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2 phosphorylation in mice
Progress in Neuro-Psychopharmacology & Biological Psychiatry 34 (2010) 895–902
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Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p
D-serine
enhances extinction of auditory cued fear conditioning via ERK1/2 phosphorylation in mice
Shingo Matsuda a, Daisuke Matsuzawa a, Ken Nakazawa a, Chihiro Sutoh a, Hiroyuki Ohtsuka a, Daisuke Ishii a, Haruna Tomizawa a, Masaomi Iyo b, Eiji Shimizu a,⁎ a b
Department of Integrative Neurophysiology, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670, Japan Department of Psychiatry, Chiba University Graduate School of Medicine, Japan
a r t i c l e
i n f o
Article history: Received 19 October 2009 Received in revised form 1 April 2010 Accepted 14 April 2010 Available online 21 April 2010 Keywords: D-serine ERK Fear extinction NMDA receptor
a b s t r a c t Several lines of evidence suggest that the N-methyl-D-aspartate (NMDA) receptor plays a significant role in fear conditioning and extinction. However, our knowledge of the role of D-serine, an endogenous ligand for the glycine site of the NMDA receptor, in fear extinction is quite limited compared to that of D-cycloserine, an exogenous partial agonist for the same site. In the current study, we examined the effects of D-serine on fear extinction and phosphorylation of extracellular signal-regulated kinase (ERK) in the hippocampus, basolateral amygdala (BLA), and medial prefrontal cortex (mPFC) during the process of fear extinction. Systemic administrations of D-serine (2.7 g/kg, i.p.) with or without the ERK inhibitor SL327 (30 mg/kg, i.p.) to C57BL/6 J mice were performed before fear extinction in a cued fear conditioning and extinction paradigm. Cytosolic and nuclear ERK 1/2 phosphorylation in the hippocampus, BLA, and mPFC were measured 1 h after extinction (E1h), 24 h after extinction (E24h), and 1 h after recall (R1h) by Western blotting. We found that D-serine enhanced the extinction of fear memory, and the effects of D-serine were reduced by the ERK phosphorylation inhibitor SL327. The Western blot analyses showed that D-serine significantly increased cytosolic ERK 2 phosphorylation at E1h in the hippocampus and cytosolic ERK 1/2 phosphorylation at R1h in the BLA. The present study suggested that D-serine might enhance fear extinction through NMDA receptorinduced ERK signaling in mice, and that D-serine has potential clinical importance for the treatment of anxiety disorders. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Fear extinction is an active form of learning in which the expression of a conditioned fear response is reduced after repeated experience of the conditioned stimulus in the absence of the unconditioned, aversive stimulus. The hippocampus, basolateral amygdala (BLA), and medial prefrontal cortex (mPFC) play a substantial role in fear extinction (Barad et al., 2006; Bouton et al., 2006; Quirk et al., 2006; Sotres-Bayon et al., 2006). Impaired fear extinction is a major symptom of anxiety disorders such as posttraumatic stress disorder (PTSD) (Peri et al., 2000). An improved understanding of the molecular mechanism of fear extinction would thus be useful for the development of new treatments for anxiety disorders. Abbreviations: BLA, basolateral amygdala; CS, conditioned stimulus; DAO, D-amino acid oxidase; DCS, D-cycloserine; DMSO, dimethyl sulfoixde; ERK, extracellular signalregulated kinase; i.p., intraperitoneal; MEK, mitogen-activated protein kinase kinases; mPFC, medial prefrontal cortex; NMDA receptor, N-methyl-D-aspartate receptor; pERK, phosphorylated ERK; PTSD, posttraumatic stress disorder; tERK, total ERK; US, unconditioned stimulus. ⁎ Corresponding author. Tel.: + 81 43 226 2027; fax: + 81 43 226 2028. E-mail address: [email protected] (E. Shimizu). 0278-5846/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2010.04.013
There is substantial evidence that the N-methyl-D-aspartate (NMDA) receptor plays an important role in fear extinction. Preclinical studies suggest that extinction learning can be blocked by antagonists at the glutamatergic NMDA receptor (Falls et al., 1992; Santini et al., 2001; Langton et al., 2007) and facilitated with Dcycloserine, a partial agonist at the glycine recognition site of the NMDA receptor in the amygdala (Walker et al., 2002). A recent metaanalysis showed that D-cycloserine enhances fear extinction/exposure therapy in both animals and anxiety-disordered humans (Norberg et al., 2008). However, D-cycloserine is only a partial and exogenous agonist for the glycine site of the NMDA receptor and may be less effective than other endogenous agonists. D-serine, one of the D-isomers of amino acids, works primarily in the brain (Hashimoto et al., 1993; Schell et al., 1995) and has been suggested to be an endogenous ligand for the glycine site of the NMDA receptor (Schell et al., 1995). In addition, immunohistochemical studies have revealed an overlapping distribution of D-serine and NMDA receptor immunoreactivity in the forebrain (Schell et al., 1997). The important brain areas for fear expression and extinction learning are the amygdala, hippocampus, and medial prefrontal cortex (Quirk and Mueller, 2008), and D-serine is also highly distributed in the forebrain in addition to these other brain regions
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(Nagata et al., 1994; Morikawa et al., 2001; Xia et al., 2004). In a recent clinical study, D-serine treatment for outpatients with PTSD significantly improved the scores of the Hamilton Anxiety Scale and the Mississippi Scale for Combat-Related PTSD (Heresco-Levy et al., 2009). However, the effects and molecular mechanisms of D-serine on fear extinction are still unclear. Through investigation of NMDA receptor-mediated intracellular signaling, Atkins et al. (1998) showed that hippocampal extracellular signal-regulated kinase (ERK) is required for consolidation of cued and contextual fear memory in rats. Furthermore, Herry et al. (2006) demonstrated that extinction of cued conditioned fear requires ERK activation in the basolateral amygdala. Extinction of contextual conditioned fear triggered a rapid activation of ERK-1/2 showing a distinctive time-course, nuclear localization, and cytosolic isoform distribution (Fischer et al., 2007). Taken together, these findings led us to hypothesize that D-serine would facilitate the extinction of fear memory through NMDA receptor-mediated ERK phosphorylation in mice. We first investigated the effect of systemic administration of D-serine with or without the ERK inhibitor SL327 before fear extinction in C57BL/6 J mice on the cued fear conditioning and extinction paradigm. Secondly, in order to better clarify the role of the phosphorylation of ERK in the hippocampus, BLA, and mPFC in fear extinction, we investigated the levels of ERK phosphorylation in those brain regions of C57BL/6 J mice at three time points, 1 h after the extinction phase, 24 h after the extinction phase and 1 h after the recall phase.
2. Materials and methods 2.1. Subjects C57BL/6 J male mice (9–12 weeks of age) were housed separately and provided with food and water ad libitum. They were kept on a 12h light/dark cycle throughout the experiments. Their weights were in the range between 20 and 28 g. All mice were handled for five days before the start of training for approximately 1–2 min. Animal use procedures were approved in advance by the Guide for Animal Experimentation of the Chiba University Graduate School of Medicine.
2.2. Drugs D-serine (Sigma-Aldrich, Steinheim, Germany) was dissolved in physiological saline. A selective MEK inhibitor, SL327 (Z-& E-α-(Amino-((4-aminophenyl)thio)methylene)-2-(trifluoromethyl)benzeneacetonitrile) (Sigma-Aldrich,Saint Louis Missouri, USA), was dissolved in 50% DMSO (Vehicle). The dose of SL327 (30 mg/kg, 2 ml/kg) and D-serine (2.7 g/kg, 10 ml/kg) was chosen based on the results of other behavioral studies, respectively (Selcher et al., 1999; Kanahara et al., 2008). Vehicle or SL327 was injected i.p. at 45 min before the onset of acclimation. Saline or D-serine was injected i.p. at 30 min before the onset of acclimation time.
2.3. Cued fear conditioning and extinction Fig. 1 shows the diagram of the experimental paradigm. On day 1, mice were placed in conditioning chambers 22.8 × 19.7 × 13 cm, transparent walls in the front and back, stainless-steel bars, and a metal-grid floor connected to a shock scrambled and generator in sound-attenuating box, and received three pairings (60–120 s variable interpairing interval) of a conditioned stimulus (CS; 30 s, 90 dB, 2.8 kHz tone) and a unconditioned stimulus (US; 2 s, 0.75 mA scrambled footshock), after a 180 s acclimation period (conditioning phase). The US was presented during the last 2 s of the CS. After a 30 s no-stimulus consolidation period after the final CS–US pairing, mice were returned to the home cage. After matching for equivalent levels of freezing, conditioned mice were divided into the four groups; Vehicle & D-serine (n = 10), Vehicle & Saline (n = 10), SL327 & Dserine (n = 10), and SL327 & Saline (n = 10). Chambers were cleaned with a 79.5% water/19.5% ethanol/1% vanilla-extract solution. On day 2, mice were placed in novel extinction chambers (square-shaped base without shock grid and four triangular shaped profiles, one of which is made of clear plastic wall). After an initial 180 s pre-explore period (Pre), the mice received 15 presentations of the CS alone, each lasting 30 s and separated by a 5 s no-stimulus interval (extinction phase). Chambers were cleaned with a 70% ethanol/30% water solution. On day 3, mice were returned to the same extinction chamber to test for recall of conditioning or extinction. After an initial 180 s pre-explore period, the mice received the same CS alone trials (30 presentations of the CS alone, each lasting 30 s and separated by a 5 s no-stimulus interval) (recall phase).Chambers were cleaned with a 70% ethanol/30% water solution. Activity of mice was monitored by FreezeFrame (Actimetrics Software, 1621 Elmwood Ave Wilmette IL 60091, USA). Freezing (no visible movement except respiration) was scored every 5 s and converted to a percentage [(freezing observations/total observations) × 100].
2.4. Tissues, nuclear and cytosolic extracts preparation Mice were killed by cervical dislocation at three time points as follows; 1 h after the extinction training phase (E1h group), 24 h after the extinction training (E24h group) and one hour after the extinction recall phase (R1h group) (Fig. 1). The time points of 1 h after the learning (E1h and R1h) were chosen because the memory consolidation follows shortly after the extinction learning (Quirk and Mueller, 2008) and enhanced ERK phosphorylation in hippocampus was observed maximal one hour after the stress (Sananbenesi et al., 2003). E24h was chosen to see the pre-pERK/tERK level before the extinction racall phase. Then, the brains were quickly removed and immediately rinsed in ice-cold phosphate-buffered saline (PBS; pH 7.4). Slices 1 mm thick were prepared using a mouse coronal brain matrix (RBM-2000C; ASI Instruments, MI, USA), and the tissue was dissected out in ice-cold PBS under a stereoscopic microscope. Dorsal hippocampus and BLA were dissected out by a sharp scalpel, and mPFC was punched out by disposable biopsy punches (Fig. 2). These tissues were placed in dry-ice-chilled 1.5-mL microcentrifuge tubes,
Fig. 1. Experimental design of fear conditioning paradigm and brain extraction. The down arrow represents injection of drugs. Vehicle or SL327 was injected i.p. at 45 min and Saline or D-serine was injected i.p. at 30 min before extinction phase. The up arrows represent brain extraction for western blot analysis. The brain samples were extracted 1 h (E1h) and 24 h (E24h) after extinction phase and 1 h (R1h) after recall phase, respectively.
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Fig. 2. Schematic diagrams and photographs of collected tissues (a) mPFC was punched out by the disposable biopsy punches. (b) Dorsal hippocampus and BLA were dissected out by a sharp scalpel. Shematic diagrams from Bregma are + 1.94 mm (mPFC) and −1.58 mm (Hippocampus and BLA).
immediately frozen in liquid nitrogen and stored in a deep freezer (−80 °C). Then, nuclear and cytosolic extracts were prepared using the modified high salt extraction method (Thiels et al., 2002). For the preparation of nuclear extracts, the tissue samples were placed in icecold buffer A (10 mM HEPES-OH, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 1 mM NaF, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 1 mM PMSF, 10 M benzamidine, 1 g/ml leupeptin, 1 g/ml aprotinin, and 1 g/ml pepstatin) and incubated on ice for 30 min. Cells were disrupted with a sonicator until nuclei were free of cytoskeletal attachments and then centrifuged at 14,000 rpm at 4 °C for 2 min. The supernatant was decanted, saved, and used as cytosolic extract, and the nuclear pellet was resuspended in buffer B (10 mM HEPES, pH 7.0, 450 mM NaCl, 5 mM EDTA, 0.05% SDS, 1% Triton X-100, 2 mM DTT, 1 mM NaF, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 1 mM PMSF, 10 M benzamidine, 1 g/ml leupeptin, 1 g/ml aprotinin, and 1 g/ml pepstatin) and incubated for 30 min on ice with gentle rocking, followed by centrifugation at 14,000 rpm at
4 °C for 10 min. The resulting supernatant was decanted, saved, and used as nuclear extract. 2.5. Western blot analysis SDS sample buffer (200 mM Tris–HCl pH 6.8, 3% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.2% bromophenol blue) was added to each sample. 20 ng of proteins from each sample was loaded and run on a 4% acrylamide stacking gel and 12% acrylamide resolving gel. Proteins were transferred to polyvinylidene difluoride membranes which were processed for immunoblotting. Membranes were first washed with Tris-buffered saline with Tween (T-TBS) (137 mM NaCl, 20 mM Tris–HCl pH 7.6, 0.1% Tween-20) and blocked in 5% skim milk in T-TBS for 60 min at room temperature, then incubated with primary antibody overnight at 4 °C. Primary antibodies and dilutions used were phosphop44/42 ERK (1:1000, Cell Signaling, Cambridge, MA). Subsequently membranes were washed with T-TBS and
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incubated with secondary antibody for 1 h at room temperature. Secondary antibody used was horseradish peroxidase-conjugated donkey ECL anti-rabbit IgG heavy and light chain (Amersham Bioscience, Buckinghamshire, UK). Finally, membranes were washed with T-TBS and then immunolabeled by chemiluminescence (ECL plus, Amersham Bioscience, Buckinghamshire, UK). Western blots were developed to be linear in the range used for densitometry. The density of the immunoblots was determined by Light-Capture II (ATTO, Tokyo, Japan) and was analyzed by CS Analyzer ver 3.0 (ATTO, Tokyo Japan). 2.6. Data analysis First, the behavioral data were analyzed by two-way analysis of variance (ANOVA) with repeated measures to determine the interaction between drug treatments and trials. In the analysis of extinction phase and recall phase, we pooled trials into 3 bins (5 trials in the extinction training test and 10 trials in the extinction recall test) in order to avoid potential type I errors for multiple comparisons. Post hoc Bonferroni test was performed when the interaction was observed. For the recall phase, the group difference was analyzed by Dunnett's t-test on pooled data setting the Vehicle & Saline group as a control. The immunoreactivity data were analyzed by Student's t-test. For all the analyses, the level of statistical significance was set at pb 0.05. All analyses were performed with the Windows software SPSS 12.0 (Chicago, Illinois). 3. Results 3.1. Experiment 1 In the fear conditioning, two-way ANOVA with repeated measures showed no significant changes in the freezing level among each group at 3rd trial (F(3,36)=0.008, n.s.) (Fig. 3a). In the extinction training phase, two-way ANOVA with repeated measures showed significant main effect of trials (F(2.72)=27.744, p ≤0.001) and interaction between drug treatments and trials (F(6.72) = 4.229, p b 0.01) but did not show significant main effect of drug treatment (F(3.36)=2.222, n.s.). Post hoc Bonferroni test revealed that Vehicle & D-serine group significantly reduced freezing level compared to Vehcle & Saline group (pb 0.01) and SL327 & Saline group (pb 0.01) (Fig. 3a) on the 1st trial block. In addition, SL327 & D-Serine group significantly reduced freezing level compared to SL327 & Saline group (pb 0.01) (Fig. 3a) on the 1st trial block. In the recall phase, two-way ANOVA with repeated measures showed significant main effect of trials (F(2.72)=11.821, p=0.01) but did not show significant main effect of drug treatment (F(3.36)=2.021, n.s.) and interaction between drug treatments and trials (F(6.72)=0.796, n.s.). Dunnett's ttest showed that the freezing level of D-serine treated group was significantly lower than Vehicle & Saline group (p=0.038) (Fig. 3b). Whereas the freezing level of other two groups (SL327 & D-seirne and SL327 & Saline) were not significantly different compared to Vehicle & Saline group. 3.2. Experiment 2 The western blot analyses at 1 h after extinction in the hippocampus (E1h) showed that D-serine treatment produced significantly increased levels of cytosolic phospho-ERK2/total-ERK2 (t(4) = −3.15, p b .05; Fig. 4c), but no significant changes of cytosolic pERK1/tERK 1(t(4)= −0.607, n.s.; Fig. 4a) and nuclear pERK2/tERK2(t(4)=−1.106, n.s.; Fig. 4d). Although, not significant, nuclear pERK1/tERK1 showed trend level increase (t(4)=−2.459, p=0.07; Fig. 4b) in D-serine treated group. The western blot analyses at 1 h after extinction (E1h) in the BLA (Fig. 5a– d) showed that D-serine had no significant effects on the levels of cytosolic pERK1/tERK1 (t(4)=1.991, n.s.) and pERK2/tERK 2 (t(2)=−1.682, n.s.) and nuclear pERK1/tERK1 (t(2)=−0.524, n.s.) and pERK2/tERK2 (t(2)= −0.283, n.s.). The western blot analyses at 1 h after extinction (E1h) in
Fig. 3. Effects of D-serine on extinction and recall of auditory cued fear conditioning. Average percentages freezing to the tone shown in trials for Veh & Sal (Vehicle & Saline , ●; n = 10), Veh & D-ser (Vehicle & D-Serine, ; n = 10), SL327 & Sal (SL327 & Saline, ▲; n = 10), and SL327 & D-Ser (SL327 & D-Serine, △; n = 10) groups. a. Percentages of freezing at conditioning and extinction phase. Vehicle & D-serine group significantly reduced freezing level compared to Vehicle & Saline group (p ≤ 0.01) and SL327 & Saline group (p ≤ 0.01) on the 1st trial block, and SL327 & D-Serine group significantly reduced freezing level compared to SL327 & Saline group (p ≤ 0.01) on the 1st trial block. b. Percentage of freezing at recall phase. Overall freezing level of D-serine treated group was significantly lower than Vehicle & Saline group (p = 0.038). Error bars indicate SEM.
the mPFC (Fig. 6a–d) showed that D-serine had no significant effects on the levels of cytosolic pERK1/tERK1 (t(4)=0.612, n.s.) and pERK2/tERK 2 (t(4)=0.354, n.s.) and nuclear pERK1/tERK1 (t(4) = 1.042, n.s.) and pERK2/tERK2 (t(4) = −0.814, n.s.). The western blot analyses at 24 h after extinction (E24h) in the hippocampus (Fig. 4a–d), BLA (Fig. 5a–d), and mPFC (Fig. 5a–d) showed that D-serine had no significant effects on the levels of cytosolic pERK1/ tERK1 (t(4) =1.247, t(4)= 0.277, and t(4) =−1.024, respectively n.s.) and pERK2/tERK2 (t(4) =0.200, t(4)= 0.595, and t(4) = −0.543, respectively n.s.) and nuclear pERK1/tERK1 (t(4) =0.987, t(4)= −1.088, and t(4) = −1.185, respectively n.s.) and pERK2/tERK2 (t(4) =−1.6, t (4) =−0.687, and t(4) =−1.385, respectively n.s.). The western blot analyses at 1 h after recall (R1h) in the hippocampus (Fig. 4a–d) showed that D-serine had no significant effects on cytosolic pERK1/tERK1 (t(4) = 0.893, n.s.), pERK2/tERK2 (t(4) = 0.867, n.s.), nuclear pERK1/tERK1 (t(4) = −0.186, n.s.) and pERK2/tERK2 (t(4) = 1.787, n.s.) (Fig. 4a–d). The western blot analyses at 1 h after recall (R1h) in the BLA showed that D-serine treatments significantly increased the levels of cytosolic pERK1/ tERK1 (t(4) = 8.934, p b .001) and pERK2/tERK2 (t(4) = −6.470, p b .01) (Fig. 5a and c). On the other hand, there were no significant changes on nuclear pERK1/tERK1 (t(2) = −1.198, n.s.) and pERK2/ tERK2 (t(4) = −0.209, n.s.) in the BLA after D-serine treatments (Fig. 5b and d). The western blot analyses at 1 h after recall (R1h) in the mPFC (Fig. 6a–d) showed that D-serine had no significant
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Fig. 4. Effects of D-serine on the levels of ERK phosphorylation in the hippocampus. a. Quantification of immunoblot densities for the cytosolic pERK1/tERK1 of control (Sal, ■; n = 3) and D-serine(D-Ser, □; n = 3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). b. Quantification of immunoblot densities for the nuclear pERK1/tERK1 of control (Sal, ■; n = 3) and D-serine(D-Ser, □; n = 3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). c. Quantification of immunoblot densities for the cytosolic pERK2/tERK2 of control (Sal, ■; n = 3) and D-serine(D-Ser, □; n = 3) groups. D-Ser group significantly increased the rate of pERK1/tERK1, compared to Sal at E1h (*p b .05). d. Quantification of immunoblot densities for the nuclear pERK2/tERK2 of control (Sal, ■; n = 3) and D-serine(D-Ser, □; n = 3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). e. Representative cytosolic (left panel) and nuclear (right panel) immunoblots. S indicates Saline. D indicates D-serine. Error bars indicate SEM.
effects on cytosolic pERK1/tERK1 (t(4) = −1.920, n.s.), pERK2/ tERK2 (t(4) = −1.035, n.s.), nuclear pERK1/tERK1 (t(4) = −0.029, n.s.) and pERK2/tERK2 (t(4) = 1.072, n.s.) (Fig. 6a–d). 4. Discussion Several studies have shown that D-cycloserine (DCS), a partial NMDA agonist, facilitates extinction of fear memory in rodents (Ledgerwood et al., 2005; Yang and Lu. 2005; Shaw et al., 2009). However, there have been fewer studies on the effects of D-Serine, which is considered to be an endogenous NMDA receptor agonist (Schell et al., 1995), on fear extinction. An increase of D-serine is known to enhance NMDA receptor levels in the rat brain (Fadda et al., 1988). Considering these facts together, the activation of NMDA receptor by D-serine as well as by D-cycloserine may enhance the extinction of fear memory. In the present study, we found that (i) D-serine reduced the freezing level from the very early phase in the extinction learning of auditory cued fear memory, (ii) the reduced fear response to the conditioned cue was retained in the extinction recall phase, (iii) inhibition of ERK phosphorylation attenuated the retention of the reduced fear response by D-serine in the recall phase, and (iv) D-serine increased ERK phosphorylation in the hippocampus in the extinction training phase and in the BLA in the extinction recall phase.
Our results suggest that D-serine may enhance the extinction of auditory cued fear memory via NMDA receptor-mediated ERK phosphorylation. To determine whether D-serine enhances fear extinction via ERK phosphorylation, we administered D-serine with or without SL327 before the extinction training phase. D-serine-treated mice retained the acquired freezing level in the fear conditioning phase and showed significantly lower freezing response than saline-treated mice from very early stages in the extinction training (Fig. 3). In the recent study of Labrie et al. (2009), genetic inactivation of D-amino acid oxidase (DAO), a catabolic enzyme of D-serine, in mice enhanced contextual fear extinction through an increase of endogenous D-serine levels but did not enhance cued fear extinction. Further studies will be needed to explain the reason for this inconsistency, but it might be related to differences in the level of D-serine in the brain due to the different ways of increasing D-serine. It should also be noted that their study did not show whether D-serine affects the retention of cued fear extinction learning. In our study, only D-serine-treated mice showed an even lower freezing response in the recall phase (36.4% freezing level in the third trial bin of extinction phase vs. 19.8% in the first trial bin of the recall phase), which means they retained the extinction learning of auditory cued fear memory. These results suggest that Dserine treatment might not only reduce the fear response to early
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Fig. 5. Effects of D-serine on the levels of ERK phosphorylation in the BLA. a. Quantification of immunoblot densities for the cytosolic pERK1/tERK1 of control (Sal, ■; n=3) and D-serine(D-Ser, □; n=3) groups. There was a significant difference between Sal and D-Ser groups at R1h (***pb .001). b. Quantification of immunoblot densities for the nuclear pERK1/tERK1 of control (Sal, ■; n=3) and D-serine(D-Ser, □; n=3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). c. Quantification of immunoblot densities for the cytosolic pERK2/tERK2 of control (Sal, ■; n=3) and D-serine(D-Ser, □; n=3) groups. D-Ser group significantly increased the levels of pERK1/tERK1, compared to Sal at R1h (**pb .01). d. Quantification of immunoblot densities for the nuclear pERK2/tERK2 of control (Sal, ■; n=3) and D-serine(D-Ser, □; n=3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). e. Representative cytosolic (left panel) and nuclear (right panel) immunoblots. S indicates Saline. D indicates D-serine. Error bars indicate SEM.
exposure of conditioned fear but also retain the extinction learning to late exposure of conditioned fear. Contrary to our expectation, Dserine treatment with SL327 did not cancel the effect of D-serine on the fear response in the extinction training phase (Fig. 3). Because the inhibitory effects of SL327 on D-serine-enhanced extinction were observed in the recall phase, our results suggest that the retention of learned fear extinction might be acquired via ERK phosphorylation. As Fig. 3 shows, the ERK inhibitor (SL327 & Saline) showed no significant increase in inhibition of the extinction memory compared to the control (Vehicle & Saline). Previous studies have shown that ERK phosphorylation plays an important role in extinction, and injection of an ERK inhibitor into the BLA (Lu et al., 2001) and mPFC (Hugues et al., 2006) blocked extinction memory. This conflict may be partly due to the different methods of drug administration, since in the previous studies SL327 was directly injected into the target brain area but in our study it was injected into the abdominal cavity. The dose of SL327 should also be considered in a future study. Immunoblot analysis revealed the effect of D-serine on ERK phosphorylation. The pERK/tERK level was higher in the hippocampus at 1 h after extinction training (E1h) (ERK2 in the cytosol; Fig. 4) and in the BLA at 1 h after the recall phase (R1h) (both ERK1 and 2 in cytosol; Fig. 5). The pERK1/2 levels in the mPFC cytosol were considerably increased at R1h by both treatments (Fig. 6). Since the previous studies with ERK1-knockout mice (Selcher et al., 2001) and ERK2-knockdown mice (Satoh et al., 2007) showed different effects on fear conditioning, we expected different phosphorylation results between the two ERKs, but the
results showed the same trend overall. In the hippocampus, the levels of pERK2/tERK2 in the cytosol at E1h were significantly higher in the Dserine-treated group than in the saline-treated group (Fig. 4), and thus Dserine might enhance the consolidation of extinction memory by activating ERK2 in the hippocampus. However, considering that the phosphorylation ratio in the hippocampus was relatively low compared with other two regions, the meaning of the effect of D-serine on ERK phosphorylation in the hippocampus should be further examined. In general, the hippocampus is considered to have only a modulating effect on cued fear extinction through contextual memory (Sotres-Bayon et al., 2006), and thus the difference might not reflect the direct effect of Dserine on the fear response, but rather may reflect the contextual memory provoked by differences between the chambers where the mice received the stimuli. Interestingly, the pERK/tERK levels in BLA at R1h were considerably different between the cytosol and nuclear fractions. Only cytosol pERK/tERK in the BLA of D-serine-treated mice was significantly higher than that of the saline-treated mice (Fig. 5). When ERK 1/2 are activated, they are known to translocate to the nucleus from the cytosol as well as to activate downstream proteins such as ribosomal S6 kinase (RSK) (Lenormand et al., 1998; Murphy and Blenis, 2006). Partner proteins to ERK are usually in constant equilibrium and thus can be altered by changes in localization and/or the subcellular concentration of ERK (Murphy and Blenis, 2006). Moreover, activated ERK signaling regulates cell fates differently depending on whether it is sustained or transient (Marshall, 1995). Thus, the increased pERK/
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Fig. 6. Effects of D-serine on the levels of ERK phosphorylation in the mPFC. a Quantification of immunoblot densities for the cytosolic pERK1/tERK1 of control (Sal, ■; n = 3) and Dserine(D-Ser, □; n = 3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). b. Quantification of immunoblot densities for the nuclear pERK1/tERK1 of control (Sal, ■; n = 3) and D-serine(D-Ser, □; n = 3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). c. Quantification of immunoblot densities for the cytosolic pERK2/tERK2 of control (Sal, ■; n = 3) and D-serine(D-Ser, □; n = 3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). d. Quantification of immunoblot densities for the nuclear pERK2/tERK2 of control (Sal, ■; n = 3) and D-serine(D-Ser, □; n = 3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). e. Representative cytosolic (left panel) and nuclear (right panel) immunoblots. S indicates Saline. D indicates D-serine. Error bars indicate SEM.
tERK level in the BLA cytosol at R1h might be the result of sustained ERK signaling and/or altered relocation of ERK. In the cytosol of mPFC, D-serine was found to affect the trend toward an increased pERK1/2 level at R1h (Fig. 6). BLA and mPFC have been reported to have functional connectivity with respect to their role in fear extinction (Herry et al., 2006; Sotres-Bayon et al., 2006), and the infralimbic region (IL) of mPFC in particular might play a crucial role in the recall of extinction (Quirk et al., 2000: Hefner et al., 2008; Laurent and Westbrook, 2009). In addition, NMDA receptors in IL have been suggested to be involved in the consolidation of extinction learning (Laurent and Westbrook, 2008; Sotres-Bayon et al., 2009). Taken together, increased pERK/tERK in BLA accompanied by the increased pERK/tERK in mPFC (Fig. 6) might have considerable meaning in terms of the retention and/or reconsolidation of reduced fear response by D-serine. The effect of D -serine in the cytosol of mPFC at R1h (Fig. 6) might have been more clear if our punched out samples were more centered on IL, considering that the neighboring prelimbic region does not seem to play a role in fear extinction (Vidal-Gonzalez et al., 2006; Laurent and Westbrook, 2009). In the standard neural model of fear extinction, mPFC-mediated amygdala inhibition is established at some point during consolidation of extinction, and after extinction learning, mPFC suppresses activity in the amygdala when the animal is required to retrieve the already-consolidated extinction memory (Sotres-Bayon et al., 2006). Thus, according to the model,
our results of ERK phosphorylation at R1h suggest that D-serine might increase the reactivity of BLA from the inhibitory input from mPFC in the retrieval process of fear extinction memory. One of the considerations was that ERK phosphorylation in the BLA at E1h was not significantly different between the two mice groups (Fig. 5). Thus, we cannot determine whether the alteration of ERK phosphorylation in BLA and mPFC did not occur in the extinction training phase or the alteration finished very quickly. The results might reflect the difference in the number of CS presentations between the extinction training and recall phase (15 and 30, respectively). In addition, it has been reported that the pre-extinction injection of D-cycloserine enhanced the extinction and increased ERK phosphorylation in the BLA 10 min after extinction (Yang and Lu, 2005). Therefore, further studies with an earlier sampling time point after several cued conditioned signals (during the extinction training) and after the extinction learning are needed to confirm the effect of Dserine on ERK phosphorylation. Finally, limitations due to sampling should be considered. The inclusion of neighboring areas in the dissected BLA might have affected the immunoblot analysis. 5. Conclusions The present study suggests that D-serine may enhance fear extinction via ERK phosphorylation in the hippocampus and BLA in the consolidation and/or retention of fear extinction memory. These
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findings also have potential clinical importance for the treatment of anxiety disorders. Disclosure/Conflicts of interest We have no conflicts of interest to disclose. Acknowledgment This research was supported by the Grant-in-Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology, Japan. References Atkins CM, Selcher JC, Petraitis JJ, Trzaskos JM, Sweatt JD. The MAPK cascade is required for mammalian associative learning. Nat Neurosci 1998;1:602–9. Barad M, Gean PW, Lutz B. The role of the amygdala in the extinction of conditioned fear. Biol Psychiatry 2006;60:322–8. Bouton ME, Westbrook RF, Corcoran KA, Maren S. Contextual and temporal modulation of extinction: behavioral and biological mechanisms. Biol Psychiatry 2006;60: 352–60. Fadda E, Danysz W, Wroblewski JT, Costa E. Glycine and D-serine increase the affinity of N-methyl-D-aspartate sensitive glutamate binding sites in rat brain synaptic membranes. Neuropharmacology 1988;27:1183–5. Falls WA, Miserendino MJ, Davis M. Extinction of fear-potentiated startle: blockade by infusion of an NMDA antagonist into the amygdala. J Neurosci 1992;12:854–63. Fischer A, Radulovic M, Schrick C, Sananbenesi F, Godovac-Zimmermann J, Radulovic J. Hippocampal Mek/Erk signaling mediates extinction of contextual freezing behavior. Neurobiol Learn Mem 2007;87:149–58. Hashimoto A, Nishikawa T, Oka T, Takahashi K. Endogenous D-serine in rat brain: Nmethyl-D-aspartate receptor-related distribution and aging. J Neurochem 1993;60: 783–6. Hefner K, Whittle N, Juhasz J, Norcross M, Karlsson RM, Saksida LM, et al. Impaired fear extinction learning and cortico-amygdala circuit abnormalities in a common genetic mouse strain. J Neurosci 2008;28:8074–85. Heresco-Levy U, Vass A, Bloch B, Wolosker H, Dumin E, Balan L, et al. Pilot controlled trial of D-serine for the treatment of post-traumatic stress disorder. Int J Neuropsychopharmacol 2009;15:1–8. Herry C, Trifilieff P, Micheau J, Luthi A, Mons N. Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala. Eur J NeuroSci 2006;24: 261–9. Hugues S, Chessel A, Lena I, Marsault R, Garcia R. Prefrontal infusion of PD098059 immediately after fear extinction training blocks extinction-associated prefrontal synaptic plasticity and decreases prefrontal ERK2 phosphorylation. Synapse 2006;60:280–7. Kanahara N, Shimizu E, Ohgake S, Fujita Y, Kohno M, Hashimoto T, et al. Glycine and D:-serine, but not D:-cycloserine, attenuate prepulse inhibition deficits induced by NMDA receptor antagonist MK-801. Psychopharmacology (Berl) 2008;198:363–74. Labrie V, Duffy S, Wang W, Barger SW, Baker GB, Roder JC. Genetic inactivation of Damino acid oxidase enhances extinction and reversal learning in mice. Learn Mem 2009;16:28–37. Langton JM, Kim JH, Nicholas J, Richardson R. The effect of the NMDA receptor antagonist MK-801 on the acquisition and extinction of learned fear in the developing rat. Learn Mem 2007;14:665–8. Laurent V, Westbrook RF. Distinct contributions of the basolateral amygdala and the medial prefrontal cortex to learning and relearning extinction of context conditioned fear. Learn Mem 2008;15:657–66. Laurent V, Westbrook RF. Inactivation of the infralimbic but not the prelimbic cortex impairs consolidation and retrieval of fear extinction. Learn Mem 2009;16:520–9. Ledgerwood L, Richardson R, Cranney J. D-cycloserine facilitates extinction of learned fear: effects on reacquisition and generalized extinction. Biol Psychiatry 2005;57: 841–7.
Lenormand P, Brondello JM, Brunet A, Pouyssegur J. Growth factor-induced p42/p44 MAPK nuclear translocation and retention requires both MAPK activation and neosynthesis of nuclear anchoring proteins. J Cell Biol 1998;142:625–33. Lu KT, Walker DL, Davis M. Mitogen-activated protein kinase cascade in the basolateral nucleus of amygdala is involved in extinction of fear-potentiated startle. J Neurosci 2001;21:RC162. Marshall CJ. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 1995;80:179–85. Morikawa A, Hamase K, Inoue T, Konno R, Niwa A, Zaitsu K. Determination of free Daspartic acid, D-serine and D-alanine in the brain of mutant mice lacking D-amino acid oxidase activity. J Chromatogr B Biomed Sci 2001;757:119–25. Murphy LO, Blenis J. MAPK signal specificity: the right place at the right time. Trends Biochem Sci 2006;31:268–75. Nagata Y, Horiike K, Maeda T. Distribution of free D-serine in vertebrate brains. Brain Res 1994;634:291–5. Norberg MM, Krystal JH, Tolin DF. A meta-analysis of D-cycloserine and the facilitation of fear extinction and exposure therapy. Biol Psychiatry 2008;63:1118–26. Peri T, Ben-Shakhar G, Orr SP, Shalev AY. Psychophysiologic assessment of aversive conditioning in posttraumatic stress disorder. Biol Psychiatry 2000;47:512–9. Quirk GJ, Mueller D. Neural mechanisms of extinction learning and retrieval. Neuropsychopharmacology 2008;33:56–72. Quirk GJ, Russo GK, Barron JL, Lebron K. The role of ventromedial prefrontal cortex in the recovery of extinguished fear. J Neurosci 2000;20:6225–31. Quirk GJ, Garcia R, Gonzalez-Lima F. Prefrontal mechanisms in extinction of conditioned fear. Biol Psychiatry 2006;60:337–43. Sananbenesi F, Fischer A, Schrick C, Spiess J, Radulovic J. Mitogen-activated protein kinase signaling in the hippocampus and its modulation by corticotrophinreleasing factor 2: a possible link between stress and fear memory. J Neurosci 2003;23:11436–43. Santini E, Muller RU, Quirk GJ. Consolidation of extinct bion learning involves transfer from NMDA-independent to NMDA-dependent memory. J Neurosci 2001;21: 9009–17. Satoh Y, Endo S, Ikeda T, Yamada K, Ito M, Kuroki M, et al. Extracellular signal-regulated kinase 2 (ERK2) knockdown mice show deficits in long-term memory; ERK2 has a specific function in learning and memory. J Neurosci 2007;27:10765–1077. Schell MJ, Molliver ME, Snyder SH. D-serine, an endogenous synaptic modulator: localization to astrocytes and glutamate-stimulated release. Proc Natl Acad Sci U S A 1995;92:3948–52. Schell MJ, Brady Jr RO, Molliver ME, Snyder SH. D-serine as a neuromodulator: regional and developmental localizations in rat brain glia resemble NMDA receptors. J Neurosci 1997;17:1604–15. Selcher JC, Atkins CM, Trzaskos JM, Paylor R, Sweatt JD. A necessity for MAP kinase activation in mammalian spatial learning. Learn Mem 1999;6:478–90. Selcher JC, Nekrasova T, Paylor R, Landreth GE, Sweatt JD. Mice lacking the ERK1 isoform of MAP kinase are unimpaired in emotional learning. Learn Mem 2001;8:11–9. Shaw D, Norwood K, Sharp K, Quigley L, McGovern SF, Leslie JC. Facilitation of extinction of operant behaviour in mice by D-cycloserine. Psychopharmacology (Berl) 2009;202:397–402. Sotres-Bayon F, Cain CK, LeDoux JE. Brain mechanisms of fear extinction: historical perspectives on the contribution of prefrontal cortex. Biol Psychiatry 2006;60: 329–36. Sotres-Bayon F, Diaz-Mataix L, Bush DE, LeDoux JE. Dissociable roles for the ventromedial prefrontal cortex and amygdala in fear extinction: NR2B contribution. Cereb Cortex 2009;19(2):474–82. Thiels E, Kanterewicz BI, Norman ED, Trzaskos JM, Klann E. Long-term depression in the adult hippocampus in vivo involves activation of extracellular signal-regulated kinase and phosphorylation of Elk-1. J Neurosci 2002;22:2054–62. Vidal-Gonzalez I, Vidal-Gonzalez B, Rauch SL, Quirk GJ. Microstimulation reveals opposing influences of prelimbic and infralimbic cortex on the expression of conditioned fear. Learn Mem 2006;13:728–33. Walker DL, Ressler KJ, Lu KT, Davis M. Facilitation of conditioned fear extinction by systemic administration or intra-amygdala infusions of D-cycloserine as assessed with fear-potentiated startle in rats. J Neurosci 2002;22:2343–51. Xia M, Liu Y, Figueroa DJ, Chiu CS, Wei N, Lawlor AM, et al. Characterization and localization of a human serine racemase. Brain Res Mol Brain Res 2004;25:96-104. Yang YL, Lu KT. Facilitation of conditioned fear extinction by D-cycloserine is mediated by mitogen-activated protein kinase and phosphatidylinositol 3-kinase cascades and requires de novo protein synthesis in basolateral nucleus of amygdala. Neuroscience 2005;134:247–60.
Contents lists available at ScienceDirect
Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p
D-serine
enhances extinction of auditory cued fear conditioning via ERK1/2 phosphorylation in mice
Shingo Matsuda a, Daisuke Matsuzawa a, Ken Nakazawa a, Chihiro Sutoh a, Hiroyuki Ohtsuka a, Daisuke Ishii a, Haruna Tomizawa a, Masaomi Iyo b, Eiji Shimizu a,⁎ a b
Department of Integrative Neurophysiology, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670, Japan Department of Psychiatry, Chiba University Graduate School of Medicine, Japan
a r t i c l e
i n f o
Article history: Received 19 October 2009 Received in revised form 1 April 2010 Accepted 14 April 2010 Available online 21 April 2010 Keywords: D-serine ERK Fear extinction NMDA receptor
a b s t r a c t Several lines of evidence suggest that the N-methyl-D-aspartate (NMDA) receptor plays a significant role in fear conditioning and extinction. However, our knowledge of the role of D-serine, an endogenous ligand for the glycine site of the NMDA receptor, in fear extinction is quite limited compared to that of D-cycloserine, an exogenous partial agonist for the same site. In the current study, we examined the effects of D-serine on fear extinction and phosphorylation of extracellular signal-regulated kinase (ERK) in the hippocampus, basolateral amygdala (BLA), and medial prefrontal cortex (mPFC) during the process of fear extinction. Systemic administrations of D-serine (2.7 g/kg, i.p.) with or without the ERK inhibitor SL327 (30 mg/kg, i.p.) to C57BL/6 J mice were performed before fear extinction in a cued fear conditioning and extinction paradigm. Cytosolic and nuclear ERK 1/2 phosphorylation in the hippocampus, BLA, and mPFC were measured 1 h after extinction (E1h), 24 h after extinction (E24h), and 1 h after recall (R1h) by Western blotting. We found that D-serine enhanced the extinction of fear memory, and the effects of D-serine were reduced by the ERK phosphorylation inhibitor SL327. The Western blot analyses showed that D-serine significantly increased cytosolic ERK 2 phosphorylation at E1h in the hippocampus and cytosolic ERK 1/2 phosphorylation at R1h in the BLA. The present study suggested that D-serine might enhance fear extinction through NMDA receptorinduced ERK signaling in mice, and that D-serine has potential clinical importance for the treatment of anxiety disorders. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Fear extinction is an active form of learning in which the expression of a conditioned fear response is reduced after repeated experience of the conditioned stimulus in the absence of the unconditioned, aversive stimulus. The hippocampus, basolateral amygdala (BLA), and medial prefrontal cortex (mPFC) play a substantial role in fear extinction (Barad et al., 2006; Bouton et al., 2006; Quirk et al., 2006; Sotres-Bayon et al., 2006). Impaired fear extinction is a major symptom of anxiety disorders such as posttraumatic stress disorder (PTSD) (Peri et al., 2000). An improved understanding of the molecular mechanism of fear extinction would thus be useful for the development of new treatments for anxiety disorders. Abbreviations: BLA, basolateral amygdala; CS, conditioned stimulus; DAO, D-amino acid oxidase; DCS, D-cycloserine; DMSO, dimethyl sulfoixde; ERK, extracellular signalregulated kinase; i.p., intraperitoneal; MEK, mitogen-activated protein kinase kinases; mPFC, medial prefrontal cortex; NMDA receptor, N-methyl-D-aspartate receptor; pERK, phosphorylated ERK; PTSD, posttraumatic stress disorder; tERK, total ERK; US, unconditioned stimulus. ⁎ Corresponding author. Tel.: + 81 43 226 2027; fax: + 81 43 226 2028. E-mail address: [email protected] (E. Shimizu). 0278-5846/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2010.04.013
There is substantial evidence that the N-methyl-D-aspartate (NMDA) receptor plays an important role in fear extinction. Preclinical studies suggest that extinction learning can be blocked by antagonists at the glutamatergic NMDA receptor (Falls et al., 1992; Santini et al., 2001; Langton et al., 2007) and facilitated with Dcycloserine, a partial agonist at the glycine recognition site of the NMDA receptor in the amygdala (Walker et al., 2002). A recent metaanalysis showed that D-cycloserine enhances fear extinction/exposure therapy in both animals and anxiety-disordered humans (Norberg et al., 2008). However, D-cycloserine is only a partial and exogenous agonist for the glycine site of the NMDA receptor and may be less effective than other endogenous agonists. D-serine, one of the D-isomers of amino acids, works primarily in the brain (Hashimoto et al., 1993; Schell et al., 1995) and has been suggested to be an endogenous ligand for the glycine site of the NMDA receptor (Schell et al., 1995). In addition, immunohistochemical studies have revealed an overlapping distribution of D-serine and NMDA receptor immunoreactivity in the forebrain (Schell et al., 1997). The important brain areas for fear expression and extinction learning are the amygdala, hippocampus, and medial prefrontal cortex (Quirk and Mueller, 2008), and D-serine is also highly distributed in the forebrain in addition to these other brain regions
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(Nagata et al., 1994; Morikawa et al., 2001; Xia et al., 2004). In a recent clinical study, D-serine treatment for outpatients with PTSD significantly improved the scores of the Hamilton Anxiety Scale and the Mississippi Scale for Combat-Related PTSD (Heresco-Levy et al., 2009). However, the effects and molecular mechanisms of D-serine on fear extinction are still unclear. Through investigation of NMDA receptor-mediated intracellular signaling, Atkins et al. (1998) showed that hippocampal extracellular signal-regulated kinase (ERK) is required for consolidation of cued and contextual fear memory in rats. Furthermore, Herry et al. (2006) demonstrated that extinction of cued conditioned fear requires ERK activation in the basolateral amygdala. Extinction of contextual conditioned fear triggered a rapid activation of ERK-1/2 showing a distinctive time-course, nuclear localization, and cytosolic isoform distribution (Fischer et al., 2007). Taken together, these findings led us to hypothesize that D-serine would facilitate the extinction of fear memory through NMDA receptor-mediated ERK phosphorylation in mice. We first investigated the effect of systemic administration of D-serine with or without the ERK inhibitor SL327 before fear extinction in C57BL/6 J mice on the cued fear conditioning and extinction paradigm. Secondly, in order to better clarify the role of the phosphorylation of ERK in the hippocampus, BLA, and mPFC in fear extinction, we investigated the levels of ERK phosphorylation in those brain regions of C57BL/6 J mice at three time points, 1 h after the extinction phase, 24 h after the extinction phase and 1 h after the recall phase.
2. Materials and methods 2.1. Subjects C57BL/6 J male mice (9–12 weeks of age) were housed separately and provided with food and water ad libitum. They were kept on a 12h light/dark cycle throughout the experiments. Their weights were in the range between 20 and 28 g. All mice were handled for five days before the start of training for approximately 1–2 min. Animal use procedures were approved in advance by the Guide for Animal Experimentation of the Chiba University Graduate School of Medicine.
2.2. Drugs D-serine (Sigma-Aldrich, Steinheim, Germany) was dissolved in physiological saline. A selective MEK inhibitor, SL327 (Z-& E-α-(Amino-((4-aminophenyl)thio)methylene)-2-(trifluoromethyl)benzeneacetonitrile) (Sigma-Aldrich,Saint Louis Missouri, USA), was dissolved in 50% DMSO (Vehicle). The dose of SL327 (30 mg/kg, 2 ml/kg) and D-serine (2.7 g/kg, 10 ml/kg) was chosen based on the results of other behavioral studies, respectively (Selcher et al., 1999; Kanahara et al., 2008). Vehicle or SL327 was injected i.p. at 45 min before the onset of acclimation. Saline or D-serine was injected i.p. at 30 min before the onset of acclimation time.
2.3. Cued fear conditioning and extinction Fig. 1 shows the diagram of the experimental paradigm. On day 1, mice were placed in conditioning chambers 22.8 × 19.7 × 13 cm, transparent walls in the front and back, stainless-steel bars, and a metal-grid floor connected to a shock scrambled and generator in sound-attenuating box, and received three pairings (60–120 s variable interpairing interval) of a conditioned stimulus (CS; 30 s, 90 dB, 2.8 kHz tone) and a unconditioned stimulus (US; 2 s, 0.75 mA scrambled footshock), after a 180 s acclimation period (conditioning phase). The US was presented during the last 2 s of the CS. After a 30 s no-stimulus consolidation period after the final CS–US pairing, mice were returned to the home cage. After matching for equivalent levels of freezing, conditioned mice were divided into the four groups; Vehicle & D-serine (n = 10), Vehicle & Saline (n = 10), SL327 & Dserine (n = 10), and SL327 & Saline (n = 10). Chambers were cleaned with a 79.5% water/19.5% ethanol/1% vanilla-extract solution. On day 2, mice were placed in novel extinction chambers (square-shaped base without shock grid and four triangular shaped profiles, one of which is made of clear plastic wall). After an initial 180 s pre-explore period (Pre), the mice received 15 presentations of the CS alone, each lasting 30 s and separated by a 5 s no-stimulus interval (extinction phase). Chambers were cleaned with a 70% ethanol/30% water solution. On day 3, mice were returned to the same extinction chamber to test for recall of conditioning or extinction. After an initial 180 s pre-explore period, the mice received the same CS alone trials (30 presentations of the CS alone, each lasting 30 s and separated by a 5 s no-stimulus interval) (recall phase).Chambers were cleaned with a 70% ethanol/30% water solution. Activity of mice was monitored by FreezeFrame (Actimetrics Software, 1621 Elmwood Ave Wilmette IL 60091, USA). Freezing (no visible movement except respiration) was scored every 5 s and converted to a percentage [(freezing observations/total observations) × 100].
2.4. Tissues, nuclear and cytosolic extracts preparation Mice were killed by cervical dislocation at three time points as follows; 1 h after the extinction training phase (E1h group), 24 h after the extinction training (E24h group) and one hour after the extinction recall phase (R1h group) (Fig. 1). The time points of 1 h after the learning (E1h and R1h) were chosen because the memory consolidation follows shortly after the extinction learning (Quirk and Mueller, 2008) and enhanced ERK phosphorylation in hippocampus was observed maximal one hour after the stress (Sananbenesi et al., 2003). E24h was chosen to see the pre-pERK/tERK level before the extinction racall phase. Then, the brains were quickly removed and immediately rinsed in ice-cold phosphate-buffered saline (PBS; pH 7.4). Slices 1 mm thick were prepared using a mouse coronal brain matrix (RBM-2000C; ASI Instruments, MI, USA), and the tissue was dissected out in ice-cold PBS under a stereoscopic microscope. Dorsal hippocampus and BLA were dissected out by a sharp scalpel, and mPFC was punched out by disposable biopsy punches (Fig. 2). These tissues were placed in dry-ice-chilled 1.5-mL microcentrifuge tubes,
Fig. 1. Experimental design of fear conditioning paradigm and brain extraction. The down arrow represents injection of drugs. Vehicle or SL327 was injected i.p. at 45 min and Saline or D-serine was injected i.p. at 30 min before extinction phase. The up arrows represent brain extraction for western blot analysis. The brain samples were extracted 1 h (E1h) and 24 h (E24h) after extinction phase and 1 h (R1h) after recall phase, respectively.
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Fig. 2. Schematic diagrams and photographs of collected tissues (a) mPFC was punched out by the disposable biopsy punches. (b) Dorsal hippocampus and BLA were dissected out by a sharp scalpel. Shematic diagrams from Bregma are + 1.94 mm (mPFC) and −1.58 mm (Hippocampus and BLA).
immediately frozen in liquid nitrogen and stored in a deep freezer (−80 °C). Then, nuclear and cytosolic extracts were prepared using the modified high salt extraction method (Thiels et al., 2002). For the preparation of nuclear extracts, the tissue samples were placed in icecold buffer A (10 mM HEPES-OH, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 1 mM NaF, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 1 mM PMSF, 10 M benzamidine, 1 g/ml leupeptin, 1 g/ml aprotinin, and 1 g/ml pepstatin) and incubated on ice for 30 min. Cells were disrupted with a sonicator until nuclei were free of cytoskeletal attachments and then centrifuged at 14,000 rpm at 4 °C for 2 min. The supernatant was decanted, saved, and used as cytosolic extract, and the nuclear pellet was resuspended in buffer B (10 mM HEPES, pH 7.0, 450 mM NaCl, 5 mM EDTA, 0.05% SDS, 1% Triton X-100, 2 mM DTT, 1 mM NaF, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 1 mM PMSF, 10 M benzamidine, 1 g/ml leupeptin, 1 g/ml aprotinin, and 1 g/ml pepstatin) and incubated for 30 min on ice with gentle rocking, followed by centrifugation at 14,000 rpm at
4 °C for 10 min. The resulting supernatant was decanted, saved, and used as nuclear extract. 2.5. Western blot analysis SDS sample buffer (200 mM Tris–HCl pH 6.8, 3% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.2% bromophenol blue) was added to each sample. 20 ng of proteins from each sample was loaded and run on a 4% acrylamide stacking gel and 12% acrylamide resolving gel. Proteins were transferred to polyvinylidene difluoride membranes which were processed for immunoblotting. Membranes were first washed with Tris-buffered saline with Tween (T-TBS) (137 mM NaCl, 20 mM Tris–HCl pH 7.6, 0.1% Tween-20) and blocked in 5% skim milk in T-TBS for 60 min at room temperature, then incubated with primary antibody overnight at 4 °C. Primary antibodies and dilutions used were phosphop44/42 ERK (1:1000, Cell Signaling, Cambridge, MA). Subsequently membranes were washed with T-TBS and
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incubated with secondary antibody for 1 h at room temperature. Secondary antibody used was horseradish peroxidase-conjugated donkey ECL anti-rabbit IgG heavy and light chain (Amersham Bioscience, Buckinghamshire, UK). Finally, membranes were washed with T-TBS and then immunolabeled by chemiluminescence (ECL plus, Amersham Bioscience, Buckinghamshire, UK). Western blots were developed to be linear in the range used for densitometry. The density of the immunoblots was determined by Light-Capture II (ATTO, Tokyo, Japan) and was analyzed by CS Analyzer ver 3.0 (ATTO, Tokyo Japan). 2.6. Data analysis First, the behavioral data were analyzed by two-way analysis of variance (ANOVA) with repeated measures to determine the interaction between drug treatments and trials. In the analysis of extinction phase and recall phase, we pooled trials into 3 bins (5 trials in the extinction training test and 10 trials in the extinction recall test) in order to avoid potential type I errors for multiple comparisons. Post hoc Bonferroni test was performed when the interaction was observed. For the recall phase, the group difference was analyzed by Dunnett's t-test on pooled data setting the Vehicle & Saline group as a control. The immunoreactivity data were analyzed by Student's t-test. For all the analyses, the level of statistical significance was set at pb 0.05. All analyses were performed with the Windows software SPSS 12.0 (Chicago, Illinois). 3. Results 3.1. Experiment 1 In the fear conditioning, two-way ANOVA with repeated measures showed no significant changes in the freezing level among each group at 3rd trial (F(3,36)=0.008, n.s.) (Fig. 3a). In the extinction training phase, two-way ANOVA with repeated measures showed significant main effect of trials (F(2.72)=27.744, p ≤0.001) and interaction between drug treatments and trials (F(6.72) = 4.229, p b 0.01) but did not show significant main effect of drug treatment (F(3.36)=2.222, n.s.). Post hoc Bonferroni test revealed that Vehicle & D-serine group significantly reduced freezing level compared to Vehcle & Saline group (pb 0.01) and SL327 & Saline group (pb 0.01) (Fig. 3a) on the 1st trial block. In addition, SL327 & D-Serine group significantly reduced freezing level compared to SL327 & Saline group (pb 0.01) (Fig. 3a) on the 1st trial block. In the recall phase, two-way ANOVA with repeated measures showed significant main effect of trials (F(2.72)=11.821, p=0.01) but did not show significant main effect of drug treatment (F(3.36)=2.021, n.s.) and interaction between drug treatments and trials (F(6.72)=0.796, n.s.). Dunnett's ttest showed that the freezing level of D-serine treated group was significantly lower than Vehicle & Saline group (p=0.038) (Fig. 3b). Whereas the freezing level of other two groups (SL327 & D-seirne and SL327 & Saline) were not significantly different compared to Vehicle & Saline group. 3.2. Experiment 2 The western blot analyses at 1 h after extinction in the hippocampus (E1h) showed that D-serine treatment produced significantly increased levels of cytosolic phospho-ERK2/total-ERK2 (t(4) = −3.15, p b .05; Fig. 4c), but no significant changes of cytosolic pERK1/tERK 1(t(4)= −0.607, n.s.; Fig. 4a) and nuclear pERK2/tERK2(t(4)=−1.106, n.s.; Fig. 4d). Although, not significant, nuclear pERK1/tERK1 showed trend level increase (t(4)=−2.459, p=0.07; Fig. 4b) in D-serine treated group. The western blot analyses at 1 h after extinction (E1h) in the BLA (Fig. 5a– d) showed that D-serine had no significant effects on the levels of cytosolic pERK1/tERK1 (t(4)=1.991, n.s.) and pERK2/tERK 2 (t(2)=−1.682, n.s.) and nuclear pERK1/tERK1 (t(2)=−0.524, n.s.) and pERK2/tERK2 (t(2)= −0.283, n.s.). The western blot analyses at 1 h after extinction (E1h) in
Fig. 3. Effects of D-serine on extinction and recall of auditory cued fear conditioning. Average percentages freezing to the tone shown in trials for Veh & Sal (Vehicle & Saline , ●; n = 10), Veh & D-ser (Vehicle & D-Serine, ; n = 10), SL327 & Sal (SL327 & Saline, ▲; n = 10), and SL327 & D-Ser (SL327 & D-Serine, △; n = 10) groups. a. Percentages of freezing at conditioning and extinction phase. Vehicle & D-serine group significantly reduced freezing level compared to Vehicle & Saline group (p ≤ 0.01) and SL327 & Saline group (p ≤ 0.01) on the 1st trial block, and SL327 & D-Serine group significantly reduced freezing level compared to SL327 & Saline group (p ≤ 0.01) on the 1st trial block. b. Percentage of freezing at recall phase. Overall freezing level of D-serine treated group was significantly lower than Vehicle & Saline group (p = 0.038). Error bars indicate SEM.
the mPFC (Fig. 6a–d) showed that D-serine had no significant effects on the levels of cytosolic pERK1/tERK1 (t(4)=0.612, n.s.) and pERK2/tERK 2 (t(4)=0.354, n.s.) and nuclear pERK1/tERK1 (t(4) = 1.042, n.s.) and pERK2/tERK2 (t(4) = −0.814, n.s.). The western blot analyses at 24 h after extinction (E24h) in the hippocampus (Fig. 4a–d), BLA (Fig. 5a–d), and mPFC (Fig. 5a–d) showed that D-serine had no significant effects on the levels of cytosolic pERK1/ tERK1 (t(4) =1.247, t(4)= 0.277, and t(4) =−1.024, respectively n.s.) and pERK2/tERK2 (t(4) =0.200, t(4)= 0.595, and t(4) = −0.543, respectively n.s.) and nuclear pERK1/tERK1 (t(4) =0.987, t(4)= −1.088, and t(4) = −1.185, respectively n.s.) and pERK2/tERK2 (t(4) =−1.6, t (4) =−0.687, and t(4) =−1.385, respectively n.s.). The western blot analyses at 1 h after recall (R1h) in the hippocampus (Fig. 4a–d) showed that D-serine had no significant effects on cytosolic pERK1/tERK1 (t(4) = 0.893, n.s.), pERK2/tERK2 (t(4) = 0.867, n.s.), nuclear pERK1/tERK1 (t(4) = −0.186, n.s.) and pERK2/tERK2 (t(4) = 1.787, n.s.) (Fig. 4a–d). The western blot analyses at 1 h after recall (R1h) in the BLA showed that D-serine treatments significantly increased the levels of cytosolic pERK1/ tERK1 (t(4) = 8.934, p b .001) and pERK2/tERK2 (t(4) = −6.470, p b .01) (Fig. 5a and c). On the other hand, there were no significant changes on nuclear pERK1/tERK1 (t(2) = −1.198, n.s.) and pERK2/ tERK2 (t(4) = −0.209, n.s.) in the BLA after D-serine treatments (Fig. 5b and d). The western blot analyses at 1 h after recall (R1h) in the mPFC (Fig. 6a–d) showed that D-serine had no significant
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Fig. 4. Effects of D-serine on the levels of ERK phosphorylation in the hippocampus. a. Quantification of immunoblot densities for the cytosolic pERK1/tERK1 of control (Sal, ■; n = 3) and D-serine(D-Ser, □; n = 3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). b. Quantification of immunoblot densities for the nuclear pERK1/tERK1 of control (Sal, ■; n = 3) and D-serine(D-Ser, □; n = 3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). c. Quantification of immunoblot densities for the cytosolic pERK2/tERK2 of control (Sal, ■; n = 3) and D-serine(D-Ser, □; n = 3) groups. D-Ser group significantly increased the rate of pERK1/tERK1, compared to Sal at E1h (*p b .05). d. Quantification of immunoblot densities for the nuclear pERK2/tERK2 of control (Sal, ■; n = 3) and D-serine(D-Ser, □; n = 3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). e. Representative cytosolic (left panel) and nuclear (right panel) immunoblots. S indicates Saline. D indicates D-serine. Error bars indicate SEM.
effects on cytosolic pERK1/tERK1 (t(4) = −1.920, n.s.), pERK2/ tERK2 (t(4) = −1.035, n.s.), nuclear pERK1/tERK1 (t(4) = −0.029, n.s.) and pERK2/tERK2 (t(4) = 1.072, n.s.) (Fig. 6a–d). 4. Discussion Several studies have shown that D-cycloserine (DCS), a partial NMDA agonist, facilitates extinction of fear memory in rodents (Ledgerwood et al., 2005; Yang and Lu. 2005; Shaw et al., 2009). However, there have been fewer studies on the effects of D-Serine, which is considered to be an endogenous NMDA receptor agonist (Schell et al., 1995), on fear extinction. An increase of D-serine is known to enhance NMDA receptor levels in the rat brain (Fadda et al., 1988). Considering these facts together, the activation of NMDA receptor by D-serine as well as by D-cycloserine may enhance the extinction of fear memory. In the present study, we found that (i) D-serine reduced the freezing level from the very early phase in the extinction learning of auditory cued fear memory, (ii) the reduced fear response to the conditioned cue was retained in the extinction recall phase, (iii) inhibition of ERK phosphorylation attenuated the retention of the reduced fear response by D-serine in the recall phase, and (iv) D-serine increased ERK phosphorylation in the hippocampus in the extinction training phase and in the BLA in the extinction recall phase.
Our results suggest that D-serine may enhance the extinction of auditory cued fear memory via NMDA receptor-mediated ERK phosphorylation. To determine whether D-serine enhances fear extinction via ERK phosphorylation, we administered D-serine with or without SL327 before the extinction training phase. D-serine-treated mice retained the acquired freezing level in the fear conditioning phase and showed significantly lower freezing response than saline-treated mice from very early stages in the extinction training (Fig. 3). In the recent study of Labrie et al. (2009), genetic inactivation of D-amino acid oxidase (DAO), a catabolic enzyme of D-serine, in mice enhanced contextual fear extinction through an increase of endogenous D-serine levels but did not enhance cued fear extinction. Further studies will be needed to explain the reason for this inconsistency, but it might be related to differences in the level of D-serine in the brain due to the different ways of increasing D-serine. It should also be noted that their study did not show whether D-serine affects the retention of cued fear extinction learning. In our study, only D-serine-treated mice showed an even lower freezing response in the recall phase (36.4% freezing level in the third trial bin of extinction phase vs. 19.8% in the first trial bin of the recall phase), which means they retained the extinction learning of auditory cued fear memory. These results suggest that Dserine treatment might not only reduce the fear response to early
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Fig. 5. Effects of D-serine on the levels of ERK phosphorylation in the BLA. a. Quantification of immunoblot densities for the cytosolic pERK1/tERK1 of control (Sal, ■; n=3) and D-serine(D-Ser, □; n=3) groups. There was a significant difference between Sal and D-Ser groups at R1h (***pb .001). b. Quantification of immunoblot densities for the nuclear pERK1/tERK1 of control (Sal, ■; n=3) and D-serine(D-Ser, □; n=3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). c. Quantification of immunoblot densities for the cytosolic pERK2/tERK2 of control (Sal, ■; n=3) and D-serine(D-Ser, □; n=3) groups. D-Ser group significantly increased the levels of pERK1/tERK1, compared to Sal at R1h (**pb .01). d. Quantification of immunoblot densities for the nuclear pERK2/tERK2 of control (Sal, ■; n=3) and D-serine(D-Ser, □; n=3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). e. Representative cytosolic (left panel) and nuclear (right panel) immunoblots. S indicates Saline. D indicates D-serine. Error bars indicate SEM.
exposure of conditioned fear but also retain the extinction learning to late exposure of conditioned fear. Contrary to our expectation, Dserine treatment with SL327 did not cancel the effect of D-serine on the fear response in the extinction training phase (Fig. 3). Because the inhibitory effects of SL327 on D-serine-enhanced extinction were observed in the recall phase, our results suggest that the retention of learned fear extinction might be acquired via ERK phosphorylation. As Fig. 3 shows, the ERK inhibitor (SL327 & Saline) showed no significant increase in inhibition of the extinction memory compared to the control (Vehicle & Saline). Previous studies have shown that ERK phosphorylation plays an important role in extinction, and injection of an ERK inhibitor into the BLA (Lu et al., 2001) and mPFC (Hugues et al., 2006) blocked extinction memory. This conflict may be partly due to the different methods of drug administration, since in the previous studies SL327 was directly injected into the target brain area but in our study it was injected into the abdominal cavity. The dose of SL327 should also be considered in a future study. Immunoblot analysis revealed the effect of D-serine on ERK phosphorylation. The pERK/tERK level was higher in the hippocampus at 1 h after extinction training (E1h) (ERK2 in the cytosol; Fig. 4) and in the BLA at 1 h after the recall phase (R1h) (both ERK1 and 2 in cytosol; Fig. 5). The pERK1/2 levels in the mPFC cytosol were considerably increased at R1h by both treatments (Fig. 6). Since the previous studies with ERK1-knockout mice (Selcher et al., 2001) and ERK2-knockdown mice (Satoh et al., 2007) showed different effects on fear conditioning, we expected different phosphorylation results between the two ERKs, but the
results showed the same trend overall. In the hippocampus, the levels of pERK2/tERK2 in the cytosol at E1h were significantly higher in the Dserine-treated group than in the saline-treated group (Fig. 4), and thus Dserine might enhance the consolidation of extinction memory by activating ERK2 in the hippocampus. However, considering that the phosphorylation ratio in the hippocampus was relatively low compared with other two regions, the meaning of the effect of D-serine on ERK phosphorylation in the hippocampus should be further examined. In general, the hippocampus is considered to have only a modulating effect on cued fear extinction through contextual memory (Sotres-Bayon et al., 2006), and thus the difference might not reflect the direct effect of Dserine on the fear response, but rather may reflect the contextual memory provoked by differences between the chambers where the mice received the stimuli. Interestingly, the pERK/tERK levels in BLA at R1h were considerably different between the cytosol and nuclear fractions. Only cytosol pERK/tERK in the BLA of D-serine-treated mice was significantly higher than that of the saline-treated mice (Fig. 5). When ERK 1/2 are activated, they are known to translocate to the nucleus from the cytosol as well as to activate downstream proteins such as ribosomal S6 kinase (RSK) (Lenormand et al., 1998; Murphy and Blenis, 2006). Partner proteins to ERK are usually in constant equilibrium and thus can be altered by changes in localization and/or the subcellular concentration of ERK (Murphy and Blenis, 2006). Moreover, activated ERK signaling regulates cell fates differently depending on whether it is sustained or transient (Marshall, 1995). Thus, the increased pERK/
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Fig. 6. Effects of D-serine on the levels of ERK phosphorylation in the mPFC. a Quantification of immunoblot densities for the cytosolic pERK1/tERK1 of control (Sal, ■; n = 3) and Dserine(D-Ser, □; n = 3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). b. Quantification of immunoblot densities for the nuclear pERK1/tERK1 of control (Sal, ■; n = 3) and D-serine(D-Ser, □; n = 3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). c. Quantification of immunoblot densities for the cytosolic pERK2/tERK2 of control (Sal, ■; n = 3) and D-serine(D-Ser, □; n = 3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). d. Quantification of immunoblot densities for the nuclear pERK2/tERK2 of control (Sal, ■; n = 3) and D-serine(D-Ser, □; n = 3) groups. There was no significant difference between Sal and D-Ser groups (n.s.). e. Representative cytosolic (left panel) and nuclear (right panel) immunoblots. S indicates Saline. D indicates D-serine. Error bars indicate SEM.
tERK level in the BLA cytosol at R1h might be the result of sustained ERK signaling and/or altered relocation of ERK. In the cytosol of mPFC, D-serine was found to affect the trend toward an increased pERK1/2 level at R1h (Fig. 6). BLA and mPFC have been reported to have functional connectivity with respect to their role in fear extinction (Herry et al., 2006; Sotres-Bayon et al., 2006), and the infralimbic region (IL) of mPFC in particular might play a crucial role in the recall of extinction (Quirk et al., 2000: Hefner et al., 2008; Laurent and Westbrook, 2009). In addition, NMDA receptors in IL have been suggested to be involved in the consolidation of extinction learning (Laurent and Westbrook, 2008; Sotres-Bayon et al., 2009). Taken together, increased pERK/tERK in BLA accompanied by the increased pERK/tERK in mPFC (Fig. 6) might have considerable meaning in terms of the retention and/or reconsolidation of reduced fear response by D-serine. The effect of D -serine in the cytosol of mPFC at R1h (Fig. 6) might have been more clear if our punched out samples were more centered on IL, considering that the neighboring prelimbic region does not seem to play a role in fear extinction (Vidal-Gonzalez et al., 2006; Laurent and Westbrook, 2009). In the standard neural model of fear extinction, mPFC-mediated amygdala inhibition is established at some point during consolidation of extinction, and after extinction learning, mPFC suppresses activity in the amygdala when the animal is required to retrieve the already-consolidated extinction memory (Sotres-Bayon et al., 2006). Thus, according to the model,
our results of ERK phosphorylation at R1h suggest that D-serine might increase the reactivity of BLA from the inhibitory input from mPFC in the retrieval process of fear extinction memory. One of the considerations was that ERK phosphorylation in the BLA at E1h was not significantly different between the two mice groups (Fig. 5). Thus, we cannot determine whether the alteration of ERK phosphorylation in BLA and mPFC did not occur in the extinction training phase or the alteration finished very quickly. The results might reflect the difference in the number of CS presentations between the extinction training and recall phase (15 and 30, respectively). In addition, it has been reported that the pre-extinction injection of D-cycloserine enhanced the extinction and increased ERK phosphorylation in the BLA 10 min after extinction (Yang and Lu, 2005). Therefore, further studies with an earlier sampling time point after several cued conditioned signals (during the extinction training) and after the extinction learning are needed to confirm the effect of Dserine on ERK phosphorylation. Finally, limitations due to sampling should be considered. The inclusion of neighboring areas in the dissected BLA might have affected the immunoblot analysis. 5. Conclusions The present study suggests that D-serine may enhance fear extinction via ERK phosphorylation in the hippocampus and BLA in the consolidation and/or retention of fear extinction memory. These
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