinomycin or anisomycin before postextinction test 2 showed a slight, but non-signi? cant, decrease in fear-potentiated startle from post-extinction test 1 to post-extinction test 2. For control rats (n 8), fear-potentiated startle was signi? cantly lower during post-extinction test 2 than post-extinction test 1 (t(7) 2. 455; P 0. 05). The lost of fear-potentiated startle in both groups probably re? cted incidental extinction produced by the 30 non-reinforced CS presentations of post-extinction test 1. The failure of rats infused before Fig . 4. MAPK and PI-3K inhibitors blocked extinction training activation of MAPK and PI-3K. (A) Representative Western blots and densitometric analysis of the activation of MAPK in the BLA under different treatments (values are mean SEM, * P 0. 05 versus VEH SAL group). (B) Representative Western blots and densitometric analysis Akt phosphorylation as an index of PI-3K activity in the BLA under different treatments (values are mean SEM, * P 0. 05 versus VEH DCS group). Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247â€260 255 Fig. 5.Intra-amygdala infusion of the transcription inhibitor or translation inhibitor blocks the facilitation effect of DCS on extinction of conditioned fear. (A) Timeline of behavioral procedures for experiment 5. (B) Cannula was placed in the BLA. Percent fear-potentiated startle measured 24 h before (pre-extinction test) and 24 h after (post-extinction test 1) extinction training. Rats underwent treatment with VEH SAL, VEH DCS, ACT DCS, ANI DCS, ACT SA L, or ANI SAL prior to a single session of extinction training. Twenty-four hours later, animals were tested for fear-potentiated startle in the absence of drugs (values are mean SEM, * P 0. 05 comparing with the VEH SAL group; # P 0. 05 compared with the VEH DCS group). C) Cannula tip placements transcribed onto atlas plates capable from Paxinos and Watson (1997). 256 Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247â€260 Fig. 6. The disruptive effects of intra-amygdala infusion of actinomycin D and anisomycin on extinction were not attributed to lasting damage to the BLA. (A) Timeline of behavioral procedures for experiment 6. The same animals used in experiment 3 were subjected for two more trials of extinction training. (B) Twenty-four hours after the last extinction training, animals were tested for fear-potentiated startle in the absence of drugs (post-extinction test-2) (values are mean SEM, * P 0. 05 versus the equivalent post-extinction test-2). esting with the tra nscription and translation inhibitors before testing to show a breathing out of fear-incidental extinction suggested that state dependency was not a major factor in the effects of actinomycin D and anisomycin. Experiment 8: effect of pretest PD98059, U0-126, wortmannin, actinomycin, and anisomycin administration on fear-potentiated startle This experiment was designed to evaluate whether the effect of the fighting(a) drugs used has had a secondary effect on fear itself or on CS processing. For example, if MAPK inhibitor U0-126 reduced CS-elicited fear, this might washed-out extinction by decreasing the divergence between CS predictions and what actually occurred. If actinomycin D or anisomycin interfered with visual processing, this might block extinction produced by non-reinforced exposures to the visual CS.To evaluate these possibilities, 42 amygdala-cannulated rats received acclimation, baseline startle test, and fear conditioning. Initially, 50 rats were used, but octette of them were excluded. After 24 h, rats were infused with PD98059, U0-126, wortmannin, actinomycin, and anisomycin. At 25 min after the infusions, rats were tested for fear-potentiated startle. As shown in Fig. 8, none of the active drugs we used here signi? cantly in? uenced fearpotentiated startle (F(6,35) 0. 993). Thus, it is improbable that these drugs in? uenced extinction by increase fear or by disrupting CS processing. Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247â€260 57 Fig. 7. The disruptive effect of intra-amygdala infusion of actinomycin D and anisomycin were not attributed to state dependency. (A) Timeline of behavioral procedures for experiment 7. (B) Cannula was placed in the BLA. Percent fear-potentiated startle measured 24 h before (pre-extinction test), 24 h after (post-extinction test 1), and 48 h after (post-extinction test 2) extinction training. Rats in each group underwent VEH SAL, ACT DCS, or ANI DCS prior to a single session of extinction trainin g and prior to post-extinction test 2. Animals were tested for fear-potentiated startle in the absence of drugs (values are mean SEM, * P 0. 05). C) Cannula tip placements transcribed onto atlas plates adapted from Paxinos and Watson (1997). 258 Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247â€260 Fig. 8. event of pretest PD98059, U0-126, wortmannin, actinomycin, and anisomycin administration on fear-potentiated startle. (A) Timeline of behavioral procedures for experiment 8. (B) Cannula was placed in the BLA. Percent fear-potentiated startle was measured 24 h after fear conditioning. Rats were treated with DMSO, PD98059 (PD), U0-126 (U0), wortmannin (WH), vehicle (VEH), actinomycin (ACT), or anisomycin (ANI) 25 min prior to the fear-potentiated startle test (values are mean SEM). C) Cannula tip placements transcribed onto atlas plates adapted from Paxinos and Watson (1997). DISCUSSION We build on the previous ? ndings that DCS facilitated extinction of conditioned fear (W alker et al. , 2002; Ledgerwood et al. , 2003, 2004; Ressler et al. , 2004). Here, we show for the ? rst time that the DCS effect was prevented by co-administration of MAPK, PI-3K, transcription, and translation inhibitors. Control experiments indicated that the blocking effects of actinomycin D and anisomycin on extinction were not due to lasting damage to the BLA or state dependency. In addition, none of active drugs we used in this study altered the expression of conditioned fear.These results suggest that PI-3K and MAPK-dependent signaling cascades and de novo protein synthesis within the BLA were important for DCS facilitation. first behavioral studies by Pavlov (1927) and Konorski (1948) de? ned extinction as an active process involving formation of new inhibitory associations as equalised to forgetting previously conditioned associations. Numerous studies since have con? rmed and elaborated these early ? ndings (reviewed in Falls and Davis, 1995; Davis et al. , 2000). It i s now well accepted that extinction occurs with repeated presentation of a CS in the absence of the pre- Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247â€260 259 viously paired US.This reduces the conditioned response elicited by the CS. In contrast to forgetting which implies the passive loss of stock, extinction implies active formation of new inhibitory associations competing with and overpowering original stimulative associations. Evidence is growing that extinction may involve circuits and use mechanisms of synaptic plasticity similar to those of conditioned fear encyclopedism (Falls and Davis, 1992; Cox and Westbrook, 1994; Baker and Azorlosa, 1996; Davis et al. , 2000). NMDA-dependent synaptic plasticity appears to mediate many forms of active study (Morris, 1989; Staubli et al. , 1989; fountain et al. , 1990; Collinridge and comfort, 1995).It is likely that conditioned fear learning depends on CSâ€US pairing mediated by NMDA receptors within the BLA (Misere ndino et al. , 1990; Fanselow and LeDoux, 1999). Extinction also appears to require active, NMDA-dependent learning within the amygdala. This was demonstrated by blockage of extinction by microinjections of APV into the BLA in both fear-potentiated startle (Falls and Davis, 1992) and freezing paradigms ( lee(prenominal) and Kim, 1998). Furthermore, systemic administration of a different NMDA antagonist, MK801, blocks the extinction process in a range of different learning paradigms (Cox and Westbrook, 1994; Baker and Azorlosa, 1996; Kehoe et al. , 1996).Recently, DCS, a partial agonist acting at the strychnine-insensitive glycine- realisation site of the NMDA receptor complex, has repeatedly been shown to facilitate learning in various cue and context association paradigms (Monahan et al. , 1989; Flood et al. , 1992; Thompson and Disterhoft, 1997). Walker et al. (2002) account the ? rst evidence that DCS facilitates extinction of learned fear. Since then, come along studies con? r med and elaborated this early ? nding (Ledgerwood et al. , 2003, 2004; Ressler et al. , 2004). These studies inform that DCS is more effective at facilitating extinction when given after extinction training, rather than before. They interpret these ? dings as evidence that DCS facilitates the integration of a new memory acquired during extinction. It is important to note that although some studies have shown DCS to be effective in improving memory impairment due to Alzheimer’s illness (Schwartz et al. , 1996; Tsai et al. , 1999) and schizophrenia (Javitt et al. , 1994; Goff et al. , 1999), other studies ensnare unforesightful or no improvement (Tsai et al. , 1998; van Berckel et al. , 1999). This may be related to the fact that acute treatment with DCS may have a more pronounced facilitation than continuing treatment (Quartermain et al. , 1994; Ledgerwood et al. , 2003; Richardson et al. , 2004). Ledgerwood et al. (2003, 2004) reported that DCStreated animals fail to sh owing reinstatement effects.That DCS enhances extinction may be through some processes different from extinction induced by repeat representation of CS. Lin et al. (2003) investigated the similarities and differences between consolidation of conditioning and consolidation of extinction. They found that both processes depend on activation of NMDA receptors, PI-3K, MAPK, and require synthesis of new proteins within the amygdala. They also found that different characteristics show differential sensitivity to the transcription inhibitor actinomycin D. Our results were consonant with the model that the extinc- tion process involved active learning of new inhibitory associations.Here we showed that DCS facilitation of extinction could be blocked by actinomycin D and anisomycin. These seemingly con? icting results could be imputable to our extinction protocol. Our protocol resembled betweensession extinction, presumably equivalent to long-term extinction memory. In addition, we used DCS to facilitate the extinction process and tested the animals in a drug free condition. Acquisition or consolidation of long-term memory requires activation of protein kinase, transcription of genes, new protein synthesis, and synapse formation (Schafe and LeDoux, 2000). Similar mechanisms were involved in the DCS facilitation of extinction. The DCS activated NMDA receptors, resulted in Ca2 in? x into the cell, and led to the PI-3K and MAPK activation. Activated MAPK can translocate to the nucleus, subsequently activating transcription factors to crusade gene transcription and new protein synthesis. Thus, combinations of drugs and extinction training may weaken or erase original memory. There is increase evidence that learning of CSâ€US associations involves synaptic plasticity within the BLA, in the lead to differential activation of this circuit by sensory afferents (Davis, 1997; Rogan et al. , 1997; Lee and Kim, 1998; Fanselow and LeDoux, 1999). Our results suggested that the extinction of conditioned fear also involved NMDA-dependent plasticity, but speci? inhibitory circuits may be activated by extinction learning. We hypothesize that this impertinently activated inhibitory circuit would oppose conditioned excitatory pathways normally take to activation of the underlying nucleus of the amygdala, resulting in the elicitation of fear responses. CONCLUSION This may be the ? rst study to show that PI-3K and MAPKdependent signaling cascade and de novo protein synthesis within the BLA were essential to the DCS facilitation of the extinction of conditioned fear. Acknowledgmentsâ€The work was support by grants from the National Science Council (NSC 90-2320-B-003-007, NSC 902320-B-006-038, NSC 93-2320-B-003-003).Our gratitude (also) goes to the schoolman Paper Editing Clinic, NTNU. REFERENCES Baker J, Azorlosa J (1996) The NMDA antagonist MK-801 blocks the extinction of Pavlovian fear conditioning. Behav Neurosci 110:618â€620. Campeau S, Davis M (199 5) Involvement of the central nucleus and basolateral complex of the amygdala in fear conditioning measured with fear-potentiated startle in rats trained concurrently with auditory and visual conditioned stimuli. J Neurosci 15:2301â€2311. Cassella J, Davis M (1986) The design and calibration of a startle measurement system. Physiol Behav 36:377â€383. Collinridge G, Bliss T (1995) Memories of NMDA receptors and LTP. Trends Neurosci 18:54 â€56.Cox J, Westbrook R (1994) The NMDA receptor antagonist MK-801 blocks acquisition and extinction of conditioned hypoalgesia responses in the rat. Q J Exp Psychol 47B:187â€210. Davis M (1997) Neurobiology of fear responses: The role of the amygdala. J Neuropsychol Clin Neurosci 9:382†402. Davis M (2000) The role of the amygdala in conditioned and unconditioned fear and anxiety. In: The amygdala, Vol. 2 (Aggleton JP, ed), pp 213â€287. Oxford, UK: Oxford University Press. 260 Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247 â€260 visual recognition memory in rhesus monkeys. J Pharmacol Exp Ther 278:891†897. McAllister WR, McAllister DE (1971) behavioral measurements of conditioned fear. In: aversive conditioning and learning (Brush FR, ed), pp one hundred fiveâ€179. New York: schoolman Press.Miserendino MJD, Sananes CB, Melia KR, Davis M (1990) mental block of acquisition but not expression of conditioned fear-potentiated startle by NMDA antagonists in the amygdala. Nature 345:716â€718. Monahan J, Handelmann G, Hood W, Cordi A (1989) D-cycloserine, a positive modulator of the N-methyl-D-aspartate receptor, enhances performance of learning tasks in rats. Pharmacol Biochem Behav 34:649 †653. Morris R (1989) Synaptic plasticity and learning: selective impairment of learning in rats and jam of long-term potentiation in vivo by the N-methyl-D-aspartate antagonist AP5. J Neurosci 9:3040â€3057. Olney JW (1994) New mechanisms of excitatory transmitter neurotoxicity.J flighty Transm Suppl 43:47â€51. Pavlov I (1927) knowledgeable re? exes. Oxford: Oxford University Press. Paxinos G, Watson C (1997) The rat brain in stereotaxic coordinates, 3rd ed. New York: Academic. Pussinen R, Niememinen S, Koivisto E, Haapalinna A, Riekkinen S, Sirvio J (1997) Enhancement of intermediate-term memory by an alpha-1 agonist or a partial agonist at the glycine site of the NMDA receptor. Neurobiol specify Mem 67:69 â€74. Quartermain D, Mower J, Rafferty M, Herting R, Lanthorn T (1994) Acute but not chronic activation of the NMDA-coupled glycine receptor with D-Cycloserine facilitates learning and retention. Eur J Pharmacol 257:7â€12.Ressler KJ, Rothbaum BO, Tannebaum L, Anderson P, Graap K, Zimand E, Hodges L, Davis M (2004) cognitive enhancers as adjuncts to psychotherapy: Use of D-Cycloserine in phobics to facilitate extinction of fear. Arch Gen psychiatry 61:1136 â€1144. Richardson R, Ledgerwood L, Cranney J (2004) Facilitation of fear extinction by D-cycloserine : Theoretical and clinical implications. peck Mem 11:510 â€516. Rogan MT, Staubli UV, LeDoux JE (1997) Fear conditioning induces associable long-term potentiation in the amygdala. Nature 390:604â€607. Schafe GE, LeDoux JE (2000) storage consolidation of auditory Pavlovian fear conditioning requires protein synthesis and protein kinase A in the amygdala. J Neurosci 20:8177†8187. Schwartz B, Hashtroudi S, Herting R, Schwartz P, Deutsch S (1996) D-Cycloserine enhances implicit memory in Alzheimer patients.Neurology 46:420 †424. Staubli U, Thibaoult O, DiLorenzo M, lynch G (1989) Antagonism of NMDA receptors impairs acquisition but not retention of olfactory memory. Behav Neurosci 103:54 †60. Thompson L, Disterhoft J (1997) Age- and dose-dependent facilitation of associative eyeblink conditioning by D-cycloserine in rabbits. Behav Neurosci 111:1303â€1312. Tsai G, Falk W, Gunther J (1998) A approach study of D-cycloserine treatment in Alzheimer’s diseas e. J Neuropsychiatry Clin Neurosci 10:224â€226. Tsai G, Falk W, Gunther J, Coyle J (1999) better cognition in Alzheimer’s disease with short-term D-cycloserine treatment. Am J psychopathology 156:467†469. an Berckel B, Evenblij C, van Loon B, Maas M, van der veer M, Wynne J, van Ree J, Kahn R (1999) D-cycloserine increases positive symptoms in chronic schizophrenic patients when administered in addition to antipsychotics: a double-blind, parallel, placebo-controlled study. Neuropsychopharmacology 21:203â€210. Walker DL, Ressler KJ, Lu KT, Davis M (2002) Facilitation of conditioned fear extinction by systemic administration or intra-amygdala infusion of D-cycloserine as assessed with fear-potentiated startle. J Neurosci 22:2343â€2351. Davis M, Falls WA, Gewirtz J (2000) Neural systems involved in fear proscription: Extinction and conditioned inhibition. In: Contemporary issues in modeling psychopathology (Myslobodsky M, Weiner I, eds), pp 113â€142. Boston: Kluwer Academic Publishers.Falls W, Davis M (1992) Extinction of fear-potentiated startle: blockade by infusion of an NMDA antagonist into the amygdala. J Neurosci 12:854 †863. Falls W, Davis M (1995) Behavioral and physiological analysis of fear inhibition: extinction and conditioned inhibition. In: neurobiological and clinical consequences of stress: From normal readjustment to PTSD (Friedman MJ, Charney DS, Deutch AY, eds), pp 177â€202. Philadelphia: Lippincott-Raven. Fanselow MS, LeDoux JE (1999) Why we think plasticity underlying pavlovian fear conditioning occurs in the basolateral amygdala. Neuron 23:229 â€232. Flood J, Baker M, Davis J (1990) Modulation of memory processing by glutamic acid receptor antagonists. Brain Res 521:197â€202.Flood J, Morley J, Lanthorn T (1992) Effect on memory processing by D-cycloserine, an agonist of the NMDA/glycine receptor. Neurosci Lett 146:215â€218. Gewirtz JC, Davis M (1997) Second-order fear conditioning prevented by blo cking NMDA receptors in amygdala. Nature 388:471†474. Goff D, Tsai G, Levitt J, Amico E, Manoach D, Schoenfeld D, Hayden D, McCarley R, Coyle J (1999) A placebo-control trial of D-cycloserine added to conventional neuroleptics in patients with schizophrenia. Arch Gen psychological medicine 56:21â€27. Javitt D, Aylberman I, Zukin S, Heresco L, Lindenmayer J (1994) betterment of negative symptoms in schizophrenia by glycine. Am J Psychiatry 151:1234 â€1236.Kehoe E, Macrae M, Hutchinson C (1996) MK-801 protects conditioned response from extinction in the rabbit nictitating membrane preparation. Psychobiology 24:127â€135. Kim J, DeCola J, Landeira-Fernandez J, Fanselow M (1991) N-methylD-aspartate receptor antagonist APV blocks acquisition but not expression of fear conditioning. Behav Neurosci 105:126 â€133. Konorski J (1948) Conditioned re? exes and neural organization. London: Cambridge University Press. Land C, Riccio D (1999) D-cycloserine: cause on long-term ret ention of a conditioned response and on memory for contextual attributes. Neurobiol Learn Mem 72:158 â€168. Lawler BA, Davis KL (1992) Does inflexion of glutamatergic function represent a feasible therapeutic strategy in Alzheimer’s disease?Biol Psychiatry 31:337â€350. Ledgerwood L, Richardson R, Cranney J (2003) Effects of D-cycloserine on extinction of conditioned freezing. Behav Neurosci 117:341â€349. Ledgerwood L, Richardson R, Cranney J (2004) D-cycloserine and the facilitation of extinction of conditioned fear. Consequences for reinstatement. Behav Neurosci 118:505â€513. Lee H, Kim J (1998) Amygdalar NMDA receptors are critical for new fear learning in previously fear-conditioned rats. J Neurosci 18:8444â€8454. Lin CH, Yeh SH, Lin CH, Lu KT, Leu TH, Cheung WC, mazzard cherry PW (2001) A role for the PI-3 kinase signaling pathway in fear conditioning and synaptic plasticity in the amygdala. Neuron 31:845â€851.Lin CH, Yeh SH, Lu HY, Gean PW (2003) The similarities and diversities of signal pathways leading(a) to consolidation of conditioning and consolidation of extinction of fear memory. J Neurosci 23:8310â€8317. Lu KT, Walker DL, Davis M (2001) Mitogen-activated protein kinase cascade in the basolateral nucleus of amygdala is involved in extinction of fear-potentiated startle. J Neurosci 21(RC162):1â€5. Maren S, Aharonov G, Stote D, Fanselow M (1996) N-methyl-Daspartate receptors in the basolateral amygdala are required for both acquisition and expression of conditional fear in rats. Behav Neurosci 110:1365â€1374. Matsuoka N, Aigner T (1996) D-cycloserine, a partial agonist at the glycine site coupled to N-methyl-D-aspartate receptors, improves (Accepted 1 April 2005)\r\n'