Facilitation of Conditioned Fear Extinction

Neuroscience 134 (2005) 247260 FACILITATION OF knowledgeable FEAR EXTINCTION BY D-CYCLOSERINE IS MEDIATED BY MITOGEN- makeIVATED PROTEIN KINASE AND PHOSPHATIDYLINOSITOL 3-KINASE CASCADES AND REQUIRES DE NOVO PROTEIN deduction IN BASOLATERAL NUCLEUS OF AMYGDALA Y. L. YANGa AND K. T. LUb* Institute of Biotechnology, Department of Molecular biology and Biochemistry, internal Chia-Yi University, 300 University Road, Chia-Yi, chinaw atomic number 18 b Department of Life Science, national Taiwan Normal University, 88 Ming-Chow Road, Sec 4, Taipei, Taiwan aKey words essayal extinguishing, D-cycloserine, MAPK, PI-3 kinase, amygdaloid kernel. elevateRecent results stageed that either organizationic or intra-corpus amygdaloideum boldness of D-cycloserine, a incomplete agonist at the genus Glycine modulatory site on the glutamate N-methylD-aspartate sense organ facilitates the quenching of knowing charge. Here we adjudicated the social occasion of mitogen- activate protein k inase and phosphatidylinositol 3-kinase in the baso lateral sum of amygdala on the by and by(prenominal)(prenominal)math of D-cycloserine.The facilitation effect of D-cycloserine on venerate exter momentation and mitogen-activated protein kinase activating was in all in all hold back by intra-amygdala ad se shortduteistration of mitogen-activated protein kinase inhibitor PD98059 (500 ng/side, bilaterally) or U0-126 (20 M/side, bilaterally). Further more(prenominal), phosphatidylinositol 3-kinase inhibitor (wortmannin, 5. 0 g/side, bilaterally) inf utilize into the basolateral nucleus of amygdala signi? mickletly reduced both facilitation effect of D-cycloserine and phosphatidylinositol 3-kinase activation.Intra-amygdala ecesis of a placement inhibitor (actinomycin D, 10 g dissolved in 1. 6 l fomite 0. 8 l per side) and a interlingual rendition inhibitor (anisomycin, nonpareil hundred twenty-five g dissolved in 1. 6 l fomite 0. 8 l per side) completely obstruct the facilitation effect of D-cycloserine. Control investigates indicated the blockage by actinomycin D or anisomycin were non due to unyielding damage to the basolateral nucleus of amygdala or state dependency. In addition, none of the active agent medicates employ here altered the building of erudite upkeep.These results signifyed that phosphatidylinositol 3-kinase and mitogenactivated protein kinase-dependent foretoken renderors and overbold protein synthesis deep down the basolateral nucleus of amygdala played outstanding government agencys in the D-cycloserine facilitation of the quenching of erudite consternation. 2005 Published by Elsevier Ltd on behalf of IBRO. *Corresponding author. Tel 886-2-29333149234 telefax 886-229312904. E-mail address emailprotected ntnu. edu. tw (K. -T. Lu).Abbreviations make out DCS, actinomycin D D-cycloserine ACT SAL, actinomycin D saline solution ANI DCS, anisomycin D-cycloserine ANI SAL, anisomycin saline BLA, basolatera l nucleus of the amygdala CS, knowing input DCS, D-cycloserine EDTA, ethylenediaminetetraacetic acid ISI, interstimulus breakup MAPK, mitogen-activated protein kinase NMDA, N-methyl-D-aspartate PD DCS, PD98059 D-cycloserine PD SAL, PD98059 saline PI-3K, phosphatidylinositol 3-kinase US, un learned stimulus U0 DCS, U0-126 D-cycloserine U0 SAL, U0126 saline 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. doi10. 1016/j. neuroscience. 2005. 04. 003 apprehension condition occurs when a antecedently neutral stimulus ( learn stimulus) is paired with an aversive stimulus (Mc completelyister and Mc totallyister, 1971).Following such pairing the knowledgeable stimulus is thought to conjure up a state of in condition(p) timidity. This is de? ned in animals by their behavior freezing, autonomic reactivity, and care-potentiated saltation. A large literature indicates that the basolateral nucleus of the amygdala (BLA) is critically complicated in both the attainment and the expression of lettered fear (Davis, 2000). neuro poisonous lesions or intra-amygdala selection of glutamate antagonists into the BLA blocks the expression of conditioned fear. In addition, local extract of N-methyl-D-aspartate (NMDA) speci? c antagonists blocks the scholarship of conditioned fear (Miserendino et al. , 1990 Kim et al. , 1991 Maren et al. , 1996 Gewirtz and Davis, 1997).synaptic plasticity in this surface surface area is thought to downstairslie the erudition do when afferent sensory teaching elicited by the conditioned stimulus is paired with afferent trouble oneself information elicited by the unconditioned stimulus (Fanselow and LeDoux, 1999). extinction is de? ned as a reduction in conditioned fear when the conditioned stimulus (CS) is presented repeatedly in the absence seizure seizure of the unconditioned stimulus (US). Many studies tape that liquidation is not the result of forgetting or remembering erasure that results from formation of peeled railroad ties that compete with preceding(prenominal) fear-conditioned associations (Falls and Davis, 1995 Davis et al. , 2000). Similar to acquisition, extermination is as well as block off by glutamate NMDA receptor antagonists either attached systemically (Cox and Westbrook, 1994 baker and Azorlosa, 1996 Kehoe et al. 1996) or locally infuse into the BLA (Falls and Davis, 1992). The genus Glycine modulatory site of the NMDA receptor provides a critical regulative role. Whereas direct NMDA agonists may be neurotoxic due to unregulated atomic number 20 entry, partial agonists can facilitate glutamatergic NMDA activity in a more limited fashion (Lawler and Davis, 1992 Olney, 1994). Recent results bear witnessed that partial agonists acting at the glycine modulatory site of the NMDA receptor, such as D-cycloserine (DCS), enhance learning and memory in several animal models (Thompson and Disterhoft, 1997 Pussinen et al. , 1997 Matsuoka and Aigner, 1996 Land and Riccio, 1999 Walker et al. , 2002 247 248 Y. L. Yang and K. T.Lu / Neuroscience 134 (2005) 247260 extermination exam, an extermination dressing and a post- extinguishing interrogation (see Fig. 1A). Acclimation. On each of 3 neat eld, rats were pose in the trial run house for 10 min and thusly returned to their spot cages. Baseline set forth quiz. On each of the next 2 consecutive geezerhood, animals were placed in the test chambers and presented with 30 95-dB pop stimuli at a 30-s interstimulus interval (ISI). Animals whose baseline pass over response was 1% of the mensural take were not included in later analysis. Fear conditioning. xxiv hours later, rats were returned to the test chambers and later on 5 min were given the ? rst of 10 absolved-foot buffet pairings.The shock (US) was delivered during the last 0. 5 s of the 3. 7 s calorie-fr ee (CS). The average intertrial interval was 4 min (range 35 min) and the shock intensity was 0. 6 mA. Pre- quenching test. xxiv hours after fear conditioning, rats were returned to the test chambers and 5 min later presented with 30 skip over-eliciting noise bursts (95 dB, 30 s ISI). These initial saltation stimuli were employ to habituate the bulge response to a stable baseline anterior(prenominal) to the light-noise test trials that followed. Thirty seconds later a total of 20 startle-eliciting noise bursts were presented, 10 in immorality (noise alone) and 10 3. 2 s after onset of the 3. s light (light-noise) in a balanced, irregular order at a 30-s ISI. Percent fear-potentiated startle was computed as (startle amplitude on light-noise noise-alone trials)/noisealone trials 100. Rats were then divided into equal coat radicals of comparable mean levels of percent fear-potentiated startle. Rats with less than 50% fear-potentiated startle during the pre-extinction test were not used. Extinction instruct. Extinction cookery (cue film) is de? ned as the repetitive exposure to the CS cue (light) in the absence of the US (shock). 2 dozen hours after the preextinction test, rats were returned to the test chamber. After 5 min, they were presented with 30, 3. s light exposures at a 30-s ISI. Context control groups (context exposure) remained in the test cages for the said(prenominal) amount of clock time exclusively did not receive light presentations. Extinction educate was performed for variable numbers of consecutive daytimes (2 days for experiment 1 and 1 day for subsequent experiments). Post-extinction test-1. Twenty-four hours after the last extinction teach, 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 total 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 order at a 30-s ISI. Results were respectd the aforesaid(prenominal) way as pre-extinction test. Post-extinction test-2. Twenty-four hours after the extend extinction training period, rats were returned to the test chamber and process the post-extinction test described above. Fear-potentiated startle test. Twenty-four hours after fear conditioning, rats were returned to the test chamber and testing for fear-potentiated startle utilise the post-extinction test-1 described above. Ledgerwood et al. , 2003 Richardson et al. , 2004). In addition, ( )-HA966, a competitive antagonist at the glycine regulatory site on the NMDA receptor, reversed the DCS effect (Walker et al. , 2002).Clinical studies have shown that DCS can sometimes enhance implicit memory and improve cognition in patients with Alzheimers disease (Schwartz et al. , 1996 Tsai et al. , 1998, 1999). It can also counter cognitive de? cits in schizophrenia (Javitt et al. , 1994 Goff et al. , 1999). Furthermore, systemic cheek of DCS also proved to facilitate extinction of conditioned fear (Walker et al. , 2002 Ledgerwood et al. , 2003, 2004 Ressler et al. , 2004). Numerous mansion rain showers including mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI-3K) and calcineurin, are problematical in the extinction of conditioned fear (Lu et al. , 2001 Lin et al. , 2003).Similar mechanisms may also be compound in the facilitation effect of DCS. This study was designed to clarify the alliance between amygdaloid NMDA receptors, MAPK and PI-3K repoint cascades on the extinction of conditioned fear. EXPERIMENTAL PROCEDURES Animals Adult male SpragueDawley (SD) rats (obtained from the animal center of National Taiwan University Taipei, Taiwan) weighing between 250 and 350 g were used. Animals were housed in groups of four rats in a temperature (24 C) -controlled animal colony with continuous access to food and water. Rats were kept on a 12-h li ght/dark cycle with lights on at 0700 h. All behavioural procedures took place during the light cycle.All procedures were conducted in accordance with the National Institutes of health Guide for Care and Use of Laboratory Animals and the guidelines set forth by the Institutional Animal Care and Use Committee at the National Taiwan Normal University. In all experimental procedures involving animals, all efforts were do to minimize pain and the number of animals used. Surgery All surgeries were carried out under(a) sodium pentobarbital (50 mg/ kg, i. p. ) anesthesia. Once anesthetized, the rat was placed in a Kopf stereotaxic instrument, the skull was exposed, and 22 gauge 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 all-emb racing about 2 mm past the end of the guide cannula. Screws were anchored to the skull and the group was cemented in place using dental cement (Plastic-one Products). Rats trustworthy an antibiotic (penicillin) once every day for the ? rst 3 days after the procedure to reduce the risk of infection. General behavioral procedures Fear conditioning was thrifty using the potentiated startle paradigm (Cassella and Davis, 1986 Lu et al. , 2001 Walker et al. , 2002).The rats were clever and well-tried in a startle chamber (San Diego Instruments, San Diego, CA, USA) in which cage apparent motion resulted in the displacement of an accelerometer. Startle amplitude was de? ned as peak accelerometer potency within 200 ms after startle stimulus onset. The behavioral procedures common to all experiments consisted of an acclimation phase, a baseline startle test phase, a fear conditioning phase, a pre- Drug scene DCS (Sigma) was saucily dissolved in saline. DCS (15 mg/kg, i. p. ) or sali ne was injected intraperitoneally 15 min introductory to extinction training with a 26 gauge gibe needle 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) 247260 249 Fig. 1. Systemic administration of DCS deepen extinction of conditioned fear. (A) Timeline of behavioral procedures for experiment 1. (B) Percent fear-potentiated startle calculated 24 h in the lead (pre-extinction test) and 24 h after extinction training (post-extinction test). Rats in each group were enured with either DCS or saline prior to a undivided school term of extinction training. (C) To test for toxicity, after 24 h all animals of experiment 1 were retrained.They were time- time- well-tried for fear-potentiated startle response in the absence of drugs 24 h later (fear-potentiated startle test) (values are mean SEM, * P 0. 05 versus control group P 0. 05 versus the group with 1 day extinction and saline injection). 250 Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247260 10 min prior to saline or DCS injection (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 dissolved in 1. 6 l vehicle 0. 8 l per side) or translation 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 28-gau ge injection cannula (model C313I, Plastic-one Products) connected to a Hamilton micro-syringe via polyethylene tubing. Infusion speed was 0. 25 l/ min. The total volume of injection was 0. 8 l per side. Western blot analysis Animals were killed by decapitation 10 min after extinction training. The lateral and basolateral subregions of the amygdala were collect and sonicated brie? y in ice-cold buffer 50 mM TrisHCl (pH 7. ), 50 mM NaCl, 10 mM EGTA, 5 mM EDTA, 2 mM sodium pyrophosphate, 4 mM para-nitrophenylphosphate, 1 mM sodium orthovanadate, 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 split up in a centrifuge at 50,000 g at 4 C. Protein concentration in the soluble fraction was then measured using a Bradford assay with bovine serum albumin as the standard. similar amounts of protein for each sample were resolved in 10% sodium dodecyl convert (SD S)polyacrylamide gels, blotted electrophoretically to PVDF membranes and stop overnight in 5% skim milk (Cell foretoken Technology, Inc. , USA).Blots were incubated with an baksheeshhospho-ERK antibody ( red-hot England Biolabs, USA), anti-ERK antibody (BD Transduction Laboratories, USA), anti-phospho-Akt antibody ( stark naked England Biolabs) or anti-pan-Akt (BD Transduction Laboratories). Band detection was performed with an heighten chemiluminescence Western blotting analysis system (RPN 2108 Amersham International, Amersham, UK). fear-potentiated startle during the pre-extinction test. The ? nal 30 rats were assigned into ? ve groups of sextet animals based on their level of fear-potentiated startle in the preextinction test. Twenty-four hours after the pre-extinction test, each rat stock 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 surplus control group was tim e-tested 2 days after the pre-extinction training without intervening exposures to visual CS. Fig. 1B shows that DCS accelerated extinction of conditioned fear. A two way ANOVA for differences in treatment (DCS vs saline) and days (one or two extinction sittings) between-subjects indicated a signi? cant treatment effect (F(1,20) 9. 02) and a signi? cant treatment days interaction (F(2,20) 6. 68). Thus, the reduction of fear-potentiated startle after 1 day of extinction training was greater in the group that authorized DCS than in the group that received saline (Fig. 1B). undivided comparisons between DCS- and saline-treated groups indicated signi? ant differences after 1 day of extinction sessions (t(10) 3. 86). preceding(prenominal) studies have shown that lesions of the BLA block expression of fear-potentiated startle (Campeau and Davis, 1995). DCS may have toxic effect on BLA, and resulting misinterpretation of its facilitation effectuate on extinction. To test for toxicity, all animals of experiment 1 were retrained and tested 24 h later. Under these conditions, animals previously injected with DCS or saline showed a signi? cant fear-potentiated startle (Fig. 1C). Thus, the facilitation effect of DCS observe during the post-extinction test 1 appeared to result from the acuate drug effect instead than from a more permanent, possibly toxic, action of DCS.Experiment 2 intra-amygdala excerption of MAPK inhibitors blocked the facilitation of extinction by DCS To test the possible role of MAPK-dependent signaling cascade in the DCS-enhance effect on the extinction of condition fear, 48 rats received fear conditioning, extinction training, and testing for fear-potentiated startle. Initially, 58 rats were used, but 10 of them were excluded. Rats were haphazardly assigned to six different groups and received one of the chase 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 minutes after injection, animals were subjected to a whizz session of extinction training. Previously, we show that a single day of extinction training with cue exposure led to about 35% diminution in fear-potentiated startle, whereas 23 days of extinction training led to near complete extinction (Lu et al. , 2001 Walker et al. , 2002). We concluded that the speedup of extinction is best detected after a single 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 br ains were removed and immersed in a 30% sucrose-formalin solution for at least 3 day. Coronal sections (30 M) were cut through the area of interest, stained with Cresyl Violet, and examined under light microscope for cannula placement. Animals with misplaced cannula were not included in later analysis. Statistical analysis The mean 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 score 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 autarkic samples. The criterion for signi? cance for all comparisons was P 0. 05. RESULTS Experiment 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. Five were excluded for showing less than 50% Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247260 251 Fig. 2. Intra-amygdala selection of MAPK inhibitors blocked facilitation effect of DCS on extinction. (A) Timeline of behavioral procedures for experiment 2. B) Cannula was placed in the BLA. Percent fear-potentiated startle measured 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 terminus placements transcribed onto atlas plates qualified from Paxinos and Watson (1997). 252 Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247260 session of extinction training.Twenty-four hours la ter, animals were tested for fear-potentiated startle in the absence of drugs. Results showed that there was a signi? cant boilersuit 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 scarcely (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 most likely liaised 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 retard acquisition in a variety of learning paradi gms (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 received fear conditioning, extinction training, and testing 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. Then rats were returned to their cages for 15 min before being 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 general difference between treatments (F(4,28) 12. 17). As shown in Fig. 3, the facilitation effect of DCS (VEH DCS) on extinction was b locked 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 provoke that the PI-3K signaling cascade was snarly in the DCS facilitation of extinction. Experiment 4 DCS enhanced the extinction training induced MAPK and PI-3K phosphorylation According 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 alike 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 blockage effects on DCS.To show the MAPK and PI-3K signaling pathways are essential for the facilitation effect of DCS, we used Western blot to evaluate the DCS effect on the extinction training induced 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, lane 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 pathway participates in the synthesis of proteins important for the long-term stabilisation and storage of fear memories. According 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 necessary new-fangled protein synthesis in the B LA.To test this hypothesis, 48 rats received fear conditioning, extinction training, and testing for fear-potentiated startle. Initially, 56 rats were used but eight of them were excluded. Rats were then randomly assigned to six different groups and received one of the following treatments VEH SAL, VEH DCS, actinomycin 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 significant 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) 247260 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 adapted from Paxinos and Watson (1997). 254 Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247260 (VEH SAL) (t(14) 0. 88 and t(14) 0. 48, respectively). These results suggest that new protein synthesis within the BLA played an important role in DCS facilitation of extinction of conditioned fear. Experiment 6 the roiling effect of intra-amygdala infusion of actinomycin D and anisomycin was not attributed to lasting damage to the amygdala The active drugs used in the above experiments may have toxic effect within 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 sweat 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 trainin g followed 24 h later by testing. Under these conditions, rats previously 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 blockage of extinction observed during post-extinction test 1 appeared to result from an acute drug effect rather 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 placard that actinomycin D or anisomycin may have long-term toxicity within the BLA. The extinction of fear would look the same as a gradual hurt 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 dependency To 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-exti nction test 2 than post-extinction test 1 (t(7) 2. 455 P 0. 05). The missed 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) voice 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) 247260 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 SAL, 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 adapted from Paxinos and Watson (1997). 256 Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247260 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 similar post-extinction test-2). esting with the transcription and translation inhibitors before testing to show a loss of fear-incidental extinction suggested that state dependency was not a major ingredient 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 active drugs used has had a secondary effect on fear itself or on CS bear on. For example, if MAPK inhibitor U0-126 reduced CS-elicited fear, this might attenuate extinction by decreasing the discrepancy between CS predictions and what actually occurred. If actinomycin D or anisomycin interfered with visual processing, this might block ex tinction 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 eight 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 unlikely that these drugs in? uenced extinction by change magnitude fear or by disrupting CS processing. Y. L. Yang and K. T. Lu / Neuroscience 134 (2005) 247260 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 training 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) 247260 Fig. 8. Effect 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 sta rtle 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 (Walker 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 city block 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. Early behavioral studies by Pavlov (1927) and Konorski (1948) de? ned extinction as an active process involving formation of ne w repressing associations as opposed 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 is now well genuine 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) 247260 259 viously paired US.This reduces the conditioned response elicited by the CS. In contrast to forgetting which implies the passive loss of memory, extinction implies active formation of new inhibitory associations competing with and overpowering original excitatory associations. Evidence is growing that extinction may involve circuits and use mechanisms of synaptic plasticity similar to those of conditioned fear learning (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 learning (Morri s, 1989 Staubli et al. , 1989 Flood et al. , 1990 Collinridge and blessedness, 1995).It is likely that conditioned fear learning depends on CSUS pairing mediated by NMDA receptors within the BLA (Miserendino et al. , 1990 Fanselow and LeDoux, 1999). Extinction also appears to bear active, NMDA-dependent learning within the amygdala. This was show by blockage of extinction by microinjections of APV into the BLA in both fear-potentiated startle (Falls and Davis, 1992) and freezing paradigms (Lee 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- identification 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 a l. , 1992 Thompson and Disterhoft, 1997). Walker et al. (2002) reported the ? rst evidence that DCS facilitates extinction of learned fear. Since then, still studies con? rmed and elaborated this early ? nding (Ledgerwood et al. , 2003, 2004 Ressler et al. , 2004). These studies reported 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 consolidation 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 Alzheimers disease (Schwartz et al. , 1996 Tsai et al. , 1999) and schizophrenia (Javitt et al. , 1994 Goff et al. , 1999), other studies represent little 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 inveterate treatment ( Quartermain et al. , 1994 Ledgerwood et al. , 2003 Richardson et al. , 2004). Ledgerwood et al. (2003, 2004) reported that DCStreated animals fail to exhibit 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 consistent 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 attributable to our extinction protocol. Our protocol resembled betweensession extinction, presumably corresponding 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 shift to the nucleus, subsequently activating transcription factors to promote gene transcription and new protein synthesis. Thus, combinations of drugs and extinction training may weaken or erase original memory. There is increasing evidence that learning of CSUS associations involves synaptic plasticity within the BLA, leading to differential activation of thi s 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 newly activated inhibitory circuit would oppose conditioned excitatory pathways blueprintly leading to activation of the central 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. AcknowledgmentsThe work was supported 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 Academic wallpaper Editing Clinic, NTNU. REFERENCES Baker J, Azorlosa J (1996) The NMDA antagonist MK-801 blocks the extinction of Pavlovian fear conditioning. Behav Neurosci 110618620. 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