Behavioral Neuroscience 2014, Vol. 128, No. 4, 468-473
© 2014 American Psychological Association 0735-7044/14/$ 12.00 http://dx.doi.org/10.1037/a0037020
BRIEF COMMUNICATION
Enhanced Extinction of Contextual Fear Conditioning in ClockA\9 Mutant Mice Rick E. Bernardi and Rainer Spanagel Central Institute of Mental Health, Mannheim, Germany
Clock genes have been implicated in several disorders, such as schizophrenia, bipolar disorder, autism spectrum disorders, and drug dependence. However, few studies to date have examined the role of clock genes in fear-related behaviors. The authors used mice with the ClockA\9 mutation to assess the involvement of this gene in contextual fear conditioning. Male wild-type (WT) and ClockA\9 mutant mice underwent a single session of contextual fear conditioning (12 min, 4 unsignaled shocks), followed by daily 12-min retention trials. There were no differences between mutant and WT mice in the acquisition of contextual fear, and WT and mutant mice demonstrated similar freezing during the first retention session. However, extinction of contextual fear was accelerated in mutant mice across the remaining retention sessions, as compared to WT mice, suggesting a role for Clock in extinction following aversive learning. Because the ClockA\9 mutation has previously been demonstrated to result in an increase in dopamine signaling, the authors confirmed the role of dopamine in extinction learning using preretention session administration of a low dose of the dopamine transport reuptake inhibitor modafinil (0.75 mg/kg), which resulted in decreased freezing across retention sessions. These findings are consistent with an emerging portrayal of the importance of Clock genes in noncircadian functions, as well as the important role of dopamine in extinction learning. Keywords: circadian, clock, ClockA 19 mutant mice, contextual fear conditioning, dopamine, extinction, modafinil
been demonstrated to result in an increase in the sensitivity to psychostimulants (McClung et al., 2005; Ozbum, Larson, Self, & McClung, 2012; but see Bernardi & Spanagel, 2013) and ethanol (Ozbum et al., 2013), as well as an increase in mania-like behavior (Roybal et al., 2007). These behavioral alterations in mutant mice have been attributed to an increase in the expression and phos phorylation of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of dopamine (DA), in the ventral tegmental area of mutant mice (McClung et al., 2005), and increased DA release and a subsequent alteration in DA receptor levels (Spencer et al.,
Clock genes have been well-evaluated for their role in pace maker functions, in which oscillatory transcriptional and transla tional autoregulatory feedback loops of these genes mediate cir cadian time-keeping (Ko & Takahashi, 2006). A great deal of recent evidence, however, has shown that the involvement of clock genes is not limited to circadian functions. Clock genes have been demonstrated to be involved in a variety of noncircadian functions and influence a variety of processes and have been implicated in several disorders, such as schizophrenia, bipolar disorder, autism spectrum disorders, and drug dependence (Albrecht, 2013; Perreau-Lenz & Spanagel, 2008; Rosenwasser, 2010). One of the clock genes, Clock, has primarily been the focus of studies involving drugs of abuse and mania-like symptomology. A dominant negative mutation of the Clock gene, the ClockA 19 mutation, in mice (King et al., 1997; Vitatema et al., 1994) has
2012). To date, no studies have examined a role for Clock in fearmediated behaviors, which have also been demonstrated to be sensitive to alterations in DA signaling. For example, peripheral (Inoue, Izumi, Maki, Muraki, & Koyama, 2000) or bilateral in traamygdala (Guarraci, Frohardt, & Kapp, 1999) administration of the DAI (Dl) receptor antagonist SCH 23390 prior to the acqui sition of contextual fear conditioning impaired conditioned fear in rats, while intraamygdala infusion of the D l agonist SKF 82958 prior to acquisition facilitated subsequent conditioned fear (Guar raci et al., 1999). These and other studies suggest DA involvement in the learning of contextual fear. The extinction of contextual fear conditioning has also been demonstrated to be DA-dependent, with increases in DA signaling enhancing, and decreases in DA signal ing impairing, the extinction of contextual fear (e.g., Abraham, Cunningham, & Lattal, 2012; El-Ghundi, O’Dowd, & George, 2001; Fernandez Espejo, 2003).
This article was published Online First May 26, 2014. Rick E. Bernardi and Rainer Spanagel, Institute of Psychopharmacology, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University. Rick E. Bernardi was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG, SPP1226, SP 383/4-1). We report no con flicts of interest. Correspondence concerning this article should be addressed to Rick E. Bernardi, Central Institute of Mental Health, Institute of Psychopharmacology, J5, 68159, Mannheim, Germany. E-mail: [email protected]
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CLOCKA19 MUTATION ENHANCES EXTINCTION The goal of the current study was to determine whether Clock A19 mutant mice showed alterations in contextual fear con ditioning and subsequent extinction of contextual fear. We report here that although the acquisition and expression of contextual fear did not differ between WT and ClockA19 mutant mice, extinction of contextual fear was significantly enhanced in mutant mice following the initial retention test. These results suggest a role for the Clock gene in extinction learning, likely becaue of enhanced DA signaling previously reported in mutant mice. To confirm the role of DA in extinction learning, we also demonstrated that treatment with the DA transporter blocker, modafinil, prior to retention sessions, resulted in a decrease in freezing behavior across sessions.
Methods Subjects Male Clock A \9 mutant mice and their WT littermates (back ground C57B1/6; Jackson Laboratories, Bar Harbor, ME; bred in-house) and male C57B1/6 mice (Charles River, Sulzfeld, Ger many) aged 10-12 weeks at the start of experiments served as subjects. Mice were single housed in a temperature-controlled (21°C) environment maintained on a 12-hr light-dark cycle (lights on at 6 a.m.). Food and water were available ad libitum. All experiments were performed in accordance with EU guidelines on the care and use of laboratory animals and were approved by the local animal care committee (RegierungsprAsidium, Karlsruhe, Germany). All behavioral experiments were conducted during the light phase.
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context. Freezing was defined as bouts of inactivity that lasted at least 1 s. Total time meeting this criterion was divided by the total time of the bin (acquisition) or session (retention) to calculate percent time freezing. Five days following the final conditioned fear-retention session, mice were given a 30-min locomotor activ ity test. Data were recorded as distance traveled (cm). Experiment 2. C57B1/6 mice received a single fear acquisi tion session as described above. Beginning 24 hr later, mice received six daily 12-min nonreinforced exposures to the context. Extinction Sessions 1 -4 were preceded by administration of 0.75 mg/kg modafinil (Tocris, Wiesbaden-Nordenstadt, Germany) or vehicle (10% Tween 80; 10 ml/kg; Sigma-Aldrich, Steinheim, Germany). Freezing was defined as described above. Again, 6 days following the final conditioned fear-retention session, mice were placed into the activity chambers for a 30-min locomotor activity test preceded by vehicle or modafinil (0.75 mg/kg) administration in mice that received vehicle or modafinil, respectively, during fear extinction. To control for potential toler ance or sensitization to the repeated administration of modafinil in a duration identical to that of the fear conditioning retention sessions, a second group of drug-nave mice were given a single habituation trial, then administered vehicle or 0.75 mg/kg modafinil prior to 12-min locomotor sessions on subsequent days. Data were recorded as distance traveled (cm) during the session.
Statistical Analysis Data were analyzed using analysis of variance (ANOVA). Sig nificance was set at p < .05.
Results Apparatus Fear conditioning. One Coulbourn Instruments mouse con ditioning chamber (H10-35M-04; Coulbourn Instruments, Allen town, PA) was used, which consists of a clear Plexiglas arena (17.8 cm X 17.8 cm X 30.5) with a stainless steel floor of grid rods spaced 6.4 mm apart. The chamber was housed in a sound-attenuating shell, with a light providing illumination and a fan providing background noise. Scrambled shocks were delivered to the grid floor by a computer-controlled shock generator (Coulbourn H13-15). Mounted above the floor of the chamber was a camera that recorded activity in real-time. Experimental events were controlled by FreezeFrame (Coulbourn Instruments, Allentown, PA) software, and freezing data were compiled using FreezeView software. Locomotor activity. Locomotor activity was assessed in six TruScan activity monitors (Coulbourn Instruments, Allentown, PA). Each monitor consists of a clear acrylic plastic test cage (22 X 22 X 40 cm) placed inside a monitoring unit that records via computer ambulatory beam interruptions from infrared photocell emitter/detector pairs evenly spaced along each axis.
Behavioral Procedures Experiment 1. During a single fear acquisition session, mice received a 12-min exposure to the context with four unsignaled shocks (2 s, 0.35 mA; Bemardi & Lattal, 2010; Lattal & Maughan, 2012) delivered at 70 s, 278 s, 510 s, and 642 s. Beginning 24 hr later, mice received 5 daily 12-min nonreinforced exposures to the
Experiment 1 During acquisition, ClockA 19 mutant and WT mice demon strated a freezing profile that did not differ as a function of genotype. Figure 1A shows the mean (± SEM) percent freezing across four 3-min time bins during the 12-min session. A two-way ANOVA (Genotype X Time Bin) revealed a significant main effect of time bin, F(1.9,19.5) = 56.9, p < .001, but no significant main effect of genotype, F (l, 10) = 0.05, p = .83, and no interaction, F(1.9,19.5) = 0.15, p = .85. Thus, WT and ClockA\9 mutant mice demonstrated a similar acquisition of conditioned fear. Across retention sessions, ClockA\9 mutant mice demonstrated an accelerated decline in freezing as compared to WT mice. Figure IB shows the mean (± SEM) percent freezing across five 12-min session nonreinforced retention sessions. A two-way ANOVA (Genotype X Retention Session) revealed significant main effects of genotype, F (l, 10) = 6.1, p < .05, and retention session, F(4, 40) = 35.0, p < .001, and a significant Genotype X Retention Session interaction, F(4, 40) = 2.8, p < .05. One-way ANOVAs conducted for each retention session revealed that the two groups did not differ in freezing behavior during the first retention session, F (l, 10) = 0.47, p = .51, but differed significantly on all subse quent retention tests—Test 2: F (l, 10) = 4.9, p = .05; Tests 3-5: Fs(l,10) > 6.5, ps < .05. These results indicate that after showing similar levels of freezing during the first retention session as compared to WT mice, Clock A 19 mutant mice demonstrated pro-
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Figure 1. Effects of the ClockA\9 mutation in mice on the acquisition and extinction of contextual fear. A: There were no differences in the acquisition of contextual fear between wild-type (WT) and ClockA 19 mutant mice. Data represent mean (± SEM) percentage freezing during four 180-s time bins during the 12-min conditioning trial. B: There was no difference in the expression of conditioned fear during the first 12-min retention session between WT and ClockA 19 mutant mice. However, during retention Sessions 2-5, ClockA\9 mutant mice demonstrated progressively enhanced extinction relative to WT mice. Data represent mean (± SEM) percentage freezing during the five 12-min retention sessions (T1-T5). (C) ClockA\9 mutant mice demonstrated higher locomotor activity as compared to WT mice during a test for locomotion following the conclusion of fear procedures. Data represent mean (± SEM) distance traveled during a 30-min session. * p < .05.
gressively lower levels of conditioned fear during subsequent sessions. ClockA\9 mutant mice demonstrated increased locomotor ac tivity relative to WT mice. Figure 1C shows the mean (± SEM) distance traveled (cm) during the 30-min locomotor activity tests for WT and ClockA\9 mutant mice. A one-way ANOVA revealed significant a significant effect of distance traveled, F (l, 10) = 16.2, p < .005.
Experiment 2 During acquisition, C57BL/6 mice demonstrated a freezing pro file that did not differ as a function of subsequent modafinil treatment. Figure 2A shows the mean (± SEM) percent freezing across four 3-min time bins during the 12-min session. A two-way ANOVA (Treatment Group X Time Bin) revealed a significant main effect of time bin, F(2.1,27.1) = 51.5, p < .001, but no significant main effect of genotype, F (l, 13) = 0.13, p — .73, and no interaction, F(2.1,27.1) = 0.07, p = .94, suggesting no differ ence between the two groups during the acquisition of contextual fear. Across retention sessions, modafinil-treated mice demonstrated a decrease in freezing as compared to WT mice. Figure 2B shows the mean (± SEM) percent freezing across four 12-min session nonreinforced extinction sessions preceded by modafinil treatment and two drug-free test sessions. Across Extinction Sessions 1-4, a two-way ANOVA (Treatment Group X Retention Session) re vealed significant main effects of treatment, F (l, 13) = 5.3, p < .05, and retention session, F(3, 39) = 42.0, p < .001, but no Genotype X Retention Session interaction, F(3, 39) = 0.35, p = .79. One-way ANOVAs conducted for each retention session revealed that the two groups did not differ in freezing behavior during the first two retention sessions, Fs( 1,13) < 3.3, ps > .09, but differed significantly during subsequent retention tests— Ext 3: F (l, 13) = 4.6, p = .05; Ext 4: F (l, 13) = 6.3, p < .05. For
drug-free Test Sessions 1 and 2, one-way ANOVAs indicated significant differences between vehicle- and modafinil-treated mice in a drug-free state, Fs(l,13) > 6.2, ps < .05. These results indicate enhanced extinction in modafinil-treated mice relative to vehicle-treated mice, including in a drug-free state. There was no difference in locomotor activity between the two groups, suggesting that a modafinil-induced increase in locomotion likely does not explain the decrease in freezing in modafinil-treated mice. Figure 2C shows the mean (± SEM) distance traveled (cm) during the 30-min locomotor activity tests for vehicle- and modafinil-treated mice. A one-way ANOVA revealed no differ ence in distance traveled, F (l, 13) = 0.52, p = .48. Furthermore, in a separate group of drug-nave mice, there was also no difference in locomotor activity between vehicle and modafinil treatment (Figure 2D), F (l, 5) = 3.4, p = .13.
Discussion The current data indicates that the ClockA 19 mutation may also be involved in the extinction of aversive memories, in addition to its prior demonstrated role in reward-related behaviors involving drugs of abuse. ClockA\9 mutant mice demonstrated normal ac quisition and expression of contextual fear, as shown by levels of freezing similar to WT mice during the conditioning session and first retention test. However, during subsequent retention sessions, the loss of the freezing response in ClockA 19 mutant mice was progressively accelerated relative to WT mice. Thus, the learning and recall of the fear response was not affected by the ClockA\9 mutation, but extinction of conditioned fear was enhanced. DA has long been considered a mediator of learning (Harley, 2004). Because the DA system has been implicated as the primary target of the ClockA\9 mutation (McClung et al., 2005; Spencer et al., 2012), the effects of the ClockA 19 mutation in male mice demonstrated here likely involve a DA-dependent increase in extinction learning. We saw no difference in contextual fear learn-
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Figure 2. Effects of modafinil (MOD) on the extinction of contextual fear. A: There were no differences in the acquisition of contextual fear between vehicle (VEH) and MOD groups prior to treatment. Data represent mean (± SEM) percentage freezing during four 180-s time bins during the 12-min conditioning trial. B: MOD-treated mice demonstrated a decrease in freezing across four extinction trials (EXT 1-4) preceded by MOD treatment (0.75 mg/kg; indicated by j ), as well as on two drug-free tests (T1 and T2), as compared to VEH treatment. Data represent mean (± SEM) percentage freezing during the six 12-min retention sessions. C: VEH- and MOD-treated mice showed no difference in locomotor activity during a test for locomotion following the conclusion of fear procedures. Data represent mean (± SEM) distance traveled during a 30-min session. D: A separate group of drug-nave mice showed no difference in locomotor activity between VEH and MOD treatment during a test for locomotion. Data represent mean (± SEM) distance traveled during a 12-min session. *p < .05.
ing or retention during the first extinction trial but a subsequent acceleration of extinction in ClockM9 mutant mice relative to WT mice. Interestingly, a previous study examined fear in response to contextual cues in mice possessing the NPAS2 gene, the human analog of the Clock, and demonstrated that NPAS2 (-/-) mice showed normal freezing 30 min following fear conditioning, but decreased freezing during a 24-hr retention test, relative to WT mice (Garcia et al., 2000). Although seemingly contrasting our findings here, in which we demonstrated no difference in freezing during the first extinction Session 24 hr after conditioning, this previous study used an auditory cue that preceded each of the repeated shocks during conditioning. Thus, the difference in freez ing during the first retention session may be more related to differences in valence attributed to the varying conditioned stimuli rather than a specific deficit in contextual fear. Furthermore, this study did not examine the time-course of extinction learning. To confirm our findings in C/ockA19 mutant mice, we admin istered modafinil, a DA reuptake inhibitor, prior to extinction sessions. We used a low dose (0.75 mg/kg) previously reported to enhance retention of contextual fear when administered prior to acquisition (Shuman, Wood, & Anagnostaras, 2009). In our study, this low dose resulted in enhanced extinction as compared to vehicle-treated mice, even when treatment was withdrawn (Tests
1-2). This is the first study to report enhanced extinction of contextual fear using modafinil. Our findings are consistent with a recent study by Abraham et al. (2012), who demonstrated that methylphenidate, a DA and norepinephrine reuptake inhibitor, administered prior to or following extinction sessions, enhanced the extinction of contextual fear in mice. Furthermore, D1 receptor knockout (KO) mice demonstrated no difference in the acquisition or expression of contextual fear relative to WT mice but an impairment of extinction during subsequent trials (El-Ghundi et al., 2001), suggesting that DA functioning through D1 receptors is necessary for normal extinction learning following fear condition ing. Other studies have also demonstrated a role for DA in extinc tion learning following fear conditioning (Abraham, Neve, & Lattal, 2014). Some data exists with regards to other clock genes, Period {Per) and Cryptochrome {Cry), in fear-related behaviors. Mechanisti cally, the CLOCK protein dimerizes with brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein 1 (BMAL1) to activate the transcription of PER and CRY proteins to influence intracellular and extracellular targets. A PER-CRY het erodimer acts via negative feedback on the CLOCK-BMAL1 heterodimer to repress further transcription (Dibner, Schibler, & Albrecht, 2010). Perl and Perl KO mice showed no differences
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relative to WTs in the acquisition or expression of conditioned fear in a contextual fear paradigm (Zueger et al., 2006; see also Wang et al., 2009), and although Cryl/Cry2 KO mice demonstrated higher freezing during the acquisition of auditory fear condition ing, they showed no difference in freezing relative to WT mice during subsequent expression testing (De Bundel, Gangarossa, Biever, Bonnefont, & Valjent, 2013). Our data are consistent with these previous findings, in that we found no difference in the ability of ClockAY) mutant mice to learn and express conditioned fear relative to WT mice. These previous studies did not examine further extinction trials in Per and Cryl/Cry2 KO mice, however, and thus the role of these clock genes in extinction is yet unknown. It is important to note that previous studies have demonstrated locomotor hyperactivity in ClockA\9 mutant mice, as compared to WT mice (e.g., Bernardi & Spanagel, 2013). We also demonstrated here that following the completion of fear conditioning, ClockA\9 mutant mice showed higher locomotor activity than WT mice. Thus, it is impossible to eliminate increased locomotion as an explanation for the significant decrease in freezing across retention sessions in mutant mice in the current study. However, we saw no difference between WT and mutant mice in the acquisition of contextual fear, and importantly, no difference in responding dur ing the first retention trial. Thus, potential differences in locomo tion cannot fully explain the significant interaction between WTs and mutants in freezing behavior achieved across retention trials, which is likely primarily due to an enhanced extinction in ClockA\9 mutant mice. Preretention session administration of modafinil resulted in an immediate and long-lasting decrease in freezing relative to vehicle treatment. Although previous studies have demonstrated locomotor activation to modafinil at higher doses, the 0.75 mg/kg dose has previously been shown not to result in either acute or sensitized locomotor activation (Shuman, Cai, Sage, & Anagnostaras, 2012). Furthermore, we demonstrated in mice that underwent fear conditioning, as well as a separate group of nave mice, that this dose failed to increase locomotor activity. Thus, we suggest that modafinil administered prior to extinction increased extinction learning. In conclusion, we report here that the ClockA19 mutation re sulted in an enhanced extinction of contextual fear, an effect mimicked using the DA reuptake inhibitor modafinil. These find ings demonstrate a specific role for Clock in extinction in aversive paradigms, and further demonstrate the importance of clock genes in general in noncircadian functions, as well as the importance of DA signaling in extinction learning.
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CL0CKM9 MUTATION ENHANCES EXTINCTION Roybal, K., Theobold, D., Graham, A., DiNieri, J. A., Russo, S. J., Krishnan, V .,. . . McClung, C. A. (2007). Mania-like behavior induced by disruption of CLOCK. Proceedings o f the National Academy of Sciences of the United States of America, 104, 6406 - 6411. doi: 10.1073/ pnas.0609625104 Shuman, T., Cai, D. J., Sage, J. R., & Anagnostaras, S. G. (2012). Interactions between modafinil and cocaine during the induction of conditioned place preference and locomotor sensitization in mice: Im plications for addiction. Behavioural Brain Research, 235, 105-112. doi:10.1016/j.bbr.2012.07.039 Shuman, T., Wood, S. C., & Anagnostaras, S. G. (2009). Modafinil and memory: Effects of modafinil on Morris water maze learning and Pavlovian fear conditioning. Behavioral Neuroscience, 123, 257-266. doi: 10.1037/a0014366 Spencer, S., Torres-Altoro, M. I., Falcon, E., Arey, R., Marvin, M., Goldberg, M .,. . . McClung, C. A. (2012). A mutation in CLOCK leads to altered dopamine receptor function. Journal o f Neurochemistry, 123, 124-134. doi: 10.1111/j.1471-4159.2012.07857.X
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Received December 12, 2013 Revision received April 15, 2014 Accepted April 23, 2014 ■
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© 2014 American Psychological Association 0735-7044/14/$ 12.00 http://dx.doi.org/10.1037/a0037020
BRIEF COMMUNICATION
Enhanced Extinction of Contextual Fear Conditioning in ClockA\9 Mutant Mice Rick E. Bernardi and Rainer Spanagel Central Institute of Mental Health, Mannheim, Germany
Clock genes have been implicated in several disorders, such as schizophrenia, bipolar disorder, autism spectrum disorders, and drug dependence. However, few studies to date have examined the role of clock genes in fear-related behaviors. The authors used mice with the ClockA\9 mutation to assess the involvement of this gene in contextual fear conditioning. Male wild-type (WT) and ClockA\9 mutant mice underwent a single session of contextual fear conditioning (12 min, 4 unsignaled shocks), followed by daily 12-min retention trials. There were no differences between mutant and WT mice in the acquisition of contextual fear, and WT and mutant mice demonstrated similar freezing during the first retention session. However, extinction of contextual fear was accelerated in mutant mice across the remaining retention sessions, as compared to WT mice, suggesting a role for Clock in extinction following aversive learning. Because the ClockA\9 mutation has previously been demonstrated to result in an increase in dopamine signaling, the authors confirmed the role of dopamine in extinction learning using preretention session administration of a low dose of the dopamine transport reuptake inhibitor modafinil (0.75 mg/kg), which resulted in decreased freezing across retention sessions. These findings are consistent with an emerging portrayal of the importance of Clock genes in noncircadian functions, as well as the important role of dopamine in extinction learning. Keywords: circadian, clock, ClockA 19 mutant mice, contextual fear conditioning, dopamine, extinction, modafinil
been demonstrated to result in an increase in the sensitivity to psychostimulants (McClung et al., 2005; Ozbum, Larson, Self, & McClung, 2012; but see Bernardi & Spanagel, 2013) and ethanol (Ozbum et al., 2013), as well as an increase in mania-like behavior (Roybal et al., 2007). These behavioral alterations in mutant mice have been attributed to an increase in the expression and phos phorylation of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of dopamine (DA), in the ventral tegmental area of mutant mice (McClung et al., 2005), and increased DA release and a subsequent alteration in DA receptor levels (Spencer et al.,
Clock genes have been well-evaluated for their role in pace maker functions, in which oscillatory transcriptional and transla tional autoregulatory feedback loops of these genes mediate cir cadian time-keeping (Ko & Takahashi, 2006). A great deal of recent evidence, however, has shown that the involvement of clock genes is not limited to circadian functions. Clock genes have been demonstrated to be involved in a variety of noncircadian functions and influence a variety of processes and have been implicated in several disorders, such as schizophrenia, bipolar disorder, autism spectrum disorders, and drug dependence (Albrecht, 2013; Perreau-Lenz & Spanagel, 2008; Rosenwasser, 2010). One of the clock genes, Clock, has primarily been the focus of studies involving drugs of abuse and mania-like symptomology. A dominant negative mutation of the Clock gene, the ClockA 19 mutation, in mice (King et al., 1997; Vitatema et al., 1994) has
2012). To date, no studies have examined a role for Clock in fearmediated behaviors, which have also been demonstrated to be sensitive to alterations in DA signaling. For example, peripheral (Inoue, Izumi, Maki, Muraki, & Koyama, 2000) or bilateral in traamygdala (Guarraci, Frohardt, & Kapp, 1999) administration of the DAI (Dl) receptor antagonist SCH 23390 prior to the acqui sition of contextual fear conditioning impaired conditioned fear in rats, while intraamygdala infusion of the D l agonist SKF 82958 prior to acquisition facilitated subsequent conditioned fear (Guar raci et al., 1999). These and other studies suggest DA involvement in the learning of contextual fear. The extinction of contextual fear conditioning has also been demonstrated to be DA-dependent, with increases in DA signaling enhancing, and decreases in DA signal ing impairing, the extinction of contextual fear (e.g., Abraham, Cunningham, & Lattal, 2012; El-Ghundi, O’Dowd, & George, 2001; Fernandez Espejo, 2003).
This article was published Online First May 26, 2014. Rick E. Bernardi and Rainer Spanagel, Institute of Psychopharmacology, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University. Rick E. Bernardi was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG, SPP1226, SP 383/4-1). We report no con flicts of interest. Correspondence concerning this article should be addressed to Rick E. Bernardi, Central Institute of Mental Health, Institute of Psychopharmacology, J5, 68159, Mannheim, Germany. E-mail: [email protected]
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CLOCKA19 MUTATION ENHANCES EXTINCTION The goal of the current study was to determine whether Clock A19 mutant mice showed alterations in contextual fear con ditioning and subsequent extinction of contextual fear. We report here that although the acquisition and expression of contextual fear did not differ between WT and ClockA19 mutant mice, extinction of contextual fear was significantly enhanced in mutant mice following the initial retention test. These results suggest a role for the Clock gene in extinction learning, likely becaue of enhanced DA signaling previously reported in mutant mice. To confirm the role of DA in extinction learning, we also demonstrated that treatment with the DA transporter blocker, modafinil, prior to retention sessions, resulted in a decrease in freezing behavior across sessions.
Methods Subjects Male Clock A \9 mutant mice and their WT littermates (back ground C57B1/6; Jackson Laboratories, Bar Harbor, ME; bred in-house) and male C57B1/6 mice (Charles River, Sulzfeld, Ger many) aged 10-12 weeks at the start of experiments served as subjects. Mice were single housed in a temperature-controlled (21°C) environment maintained on a 12-hr light-dark cycle (lights on at 6 a.m.). Food and water were available ad libitum. All experiments were performed in accordance with EU guidelines on the care and use of laboratory animals and were approved by the local animal care committee (RegierungsprAsidium, Karlsruhe, Germany). All behavioral experiments were conducted during the light phase.
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context. Freezing was defined as bouts of inactivity that lasted at least 1 s. Total time meeting this criterion was divided by the total time of the bin (acquisition) or session (retention) to calculate percent time freezing. Five days following the final conditioned fear-retention session, mice were given a 30-min locomotor activ ity test. Data were recorded as distance traveled (cm). Experiment 2. C57B1/6 mice received a single fear acquisi tion session as described above. Beginning 24 hr later, mice received six daily 12-min nonreinforced exposures to the context. Extinction Sessions 1 -4 were preceded by administration of 0.75 mg/kg modafinil (Tocris, Wiesbaden-Nordenstadt, Germany) or vehicle (10% Tween 80; 10 ml/kg; Sigma-Aldrich, Steinheim, Germany). Freezing was defined as described above. Again, 6 days following the final conditioned fear-retention session, mice were placed into the activity chambers for a 30-min locomotor activity test preceded by vehicle or modafinil (0.75 mg/kg) administration in mice that received vehicle or modafinil, respectively, during fear extinction. To control for potential toler ance or sensitization to the repeated administration of modafinil in a duration identical to that of the fear conditioning retention sessions, a second group of drug-nave mice were given a single habituation trial, then administered vehicle or 0.75 mg/kg modafinil prior to 12-min locomotor sessions on subsequent days. Data were recorded as distance traveled (cm) during the session.
Statistical Analysis Data were analyzed using analysis of variance (ANOVA). Sig nificance was set at p < .05.
Results Apparatus Fear conditioning. One Coulbourn Instruments mouse con ditioning chamber (H10-35M-04; Coulbourn Instruments, Allen town, PA) was used, which consists of a clear Plexiglas arena (17.8 cm X 17.8 cm X 30.5) with a stainless steel floor of grid rods spaced 6.4 mm apart. The chamber was housed in a sound-attenuating shell, with a light providing illumination and a fan providing background noise. Scrambled shocks were delivered to the grid floor by a computer-controlled shock generator (Coulbourn H13-15). Mounted above the floor of the chamber was a camera that recorded activity in real-time. Experimental events were controlled by FreezeFrame (Coulbourn Instruments, Allentown, PA) software, and freezing data were compiled using FreezeView software. Locomotor activity. Locomotor activity was assessed in six TruScan activity monitors (Coulbourn Instruments, Allentown, PA). Each monitor consists of a clear acrylic plastic test cage (22 X 22 X 40 cm) placed inside a monitoring unit that records via computer ambulatory beam interruptions from infrared photocell emitter/detector pairs evenly spaced along each axis.
Behavioral Procedures Experiment 1. During a single fear acquisition session, mice received a 12-min exposure to the context with four unsignaled shocks (2 s, 0.35 mA; Bemardi & Lattal, 2010; Lattal & Maughan, 2012) delivered at 70 s, 278 s, 510 s, and 642 s. Beginning 24 hr later, mice received 5 daily 12-min nonreinforced exposures to the
Experiment 1 During acquisition, ClockA 19 mutant and WT mice demon strated a freezing profile that did not differ as a function of genotype. Figure 1A shows the mean (± SEM) percent freezing across four 3-min time bins during the 12-min session. A two-way ANOVA (Genotype X Time Bin) revealed a significant main effect of time bin, F(1.9,19.5) = 56.9, p < .001, but no significant main effect of genotype, F (l, 10) = 0.05, p = .83, and no interaction, F(1.9,19.5) = 0.15, p = .85. Thus, WT and ClockA\9 mutant mice demonstrated a similar acquisition of conditioned fear. Across retention sessions, ClockA\9 mutant mice demonstrated an accelerated decline in freezing as compared to WT mice. Figure IB shows the mean (± SEM) percent freezing across five 12-min session nonreinforced retention sessions. A two-way ANOVA (Genotype X Retention Session) revealed significant main effects of genotype, F (l, 10) = 6.1, p < .05, and retention session, F(4, 40) = 35.0, p < .001, and a significant Genotype X Retention Session interaction, F(4, 40) = 2.8, p < .05. One-way ANOVAs conducted for each retention session revealed that the two groups did not differ in freezing behavior during the first retention session, F (l, 10) = 0.47, p = .51, but differed significantly on all subse quent retention tests—Test 2: F (l, 10) = 4.9, p = .05; Tests 3-5: Fs(l,10) > 6.5, ps < .05. These results indicate that after showing similar levels of freezing during the first retention session as compared to WT mice, Clock A 19 mutant mice demonstrated pro-
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Figure 1. Effects of the ClockA\9 mutation in mice on the acquisition and extinction of contextual fear. A: There were no differences in the acquisition of contextual fear between wild-type (WT) and ClockA 19 mutant mice. Data represent mean (± SEM) percentage freezing during four 180-s time bins during the 12-min conditioning trial. B: There was no difference in the expression of conditioned fear during the first 12-min retention session between WT and ClockA 19 mutant mice. However, during retention Sessions 2-5, ClockA\9 mutant mice demonstrated progressively enhanced extinction relative to WT mice. Data represent mean (± SEM) percentage freezing during the five 12-min retention sessions (T1-T5). (C) ClockA\9 mutant mice demonstrated higher locomotor activity as compared to WT mice during a test for locomotion following the conclusion of fear procedures. Data represent mean (± SEM) distance traveled during a 30-min session. * p < .05.
gressively lower levels of conditioned fear during subsequent sessions. ClockA\9 mutant mice demonstrated increased locomotor ac tivity relative to WT mice. Figure 1C shows the mean (± SEM) distance traveled (cm) during the 30-min locomotor activity tests for WT and ClockA\9 mutant mice. A one-way ANOVA revealed significant a significant effect of distance traveled, F (l, 10) = 16.2, p < .005.
Experiment 2 During acquisition, C57BL/6 mice demonstrated a freezing pro file that did not differ as a function of subsequent modafinil treatment. Figure 2A shows the mean (± SEM) percent freezing across four 3-min time bins during the 12-min session. A two-way ANOVA (Treatment Group X Time Bin) revealed a significant main effect of time bin, F(2.1,27.1) = 51.5, p < .001, but no significant main effect of genotype, F (l, 13) = 0.13, p — .73, and no interaction, F(2.1,27.1) = 0.07, p = .94, suggesting no differ ence between the two groups during the acquisition of contextual fear. Across retention sessions, modafinil-treated mice demonstrated a decrease in freezing as compared to WT mice. Figure 2B shows the mean (± SEM) percent freezing across four 12-min session nonreinforced extinction sessions preceded by modafinil treatment and two drug-free test sessions. Across Extinction Sessions 1-4, a two-way ANOVA (Treatment Group X Retention Session) re vealed significant main effects of treatment, F (l, 13) = 5.3, p < .05, and retention session, F(3, 39) = 42.0, p < .001, but no Genotype X Retention Session interaction, F(3, 39) = 0.35, p = .79. One-way ANOVAs conducted for each retention session revealed that the two groups did not differ in freezing behavior during the first two retention sessions, Fs( 1,13) < 3.3, ps > .09, but differed significantly during subsequent retention tests— Ext 3: F (l, 13) = 4.6, p = .05; Ext 4: F (l, 13) = 6.3, p < .05. For
drug-free Test Sessions 1 and 2, one-way ANOVAs indicated significant differences between vehicle- and modafinil-treated mice in a drug-free state, Fs(l,13) > 6.2, ps < .05. These results indicate enhanced extinction in modafinil-treated mice relative to vehicle-treated mice, including in a drug-free state. There was no difference in locomotor activity between the two groups, suggesting that a modafinil-induced increase in locomotion likely does not explain the decrease in freezing in modafinil-treated mice. Figure 2C shows the mean (± SEM) distance traveled (cm) during the 30-min locomotor activity tests for vehicle- and modafinil-treated mice. A one-way ANOVA revealed no differ ence in distance traveled, F (l, 13) = 0.52, p = .48. Furthermore, in a separate group of drug-nave mice, there was also no difference in locomotor activity between vehicle and modafinil treatment (Figure 2D), F (l, 5) = 3.4, p = .13.
Discussion The current data indicates that the ClockA 19 mutation may also be involved in the extinction of aversive memories, in addition to its prior demonstrated role in reward-related behaviors involving drugs of abuse. ClockA\9 mutant mice demonstrated normal ac quisition and expression of contextual fear, as shown by levels of freezing similar to WT mice during the conditioning session and first retention test. However, during subsequent retention sessions, the loss of the freezing response in ClockA 19 mutant mice was progressively accelerated relative to WT mice. Thus, the learning and recall of the fear response was not affected by the ClockA\9 mutation, but extinction of conditioned fear was enhanced. DA has long been considered a mediator of learning (Harley, 2004). Because the DA system has been implicated as the primary target of the ClockA\9 mutation (McClung et al., 2005; Spencer et al., 2012), the effects of the ClockA 19 mutation in male mice demonstrated here likely involve a DA-dependent increase in extinction learning. We saw no difference in contextual fear learn-
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Figure 2. Effects of modafinil (MOD) on the extinction of contextual fear. A: There were no differences in the acquisition of contextual fear between vehicle (VEH) and MOD groups prior to treatment. Data represent mean (± SEM) percentage freezing during four 180-s time bins during the 12-min conditioning trial. B: MOD-treated mice demonstrated a decrease in freezing across four extinction trials (EXT 1-4) preceded by MOD treatment (0.75 mg/kg; indicated by j ), as well as on two drug-free tests (T1 and T2), as compared to VEH treatment. Data represent mean (± SEM) percentage freezing during the six 12-min retention sessions. C: VEH- and MOD-treated mice showed no difference in locomotor activity during a test for locomotion following the conclusion of fear procedures. Data represent mean (± SEM) distance traveled during a 30-min session. D: A separate group of drug-nave mice showed no difference in locomotor activity between VEH and MOD treatment during a test for locomotion. Data represent mean (± SEM) distance traveled during a 12-min session. *p < .05.
ing or retention during the first extinction trial but a subsequent acceleration of extinction in ClockM9 mutant mice relative to WT mice. Interestingly, a previous study examined fear in response to contextual cues in mice possessing the NPAS2 gene, the human analog of the Clock, and demonstrated that NPAS2 (-/-) mice showed normal freezing 30 min following fear conditioning, but decreased freezing during a 24-hr retention test, relative to WT mice (Garcia et al., 2000). Although seemingly contrasting our findings here, in which we demonstrated no difference in freezing during the first extinction Session 24 hr after conditioning, this previous study used an auditory cue that preceded each of the repeated shocks during conditioning. Thus, the difference in freez ing during the first retention session may be more related to differences in valence attributed to the varying conditioned stimuli rather than a specific deficit in contextual fear. Furthermore, this study did not examine the time-course of extinction learning. To confirm our findings in C/ockA19 mutant mice, we admin istered modafinil, a DA reuptake inhibitor, prior to extinction sessions. We used a low dose (0.75 mg/kg) previously reported to enhance retention of contextual fear when administered prior to acquisition (Shuman, Wood, & Anagnostaras, 2009). In our study, this low dose resulted in enhanced extinction as compared to vehicle-treated mice, even when treatment was withdrawn (Tests
1-2). This is the first study to report enhanced extinction of contextual fear using modafinil. Our findings are consistent with a recent study by Abraham et al. (2012), who demonstrated that methylphenidate, a DA and norepinephrine reuptake inhibitor, administered prior to or following extinction sessions, enhanced the extinction of contextual fear in mice. Furthermore, D1 receptor knockout (KO) mice demonstrated no difference in the acquisition or expression of contextual fear relative to WT mice but an impairment of extinction during subsequent trials (El-Ghundi et al., 2001), suggesting that DA functioning through D1 receptors is necessary for normal extinction learning following fear condition ing. Other studies have also demonstrated a role for DA in extinc tion learning following fear conditioning (Abraham, Neve, & Lattal, 2014). Some data exists with regards to other clock genes, Period {Per) and Cryptochrome {Cry), in fear-related behaviors. Mechanisti cally, the CLOCK protein dimerizes with brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein 1 (BMAL1) to activate the transcription of PER and CRY proteins to influence intracellular and extracellular targets. A PER-CRY het erodimer acts via negative feedback on the CLOCK-BMAL1 heterodimer to repress further transcription (Dibner, Schibler, & Albrecht, 2010). Perl and Perl KO mice showed no differences
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relative to WTs in the acquisition or expression of conditioned fear in a contextual fear paradigm (Zueger et al., 2006; see also Wang et al., 2009), and although Cryl/Cry2 KO mice demonstrated higher freezing during the acquisition of auditory fear condition ing, they showed no difference in freezing relative to WT mice during subsequent expression testing (De Bundel, Gangarossa, Biever, Bonnefont, & Valjent, 2013). Our data are consistent with these previous findings, in that we found no difference in the ability of ClockAY) mutant mice to learn and express conditioned fear relative to WT mice. These previous studies did not examine further extinction trials in Per and Cryl/Cry2 KO mice, however, and thus the role of these clock genes in extinction is yet unknown. It is important to note that previous studies have demonstrated locomotor hyperactivity in ClockA\9 mutant mice, as compared to WT mice (e.g., Bernardi & Spanagel, 2013). We also demonstrated here that following the completion of fear conditioning, ClockA\9 mutant mice showed higher locomotor activity than WT mice. Thus, it is impossible to eliminate increased locomotion as an explanation for the significant decrease in freezing across retention sessions in mutant mice in the current study. However, we saw no difference between WT and mutant mice in the acquisition of contextual fear, and importantly, no difference in responding dur ing the first retention trial. Thus, potential differences in locomo tion cannot fully explain the significant interaction between WTs and mutants in freezing behavior achieved across retention trials, which is likely primarily due to an enhanced extinction in ClockA\9 mutant mice. Preretention session administration of modafinil resulted in an immediate and long-lasting decrease in freezing relative to vehicle treatment. Although previous studies have demonstrated locomotor activation to modafinil at higher doses, the 0.75 mg/kg dose has previously been shown not to result in either acute or sensitized locomotor activation (Shuman, Cai, Sage, & Anagnostaras, 2012). Furthermore, we demonstrated in mice that underwent fear conditioning, as well as a separate group of nave mice, that this dose failed to increase locomotor activity. Thus, we suggest that modafinil administered prior to extinction increased extinction learning. In conclusion, we report here that the ClockA19 mutation re sulted in an enhanced extinction of contextual fear, an effect mimicked using the DA reuptake inhibitor modafinil. These find ings demonstrate a specific role for Clock in extinction in aversive paradigms, and further demonstrate the importance of clock genes in general in noncircadian functions, as well as the importance of DA signaling in extinction learning.
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Received December 12, 2013 Revision received April 15, 2014 Accepted April 23, 2014 ■
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