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d -Cycloserine acts via increasing the GluN1 protein expressions in the frontal cortex and decreases the avoidance and risk assessment behaviors in a rat traumatic stress model
Accepted Manuscript Title: D-cycloserine acts via increasing the GluN1 protein expressions in the frontal cortex and decreases the avoidance and risk assessment behaviors in a rat traumatic stress model Author: G¨okc¸e Elif Sarıdo˘gan Aslı Aykac¸ H¨ulya Cabadak Cem Cerit Mecit C ¸ alıs¸kan M. Zafer G¨oren PII: DOI: Reference:
S0166-4328(15)30121-2 http://dx.doi.org/doi:10.1016/j.bbr.2015.07.050 BBR 9738
To appear in:
Behavioural Brain Research
Received date: Revised date: Accepted date:
8-5-2015 21-7-2015 24-7-2015
Please cite this article as: Sarido˘gan Gddotokc¸e Elif, Aykac¸ Asli, Cabadak H¨ulya, Cerit Cem, C ¸ alis¸kan Mecit, Gddotoren M.Zafer.D-cycloserine acts via increasing the GluN1 protein expressions in the frontal cortex and decreases the avoidance and risk assessment behaviors in a rat traumatic stress model.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2015.07.050 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
D-cycloserine acts via increasing the GluN1 protein expressions in the frontal cortex and decreases the avoidance and risk assessment behaviors in a rat traumatic Stress Model
Gökçe Elif Sarıdoğan1, Aslı Aykaç2, Hülya Cabadak2, Cem Cerit3, Mecit Çalışkan4, M Zafer Gören5,* 1
Erenköy State Hospital, Psychiatry Clinics, 2Department of Biophysics, Marmara University,
School of Medicine, Istanbul, 3Department of Psychiatry, Kocaeli University, School of Medicine, Kocaeli, 4Haydarpaşa State Hospital, Psychiatry Clinics, 5Department of Medical Pharmacology, Marmara University, School of Medicine, *
Corresponding author: M. Zafer Gören, Marmara University, School of Medicine,
Department of Medical Pharmacology, Başıbüyük Health Campus, Basic Medical Sciences Building, Maltepe, Istanbul, 34854, Turkey e-mail: [email protected] Tel:+90 216 421 22 22 (1638), Fax: +90 216 414 47 31 Highlights ► Three day D-cycloserine (DCS) served as an adjuvant to extinction therapy in predator scent test in rats. ► GluN1 expressions were decreased in the amygdala and the dorsal hippocampus of rats with cat odor. ► Extinction training together with DCS upregulated GluN1 in the affected brain regions of rats. ► DCS with extinction training increased GluN1 protein levels in the frontal cortex.
Abstract
D-cycloserine (DCS), an FDA approved anti-tuberculosis drug has extensively been studied for its cognitive enhancer effects in psychiatric disorders. DCS may enhance the effects of fear extinction trainings in animals during exposure therapy and hence we investigated the effects of DCS on distinct behavioral parameters in a predator odor stress model and tested the optimal duration for repeated daily administrations of the agent. Cat fur odor blocks were used to produce stress and avoidance and risk assessment behavioral parameters were used where DCS or saline were used as treatments in adjunct to extinction trainings.
We observed that DCS facilitated extinction training by providing further extinction of avoidance responses, risk assessment behaviors and increased the contact with the cue in a setting where DCS was administered before extinction trainings for 3 days without producing a significant tolerance. In amygdala and hippocampus, GluN1 protein expressions decreased 72 hours after the fear conditioning in the traumatic stress group suggesting a possible downregulation of NMDARs. We observed that extinction learning increased GluN1 proteins both in the amygdaloid complex and the dorsal hippocampus of the rats receiving extinction training or extinction training with DCS.
Our findings also indicate that DCS with extinction training increased GluN1 protein levels in the frontal cortex. We may suggest that action of DCS relies on enhancement of the consolidation of fear extinction in the frontal cortex.
Keywords Amygdaloid complex . Dorsal hippocampus . Predator scent test . Post-traumatic stress disorder
1.Introduction
Post-traumatic stress disorder (PTSD) is a unique mental disorder owing to the fact that the disorder naturally requires an etiological factor, a traumatic experience as a stressor to elicit the associated intrusive re-experiencing symptoms, avoidance behaviors, negative alterations in cognition and mood, alterations in arousal and reactivity levels which finally result in significant distress and functional impairment according to its recent definition in Diagnostic and Statistical Manual of Mental Disorders, 5th edition (American Psychiatry Association, 2013). PTSD was moved from the category of anxiety disorders to a traumatic or stressor related disorders with the introduction of the DSM V. However the description of the disease extends to the second half of 19th century as hysteria defined by Pierre Briquet, traumatic neurosis by Hermann Oppenheim and choc nerveux by Jean Martin Charcot (Schestatsky et al, 2003). Despite the changes in the historical efforts of classification, the disorder has always been associated with a triggering etiologic factor. Therefore it is no surprise that PTSD was considered as the only mental disorder that involves an explicit conditioning session. Furthermore, it is closely associated with the “conditioning frame-work” (Vanelzakker et al, 2014). From this view, a neutral stimulus may result in an unadaptive fear response when it was previously associated with an earlier traumatic experience. In other words, a memory trace may be formed between the neutral and the threatening events. Thus neutral cues may activate the threatening representations, triggering the anticipatory behaviors and anxiety (Vervliet, 2008). A variety of pharmacological agents are available in the pharmacotherapy of the posttraumatic stress disorder. Current evidence is not sufficient to support the efficacy of any specific agent in the mainstream symptoms of PTSD, however the efficacy of cognitive behavioral therapy (CBT) has been well documented (Baker et al, 2009; Mendes et al, 2008; Rothbaum et al, 2014). Therefore a tendency towards identifying pharmacological agents which can enhance the overall effectiveness of CBT and target the fear memory arose in the last
decade.
Hydrocortisone,
propranolol,
3,4-methylenedioxy-N-methylamphetamine,
riluzole and D-4-amino-3-isoxazolidone (D-cycloserine; DCS) are some of the promising agents (Kleine et al, 2013; Nandhra et al, 2013). DCS is an FDA approved anti-tuberculosis drug which has extensively been studied for its cognitive enhancer effects in psychiatric
disorders. DCS may enhance the effects of fear extinction trainings in animals and exposure therapy in human studies (Norberg et al, 2008). Mechanism of action of DCS is through the N-methyl-d-aspartate subtype of glutamatergic receptors (NMDARS). The crucial role of the NMDARs in the fear extinction process has been reported by several researchers (Falls et al, 1992; Ledgerwood et al, 2003; Ledgerwood et al, 2004, 2005, Walker et al, 2002 ).
The aim of this current study is to show the efficacy of DCS on different aspects of the fear related behaviors in rats subjected to predator scent in extinction sessions as an animal model for exposure therapy and the expression of GluN1 (NMDAR NR1 subunit) in critical brain regions involved in PTSD. 2.Methods 2.1 Subjects Thirty adult Wistar rats of both sexes supplied from Experimental Animal Research Center (DEHAMER, Marmara University, Istanbul) were used in the study. The number of male and female rats were equal in all groups. Upon getting approval from the University Local Ethical Committee, the rats were maintained in 12 h reverse light-dark circle (lights on 7 p.m.), at a constant room temperature of 22±2º C and a humidity ratio of 50±5 %. The reason that reverse light-dark cycle to be used is that the rats are nocturnal animals so that the procedures would be performed during the active period of the rats. Rats were fed ad libitum with standardized rat food and water. Each rat was handled once a day for 10 minutes by the same researcher. During this period, 6 rats were housed per cage but following stress procedures the rats were housed singly. Each experimental group consisted of 6 rats. The rats were assigned into 5 groups where the first group consisted of non-traumatized controls and the second traumatized controls. These 2 groups received no treatment. The other three groups were also traumatized and received either physiological saline or DCS for 3 or 5 days together with extinction procedures.
2.2 Experimental Apparatus The apparatus in which the rats were exposed to odor or no-odor blocks was a plexiglass box with 100x15x50 cm dimensions allowing the rat a back and forth movement. The apparatus was reproduced according to Blanchard et al (2001). The box had clear walls enabling video recording and the front wall of the box was divided into 3 equal segments with a red marker.
Wooden blocks covered with cotton fabric were used to serve as predator odor stress stimulus. Blocks were left in the bed of street cats for 2 days prior to testing and rubbed against the fur around the cats neck prior to the experiment. Identical clean blocks were used as no odor control blocks.
2.3 Procedure Stimulus block was placed at one end of the box. A rat was placed at the other end and allowed to move freely in the box for 10 minutes. Sessions were recorded by a video device under red light (7 Watts). Behavioral parameters were assessed according to the cat fur odor model (Blanchard et al, 2001). The analyzed behavioral parameters included were: Far location: Time spent in the far segment of the experimental apparatus from the compartment where block is placed. Contact duration: Time spent in contact with the block. Far location and contact duration; former being directly and the later inversely asseses the avoidance. Freezing time: Total immobility time. Location change: Total number of location change in between 3 compartments. This parameter is scored when all 4 limbs of the animals move to another segment. Activity is measured by this parameter. Stretch Attend Duration: Duration of the subject’s stationary position where the body is elongated with a low back when the subject is facing the stimulus block with placing fore and hind limbs far apart . Stretch Approach Duration: Duration of the subject’s movement toward stimulus block with a low back and elongated posture. Stretch attend and stretch approach were considered as the risk assessment behaviors. The phases of the procedure were as described:
Habituation: From day 1 to day 4, each rat was placed in the test apparatus for 10 min for 4 consecutive days in the absence of the stimulus block.
Cat odor block exposure: On day 5, the rats were exposed to cat odor block while control group rats were exposed to no-odor blocks. Each session for a subject was videotaped for 10 minutes and the behaviors were assessed.
Extinction: Days 6 through 8, 3 groups of rats; days 6 through 10, another extra group of rats were exposed to no-odor block in the apparatus for 10 min daily. Two groups of rats experiencing the extinction procedure also received subcutaneous 15 mg/kg DCS (Sigma, USA) 15 min before each session while the other extinction group as well as the nonextinction groups were treated with subcutaneous physiological saline (0.1 ml/100 g rat).
Test: On day 9, 4 groups were tested in the same apparatus with a no-odor block. On day 11, only the rats that received DCS injections for 5 days were exposed to no-odor blocks. The procedures and treatments of all groups were summarized in Table.1. 2.4 Tissue Preparation and Immunoblotting All chemicals were purchased from Sigma (St Louis, MO, USA) unless indicated. The rats were decapitated following high dose subcutaneous thiopental sodium (Pental®, İE Ulagay, Turkey) administration. The amygdaloid complex, the dorsal hippocampus and the frontal cortex were removed in accordance with the Rat Brain Atlas (Paxinos and Watson, 1986) and were frozen at -80oC until further processed. The slides in the anteroposterior planes located between 6.70- 4.70 mm, 13.20-11.20 mm and 7.20-5.70 mm anterior to the interaural line were taken for the separation of the amygdaloid complex, the dorsal hippocampus and the frontal cortex, respectively. The distance between interaural line and bregma was accepted as 9.00 mm, posterior to the tip of the brain. The frozen tissues were weighed and homogenized in ice cold 10 mM EDTA and protease inhibitors (0.2 mM phenylmethanesulfonyl fluoride, 1 µM pepstatin, 10 µg/ml soybean trypsin inhibitors) with T25 Ultraturrax homogenizer (Janke&Kunkel IKA-Labortechnik, Germany). Whole homogenates were used in western blots. The protein content of the crude homogenate was determined as indicated (Lowry et al, 1951). One hundred µg of protein was loaded onto 12% sodium dodecyl sulfate-polyacrylamide gels and electrophoretically transferred onto 0.45 µm nitrocellulose membranes (Schleicher and Schuell, Germany) for 120 min at 80 V. The membranes were blocked with tris buffered saline containing 1% bovine serum albumin and 0.05% Tween-20 at room temperature for 60 min and incubated overnight at 4oC with antibody against GluN1 (NMDAR subunit NR1; added in concentration 1:100). Results were standardized using β-actin (Santa Cruz, CA, USA; 1:200 dilution) as the control protein, which was detected by evaluating the band density at 43 kDa.
Membranes were incubated with the rabbit polyclonal anti-GluN1 antibody at a (1:100) dilution. The size of the GluN1 protein is 115 kDa. The GluN1 specific antibody were supplied by Santa Cruz Biotechnology, Inc. (Santa Cruz, USA). The secondary antibodies were purchased from Sigma (USA). All chemicals were obtained from Sigma, unless stated otherwise. The blots were washed three times with Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and incubated with alkaline phosphatase-conjugated secondary antibodies for 1 h at room temperature (20oC). The antibody–antigen complex was detected with NBT/BCIP (nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indolyphosphate; Promega, Wisconsin, USA). The densitometric analyses were carried out with Bio-Rad Molecular Analyst software (free edition, www.totallab.com). 2.5 Statistical Analysis Unpaired Student's t-test was used for comparing the behavioral effects on the test day and the control group. One-way analysis of variance followed by Tukey's multiple comparison post hoc test were used for the remaining comparisons. Statistical significance was accepted where p<0.05. 3.Results 3.1 The Behavioral Consequences of the Traumatic Stress On the fifth day, the data of the behavioral parameters of the traumatized rats who were exposed to cat odor blocks (4 groups) were pooled and compared to that of control rats (the rats exposed to no odor blocks). The traumatized rats were then assigned into different treatment and extinction training protocols. The means±sem values of the behavioral data and p value were presented in Table.2. The analyses have shown that risk assessment behaviours were distorted in rats exposed to odor blocks. 3.2 The Effects of DCS Treatment and Cue Extinction in Trauma Reminding Sessions On the test days, all subjects were exposed to no-odor blocks in the same test apparatus. The sessions were recorded and the behavioral parameters were calculated. Contact durations: One-way analysis of variance produced significant variance within the group (F=9.446, df=4, p=0.0002). Traumatized rats receiving a 3 day DCS treatment with extinction training touched the block for a longer duration, being significantly different from saline treated group (p<0.05; Fig.1a). No other statistically significant difference was observed in the contact durations between other groups.
Far location: One-way analysis of variance yielded a significant variance in the far location parameter (F=5.694, df=4; p=0.0038). The mean of far location duration of the control group rats exposed to no-odor blocks treated with physiological saline was 103.0±2.5 s while the rats exposed to odor blocks treated with physiological saline stayed 289.3±35.3 s in the far segment producing a statistical significance (p<0.05). Far location duration of the traumatized 3 day extinction training group treated with physiological saline was 255.2±43.8 s. Post hoc test also revealed a significant difference in the far location parameter between nontraumatized group and traumatized group experiencing a 3 day extinction training (p<0.05). The far location duration of the traumatized rats that received 3 day DCS injection and extinction training was 83.50±25.7 s yielding statistically significant decrease in the time spent in the far segment compared to that of their matching controls (p<0.05; Fig.1b). Far location duration of the traumatized rats that received 5 day DCS treatment with extinction training was 188.6±40.0 s. There was no statistical significance between traumatized rats that received 5 day DCS treatment with extinction training and non- traumatic control group treated with physiological saline (p>0.05; Fig.1b). The far location duration of the traumatized rats that received 3 day DCS and extinction training showed a statistically significant decrease, compared to that of their controls (p<0.01). No statistically significant difference was found between 5 day DCS and extinction training group and 3 day physiological saline treated control group (p>0.05). Stretch attend duration: One-way analysis of variance yielded significant variances (F=6.7570, df=4; p=0.0008). Traumatized rats treated with physiological saline displayed a longer stretch attend duration compared to traumatized rats that received 3 or 5 day DCS injections and extinction sessions (p<0.05; Fig.1c). Stretch approach duration: No statistically significant difference was observed among groups (F=0.7286, df=4, p=0.5809; Fif.1d). Freezing times: The freezing times of all experimental groups did not produce statistical significant change (F=1.6100, df=4; p=0.5809; Fig.1e). Location change: No statistically significant difference was found between groups (F=1.3990, df=4; p=0.2630; Fig.1f). 3.3 Densitometric Analyses of GluN1 Protein Immunoblots Performed in Different Brain Regions
Analysis of western-blots of the frontal cortex homogenates showed significant changes (F=358.7, df=3; p<0.0001). The results imply that the rats exposed to cat odor had increased GluN1 protein and extinction training alone didn’t produce a significant change. Adding DCS to extinction sessions again resulted in increased GluN1 protein levels (Fig.2a; p<0.05). One way analysis of variance revealed significant variances in the dorsal hippocampus (F=141.4, df=3, p<0.0001) where GluN1 expression decreased in rats subjected to cat odor (Fig.2b; p<0.01) extinction training alone or with DCS injection for 3 days restored these decreases. Similarly, GluN1 protein expression in the amygdaloid complex decreased in response to cat odor (p<0.05) and both treatment approaches corrected these changes (Fig.2c; F=30.12, df=3, p<0.0001). A representative immunoblots for GluN1 and β-actin were presented in Fig.3 4. Discussion In this study, we demonstrate that cat odor can generate traumatic stress in rats and the noodor blocks can trigger trauma related behavioral changes. The conditioning process in the PTSD and emerging anxiety have long been attributed to the impaired extinction of the previously conditioned fear responses (Eysenck, 1979; Pitman and Orr, 1986). We demonstrate that extinction procedures restored the worsened behavioral parameters. DCS administration immediately before the extinction procedures produced better outcomes. Our recent results show that a 3 day extinction training with DCS injection protocol significantly decreased the duration of avoidance and the risk assessment behaviors. Furthermore, the duration of contact with the block also increased with extinction procedures and DCS adjunct. These findings may suggest that DCS can serve as an adjuvant in decreasing the avoidance and the perceived risk related to the threatening cue, while facilitating the reconsideration of the perceived risk. In clinical settings this finding may be help patients to cope with the desired tasks and work with the trauma related cue exposure sessions. It was previously shown that DCS alone did not produce any amelioration in the conditioned fear responses without the extinction learning (Walker et al, 2002; David et al, 2002). Other behavioral parameters apart from the anxiety levels should be considered in further preclinical and clinical studies to clarify the controversial results in the literature. The augmentation effect of DCS in fear extinction was shown in experimental animals by measuring the fear-potentiated startle in a conditioning paradigm (Walker et al, 2002). Surprisingly, the group showed that this effect could only be observed in the rats receiving DCS together with extinction training. The authors used intraamygdaloid injections of DCS
and HA-966, an antagonist for NMDAR glycine-recognition site. HA-966 blocked the effects of the extinction sessions. DCS alone could not augment the fear extinction. DCS or HA-966 failed to affect fear-potentiated startle if injected before testing. The findings were replicated by subsequent studies (Ledgerwood et al, 2003, Ledgerwood et al, 2004). Previous studies indicated that the timing of administration, the dose, the dosing intervals of DCS, the quality and the number of the training/exposure sessions, the distribution of DCS administrations over the training sessions, previous exposure to DCS affect the overall success of the drug. Timing of DCS administration was found to be critical for the extinction retention effect (Ledgerwood et al, 2003). In that study, a linear decrease in the extinction effect was observed if long intervals were given between the extinction sessions and the drug administration (Ledgerwood et al, 2003). It was also demonstrated that insufficient number of extinction training sessions may result in an enhanced reconsolidation of fear acquisition (Lee et al, 2006). In the mentioned study while short extinction resulted in augmented fear long extinction training resulted in augmentation of extinction. In another study, 5 repeated DCS administrations once every other day and a later post extinction DCS injection produced a right shift in the dose-response curve indicating a reduced facilitation of extinction effect due to a possible development of tolerance (Parnas et al, 2004). In our study we examined the optimum number of repeated DCS administrations needed which can effectively enhance extinction. We demonstrate that 3 day regimen had a better outcome than 5 day regimen. Likewise, 5 day treatment group showed indifferent results from the non-traumatized control group in our study. It was previously reported that habituation took place on the 5th day in a predator stress model where worn cat collar was used 20 min per day for extinction training (Dielenberg and McGregor, 1999). Similarly in our study this effect is likely to be caused by the nature of predator stress model rather than tolerance. In our study far location duration on the 5 day DCS group stayed in the far segment from the block according to 3 day DCS group. This result is possibly related with the habituation itself where rats finally start freely exploring the apparatus and the compartment of the block became insignificant since we observed free exploration behaviour around the 5th day in all groups. According to our findings we propose that 5 day extinction may be too long since habituation to predator stress may take place and tolerance to DCS can be observed in repeated daily administrations leading to a reduced facilitation of extinction in another animal model of severe traumatic stress where habituation itself takes longer to appear. 3 day
repeated administrations may show superior and adequate results for mild to moderate traumatic stresses such as the model we used in this study. Many reports indicated that DCS administration prior to (Walker et al, 2002, Parnas et al., 2005) or shortly after (Ledgerwood et al., 2003; Ledgerwood et al., 2004; Ledgerwood et al., 2005) the extinction training facilitated extinction. Walker et al. reported that this enhancement occurs in a dose dependent manner where the rats receiving 15 mg or 30 mg of DCS prior to extinction training exhibited facilitation of extinction while the rats given 3.25 mg responded like the saline given rats (2002). We also conducted our study with a dose of 15 mg/kg as used by Walker et al. (2002) and Ledgerwood et al. (2003) and prior to extinction training. Our results support those reported by Walker et al. (2002), and also show that DCS is effective at facilitating extinction when administered prior to extinction trials. Two published clinical trials designed to assess the effects of DCS in PTSD patients failed to show an augmentation of exposure effect. First trial didn’t report enhancement of the overall treatment effect (de Kleine et al., 2012) and moreover the second trial reported greater symptoms in the DCS group (Litz et al., 2012). Further clinical studies with different study designs are needed to underline the changes in different behavioral aspects of PTSD.
Freezing time is also a commonly used behavioral parameter in previous anxiety and stress studies (Ledgerwood et al., 2003; Walker et al., 2002; Lee et al., 2006). However, we didn't observe high levels of freezing in all groups in our experiments. Correspondingly Blanchard et. al reported that the immobile crouch/freeze do not increase with exposure to cat odor (2001). A possible explanation for this finding may be that cat odor is a milder traumatic stress to produce significant freezing levels. It was also reported that freezing occurs as low as 4% of the time when an attacking cat is not present in the experiment settings (Fanselow, 1989). In the traumatized rats, we found that the freezing time constituted 8% of the experimental observation period. This may also indicate that freezing time is not a suitable parameter itself for predator odor stress models. The role of NMDARs in the long term potentiation (LTP) is well known. LTP is widely accepted to take place in the first 24 hours and can be described as a “capacity to reactivate” instead of presence of an “active state” (Dudai et al, 2002). Different types of NMDA transmission patterns were observed in this process (Zinebi et al. 2003). Another human study has shown that DCS facilitated recall of the fear memory 72 hours after acquisition of cued
conditioned fear. Authors attributed a role for NMDA in consolidation of fear to explain this finding (Kalisch et al., 2009). The mechanism of action of DCS is not known, but the affinity of DCS to the glycine modulatory site on the GluN1 seems to be the most logical explanation (Sheinin et al., 2001). The distribution of NMDARs in the brain is widespread (Myers et al, 2011) and their functions learning and memory processes have been long shown. There are two separate subunit families that join to make up functional NMDARs; the GluN1 and GluN2 subunits which are coded with different genes. The GluN1 subunit is an essential component of all NMDAR complexes while there are four GluN2 (GluN2A-D) and recently found GluN3 (GluN3A-B) subunits. Both subunit types are required to generate a fully functioning receptor. The modulatory polyamines bind to GluN2B subunit and DCS is suggested to be a GluN1 partial agonist where its efficacy determined by GluN2 subunits (Myers et al, 2011; C The activity of the NMDARs located in the basolateral amygdala (de Kleine et al, 2013; Norberg et al, 2008), hippocampus and subsequent signaling pathways (Bevilaqua et al, 2005; Szapiro et al, 2003) has been linked to fear extinction. Extinction is a learning process composed of acquisition, consolidation and retrieval phases. The amygdala plays the leading role in the encoding, consolidation and expression of extinction memory and works interconnectedly with the hippocampus and the medial prefrontal cortex (Quirk and Mueller, 2008). Evidence regarding the involvement of medial prefrontal cortex in fear extinction includes both positive findings indicating the involvement of this area (Milad and Quirk 2002,1) and non-validating negative results ( Gewirtz et al. 1997,). Animal studies established the interaction between the amygdala and the medial prefrontal cortex as crucial for the extinction learning. Furthermore the infralimbic prefrontal cortex have been revealed to have a role in fear consolidation (Gupta et. al. 2013; Ciabarra et al, 1995; Forcina et al, 1995; Low et al, 2010; Dravid et al, 2010).
We also performed immunoblotting in order to have a better understanding of the fear and extinction neurocircuitry, together with the mechanism and the target brain sites of DCS action. Gupta et al. suggested that DCS may have temporally changing effects over the cortico-amygdala circuitry which may be explained as a more short term effect in amygdala and a longer lasting effect in PFC. In the mentioned study extinction training resulted in increased GluA1, GluN2A and GluN1 expression in the mPFC compared to the groups that
did not undergo extinction training suggesting an induced LTP in the mPFC. However authors reported no alteration in the protein expression in spite of the increased pERK activity in the mPFC 6 hours after DCS was administered. Authors related this finding with saturation of increasing in the protein levels after extinction training and short duration that may not allow to induce the protein level change in the mPFC. DCS administration was also reported to cause dramatic increase in iGluR levels in the amygdala and GluN2B expression was also suggested to facilitate fear extinction consolidation in this region. (Gupta et al., 2013).
We demonstrated that GluN1 protein decreased in the amygdaloid complex of traumatized control rats. GluN1 protein levels were restored in rats that receiving extinction training or DCS with training. Our findings were consistent with that of Zinebi et al. (Zinebi et al., 2003). They reported three to four fold decrease in GluN1, GluN2A and GluN2B subunit protein expressions in the amygdala of the fear conditioned rats. Therefore, they suggested that NMDAR-mediated transmission in the lateral amygdala down-regulates while the fear memory is being maintained. They suggested that this process occurs to allow a capacity to reactivate the fear memories and provide protection against excitotoxicity from NMDA receptor recruitment and lead to long term depression after fear acquisition. The effects of extinction training to the cue probably continue to take place both in the amydaloid complex and in the dorsal hippocampus immediately after the last extinction session. This may suggest a role for dorsal hippocampus for the extinction learning to cues as well as contextualized extinction learning. Our findings show no superior results in terms of GluN1 expressions in both brain regions when DCS was administered together with the extinction sessions. Instead, we observed slightly decreased level of GluN1 protein expressions in the dorsal hippocampus in the group receiving DCS and extinction training. In addition, we found increased GluN1 protein levels in the frontal cortex of rats that received 3 day DCS plus extinction training suggesting a consolidation phase of the extinction memory. Thus increased levels of GluN1 proteins possibly could have been observed in this area within the first days of the extinction training. We may also indirectly suggest that the decreased levels of GluN1 protein in the hippocampus of the DCS plus extinction group may be another indicator of a down-regulation due to the onset of consolidation phase that likely takes place in the frontal cortex. However, phases of maintenance of the fear memory should be further studied to clarify the temporal roles of the key structures in the fear neurocircuitry. According to our findings, we may suggest that DCS administration possibly facilitates extinction memory consolidation by enhancing the expression of new NMDARs in the frontal
cortex. Another recent study found increased expressions of GLuA1, GluN1, GluN2A, GluN2B in the medial prefrontal cortex (mPFC) of untrained rats following intraperitoneal DCS injection while they observed no significant difference in the protein levels when DCS was administered in conjunction with extinction training (Gupta et al., 2013). Our study demonstrates increased GluN1 protein expressions in the DCS with extinction group indicating mPFC as a major target for DCS action. Conclusion In conclusion, our study suggests that DCS may facilitate the outcomes of cognitive behavioral therapies in PTSD by decreasing the avoidance and the attributed risk to the threatening cue while facilitating the reconsideration of the perceived risk. In amygdala and hippocampus GluN1 protein expressions decreased 72 h after the cued fear conditioning in traumatic stress group indicating a possible downregulation of NMDARs. We suggest that this process may occur in order to enable formation of either new fear memories or extinction learning and to impede further excitotoxicity. We observed the expression of GluN1 protein increased in the amygdaloid complex and the dorsal hippocampal regions of groups that received extinction trainining alone or with DCS. According to our findings, DCS action may eventually occur in the frontal cortex resulting in enhancement of the consolidation of fear extinction and further extinction retention.
Acknowledgements This study was sponsored by Santa Cruz Biotechnology Inc., Turkey and partly by a grant supplied from Marmara University (Project number: SAG-A-110913-0363). References American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders, 5th ed. Arlington VA: American Psychiatric Publishing Arlington; 2013. Baker DG, Nievergelt CM, Risbrough VB. Posttraumatic Stress Disorder: Emerging Concepts of Pharmacotherapy. Expert Opin Emerg Drugs. 2009;14:251–272. Blanchard RJ, Yang M, Li CI, Gervacio A, Blanchard DC. Cue and context conditioning of defensive behaviors to cat odor stimuli. Neurosci Biobehav Rev. 2001;25:587-95. Bevilaqua LR, da Silva WN, Medina JH, Izquierdo I, Cammarota M. Extinction and reacquisition of a fear-motivated memory require activity of the Src family of tyrosine kinases in the CA1 region of the hippocampus. Pharmacol Biochem Behav. 2005;81:139–145.
Ciabarra AM, Sullivan JM, Gahn LG, Pecht G, Heinemann S, Sevarino KA. Cloning and characterization of chi-1: a developmentally regulated member of a novel class of the ionotropic glutamate receptor family. J Neurosci. 1995;15:6498–6508. Walker DL, Ressler KJ, Lu KT, Davis M. Facilitation of Conditioned Fear Extinction by SystemicAdministration or Intra-Amygdala Infusions of D-Cycloserine as Assessed with Fear-Potentiated Startle in Rats. The Journal of Neuroscience 2002;22:2343–2351. Dravid SM, Burger PB, Prakash A, Geballe MT, Yadav R, Le P, Vellano K , Snyder JP , Traynelis SF. Structural determinants of D-cycloserine efficacy at the NR1/NR2C NMDA receptors. J Neurosci. 2010;30:2741-54. Dudai Y. Molecular bases of long-term memories: a question of persistence. Curr Opin Neurobiol. 2002;12:211-6. De Kleine RA, Hendriks GJ, Kusters WJ, Broekman TG, van Minnen A. A randomized placebo-controlled trial of D-cycloserine to enhance exposure therapy for posttraumatic stress disorder. Biol Psychiatry. 2012;71:962-8. De Kleine RA, Rothbaum BO, van Minnen A. Pharmacologic enhancement of exposure based treatment in PTSD: a qualitative review. Eur J Psychotraumatol. 2013;17;4. Dielenberg RA, McGregor IS. Habituation of the hiding response to cat odors in rats (Rattus norvegicus). J Comp Psychol. 1999;113:376-87. Dravid SM, Burger PB, Prakash A, Geballe MT, Yadav R, Le P et al. Structural determinants of D-cycloserine efficacy at the NR1/NR2C NMDA receptors. J Neurosci. 2010;30:2741-54. Eysenck HJ. The conditioning model of neurosis. The Behavioral and Brain Sciences. 1999;2:155–199. Falls WA, Miserendino MJ, Davis M. Extinction of fear potentiated startle: blockade by infusion of an NMDA antagonist into the amygdala. J Neurosci. 1992;12:854–863. Fanselow MS. The adaptive function of conditioned defensive behaviour: an etiologic approach to Pavlovian stimulus-substitution theory. In: Blanchard RJ, Brain PF, Blanchard DC, Parmigiani S, editors. Ethoexperimental approaches to the study of behavior. Dordrecht: Kluwer Academic Publishers; 1989, p.114-36. Forcina MS, Ciabarra AM, Seravino KA. Cloning of chi-2: a putative member of the ionotropic glutamate receptor superfamily. Soc Neurosci Abstr. 1995;21:438.3. Gewirtz JC, Falls WA, Davis M. Normal conditioned inhibition and extinction of freezing and fear-potentiated startle following electrolytic lesions of medial prefrontal cortex in rats. Behav Neurosci. 1997;111:712–726. Gupta SC, Hillman BG, Prakash A, Ugale RR, Stairs DJ, Dravid SM. Effect of D-cycloserine in conjunction with fear extinction training on extracellular signal-regulated kinase activation in medial prefrontal cortex and amygdala in rat. Eur J Neurosci. 2013;37:1811–1822. Kalisch R, Holt B, Petrovic P, De Martino B, Klöppel S, Büchel C, Dolan RJ. The NMDA agonist D-cycloserine facilitates fear memory consolidation in humans. Cereb Cortex. 2009;19:187-96.
Ledgerwood L, Richardson R, Cranney J. Effects of D-cycloserine on extinction of conditioned freezing. Behav Neurosci. 2003;117:341–349. Ledgerwood L, Richardson R, Cranney J. D-cycloserine and the facilitation of extinction of conditioned fear: consequences for reinstatement. Behav Neurosci. 2004;118:505–513. Ledgerwood L, Richardson R, Cranney J. D-cycloserine facilitates extinction of learned fear: effects on reacquisition and generalized extinction. Biol Psychiatry. 2005;57:841–847. Lee JLC, Milton AL, Everitt BJ. Reconsolidation and extinction of conditioned fear: Inhibition and potentiation. The Journal of Neuroscience. 2006;26:10051–10056. Litz BT, Salters-Pedneault K, Steenkamp MM, Hermos JA, Bryant RA, Otto MW, Hoffman SG . A randomized placebo-controlled trial of D-cycloserine and exposure therapy for posttraumatic stress disorder. J Psychiatr Res. 2012;46:1184-9 Low CM, Wee KS. New insights into the not-so-new NR3 subunits of N-methyl-D-aspartate receptor: localization, structure, and function. Mol Pharmacol. 2010;78: 1-11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry. 1951;193:265–327. Mendes DD, Mello MF, Ventura P, Passarela Cde M, Mari Jde J. A systematic review on the effectiveness of cognitive behavioral therapy for posttraumatic stress disorder. Int J Psychiatry Med. 2008;38:241-259. Milad MR, Quirk GJ.Neurons in medial prefrontal cortex signal memory for fear extinction. Nature. 2002;420:70-4 Myers KM, Carlezon WA Jr, Davis M. Glutamate Receptors in Extinction and ExtinctionBased Therapies for Psychiatric Illness. Neuropsychopharmacology Reviews. 2011;36:274– 293 Nandhra HS, Murphy CL, Sule A. Novel pharmacological agents targeting memory and cognition in the treatment of anxiety disorders. Hum Psychopharmacol Clin Exp 2013;28:538–543. Norberg MM, Krystal JH, Tolin DF. A Meta-Analysis of D-Cycloserine and the Facilitation of Fear Extinction and Exposure Therapy. Biol Psychiatry. 2008;63:1118–1126. Sheinin A, Shavit S, Benveniste M. Subunit specificity and mechanism of action of NMDA partial agonist D-cycloserine. Neuropharmacology. 2001;41:151-8. Szapiro G, Vianna MR, McGaugh JL, Medina JH, Izquierdo I. The role of NMDA glutamate receptors, PKA, MAPK, and CAMKII in the hippocampus in extinction of conditioned fear. Hippocampus. 2003;3:53–58. Parnas AS, Weber M, Richardson R. Effects of multiple exposures to D-cycloserine on extinction of conditioned fear in rats. Neurobiol Learn Mem. 2005;83:224–231. Paxinos G, Watson C. The rat brain in stereotaxic coordinates, 2nd ed. London: Academic Press, 1986
Pitman RK, Orr SP. Test of the conditioning model of neurosis: Differential aversive conditioning of angry and neutral facial expressions in anxiety disorder patients. Journal of Abnormal Psychology. 1986;95:208–213. Rothbaum BO, Price M, Jovanovic T, Norrholm SD, Gerardi M, Dunlop B, Davis M, Bradley B, Duncan EJ, Rizzo A, Ressler KJ. A randomized, double-blind evaluation of D-cycloserine or alprazolam combined with virtual reality exposure therapy for posttraumatic stress disorder in Iraq and Afghanistan War veterans. Am J Psychiatry. 2014;171:640-648. Quirk GJ, Mueller D. Neural mechanisms of extinction learning and retrieval. Neuropsychopharmacology. 2008;33:56–72. Schestatsky S, Shansis F, Ceitlin LH, Abreu PB, Hauck S. Historical evolution of the concept of posttraumatic stress disorder. Rev Bras Psiquiatr. 2003;25:8-11. Vanelzakker MB, Dahlgren MK, Davis FC, Dubois S, Shin LM. From Pavlov to PTSD: the extinction of conditioned fear in rodents, humans, and anxiety disorders. Neurobiol Learn Mem. 2014;113:3-18. Vervliet B. Learning and memory in conditioned fear extinction: effects of D-cycloserine. Acta Psychol (Amst). 2008;127:601-613. Walker DL, Ressler KJ, Lu KT, Davis M. Facilitation of conditioned fear extinction by systemic administration or intraamygdala infusions of D-cycloserine as assessed with fear potentiated startle in rats. J Neurosci. 2002;22:2343–2351. Zinebi F, Xie J, Liu J, Russell RT, Gallagher JP, McKernan MG et al. NMDA currents and receptor protein are downregulated in the amygdala during maintenance of fear memory. J Neurosci. 2003;23:10283-91.
All rats were habituated and exposed to no odor block as a trauma reminder on the corresponding test days.
All assesments were done on day 5, following habituation period.
Legends for figures
Fig. 1 The behavioral parameters (contact duration (a), far location (b), stretch approach (c), stretch attend (d), freezing time (e), total location change count (f), in the insets) calculated from the video recordings of rats re-exposed to trauma reminder, the no odor block who were previously traumatized with cat odor (thick bordered boxes) or no odor (thin bordered box) blocks. The groups that received extinction protocol were marked with a line. Subcutaneous D-cycloserine treatments were given 15 min prior to sessions (15mg/kg; n=6/group; *, p< 0.05, **, p< 0.01, as indicated). The data are expressed as means ± s.e.m. Fig. 2 The densitometric analyses obtained from photographs of western immunoblots of 3 different brain regions of rats exposed to cat odor (the thick bordered boxes) where the control rats were exposed to no odor blocks. Traumatized rats were either treated with physiological saline or subjected to extinction training sessions with or without d-cycloserine injections (15 mg/kg) for 3 days. The brain slides were taken upon completion of behavioral experiments where the rats were exposed to trauma reminder. *, p<0.05,**, p<0.01, as indicated. The data are expressed as means ± s.e.m. Fig. 3 The
photographs of nitrocellulose membranes collected from immunoblotting
experiments of non-traumatized control rats (a) and traumatized rats with cat odor treated with physiological saline (b), extinction training (c) or D-cycloserine (d). The blots used were for GluN1 as indicated. β-actin was used to normalize the amount of protein loaded in each lane.
Figure-1 .
Figure-2 .
Figure-3 .
Table.1. The summary of experimental groups and the procedures Type of block exposed
Number of extinction days with no odor block
Test day
Groups Non traumatized control Traumatized control Traumatized and subjected to extinction procedures and treated with physiological saline for 3 days Traumatized and subjected to extinction procedures and treated with DCS for 3 days Traumatized and subjected to extinction procedures and treated with DCS for 5 days
No odor block Odor block Odor block
9 -
9
3
9
3
9
Odor block
Odor block
11 5
Table.2. The change in the behavioral parameters in control rats (exposed to no odor blocks) and the rats traumatized with cat odor (odor block). The type of block exposed The risk assessment behaviors
No odor block
Odor block
Statistics summary
Contact duration
12.0±3.3 s
116.0±27.9 s
p<0.001
Far location
494±13.7s
289.7±68.8 s
p<0.01
Stretch attend duration
1.5±3.4 s
33.3±13.7 s
p<0.01
Stretch approach duration
5.8±2.71 s
23.0±2.4 s
p<0.01
Freezing
11.0±2.6 s
50.6±2.4 s
p<0.05
Location change frequency
29.9±9.0 s
15.4±3.1 s
p<0.05
S0166-4328(15)30121-2 http://dx.doi.org/doi:10.1016/j.bbr.2015.07.050 BBR 9738
To appear in:
Behavioural Brain Research
Received date: Revised date: Accepted date:
8-5-2015 21-7-2015 24-7-2015
Please cite this article as: Sarido˘gan Gddotokc¸e Elif, Aykac¸ Asli, Cabadak H¨ulya, Cerit Cem, C ¸ alis¸kan Mecit, Gddotoren M.Zafer.D-cycloserine acts via increasing the GluN1 protein expressions in the frontal cortex and decreases the avoidance and risk assessment behaviors in a rat traumatic stress model.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2015.07.050 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
D-cycloserine acts via increasing the GluN1 protein expressions in the frontal cortex and decreases the avoidance and risk assessment behaviors in a rat traumatic Stress Model
Gökçe Elif Sarıdoğan1, Aslı Aykaç2, Hülya Cabadak2, Cem Cerit3, Mecit Çalışkan4, M Zafer Gören5,* 1
Erenköy State Hospital, Psychiatry Clinics, 2Department of Biophysics, Marmara University,
School of Medicine, Istanbul, 3Department of Psychiatry, Kocaeli University, School of Medicine, Kocaeli, 4Haydarpaşa State Hospital, Psychiatry Clinics, 5Department of Medical Pharmacology, Marmara University, School of Medicine, *
Corresponding author: M. Zafer Gören, Marmara University, School of Medicine,
Department of Medical Pharmacology, Başıbüyük Health Campus, Basic Medical Sciences Building, Maltepe, Istanbul, 34854, Turkey e-mail: [email protected] Tel:+90 216 421 22 22 (1638), Fax: +90 216 414 47 31 Highlights ► Three day D-cycloserine (DCS) served as an adjuvant to extinction therapy in predator scent test in rats. ► GluN1 expressions were decreased in the amygdala and the dorsal hippocampus of rats with cat odor. ► Extinction training together with DCS upregulated GluN1 in the affected brain regions of rats. ► DCS with extinction training increased GluN1 protein levels in the frontal cortex.
Abstract
D-cycloserine (DCS), an FDA approved anti-tuberculosis drug has extensively been studied for its cognitive enhancer effects in psychiatric disorders. DCS may enhance the effects of fear extinction trainings in animals during exposure therapy and hence we investigated the effects of DCS on distinct behavioral parameters in a predator odor stress model and tested the optimal duration for repeated daily administrations of the agent. Cat fur odor blocks were used to produce stress and avoidance and risk assessment behavioral parameters were used where DCS or saline were used as treatments in adjunct to extinction trainings.
We observed that DCS facilitated extinction training by providing further extinction of avoidance responses, risk assessment behaviors and increased the contact with the cue in a setting where DCS was administered before extinction trainings for 3 days without producing a significant tolerance. In amygdala and hippocampus, GluN1 protein expressions decreased 72 hours after the fear conditioning in the traumatic stress group suggesting a possible downregulation of NMDARs. We observed that extinction learning increased GluN1 proteins both in the amygdaloid complex and the dorsal hippocampus of the rats receiving extinction training or extinction training with DCS.
Our findings also indicate that DCS with extinction training increased GluN1 protein levels in the frontal cortex. We may suggest that action of DCS relies on enhancement of the consolidation of fear extinction in the frontal cortex.
Keywords Amygdaloid complex . Dorsal hippocampus . Predator scent test . Post-traumatic stress disorder
1.Introduction
Post-traumatic stress disorder (PTSD) is a unique mental disorder owing to the fact that the disorder naturally requires an etiological factor, a traumatic experience as a stressor to elicit the associated intrusive re-experiencing symptoms, avoidance behaviors, negative alterations in cognition and mood, alterations in arousal and reactivity levels which finally result in significant distress and functional impairment according to its recent definition in Diagnostic and Statistical Manual of Mental Disorders, 5th edition (American Psychiatry Association, 2013). PTSD was moved from the category of anxiety disorders to a traumatic or stressor related disorders with the introduction of the DSM V. However the description of the disease extends to the second half of 19th century as hysteria defined by Pierre Briquet, traumatic neurosis by Hermann Oppenheim and choc nerveux by Jean Martin Charcot (Schestatsky et al, 2003). Despite the changes in the historical efforts of classification, the disorder has always been associated with a triggering etiologic factor. Therefore it is no surprise that PTSD was considered as the only mental disorder that involves an explicit conditioning session. Furthermore, it is closely associated with the “conditioning frame-work” (Vanelzakker et al, 2014). From this view, a neutral stimulus may result in an unadaptive fear response when it was previously associated with an earlier traumatic experience. In other words, a memory trace may be formed between the neutral and the threatening events. Thus neutral cues may activate the threatening representations, triggering the anticipatory behaviors and anxiety (Vervliet, 2008). A variety of pharmacological agents are available in the pharmacotherapy of the posttraumatic stress disorder. Current evidence is not sufficient to support the efficacy of any specific agent in the mainstream symptoms of PTSD, however the efficacy of cognitive behavioral therapy (CBT) has been well documented (Baker et al, 2009; Mendes et al, 2008; Rothbaum et al, 2014). Therefore a tendency towards identifying pharmacological agents which can enhance the overall effectiveness of CBT and target the fear memory arose in the last
decade.
Hydrocortisone,
propranolol,
3,4-methylenedioxy-N-methylamphetamine,
riluzole and D-4-amino-3-isoxazolidone (D-cycloserine; DCS) are some of the promising agents (Kleine et al, 2013; Nandhra et al, 2013). DCS is an FDA approved anti-tuberculosis drug which has extensively been studied for its cognitive enhancer effects in psychiatric
disorders. DCS may enhance the effects of fear extinction trainings in animals and exposure therapy in human studies (Norberg et al, 2008). Mechanism of action of DCS is through the N-methyl-d-aspartate subtype of glutamatergic receptors (NMDARS). The crucial role of the NMDARs in the fear extinction process has been reported by several researchers (Falls et al, 1992; Ledgerwood et al, 2003; Ledgerwood et al, 2004, 2005, Walker et al, 2002 ).
The aim of this current study is to show the efficacy of DCS on different aspects of the fear related behaviors in rats subjected to predator scent in extinction sessions as an animal model for exposure therapy and the expression of GluN1 (NMDAR NR1 subunit) in critical brain regions involved in PTSD. 2.Methods 2.1 Subjects Thirty adult Wistar rats of both sexes supplied from Experimental Animal Research Center (DEHAMER, Marmara University, Istanbul) were used in the study. The number of male and female rats were equal in all groups. Upon getting approval from the University Local Ethical Committee, the rats were maintained in 12 h reverse light-dark circle (lights on 7 p.m.), at a constant room temperature of 22±2º C and a humidity ratio of 50±5 %. The reason that reverse light-dark cycle to be used is that the rats are nocturnal animals so that the procedures would be performed during the active period of the rats. Rats were fed ad libitum with standardized rat food and water. Each rat was handled once a day for 10 minutes by the same researcher. During this period, 6 rats were housed per cage but following stress procedures the rats were housed singly. Each experimental group consisted of 6 rats. The rats were assigned into 5 groups where the first group consisted of non-traumatized controls and the second traumatized controls. These 2 groups received no treatment. The other three groups were also traumatized and received either physiological saline or DCS for 3 or 5 days together with extinction procedures.
2.2 Experimental Apparatus The apparatus in which the rats were exposed to odor or no-odor blocks was a plexiglass box with 100x15x50 cm dimensions allowing the rat a back and forth movement. The apparatus was reproduced according to Blanchard et al (2001). The box had clear walls enabling video recording and the front wall of the box was divided into 3 equal segments with a red marker.
Wooden blocks covered with cotton fabric were used to serve as predator odor stress stimulus. Blocks were left in the bed of street cats for 2 days prior to testing and rubbed against the fur around the cats neck prior to the experiment. Identical clean blocks were used as no odor control blocks.
2.3 Procedure Stimulus block was placed at one end of the box. A rat was placed at the other end and allowed to move freely in the box for 10 minutes. Sessions were recorded by a video device under red light (7 Watts). Behavioral parameters were assessed according to the cat fur odor model (Blanchard et al, 2001). The analyzed behavioral parameters included were: Far location: Time spent in the far segment of the experimental apparatus from the compartment where block is placed. Contact duration: Time spent in contact with the block. Far location and contact duration; former being directly and the later inversely asseses the avoidance. Freezing time: Total immobility time. Location change: Total number of location change in between 3 compartments. This parameter is scored when all 4 limbs of the animals move to another segment. Activity is measured by this parameter. Stretch Attend Duration: Duration of the subject’s stationary position where the body is elongated with a low back when the subject is facing the stimulus block with placing fore and hind limbs far apart . Stretch Approach Duration: Duration of the subject’s movement toward stimulus block with a low back and elongated posture. Stretch attend and stretch approach were considered as the risk assessment behaviors. The phases of the procedure were as described:
Habituation: From day 1 to day 4, each rat was placed in the test apparatus for 10 min for 4 consecutive days in the absence of the stimulus block.
Cat odor block exposure: On day 5, the rats were exposed to cat odor block while control group rats were exposed to no-odor blocks. Each session for a subject was videotaped for 10 minutes and the behaviors were assessed.
Extinction: Days 6 through 8, 3 groups of rats; days 6 through 10, another extra group of rats were exposed to no-odor block in the apparatus for 10 min daily. Two groups of rats experiencing the extinction procedure also received subcutaneous 15 mg/kg DCS (Sigma, USA) 15 min before each session while the other extinction group as well as the nonextinction groups were treated with subcutaneous physiological saline (0.1 ml/100 g rat).
Test: On day 9, 4 groups were tested in the same apparatus with a no-odor block. On day 11, only the rats that received DCS injections for 5 days were exposed to no-odor blocks. The procedures and treatments of all groups were summarized in Table.1. 2.4 Tissue Preparation and Immunoblotting All chemicals were purchased from Sigma (St Louis, MO, USA) unless indicated. The rats were decapitated following high dose subcutaneous thiopental sodium (Pental®, İE Ulagay, Turkey) administration. The amygdaloid complex, the dorsal hippocampus and the frontal cortex were removed in accordance with the Rat Brain Atlas (Paxinos and Watson, 1986) and were frozen at -80oC until further processed. The slides in the anteroposterior planes located between 6.70- 4.70 mm, 13.20-11.20 mm and 7.20-5.70 mm anterior to the interaural line were taken for the separation of the amygdaloid complex, the dorsal hippocampus and the frontal cortex, respectively. The distance between interaural line and bregma was accepted as 9.00 mm, posterior to the tip of the brain. The frozen tissues were weighed and homogenized in ice cold 10 mM EDTA and protease inhibitors (0.2 mM phenylmethanesulfonyl fluoride, 1 µM pepstatin, 10 µg/ml soybean trypsin inhibitors) with T25 Ultraturrax homogenizer (Janke&Kunkel IKA-Labortechnik, Germany). Whole homogenates were used in western blots. The protein content of the crude homogenate was determined as indicated (Lowry et al, 1951). One hundred µg of protein was loaded onto 12% sodium dodecyl sulfate-polyacrylamide gels and electrophoretically transferred onto 0.45 µm nitrocellulose membranes (Schleicher and Schuell, Germany) for 120 min at 80 V. The membranes were blocked with tris buffered saline containing 1% bovine serum albumin and 0.05% Tween-20 at room temperature for 60 min and incubated overnight at 4oC with antibody against GluN1 (NMDAR subunit NR1; added in concentration 1:100). Results were standardized using β-actin (Santa Cruz, CA, USA; 1:200 dilution) as the control protein, which was detected by evaluating the band density at 43 kDa.
Membranes were incubated with the rabbit polyclonal anti-GluN1 antibody at a (1:100) dilution. The size of the GluN1 protein is 115 kDa. The GluN1 specific antibody were supplied by Santa Cruz Biotechnology, Inc. (Santa Cruz, USA). The secondary antibodies were purchased from Sigma (USA). All chemicals were obtained from Sigma, unless stated otherwise. The blots were washed three times with Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and incubated with alkaline phosphatase-conjugated secondary antibodies for 1 h at room temperature (20oC). The antibody–antigen complex was detected with NBT/BCIP (nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indolyphosphate; Promega, Wisconsin, USA). The densitometric analyses were carried out with Bio-Rad Molecular Analyst software (free edition, www.totallab.com). 2.5 Statistical Analysis Unpaired Student's t-test was used for comparing the behavioral effects on the test day and the control group. One-way analysis of variance followed by Tukey's multiple comparison post hoc test were used for the remaining comparisons. Statistical significance was accepted where p<0.05. 3.Results 3.1 The Behavioral Consequences of the Traumatic Stress On the fifth day, the data of the behavioral parameters of the traumatized rats who were exposed to cat odor blocks (4 groups) were pooled and compared to that of control rats (the rats exposed to no odor blocks). The traumatized rats were then assigned into different treatment and extinction training protocols. The means±sem values of the behavioral data and p value were presented in Table.2. The analyses have shown that risk assessment behaviours were distorted in rats exposed to odor blocks. 3.2 The Effects of DCS Treatment and Cue Extinction in Trauma Reminding Sessions On the test days, all subjects were exposed to no-odor blocks in the same test apparatus. The sessions were recorded and the behavioral parameters were calculated. Contact durations: One-way analysis of variance produced significant variance within the group (F=9.446, df=4, p=0.0002). Traumatized rats receiving a 3 day DCS treatment with extinction training touched the block for a longer duration, being significantly different from saline treated group (p<0.05; Fig.1a). No other statistically significant difference was observed in the contact durations between other groups.
Far location: One-way analysis of variance yielded a significant variance in the far location parameter (F=5.694, df=4; p=0.0038). The mean of far location duration of the control group rats exposed to no-odor blocks treated with physiological saline was 103.0±2.5 s while the rats exposed to odor blocks treated with physiological saline stayed 289.3±35.3 s in the far segment producing a statistical significance (p<0.05). Far location duration of the traumatized 3 day extinction training group treated with physiological saline was 255.2±43.8 s. Post hoc test also revealed a significant difference in the far location parameter between nontraumatized group and traumatized group experiencing a 3 day extinction training (p<0.05). The far location duration of the traumatized rats that received 3 day DCS injection and extinction training was 83.50±25.7 s yielding statistically significant decrease in the time spent in the far segment compared to that of their matching controls (p<0.05; Fig.1b). Far location duration of the traumatized rats that received 5 day DCS treatment with extinction training was 188.6±40.0 s. There was no statistical significance between traumatized rats that received 5 day DCS treatment with extinction training and non- traumatic control group treated with physiological saline (p>0.05; Fig.1b). The far location duration of the traumatized rats that received 3 day DCS and extinction training showed a statistically significant decrease, compared to that of their controls (p<0.01). No statistically significant difference was found between 5 day DCS and extinction training group and 3 day physiological saline treated control group (p>0.05). Stretch attend duration: One-way analysis of variance yielded significant variances (F=6.7570, df=4; p=0.0008). Traumatized rats treated with physiological saline displayed a longer stretch attend duration compared to traumatized rats that received 3 or 5 day DCS injections and extinction sessions (p<0.05; Fig.1c). Stretch approach duration: No statistically significant difference was observed among groups (F=0.7286, df=4, p=0.5809; Fif.1d). Freezing times: The freezing times of all experimental groups did not produce statistical significant change (F=1.6100, df=4; p=0.5809; Fig.1e). Location change: No statistically significant difference was found between groups (F=1.3990, df=4; p=0.2630; Fig.1f). 3.3 Densitometric Analyses of GluN1 Protein Immunoblots Performed in Different Brain Regions
Analysis of western-blots of the frontal cortex homogenates showed significant changes (F=358.7, df=3; p<0.0001). The results imply that the rats exposed to cat odor had increased GluN1 protein and extinction training alone didn’t produce a significant change. Adding DCS to extinction sessions again resulted in increased GluN1 protein levels (Fig.2a; p<0.05). One way analysis of variance revealed significant variances in the dorsal hippocampus (F=141.4, df=3, p<0.0001) where GluN1 expression decreased in rats subjected to cat odor (Fig.2b; p<0.01) extinction training alone or with DCS injection for 3 days restored these decreases. Similarly, GluN1 protein expression in the amygdaloid complex decreased in response to cat odor (p<0.05) and both treatment approaches corrected these changes (Fig.2c; F=30.12, df=3, p<0.0001). A representative immunoblots for GluN1 and β-actin were presented in Fig.3 4. Discussion In this study, we demonstrate that cat odor can generate traumatic stress in rats and the noodor blocks can trigger trauma related behavioral changes. The conditioning process in the PTSD and emerging anxiety have long been attributed to the impaired extinction of the previously conditioned fear responses (Eysenck, 1979; Pitman and Orr, 1986). We demonstrate that extinction procedures restored the worsened behavioral parameters. DCS administration immediately before the extinction procedures produced better outcomes. Our recent results show that a 3 day extinction training with DCS injection protocol significantly decreased the duration of avoidance and the risk assessment behaviors. Furthermore, the duration of contact with the block also increased with extinction procedures and DCS adjunct. These findings may suggest that DCS can serve as an adjuvant in decreasing the avoidance and the perceived risk related to the threatening cue, while facilitating the reconsideration of the perceived risk. In clinical settings this finding may be help patients to cope with the desired tasks and work with the trauma related cue exposure sessions. It was previously shown that DCS alone did not produce any amelioration in the conditioned fear responses without the extinction learning (Walker et al, 2002; David et al, 2002). Other behavioral parameters apart from the anxiety levels should be considered in further preclinical and clinical studies to clarify the controversial results in the literature. The augmentation effect of DCS in fear extinction was shown in experimental animals by measuring the fear-potentiated startle in a conditioning paradigm (Walker et al, 2002). Surprisingly, the group showed that this effect could only be observed in the rats receiving DCS together with extinction training. The authors used intraamygdaloid injections of DCS
and HA-966, an antagonist for NMDAR glycine-recognition site. HA-966 blocked the effects of the extinction sessions. DCS alone could not augment the fear extinction. DCS or HA-966 failed to affect fear-potentiated startle if injected before testing. The findings were replicated by subsequent studies (Ledgerwood et al, 2003, Ledgerwood et al, 2004). Previous studies indicated that the timing of administration, the dose, the dosing intervals of DCS, the quality and the number of the training/exposure sessions, the distribution of DCS administrations over the training sessions, previous exposure to DCS affect the overall success of the drug. Timing of DCS administration was found to be critical for the extinction retention effect (Ledgerwood et al, 2003). In that study, a linear decrease in the extinction effect was observed if long intervals were given between the extinction sessions and the drug administration (Ledgerwood et al, 2003). It was also demonstrated that insufficient number of extinction training sessions may result in an enhanced reconsolidation of fear acquisition (Lee et al, 2006). In the mentioned study while short extinction resulted in augmented fear long extinction training resulted in augmentation of extinction. In another study, 5 repeated DCS administrations once every other day and a later post extinction DCS injection produced a right shift in the dose-response curve indicating a reduced facilitation of extinction effect due to a possible development of tolerance (Parnas et al, 2004). In our study we examined the optimum number of repeated DCS administrations needed which can effectively enhance extinction. We demonstrate that 3 day regimen had a better outcome than 5 day regimen. Likewise, 5 day treatment group showed indifferent results from the non-traumatized control group in our study. It was previously reported that habituation took place on the 5th day in a predator stress model where worn cat collar was used 20 min per day for extinction training (Dielenberg and McGregor, 1999). Similarly in our study this effect is likely to be caused by the nature of predator stress model rather than tolerance. In our study far location duration on the 5 day DCS group stayed in the far segment from the block according to 3 day DCS group. This result is possibly related with the habituation itself where rats finally start freely exploring the apparatus and the compartment of the block became insignificant since we observed free exploration behaviour around the 5th day in all groups. According to our findings we propose that 5 day extinction may be too long since habituation to predator stress may take place and tolerance to DCS can be observed in repeated daily administrations leading to a reduced facilitation of extinction in another animal model of severe traumatic stress where habituation itself takes longer to appear. 3 day
repeated administrations may show superior and adequate results for mild to moderate traumatic stresses such as the model we used in this study. Many reports indicated that DCS administration prior to (Walker et al, 2002, Parnas et al., 2005) or shortly after (Ledgerwood et al., 2003; Ledgerwood et al., 2004; Ledgerwood et al., 2005) the extinction training facilitated extinction. Walker et al. reported that this enhancement occurs in a dose dependent manner where the rats receiving 15 mg or 30 mg of DCS prior to extinction training exhibited facilitation of extinction while the rats given 3.25 mg responded like the saline given rats (2002). We also conducted our study with a dose of 15 mg/kg as used by Walker et al. (2002) and Ledgerwood et al. (2003) and prior to extinction training. Our results support those reported by Walker et al. (2002), and also show that DCS is effective at facilitating extinction when administered prior to extinction trials. Two published clinical trials designed to assess the effects of DCS in PTSD patients failed to show an augmentation of exposure effect. First trial didn’t report enhancement of the overall treatment effect (de Kleine et al., 2012) and moreover the second trial reported greater symptoms in the DCS group (Litz et al., 2012). Further clinical studies with different study designs are needed to underline the changes in different behavioral aspects of PTSD.
Freezing time is also a commonly used behavioral parameter in previous anxiety and stress studies (Ledgerwood et al., 2003; Walker et al., 2002; Lee et al., 2006). However, we didn't observe high levels of freezing in all groups in our experiments. Correspondingly Blanchard et. al reported that the immobile crouch/freeze do not increase with exposure to cat odor (2001). A possible explanation for this finding may be that cat odor is a milder traumatic stress to produce significant freezing levels. It was also reported that freezing occurs as low as 4% of the time when an attacking cat is not present in the experiment settings (Fanselow, 1989). In the traumatized rats, we found that the freezing time constituted 8% of the experimental observation period. This may also indicate that freezing time is not a suitable parameter itself for predator odor stress models. The role of NMDARs in the long term potentiation (LTP) is well known. LTP is widely accepted to take place in the first 24 hours and can be described as a “capacity to reactivate” instead of presence of an “active state” (Dudai et al, 2002). Different types of NMDA transmission patterns were observed in this process (Zinebi et al. 2003). Another human study has shown that DCS facilitated recall of the fear memory 72 hours after acquisition of cued
conditioned fear. Authors attributed a role for NMDA in consolidation of fear to explain this finding (Kalisch et al., 2009). The mechanism of action of DCS is not known, but the affinity of DCS to the glycine modulatory site on the GluN1 seems to be the most logical explanation (Sheinin et al., 2001). The distribution of NMDARs in the brain is widespread (Myers et al, 2011) and their functions learning and memory processes have been long shown. There are two separate subunit families that join to make up functional NMDARs; the GluN1 and GluN2 subunits which are coded with different genes. The GluN1 subunit is an essential component of all NMDAR complexes while there are four GluN2 (GluN2A-D) and recently found GluN3 (GluN3A-B) subunits. Both subunit types are required to generate a fully functioning receptor. The modulatory polyamines bind to GluN2B subunit and DCS is suggested to be a GluN1 partial agonist where its efficacy determined by GluN2 subunits (Myers et al, 2011; C The activity of the NMDARs located in the basolateral amygdala (de Kleine et al, 2013; Norberg et al, 2008), hippocampus and subsequent signaling pathways (Bevilaqua et al, 2005; Szapiro et al, 2003) has been linked to fear extinction. Extinction is a learning process composed of acquisition, consolidation and retrieval phases. The amygdala plays the leading role in the encoding, consolidation and expression of extinction memory and works interconnectedly with the hippocampus and the medial prefrontal cortex (Quirk and Mueller, 2008). Evidence regarding the involvement of medial prefrontal cortex in fear extinction includes both positive findings indicating the involvement of this area (Milad and Quirk 2002,1) and non-validating negative results ( Gewirtz et al. 1997,). Animal studies established the interaction between the amygdala and the medial prefrontal cortex as crucial for the extinction learning. Furthermore the infralimbic prefrontal cortex have been revealed to have a role in fear consolidation (Gupta et. al. 2013; Ciabarra et al, 1995; Forcina et al, 1995; Low et al, 2010; Dravid et al, 2010).
We also performed immunoblotting in order to have a better understanding of the fear and extinction neurocircuitry, together with the mechanism and the target brain sites of DCS action. Gupta et al. suggested that DCS may have temporally changing effects over the cortico-amygdala circuitry which may be explained as a more short term effect in amygdala and a longer lasting effect in PFC. In the mentioned study extinction training resulted in increased GluA1, GluN2A and GluN1 expression in the mPFC compared to the groups that
did not undergo extinction training suggesting an induced LTP in the mPFC. However authors reported no alteration in the protein expression in spite of the increased pERK activity in the mPFC 6 hours after DCS was administered. Authors related this finding with saturation of increasing in the protein levels after extinction training and short duration that may not allow to induce the protein level change in the mPFC. DCS administration was also reported to cause dramatic increase in iGluR levels in the amygdala and GluN2B expression was also suggested to facilitate fear extinction consolidation in this region. (Gupta et al., 2013).
We demonstrated that GluN1 protein decreased in the amygdaloid complex of traumatized control rats. GluN1 protein levels were restored in rats that receiving extinction training or DCS with training. Our findings were consistent with that of Zinebi et al. (Zinebi et al., 2003). They reported three to four fold decrease in GluN1, GluN2A and GluN2B subunit protein expressions in the amygdala of the fear conditioned rats. Therefore, they suggested that NMDAR-mediated transmission in the lateral amygdala down-regulates while the fear memory is being maintained. They suggested that this process occurs to allow a capacity to reactivate the fear memories and provide protection against excitotoxicity from NMDA receptor recruitment and lead to long term depression after fear acquisition. The effects of extinction training to the cue probably continue to take place both in the amydaloid complex and in the dorsal hippocampus immediately after the last extinction session. This may suggest a role for dorsal hippocampus for the extinction learning to cues as well as contextualized extinction learning. Our findings show no superior results in terms of GluN1 expressions in both brain regions when DCS was administered together with the extinction sessions. Instead, we observed slightly decreased level of GluN1 protein expressions in the dorsal hippocampus in the group receiving DCS and extinction training. In addition, we found increased GluN1 protein levels in the frontal cortex of rats that received 3 day DCS plus extinction training suggesting a consolidation phase of the extinction memory. Thus increased levels of GluN1 proteins possibly could have been observed in this area within the first days of the extinction training. We may also indirectly suggest that the decreased levels of GluN1 protein in the hippocampus of the DCS plus extinction group may be another indicator of a down-regulation due to the onset of consolidation phase that likely takes place in the frontal cortex. However, phases of maintenance of the fear memory should be further studied to clarify the temporal roles of the key structures in the fear neurocircuitry. According to our findings, we may suggest that DCS administration possibly facilitates extinction memory consolidation by enhancing the expression of new NMDARs in the frontal
cortex. Another recent study found increased expressions of GLuA1, GluN1, GluN2A, GluN2B in the medial prefrontal cortex (mPFC) of untrained rats following intraperitoneal DCS injection while they observed no significant difference in the protein levels when DCS was administered in conjunction with extinction training (Gupta et al., 2013). Our study demonstrates increased GluN1 protein expressions in the DCS with extinction group indicating mPFC as a major target for DCS action. Conclusion In conclusion, our study suggests that DCS may facilitate the outcomes of cognitive behavioral therapies in PTSD by decreasing the avoidance and the attributed risk to the threatening cue while facilitating the reconsideration of the perceived risk. In amygdala and hippocampus GluN1 protein expressions decreased 72 h after the cued fear conditioning in traumatic stress group indicating a possible downregulation of NMDARs. We suggest that this process may occur in order to enable formation of either new fear memories or extinction learning and to impede further excitotoxicity. We observed the expression of GluN1 protein increased in the amygdaloid complex and the dorsal hippocampal regions of groups that received extinction trainining alone or with DCS. According to our findings, DCS action may eventually occur in the frontal cortex resulting in enhancement of the consolidation of fear extinction and further extinction retention.
Acknowledgements This study was sponsored by Santa Cruz Biotechnology Inc., Turkey and partly by a grant supplied from Marmara University (Project number: SAG-A-110913-0363). References American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders, 5th ed. Arlington VA: American Psychiatric Publishing Arlington; 2013. Baker DG, Nievergelt CM, Risbrough VB. Posttraumatic Stress Disorder: Emerging Concepts of Pharmacotherapy. Expert Opin Emerg Drugs. 2009;14:251–272. Blanchard RJ, Yang M, Li CI, Gervacio A, Blanchard DC. Cue and context conditioning of defensive behaviors to cat odor stimuli. Neurosci Biobehav Rev. 2001;25:587-95. Bevilaqua LR, da Silva WN, Medina JH, Izquierdo I, Cammarota M. Extinction and reacquisition of a fear-motivated memory require activity of the Src family of tyrosine kinases in the CA1 region of the hippocampus. Pharmacol Biochem Behav. 2005;81:139–145.
Ciabarra AM, Sullivan JM, Gahn LG, Pecht G, Heinemann S, Sevarino KA. Cloning and characterization of chi-1: a developmentally regulated member of a novel class of the ionotropic glutamate receptor family. J Neurosci. 1995;15:6498–6508. Walker DL, Ressler KJ, Lu KT, Davis M. Facilitation of Conditioned Fear Extinction by SystemicAdministration or Intra-Amygdala Infusions of D-Cycloserine as Assessed with Fear-Potentiated Startle in Rats. The Journal of Neuroscience 2002;22:2343–2351. Dravid SM, Burger PB, Prakash A, Geballe MT, Yadav R, Le P, Vellano K , Snyder JP , Traynelis SF. Structural determinants of D-cycloserine efficacy at the NR1/NR2C NMDA receptors. J Neurosci. 2010;30:2741-54. Dudai Y. Molecular bases of long-term memories: a question of persistence. Curr Opin Neurobiol. 2002;12:211-6. De Kleine RA, Hendriks GJ, Kusters WJ, Broekman TG, van Minnen A. A randomized placebo-controlled trial of D-cycloserine to enhance exposure therapy for posttraumatic stress disorder. Biol Psychiatry. 2012;71:962-8. De Kleine RA, Rothbaum BO, van Minnen A. Pharmacologic enhancement of exposure based treatment in PTSD: a qualitative review. Eur J Psychotraumatol. 2013;17;4. Dielenberg RA, McGregor IS. Habituation of the hiding response to cat odors in rats (Rattus norvegicus). J Comp Psychol. 1999;113:376-87. Dravid SM, Burger PB, Prakash A, Geballe MT, Yadav R, Le P et al. Structural determinants of D-cycloserine efficacy at the NR1/NR2C NMDA receptors. J Neurosci. 2010;30:2741-54. Eysenck HJ. The conditioning model of neurosis. The Behavioral and Brain Sciences. 1999;2:155–199. Falls WA, Miserendino MJ, Davis M. Extinction of fear potentiated startle: blockade by infusion of an NMDA antagonist into the amygdala. J Neurosci. 1992;12:854–863. Fanselow MS. The adaptive function of conditioned defensive behaviour: an etiologic approach to Pavlovian stimulus-substitution theory. In: Blanchard RJ, Brain PF, Blanchard DC, Parmigiani S, editors. Ethoexperimental approaches to the study of behavior. Dordrecht: Kluwer Academic Publishers; 1989, p.114-36. Forcina MS, Ciabarra AM, Seravino KA. Cloning of chi-2: a putative member of the ionotropic glutamate receptor superfamily. Soc Neurosci Abstr. 1995;21:438.3. Gewirtz JC, Falls WA, Davis M. Normal conditioned inhibition and extinction of freezing and fear-potentiated startle following electrolytic lesions of medial prefrontal cortex in rats. Behav Neurosci. 1997;111:712–726. Gupta SC, Hillman BG, Prakash A, Ugale RR, Stairs DJ, Dravid SM. Effect of D-cycloserine in conjunction with fear extinction training on extracellular signal-regulated kinase activation in medial prefrontal cortex and amygdala in rat. Eur J Neurosci. 2013;37:1811–1822. Kalisch R, Holt B, Petrovic P, De Martino B, Klöppel S, Büchel C, Dolan RJ. The NMDA agonist D-cycloserine facilitates fear memory consolidation in humans. Cereb Cortex. 2009;19:187-96.
Ledgerwood L, Richardson R, Cranney J. Effects of D-cycloserine on extinction of conditioned freezing. Behav Neurosci. 2003;117:341–349. Ledgerwood L, Richardson R, Cranney J. D-cycloserine and the facilitation of extinction of conditioned fear: consequences for reinstatement. Behav Neurosci. 2004;118:505–513. Ledgerwood L, Richardson R, Cranney J. D-cycloserine facilitates extinction of learned fear: effects on reacquisition and generalized extinction. Biol Psychiatry. 2005;57:841–847. Lee JLC, Milton AL, Everitt BJ. Reconsolidation and extinction of conditioned fear: Inhibition and potentiation. The Journal of Neuroscience. 2006;26:10051–10056. Litz BT, Salters-Pedneault K, Steenkamp MM, Hermos JA, Bryant RA, Otto MW, Hoffman SG . A randomized placebo-controlled trial of D-cycloserine and exposure therapy for posttraumatic stress disorder. J Psychiatr Res. 2012;46:1184-9 Low CM, Wee KS. New insights into the not-so-new NR3 subunits of N-methyl-D-aspartate receptor: localization, structure, and function. Mol Pharmacol. 2010;78: 1-11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry. 1951;193:265–327. Mendes DD, Mello MF, Ventura P, Passarela Cde M, Mari Jde J. A systematic review on the effectiveness of cognitive behavioral therapy for posttraumatic stress disorder. Int J Psychiatry Med. 2008;38:241-259. Milad MR, Quirk GJ.Neurons in medial prefrontal cortex signal memory for fear extinction. Nature. 2002;420:70-4 Myers KM, Carlezon WA Jr, Davis M. Glutamate Receptors in Extinction and ExtinctionBased Therapies for Psychiatric Illness. Neuropsychopharmacology Reviews. 2011;36:274– 293 Nandhra HS, Murphy CL, Sule A. Novel pharmacological agents targeting memory and cognition in the treatment of anxiety disorders. Hum Psychopharmacol Clin Exp 2013;28:538–543. Norberg MM, Krystal JH, Tolin DF. A Meta-Analysis of D-Cycloserine and the Facilitation of Fear Extinction and Exposure Therapy. Biol Psychiatry. 2008;63:1118–1126. Sheinin A, Shavit S, Benveniste M. Subunit specificity and mechanism of action of NMDA partial agonist D-cycloserine. Neuropharmacology. 2001;41:151-8. Szapiro G, Vianna MR, McGaugh JL, Medina JH, Izquierdo I. The role of NMDA glutamate receptors, PKA, MAPK, and CAMKII in the hippocampus in extinction of conditioned fear. Hippocampus. 2003;3:53–58. Parnas AS, Weber M, Richardson R. Effects of multiple exposures to D-cycloserine on extinction of conditioned fear in rats. Neurobiol Learn Mem. 2005;83:224–231. Paxinos G, Watson C. The rat brain in stereotaxic coordinates, 2nd ed. London: Academic Press, 1986
Pitman RK, Orr SP. Test of the conditioning model of neurosis: Differential aversive conditioning of angry and neutral facial expressions in anxiety disorder patients. Journal of Abnormal Psychology. 1986;95:208–213. Rothbaum BO, Price M, Jovanovic T, Norrholm SD, Gerardi M, Dunlop B, Davis M, Bradley B, Duncan EJ, Rizzo A, Ressler KJ. A randomized, double-blind evaluation of D-cycloserine or alprazolam combined with virtual reality exposure therapy for posttraumatic stress disorder in Iraq and Afghanistan War veterans. Am J Psychiatry. 2014;171:640-648. Quirk GJ, Mueller D. Neural mechanisms of extinction learning and retrieval. Neuropsychopharmacology. 2008;33:56–72. Schestatsky S, Shansis F, Ceitlin LH, Abreu PB, Hauck S. Historical evolution of the concept of posttraumatic stress disorder. Rev Bras Psiquiatr. 2003;25:8-11. Vanelzakker MB, Dahlgren MK, Davis FC, Dubois S, Shin LM. From Pavlov to PTSD: the extinction of conditioned fear in rodents, humans, and anxiety disorders. Neurobiol Learn Mem. 2014;113:3-18. Vervliet B. Learning and memory in conditioned fear extinction: effects of D-cycloserine. Acta Psychol (Amst). 2008;127:601-613. Walker DL, Ressler KJ, Lu KT, Davis M. Facilitation of conditioned fear extinction by systemic administration or intraamygdala infusions of D-cycloserine as assessed with fear potentiated startle in rats. J Neurosci. 2002;22:2343–2351. Zinebi F, Xie J, Liu J, Russell RT, Gallagher JP, McKernan MG et al. NMDA currents and receptor protein are downregulated in the amygdala during maintenance of fear memory. J Neurosci. 2003;23:10283-91.
All rats were habituated and exposed to no odor block as a trauma reminder on the corresponding test days.
All assesments were done on day 5, following habituation period.
Legends for figures
Fig. 1 The behavioral parameters (contact duration (a), far location (b), stretch approach (c), stretch attend (d), freezing time (e), total location change count (f), in the insets) calculated from the video recordings of rats re-exposed to trauma reminder, the no odor block who were previously traumatized with cat odor (thick bordered boxes) or no odor (thin bordered box) blocks. The groups that received extinction protocol were marked with a line. Subcutaneous D-cycloserine treatments were given 15 min prior to sessions (15mg/kg; n=6/group; *, p< 0.05, **, p< 0.01, as indicated). The data are expressed as means ± s.e.m. Fig. 2 The densitometric analyses obtained from photographs of western immunoblots of 3 different brain regions of rats exposed to cat odor (the thick bordered boxes) where the control rats were exposed to no odor blocks. Traumatized rats were either treated with physiological saline or subjected to extinction training sessions with or without d-cycloserine injections (15 mg/kg) for 3 days. The brain slides were taken upon completion of behavioral experiments where the rats were exposed to trauma reminder. *, p<0.05,**, p<0.01, as indicated. The data are expressed as means ± s.e.m. Fig. 3 The
photographs of nitrocellulose membranes collected from immunoblotting
experiments of non-traumatized control rats (a) and traumatized rats with cat odor treated with physiological saline (b), extinction training (c) or D-cycloserine (d). The blots used were for GluN1 as indicated. β-actin was used to normalize the amount of protein loaded in each lane.
Figure-1 .
Figure-2 .
Figure-3 .
Table.1. The summary of experimental groups and the procedures Type of block exposed
Number of extinction days with no odor block
Test day
Groups Non traumatized control Traumatized control Traumatized and subjected to extinction procedures and treated with physiological saline for 3 days Traumatized and subjected to extinction procedures and treated with DCS for 3 days Traumatized and subjected to extinction procedures and treated with DCS for 5 days
No odor block Odor block Odor block
9 -
9
3
9
3
9
Odor block
Odor block
11 5
Table.2. The change in the behavioral parameters in control rats (exposed to no odor blocks) and the rats traumatized with cat odor (odor block). The type of block exposed The risk assessment behaviors
No odor block
Odor block
Statistics summary
Contact duration
12.0±3.3 s
116.0±27.9 s
p<0.001
Far location
494±13.7s
289.7±68.8 s
p<0.01
Stretch attend duration
1.5±3.4 s
33.3±13.7 s
p<0.01
Stretch approach duration
5.8±2.71 s
23.0±2.4 s
p<0.01
Freezing
11.0±2.6 s
50.6±2.4 s
p<0.05
Location change frequency
29.9±9.0 s
15.4±3.1 s
p<0.05