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Enhancement of acoustic prepulse inhibition by contextual fear conditioning in mice is maintained even after contextual fear extinction
Progress in Neuro-Psychopharmacology & Biological Psychiatry 34 (2010) 183–188
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Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p
Enhancement of acoustic prepulse inhibition by contextual fear conditioning in mice is maintained even after contextual fear extinction Daisuke Ishii a, Daisuke Matsuzawa a, Yuko Fujita c, Chihiro Sutoh a, Hiroyuki Ohtsuka a, Shingo Matsuda a, Nobuhisa Kanahara b, Kenji Hashimoto c, Masaomi Iyo b, Eiji Shimizu a,⁎ a b c
Department of Integrative Neurophysiology, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670, Japan Department of Psychiatry, Chiba University Graduate School of Medicine, Chiba University Graduate School of Medicine 1-8-1 Inohana, Chuouku, Chiba 260-8670 Japan Division of Clinical Neuroscience, Center of Forensic Mental Health, Chiba University, 1-8-1 Inohana, Chuouku, Chiba, 260-8670, Japan
a r t i c l e
i n f o
Article history: Received 6 August 2009 Received in revised form 31 October 2009 Accepted 31 October 2009 Available online 14 November 2009 Keywords: Contextual fear conditioning Fear extinction PPI PTSD Sensorimotor gating
a b s t r a c t Prepulse inhibition (PPI) of the acoustic startle response is one of the few and major paradigms for investigating sensorimotor gating systems in humans and rodents in a similar fashion. PPI deficits are observed not only in patients with schizophrenia, but also in patients with anxiety disorders. Previous studies have shown that PPI in rats can be enhanced by auditory fear conditioning. In this study, we evaluated the effects of contextual fear conditioning (FC) for six times a day and fear extinction (FE) for seven days on PPI in mice. C57BL/6J mice (male, 8–12 weeks) were divided into three groups; no-FC (control), FC and FC + FE. We measured PPI at the following three time points, (1) baseline before FC, (2) after FC, and (3) after FE. The results showed that PPI was increased after FC. Moreover, the enhanced PPI following FC was observed even after FE with decreased freezing behaviors. These results suggested contextual fear conditioning could enhance acoustic PPI, and that contextual fear extinction could decrease freezing behaviors, but not acoustic PPI. © 2009 Elsevier Inc. All rights reserved.
1. Introduction Prepulse inhibition (PPI) of the acoustic startle reflex, used in the current study, is an established paradigm for the assessment of sensorimotor gating systems. PPI reflects the regulation of sensory input by filtering out irrelevant or distracting signals, and PPI is defined as a substantial reduction of the amplitude of the startle reflex that occurs when a prepulse is presented 30–500 ms prior to the startling stimulus. PPI are disturbed in schizophrenia (Swerdlow et al., 1994; Braff et al., 2001; Geyer et al., 2002). Interestingly, PPI deficits were also reported in patients with anxiety disorders, including posttraumatic stress disorder (PTSD) (Grillon et al., 1996), panic disorder (Ludewig et al, 2002) and obsessive–compulsive disorder (Hoenig et al., 2005). The paradigm of fear conditioning and extinction in rodents has been considered as a valuable animal model for studying anxiety disorders such as PTSD. Fear conditioning to either a cue or a context represents a form of associative learning involving the formation of linkages between a neutral stimulus and a stimulus with innate behavioral significance (Sanders et al., 2002). As a conditioned
Abbreviations: ASR, Acoustic startle reactivity; CS, Conditioned stimulus; FC, Fear conditioning; FE, Fear extinction; PPI, Prepulse inhibition; PTSD, Posttraumatic stress disorder; US, Unconditioned stimulus. ⁎ Corresponding author. Tel.: +81 43 226 2027; fax: +81 43 226 2028. E-mail address: [email protected] (E. Shimizu). 0278-5846/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2009.10.023
stimulus (CS) such as a contextual or a tone and an aversive unconditioned stimulus (US) such as an electric footshock are repeatedly and consistently paired, CS alone begins to elicit a freezing behavior in anticipation of US presentation. Fear extinction refers to the repeated presentations of the CS in the absence of the US previously paired. It has been hypothesized that extinction does not erase the original fear memory (a previously established CS–US association) but forms a new memory of safety that inhibits fear expression (a newly established CS–no US association) (Myers and Davis, 2007). Even a completely extinguished fear can be recovered spontaneously after the passage of time or “reinstated” by the presentations of the US alone or renewed by placing the animal in a different context. Extinction might be labile and weak, compared with fear conditioning itself. Understanding the neural mechanisms of fear extinction is important for the treatment of patients with anxiety disorders. Cognitive behavior therapy including exposure to feareliciting cues in a safe setting is based on mechanisms of fear extinction. It is widely used today and remains one of the most effective therapies for pathological anxiety including PTSD, panic disorder, phobias and obsessive–compulsive disorder (Sotres-Bayon et al., 2006). In general, two types of conditioning that are typically employed are cued and contextual conditioning (Curzon et al., 2009). In this study, we focused on contextual but not auditory (cued) fear conditioning. Contextual fear conditioning is the most basic of the conditioning procedures. It involves taking an animal and placing it in
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a novel environment, providing an aversive stimulus, and then removing it. When the animal is returned to the same environment, it generally will demonstrate a freezing response if it remembers and associates that environment with the aversive stimulus. If in a conditioning context one administers a foot shock that is paired with a tone, there will be learning not only to the tone, but also to the context. On the other hand, the animal will also display the freezing response to the auditory cue when it is presented in a different context. This is termed auditory (cued) fear conditioning. The auditory cue is generally tested in a different context so that the freezing can be attributed to conditioning to the cue and not to the context in which shock occurred. In the viewpoint of the differences between both fear conditionings, contextual fear conditioning is hippocampus-dependent, whereas cued fear conditions is hippocampus-independent (Phillips and LeDoux, 1992; Rudy et al., 2004). Both these types of fear conditioning require the activation of NMDA receptors (Kim et al., 1992; Tang et al., 1999). Moreover, many studies have researched mechanism of PPI alterations through NMDA receptors. In animals, disruption of PPI can be induced by administration of either NMDA antagonists or dopamine agonists (Johansson et al., 1995; Kanahara et al., 2008; Mansbach and Geyer, 1989; Mansbach et al., 1988; Swerdlow et al., 1998; Swerdlow et al., 1994; Varty and Higgins, 1995). Moreover, patients with schizophrenia and schizotypal personality disorder usually suffer from impaired sensory gating (Braff et al., 2001; Geyer et al., 2001; Kumari et al., 2000; Swerdlow et al., 2006). Taken together, PPI and fear conditioning seem to be involved with pathophysiology of anxiety disorders. The previous studies (Zou et al., 2007; Huang et al., 2007; Li et al., 2008; Du et al., 2009) demonstrated that that auditory (cued) fear conditioning for the prepulse might enhance PPI of the auditory startle reflex. When a neutral prepulse sound is temporally combined with footshock, the prepulse sound becomes conditioned and signifies the aversive event, and consequently, enhances auditory PPI. These results indicate that PPI is enhanced when the prepulse signifies an aversive event after fear conditioning. To the best of our knowledge, there are no reports about the relation between PPI and contextual fear conditioning. It is still unclear whether PPI is enhanced when the prepulse remains a nonaversive after contextual fear conditioning. In this study, we tested the hypothesis that contextual fear conditioning could enhance PPI following neutral prepulse in mice. In addition, Du et al. (2009) reported auditory fear conditioning of the prepulse enhances PPI and this PPI enhancement largely reduces after fear extinction. On the other hand, there are no reports about the relation between PPI and contextual fear extinction. In this study, we further investigated whether PPI following neutral prepulse could be influenced by extinction of contextual conditioned fear in mice. 2. Materials and methods 2.1. Animals C57BL/6J male mice (8–12 weeks old, n = 39) were housed 5 per cage kept at a controlled temperature (23 ± 1 °C) and on a 12-h light/ dark cycle (light on at 07:00 hours). The animals were provided food and water ad libitum. All behavioral testing was conducted between 09:00 and 18:00 hours. Mice were randomized and used only once. The research and animal care were carried out according to the Guide for Animal Experimentation of the Chiba University Graduate School of Medicine. 2.2. Behavioral experiment To begin exploring effect of fear conditioning and extinction on PPI, we trained three groups of mice using a mixed fear conditioning, extinction and PPI paradigm (Fig. 1). The animals were divided into
three groups; no-FC (no received US, n = 12), FC (only fear conditioning, n = 14) and FC + FE (fear conditioning and extinction, n = 13).
2.3. Prepulse inhibition (PPI) tests of acoustic startle The mice were tested to assess their acoustic startle reactivity (ASR) in startle chambers with an automated system for measuring startle responses, containing a cylindrical animal enclosure mounted on a Plexiglas base (SR-LAB, San Diego Instruments, CA) using standard methods described by Swerdlow and Geyer (1998). Startle responses are transduced by a piezoelectric accelerometer mounted below the cylinder, rectified, digitized and recorded as data points on a computer. Background noise was set at 65 dB SPL. Three trial types were used. Pulse-alone trials (P) consisted of a single white-noise burst (120 dB, 40 ms). The prepulse + pulse trails (PP69P, PP73P, PP77P, and PP81P) consisted of a prepulse of noise (20 ms at 69, 73, 77 or 81 dB, respectively), which was followed 100 ms after the prepulse onset by a startle pulse (120 dB, 40 ms). No-stimulus (NS) trials consisted of the background noise only. Sessions were structured as follows: (1) 15-min acclimation at background noise level; (2) five P trials; (3) ten blocks, each containing all trials (P, PP69P, PP73P, PP77P, PP81P and NS) in pseudorandom order; and (4) five P trials. Intertrial intervals were distributed between 7 and 23 s. The average percent reduction in startle intensity between pulse and prepulse + pulse trials at all four prepulse levels was defined as the PPI level. The amount of PPI was calculated as a percentage score for each acoustic prepulse trial type: % PPI = [1 − (startle amplitude on prepulse trial) / (startle amplitude on pulse-alone)] × 100%. Total %PPI was calculated by adding together all of %PPI in 69, 73, 77 and 81 dB. Startle magnitude in this formula was calculated as the average response to all of the P trials, excluding the first and last blocks of five P trials. PPI at baseline (before fear conditioning), day 4 (after fear conditioning) and day 12 (after extinction) were measured.
2.4. Habituation, contextual fear conditioning and extinction The footshock chambers for fear conditioning were different from the startle chambers for prepulse inhibition. Experiments were carried out in 22.8 × 19.7 × 13 cm experimental footshock chamber with transparent walls and metal-rod floor (Actimetrics, IL, USA). The experimental footshock chamber was cleaned with 70% ethanol before and after use. Habituation started 2 days before the contextual fear conditioning and consisted of two 10-min-long pre-exposure periods to the footshock chamber. The interval between the periods was 4 h and no shock was delivery. We conducted contextual fear conditioning without any cues (tones or lights). After 180 s acclimation period, FC and FC + FE groups of mice received six pairings (20–120 s interpairing interval) of CS (context at the experimental chamber) and US (2 s, 0.75 mA footshock), in which the US was presented by the Actimetrics (Wilmette, IL) FreezeFrame System. A no-FC group of mice was exposed to the footshock chamber for the same time period but no shock was given. The testing session was performed in the last 180 s. Freezing behavior was measured using a digital video camera connected to a computer with Actimetrics FreezeFrame software (Actimetrics). Fear extinction was defined as the repetitive exposure to the same experimental chamber in the absence of footshock. Forty-eight hours after contextual fear conditioning, no-FC and FC + FE groups of mice were placed for 20 min without footshock in the same experimental chamber where the footshock was delivered. Fear extinction was performed on each of seven consecutive days following measurement of PPI after fear conditioning.
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Fig. 1. The current protocol for fear conditioning, extinction and PPI paradigm. Day 1, habituation started 2 days before the contextual fear conditioning. Day 2, PPI was measured as baseline. Day 3, contextual fear conditioning and freezing test were preformed. Day 4, PPI was measured as after FC. Day 5–11, extinction training was performed on each of seven consecutive days. Day 12, PPI was measured as after FE. Abbreviation: FC, fear conditioning; FE, fear extinction; PPI, prepulse inhibition.
2.4. Statistical analysis Comparisons of each percent of PPI and ASR among three phases (baseline before FC, after FC, after FE) were performed with two-way repeated measures of analysis of variance (ANOVA) after Levene's test was used to evaluate for homogeneity of variances. Data of fear conditioning and extinction were analyzed using one-way repeated measures of ANOVA. Bonferroni's correction was used for post hoc comparisons when ANOVA revealed statistically significant differences. Statistical significance was set at p b 0.05. All analyses were conducted with the software SPSS 12.0 for Windows (SPSS, Chicago, Illinois). Data are shown as mean ± SEM for all results. 3. Results 3.1. Freezing responses during fear conditioning and extinction In the fear conditioning phase, analysis of variance (one-way repeated ANOVA) followed by Bonferroni post hoc comparisons indicated that FC and FC + FE groups showed increased percent of freezing, compared to no-FC (control) group at 2–18 trials of freezing measurement(p b 0.01) (Fig. 2). There were no significant differences of freezing between FC group and FC + FE group at all trials (Fig. 2). In the fear extinction phase, analysis of variance (one-way repeated ANOVA) followed by Bonferroni post hoc comparisons showed that the freezing level at day11 trials of FC + FE group was significantly reduced, compared to day 5, 6, 7 trials (p b 0.01)(Fig. 3). 3.2. Effects of fear conditioning and extinction on PPI There was a significant effect of contextual fear conditioning on PPI. Post hoc comparison revealed that total %PPIs after FE in both FC (F(2, 35) = 6.638, p b 0.01) and FC + FE (F(2, 35) = 9.526, p b 0.01)
Fig. 2. Effects of contextual fear conditioning on percentages of freezing. Average percentages of freezing to the context were shown in blocks of 18 trials for no-FC, FC, FC+FE groups at fear conditioning phase. The freezing levels of FC and FC+FE groups were significantly increased when compared to that of no-FC group at 2–18 trials (⁎⁎pb 0.01). Abbreviation: FC, fear conditioning; FE, fear extinction.
groups were significantly increased when compared to those at baseline before FC. At this time, there was no significant change in total %PPIs between FC and FC + FE (Fig. 4). In addition, the magnitude of the startle reflex gradually showed a decrease. Especially, a significant difference was shown in no-FC (baseline vs. after FC; p b 0.05, baseline vs. after FE; p b 0.05) and FC groups (baseline vs. after FE; p b 0.05, after FC vs. after FE; p b 0.05). The decreasing tendency in the magnitude of the startle reflex was also shown in FC + FE group. There was no difference among all groups at each time point (Fig. 5). 4. Discussion The major findings of our study were as follows: total %PPIs were enhanced following contextual fear conditioning, and the enhanced total %PPIs were maintained even after contextual extinction training which succeeded in reducing the percent of freezing. Thus, PPI enhanced by contextual fear conditioning may not be influenced by contextual fear extinction. In no-FC group as control condition, no significant changes of total % PPI among all phases were found in the current study. The results are consistent with previous reports (Li et al., 2008), showing that PPIs were not changed at repeated measurement points. Additionally, no significant differences among all groups were found in the ASR without prepulse. There are two types of measurement of conditioned fear; freezing and startle. The fear-potentiated startle paradigm measures conditioned fear by an increase in the amplitude of a simple reflex (the acoustic startle reflex) in the presence of a cue previously paired with a shock (Davis 1986, 1992). It has also been demonstrated that the startle reflex can be increased when a context is used as the conditioned stimulus (Campeau et al., 1991). We measured contextual conditioned fear using freezing paradigm, but not fear-potentiated startle reflex. In addition, we measured ASRs including PPIs in a
Fig. 3. Effects of contextual fear extinction training on percentages of freezing. Average percentages of freezing to the context were shown in blocks of 7 trials for no-FC and FC + FE groups at fear extinction phase. The freezing level at day 11 trials of FC + FE group was significantly reduced when compared to that at day 5, 6 and 7 trials (⁎⁎pb 0.01). Abbreviation: FC, fear conditioning; FE, fear extinction.
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Fig. 4. Effects of contextual fear conditioning on prepulse inhibition (PPI). Total %PPI was showed as amount of %PPI in 69, 73, 77 and 81 dB. The total %PPIs in FC (⁎⁎p b 0.01) and FC + FE (##p b 0.01) groups at after FE were significantly increased when compared to that at baseline, respectively. No significant differences between FC and FC + FE groups were found in the total %PPI at after FE. Abbreviation: FC, fear conditioning; FE, fear extinction; total %PPI, total %prepulse inhibition.
completely different context from the conditioned context combined with footshock for contextual fear conditioning. In other words, ASRs including PPIs were measured in the non-conditioned context. Our results indicated that ASRs were not influenced by contextual fear conditioning or extinction. No enhancement of fear-potentiated startle reflex in the current study seems reasonable without conditioned cue or conditioned context. On the other hand, the previous reports in humans (Grillon and Davis, 1997a, 1997b; Greenwald et al., 1998; Vanman et al., 1996; Waters and Ornitz, 2005) and in rats (Davis 1986; Davis et al., 1993; Du et al., 2009; Li et al., 2008) suggested that enhancement of fearpotentiated startle reflex may follow the presence of conditioned cue or conditioned context. In humans, the startle stimulus was given in the same context that shock might come. In rats, CS for fearpotentiated startle reflex was a prepulse. In these previous works, the reaction to the startle stimulus was investigated in the same conditioned context or with conditioned cue, and the fear memory was recalled when ASRs were measured. Our results suggested that contextual fear conditioning may not influence ASRs if there is no conditioned fear against the auditory stimulation for ASR or if the ASRs were not measured in the context that relates to fear. Furthermore, contextual fear conditioning may enhance acoustic PPI even in the case that there is no conditioned fear against the auditory stimulation for ASR and the context. PPI is assumed to reflect sensorimotor gating. Habituation to irrelevant sensory input is an important function for the brain as the
Fig. 5. Effects of contextual fear conditioning on acoustic startle reactivity (ASR). The significant difference was shown in no-FC (baseline vs. after FC; ⁎p b 0.05, baseline vs. after FE; ⁎p b 0.05) and FC groups (baseline vs. after FE; #p b 0.05, after FC vs. after FE; #p b 0.05). The decreasing tendency in the magnitude of the startle reflex was also shown in FC + FE group. There was no difference among all groups at each time point. Abbreviation: ASR, acoustic startle reactivity; FC, fear conditioning; FE, fear extinction.
central information-processing organ of the body because the failure may be associated with mental disturbances. The term “gating” is a hypothetical psychophysiological construction in the brain and is related to a central inhibitory function which occurred at the neural level. Our results suggest that sensorimotor gating system, but not startle response system, might be physiologically associated with contextual fear conditioning. The data suggest that contextual fear conditioning might increase the sensitivity of sensorimotor gating. To our knowledge, this is the first report to show alterations of total %PPI by contextual fear conditioning in mice, even there is no conditioned fear against the auditory stimulation. It has been known that the emotional learning enhances PPI (Du et al., 2009; Grillon and Davis, 1997a, 1997b; Huang et al., 2007; Li et al., 2008; Zou et al., 2007). In the previous studies, following temporally combining the gap (CS; prepulse stimulus) with footshock in a precise manner, gapinduced PPI was enhanced. One of the possible reasons of this PPI enhancement is that conditioning the prepulse stimulus facilitates attention to the prepulse stimulus. In our study, acoustic PPI after contextual fear conditioning showed enhancement though the prepulse stimulus and CS were different. Moreover, the enhanced PPI following contextual fear conditioning was observed even after contextual fear extinction. In humans, PPI is modulated by both attentional and emotional responses to prepulse, indicating that this early-stage gating is topdown modulated by higher-order cognitive processes (Li et al., 2009). A possible explanation of the PPI enhancement after contextual conditioning in our study is that contextual fear conditioning facilitates attention to being the situations from which escape might be difficult, like agoraphobia. Agoraphobia is an anxiety disorder characterized by intense fear related to being in situations from which escape might be difficult (i.e., being on a bus or train). The sufferer with agoraphobia becomes anxious in environments where he or she perceives that they have little control. Spatial memory is the part of memory responsible for recording information about one's environment and its spatial orientation. The mice after contextual fear conditioning may have spatial memory and attention to being in situations from which escape might be difficult. Attention to the situations might lead to enhancement of sensorimotor gating including acoustic PPI. Moreover, our findings indicated that contextual fear extinction could decrease freezing behaviors, but not acoustic PPI for sensorimotor gating. The results supported that fear extinction does not erase the original fear memory but forms a new memory of safety that inhibits fear expression. Our data also suggested that the phenomena that a completely extinguished fear can be recovered spontaneously after the passage of time or “reinstated” by the presentations of the US alone in a relevant context or renewed by placing the animal in a different context might be related to the maintenance of enhanced sensorimotor gating after fear extinction. In short, context effects of extinction including spontaneous recovery, reinstatement and renewal might be due to enhanced sensorimotor gating. Many researchers reported that the hippocampus may be involved in contextual fear conditioning as the essential neural circuit (Koch, 1999; Swerdlow and Geyer, 1999; Anagnostars et al., 1999; Kim and Fanselow, 1992; Maren et al., 1997; Helmstetter and Bellgowan, 1994; Muller et al., 1997). The hippocampus is also known to be involved in contextual modulation of fear extinction including spontaneous recovery, reinstatement and renewal (Ji and Maren, 2007). While the hippocampus makes unique contributions to memory, it has also long been associated with sensorimotor processes, i.e. innate processes involving control of motor responses to sensory stimuli (Bast and Feldon, 2003). Taken together, our results suggest that the influences of contextual fear conditioning and extinction to the hippocampus might appear to reflect sensorimotor gating systems as alterations of PPI. On the other hand, the prefrontal cortex (PFC) is
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known to play an important role in fear extinction and project directly to a number of subcortical brain structures, including amygdala and hypothalamus, exerting inhibitory influences (Fuster, 1997). Stimulation of the PFC inhibits discharges of amygdaloid neurons (Quirk et al., 2003; Rosenkranz and Grace, 2001, 2002) and reduces conditioned fear responses of the animal (Milad and Quirk, 2002; Milad et al., 2004) in the process of fear extinction. Although our results suggested that the inhibitory effect of the PFC on the amygdala in fear extinction might not affect sensorimotor gating system, further researches about brain structures for association between PPI and fear conditioning will be necessary. There are many studies that investigated each mechanism of PPI or fear conditioning, respectively, but the relation between PPI and fear conditioning remains unclear. In the near future, we plan to examine the molecular connections between contextual fear conditioning and sensorimotor gating systems. 5. Conclusions In summary, the current study showed that contextual fear conditioning in mice might enhance prepulse inhibition for the auditory startle reflex, and maintain even after fear extinction. These results suggested that sensorimotor gating system may be associated with contextual fear conditioning and context effects of extinction including spontaneous recovery, reinstatement and renewal. Acknowledgments This research was supported by the Grant-in-Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology, Japan. References Anagnostars SG, Maren S, Fanselow MS. Temporally graded retrograde amnesia of contextual fear after hippocampal damage in rats: within-subjects examination. J. Neurosci. 1999;19:1106–14. Bast T, Feldon J. Hippocampal modulation of sensorimotor processes. Prog Neurobiol 2003;70(4):319–45 July. Braff DL, Geyer MA, Light GA, Sprock J, Perry W, et al. Impact of prepulse characteristics on the detection of sensorimotor gating deficits in schizophrenia. Schizophr Res 2001;49:171–8. Campeau S, Hayward MD, Hope BT, Rosen JB, Nestler EJ, Davis M. Induction of the c-fos proto-oncogene in the rat amygdala during unconditioned and conditioned fear. Brain Res 1991;565:349–52. Curzon P, Rustay NR, Browman KE. Cued and contextual fear conditioning for rodents. Methods of behavior analysis in neuroscience. In: Buccafusco Jerry J, editor. New Frontiers in Neuroscience, Second EditionTaylor & Francis Group; 2009. LLC PP.33-35. Davis M. Pharmacological and anatomical analysis of fear conditioning using the fearpotentiated startle paradigm. Behav Neurosci. 1986;100:814–24. Davis M. The role of the amygdala in fear-potentiated startle: implications for animal models of anxiety. Trends Pharmacol Sci 1992;13:35–41. Davis M, Falls WA, Campeau S, Kim M. Fear-potentiated startle: a neural and pharmacological analysis. Behav Brain Res. 1993;58:175–98. Du Y, Li J, Wu X, Li L. Precedence-effect-induced enhancement of prepulse inhibition in socially reared but not isolation-reared rats. Cogn Affect Behav Neurosci 2009;9:44–58. Fuster JM. The Prefrontal Cortex. Philadelphia: Lippincott-Raven; 1997. Geyer MA, Krebs-Thomson K, Braff DL, Swerdlow NR. Pharmacological studies of prepulse inhibition models of sensorimotor gating deficits in schizophrenia: a decade in review. Psychopharmacology (Berl). 2001;156:117–54. Geyer MA, Mcllwain KL, Paylor R. Mouse genetic models for prepulse inhibition: an early review. Mol Psychiatry 2002;7:1039–53. Greenwald MK, Bradley MM, Cuthbert BN, Lang PJ. Startle potentiation: shock sensitization, aversive learning, and affective picture modulation. Behav Neurosci 1998;112:1069–79. Grillon C, Davis M. Fear-potentiated startle conditioning in humans: explicit and contextual cue conditioning following paired versus unpaired training. Psychophysiology 1997a;34:451–8. Grillon C, Morgan CA, Southwick SM, Davis M, Charney DS. Baseline startle amplitude and prepulse inhibition in Vietnam veterans with posttraumatic stress disorder. Psychiatry Res 1996;64:169–78. Grillon C, Davis M. Effects of stress and shock anticipation on prepulse inhibition of the startle reflex. Psychophysiology 1997b;34:511–7.
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Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p
Enhancement of acoustic prepulse inhibition by contextual fear conditioning in mice is maintained even after contextual fear extinction Daisuke Ishii a, Daisuke Matsuzawa a, Yuko Fujita c, Chihiro Sutoh a, Hiroyuki Ohtsuka a, Shingo Matsuda a, Nobuhisa Kanahara b, Kenji Hashimoto c, Masaomi Iyo b, Eiji Shimizu a,⁎ a b c
Department of Integrative Neurophysiology, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670, Japan Department of Psychiatry, Chiba University Graduate School of Medicine, Chiba University Graduate School of Medicine 1-8-1 Inohana, Chuouku, Chiba 260-8670 Japan Division of Clinical Neuroscience, Center of Forensic Mental Health, Chiba University, 1-8-1 Inohana, Chuouku, Chiba, 260-8670, Japan
a r t i c l e
i n f o
Article history: Received 6 August 2009 Received in revised form 31 October 2009 Accepted 31 October 2009 Available online 14 November 2009 Keywords: Contextual fear conditioning Fear extinction PPI PTSD Sensorimotor gating
a b s t r a c t Prepulse inhibition (PPI) of the acoustic startle response is one of the few and major paradigms for investigating sensorimotor gating systems in humans and rodents in a similar fashion. PPI deficits are observed not only in patients with schizophrenia, but also in patients with anxiety disorders. Previous studies have shown that PPI in rats can be enhanced by auditory fear conditioning. In this study, we evaluated the effects of contextual fear conditioning (FC) for six times a day and fear extinction (FE) for seven days on PPI in mice. C57BL/6J mice (male, 8–12 weeks) were divided into three groups; no-FC (control), FC and FC + FE. We measured PPI at the following three time points, (1) baseline before FC, (2) after FC, and (3) after FE. The results showed that PPI was increased after FC. Moreover, the enhanced PPI following FC was observed even after FE with decreased freezing behaviors. These results suggested contextual fear conditioning could enhance acoustic PPI, and that contextual fear extinction could decrease freezing behaviors, but not acoustic PPI. © 2009 Elsevier Inc. All rights reserved.
1. Introduction Prepulse inhibition (PPI) of the acoustic startle reflex, used in the current study, is an established paradigm for the assessment of sensorimotor gating systems. PPI reflects the regulation of sensory input by filtering out irrelevant or distracting signals, and PPI is defined as a substantial reduction of the amplitude of the startle reflex that occurs when a prepulse is presented 30–500 ms prior to the startling stimulus. PPI are disturbed in schizophrenia (Swerdlow et al., 1994; Braff et al., 2001; Geyer et al., 2002). Interestingly, PPI deficits were also reported in patients with anxiety disorders, including posttraumatic stress disorder (PTSD) (Grillon et al., 1996), panic disorder (Ludewig et al, 2002) and obsessive–compulsive disorder (Hoenig et al., 2005). The paradigm of fear conditioning and extinction in rodents has been considered as a valuable animal model for studying anxiety disorders such as PTSD. Fear conditioning to either a cue or a context represents a form of associative learning involving the formation of linkages between a neutral stimulus and a stimulus with innate behavioral significance (Sanders et al., 2002). As a conditioned
Abbreviations: ASR, Acoustic startle reactivity; CS, Conditioned stimulus; FC, Fear conditioning; FE, Fear extinction; PPI, Prepulse inhibition; PTSD, Posttraumatic stress disorder; US, Unconditioned stimulus. ⁎ Corresponding author. Tel.: +81 43 226 2027; fax: +81 43 226 2028. E-mail address: [email protected] (E. Shimizu). 0278-5846/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2009.10.023
stimulus (CS) such as a contextual or a tone and an aversive unconditioned stimulus (US) such as an electric footshock are repeatedly and consistently paired, CS alone begins to elicit a freezing behavior in anticipation of US presentation. Fear extinction refers to the repeated presentations of the CS in the absence of the US previously paired. It has been hypothesized that extinction does not erase the original fear memory (a previously established CS–US association) but forms a new memory of safety that inhibits fear expression (a newly established CS–no US association) (Myers and Davis, 2007). Even a completely extinguished fear can be recovered spontaneously after the passage of time or “reinstated” by the presentations of the US alone or renewed by placing the animal in a different context. Extinction might be labile and weak, compared with fear conditioning itself. Understanding the neural mechanisms of fear extinction is important for the treatment of patients with anxiety disorders. Cognitive behavior therapy including exposure to feareliciting cues in a safe setting is based on mechanisms of fear extinction. It is widely used today and remains one of the most effective therapies for pathological anxiety including PTSD, panic disorder, phobias and obsessive–compulsive disorder (Sotres-Bayon et al., 2006). In general, two types of conditioning that are typically employed are cued and contextual conditioning (Curzon et al., 2009). In this study, we focused on contextual but not auditory (cued) fear conditioning. Contextual fear conditioning is the most basic of the conditioning procedures. It involves taking an animal and placing it in
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a novel environment, providing an aversive stimulus, and then removing it. When the animal is returned to the same environment, it generally will demonstrate a freezing response if it remembers and associates that environment with the aversive stimulus. If in a conditioning context one administers a foot shock that is paired with a tone, there will be learning not only to the tone, but also to the context. On the other hand, the animal will also display the freezing response to the auditory cue when it is presented in a different context. This is termed auditory (cued) fear conditioning. The auditory cue is generally tested in a different context so that the freezing can be attributed to conditioning to the cue and not to the context in which shock occurred. In the viewpoint of the differences between both fear conditionings, contextual fear conditioning is hippocampus-dependent, whereas cued fear conditions is hippocampus-independent (Phillips and LeDoux, 1992; Rudy et al., 2004). Both these types of fear conditioning require the activation of NMDA receptors (Kim et al., 1992; Tang et al., 1999). Moreover, many studies have researched mechanism of PPI alterations through NMDA receptors. In animals, disruption of PPI can be induced by administration of either NMDA antagonists or dopamine agonists (Johansson et al., 1995; Kanahara et al., 2008; Mansbach and Geyer, 1989; Mansbach et al., 1988; Swerdlow et al., 1998; Swerdlow et al., 1994; Varty and Higgins, 1995). Moreover, patients with schizophrenia and schizotypal personality disorder usually suffer from impaired sensory gating (Braff et al., 2001; Geyer et al., 2001; Kumari et al., 2000; Swerdlow et al., 2006). Taken together, PPI and fear conditioning seem to be involved with pathophysiology of anxiety disorders. The previous studies (Zou et al., 2007; Huang et al., 2007; Li et al., 2008; Du et al., 2009) demonstrated that that auditory (cued) fear conditioning for the prepulse might enhance PPI of the auditory startle reflex. When a neutral prepulse sound is temporally combined with footshock, the prepulse sound becomes conditioned and signifies the aversive event, and consequently, enhances auditory PPI. These results indicate that PPI is enhanced when the prepulse signifies an aversive event after fear conditioning. To the best of our knowledge, there are no reports about the relation between PPI and contextual fear conditioning. It is still unclear whether PPI is enhanced when the prepulse remains a nonaversive after contextual fear conditioning. In this study, we tested the hypothesis that contextual fear conditioning could enhance PPI following neutral prepulse in mice. In addition, Du et al. (2009) reported auditory fear conditioning of the prepulse enhances PPI and this PPI enhancement largely reduces after fear extinction. On the other hand, there are no reports about the relation between PPI and contextual fear extinction. In this study, we further investigated whether PPI following neutral prepulse could be influenced by extinction of contextual conditioned fear in mice. 2. Materials and methods 2.1. Animals C57BL/6J male mice (8–12 weeks old, n = 39) were housed 5 per cage kept at a controlled temperature (23 ± 1 °C) and on a 12-h light/ dark cycle (light on at 07:00 hours). The animals were provided food and water ad libitum. All behavioral testing was conducted between 09:00 and 18:00 hours. Mice were randomized and used only once. The research and animal care were carried out according to the Guide for Animal Experimentation of the Chiba University Graduate School of Medicine. 2.2. Behavioral experiment To begin exploring effect of fear conditioning and extinction on PPI, we trained three groups of mice using a mixed fear conditioning, extinction and PPI paradigm (Fig. 1). The animals were divided into
three groups; no-FC (no received US, n = 12), FC (only fear conditioning, n = 14) and FC + FE (fear conditioning and extinction, n = 13).
2.3. Prepulse inhibition (PPI) tests of acoustic startle The mice were tested to assess their acoustic startle reactivity (ASR) in startle chambers with an automated system for measuring startle responses, containing a cylindrical animal enclosure mounted on a Plexiglas base (SR-LAB, San Diego Instruments, CA) using standard methods described by Swerdlow and Geyer (1998). Startle responses are transduced by a piezoelectric accelerometer mounted below the cylinder, rectified, digitized and recorded as data points on a computer. Background noise was set at 65 dB SPL. Three trial types were used. Pulse-alone trials (P) consisted of a single white-noise burst (120 dB, 40 ms). The prepulse + pulse trails (PP69P, PP73P, PP77P, and PP81P) consisted of a prepulse of noise (20 ms at 69, 73, 77 or 81 dB, respectively), which was followed 100 ms after the prepulse onset by a startle pulse (120 dB, 40 ms). No-stimulus (NS) trials consisted of the background noise only. Sessions were structured as follows: (1) 15-min acclimation at background noise level; (2) five P trials; (3) ten blocks, each containing all trials (P, PP69P, PP73P, PP77P, PP81P and NS) in pseudorandom order; and (4) five P trials. Intertrial intervals were distributed between 7 and 23 s. The average percent reduction in startle intensity between pulse and prepulse + pulse trials at all four prepulse levels was defined as the PPI level. The amount of PPI was calculated as a percentage score for each acoustic prepulse trial type: % PPI = [1 − (startle amplitude on prepulse trial) / (startle amplitude on pulse-alone)] × 100%. Total %PPI was calculated by adding together all of %PPI in 69, 73, 77 and 81 dB. Startle magnitude in this formula was calculated as the average response to all of the P trials, excluding the first and last blocks of five P trials. PPI at baseline (before fear conditioning), day 4 (after fear conditioning) and day 12 (after extinction) were measured.
2.4. Habituation, contextual fear conditioning and extinction The footshock chambers for fear conditioning were different from the startle chambers for prepulse inhibition. Experiments were carried out in 22.8 × 19.7 × 13 cm experimental footshock chamber with transparent walls and metal-rod floor (Actimetrics, IL, USA). The experimental footshock chamber was cleaned with 70% ethanol before and after use. Habituation started 2 days before the contextual fear conditioning and consisted of two 10-min-long pre-exposure periods to the footshock chamber. The interval between the periods was 4 h and no shock was delivery. We conducted contextual fear conditioning without any cues (tones or lights). After 180 s acclimation period, FC and FC + FE groups of mice received six pairings (20–120 s interpairing interval) of CS (context at the experimental chamber) and US (2 s, 0.75 mA footshock), in which the US was presented by the Actimetrics (Wilmette, IL) FreezeFrame System. A no-FC group of mice was exposed to the footshock chamber for the same time period but no shock was given. The testing session was performed in the last 180 s. Freezing behavior was measured using a digital video camera connected to a computer with Actimetrics FreezeFrame software (Actimetrics). Fear extinction was defined as the repetitive exposure to the same experimental chamber in the absence of footshock. Forty-eight hours after contextual fear conditioning, no-FC and FC + FE groups of mice were placed for 20 min without footshock in the same experimental chamber where the footshock was delivered. Fear extinction was performed on each of seven consecutive days following measurement of PPI after fear conditioning.
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Fig. 1. The current protocol for fear conditioning, extinction and PPI paradigm. Day 1, habituation started 2 days before the contextual fear conditioning. Day 2, PPI was measured as baseline. Day 3, contextual fear conditioning and freezing test were preformed. Day 4, PPI was measured as after FC. Day 5–11, extinction training was performed on each of seven consecutive days. Day 12, PPI was measured as after FE. Abbreviation: FC, fear conditioning; FE, fear extinction; PPI, prepulse inhibition.
2.4. Statistical analysis Comparisons of each percent of PPI and ASR among three phases (baseline before FC, after FC, after FE) were performed with two-way repeated measures of analysis of variance (ANOVA) after Levene's test was used to evaluate for homogeneity of variances. Data of fear conditioning and extinction were analyzed using one-way repeated measures of ANOVA. Bonferroni's correction was used for post hoc comparisons when ANOVA revealed statistically significant differences. Statistical significance was set at p b 0.05. All analyses were conducted with the software SPSS 12.0 for Windows (SPSS, Chicago, Illinois). Data are shown as mean ± SEM for all results. 3. Results 3.1. Freezing responses during fear conditioning and extinction In the fear conditioning phase, analysis of variance (one-way repeated ANOVA) followed by Bonferroni post hoc comparisons indicated that FC and FC + FE groups showed increased percent of freezing, compared to no-FC (control) group at 2–18 trials of freezing measurement(p b 0.01) (Fig. 2). There were no significant differences of freezing between FC group and FC + FE group at all trials (Fig. 2). In the fear extinction phase, analysis of variance (one-way repeated ANOVA) followed by Bonferroni post hoc comparisons showed that the freezing level at day11 trials of FC + FE group was significantly reduced, compared to day 5, 6, 7 trials (p b 0.01)(Fig. 3). 3.2. Effects of fear conditioning and extinction on PPI There was a significant effect of contextual fear conditioning on PPI. Post hoc comparison revealed that total %PPIs after FE in both FC (F(2, 35) = 6.638, p b 0.01) and FC + FE (F(2, 35) = 9.526, p b 0.01)
Fig. 2. Effects of contextual fear conditioning on percentages of freezing. Average percentages of freezing to the context were shown in blocks of 18 trials for no-FC, FC, FC+FE groups at fear conditioning phase. The freezing levels of FC and FC+FE groups were significantly increased when compared to that of no-FC group at 2–18 trials (⁎⁎pb 0.01). Abbreviation: FC, fear conditioning; FE, fear extinction.
groups were significantly increased when compared to those at baseline before FC. At this time, there was no significant change in total %PPIs between FC and FC + FE (Fig. 4). In addition, the magnitude of the startle reflex gradually showed a decrease. Especially, a significant difference was shown in no-FC (baseline vs. after FC; p b 0.05, baseline vs. after FE; p b 0.05) and FC groups (baseline vs. after FE; p b 0.05, after FC vs. after FE; p b 0.05). The decreasing tendency in the magnitude of the startle reflex was also shown in FC + FE group. There was no difference among all groups at each time point (Fig. 5). 4. Discussion The major findings of our study were as follows: total %PPIs were enhanced following contextual fear conditioning, and the enhanced total %PPIs were maintained even after contextual extinction training which succeeded in reducing the percent of freezing. Thus, PPI enhanced by contextual fear conditioning may not be influenced by contextual fear extinction. In no-FC group as control condition, no significant changes of total % PPI among all phases were found in the current study. The results are consistent with previous reports (Li et al., 2008), showing that PPIs were not changed at repeated measurement points. Additionally, no significant differences among all groups were found in the ASR without prepulse. There are two types of measurement of conditioned fear; freezing and startle. The fear-potentiated startle paradigm measures conditioned fear by an increase in the amplitude of a simple reflex (the acoustic startle reflex) in the presence of a cue previously paired with a shock (Davis 1986, 1992). It has also been demonstrated that the startle reflex can be increased when a context is used as the conditioned stimulus (Campeau et al., 1991). We measured contextual conditioned fear using freezing paradigm, but not fear-potentiated startle reflex. In addition, we measured ASRs including PPIs in a
Fig. 3. Effects of contextual fear extinction training on percentages of freezing. Average percentages of freezing to the context were shown in blocks of 7 trials for no-FC and FC + FE groups at fear extinction phase. The freezing level at day 11 trials of FC + FE group was significantly reduced when compared to that at day 5, 6 and 7 trials (⁎⁎pb 0.01). Abbreviation: FC, fear conditioning; FE, fear extinction.
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Fig. 4. Effects of contextual fear conditioning on prepulse inhibition (PPI). Total %PPI was showed as amount of %PPI in 69, 73, 77 and 81 dB. The total %PPIs in FC (⁎⁎p b 0.01) and FC + FE (##p b 0.01) groups at after FE were significantly increased when compared to that at baseline, respectively. No significant differences between FC and FC + FE groups were found in the total %PPI at after FE. Abbreviation: FC, fear conditioning; FE, fear extinction; total %PPI, total %prepulse inhibition.
completely different context from the conditioned context combined with footshock for contextual fear conditioning. In other words, ASRs including PPIs were measured in the non-conditioned context. Our results indicated that ASRs were not influenced by contextual fear conditioning or extinction. No enhancement of fear-potentiated startle reflex in the current study seems reasonable without conditioned cue or conditioned context. On the other hand, the previous reports in humans (Grillon and Davis, 1997a, 1997b; Greenwald et al., 1998; Vanman et al., 1996; Waters and Ornitz, 2005) and in rats (Davis 1986; Davis et al., 1993; Du et al., 2009; Li et al., 2008) suggested that enhancement of fearpotentiated startle reflex may follow the presence of conditioned cue or conditioned context. In humans, the startle stimulus was given in the same context that shock might come. In rats, CS for fearpotentiated startle reflex was a prepulse. In these previous works, the reaction to the startle stimulus was investigated in the same conditioned context or with conditioned cue, and the fear memory was recalled when ASRs were measured. Our results suggested that contextual fear conditioning may not influence ASRs if there is no conditioned fear against the auditory stimulation for ASR or if the ASRs were not measured in the context that relates to fear. Furthermore, contextual fear conditioning may enhance acoustic PPI even in the case that there is no conditioned fear against the auditory stimulation for ASR and the context. PPI is assumed to reflect sensorimotor gating. Habituation to irrelevant sensory input is an important function for the brain as the
Fig. 5. Effects of contextual fear conditioning on acoustic startle reactivity (ASR). The significant difference was shown in no-FC (baseline vs. after FC; ⁎p b 0.05, baseline vs. after FE; ⁎p b 0.05) and FC groups (baseline vs. after FE; #p b 0.05, after FC vs. after FE; #p b 0.05). The decreasing tendency in the magnitude of the startle reflex was also shown in FC + FE group. There was no difference among all groups at each time point. Abbreviation: ASR, acoustic startle reactivity; FC, fear conditioning; FE, fear extinction.
central information-processing organ of the body because the failure may be associated with mental disturbances. The term “gating” is a hypothetical psychophysiological construction in the brain and is related to a central inhibitory function which occurred at the neural level. Our results suggest that sensorimotor gating system, but not startle response system, might be physiologically associated with contextual fear conditioning. The data suggest that contextual fear conditioning might increase the sensitivity of sensorimotor gating. To our knowledge, this is the first report to show alterations of total %PPI by contextual fear conditioning in mice, even there is no conditioned fear against the auditory stimulation. It has been known that the emotional learning enhances PPI (Du et al., 2009; Grillon and Davis, 1997a, 1997b; Huang et al., 2007; Li et al., 2008; Zou et al., 2007). In the previous studies, following temporally combining the gap (CS; prepulse stimulus) with footshock in a precise manner, gapinduced PPI was enhanced. One of the possible reasons of this PPI enhancement is that conditioning the prepulse stimulus facilitates attention to the prepulse stimulus. In our study, acoustic PPI after contextual fear conditioning showed enhancement though the prepulse stimulus and CS were different. Moreover, the enhanced PPI following contextual fear conditioning was observed even after contextual fear extinction. In humans, PPI is modulated by both attentional and emotional responses to prepulse, indicating that this early-stage gating is topdown modulated by higher-order cognitive processes (Li et al., 2009). A possible explanation of the PPI enhancement after contextual conditioning in our study is that contextual fear conditioning facilitates attention to being the situations from which escape might be difficult, like agoraphobia. Agoraphobia is an anxiety disorder characterized by intense fear related to being in situations from which escape might be difficult (i.e., being on a bus or train). The sufferer with agoraphobia becomes anxious in environments where he or she perceives that they have little control. Spatial memory is the part of memory responsible for recording information about one's environment and its spatial orientation. The mice after contextual fear conditioning may have spatial memory and attention to being in situations from which escape might be difficult. Attention to the situations might lead to enhancement of sensorimotor gating including acoustic PPI. Moreover, our findings indicated that contextual fear extinction could decrease freezing behaviors, but not acoustic PPI for sensorimotor gating. The results supported that fear extinction does not erase the original fear memory but forms a new memory of safety that inhibits fear expression. Our data also suggested that the phenomena that a completely extinguished fear can be recovered spontaneously after the passage of time or “reinstated” by the presentations of the US alone in a relevant context or renewed by placing the animal in a different context might be related to the maintenance of enhanced sensorimotor gating after fear extinction. In short, context effects of extinction including spontaneous recovery, reinstatement and renewal might be due to enhanced sensorimotor gating. Many researchers reported that the hippocampus may be involved in contextual fear conditioning as the essential neural circuit (Koch, 1999; Swerdlow and Geyer, 1999; Anagnostars et al., 1999; Kim and Fanselow, 1992; Maren et al., 1997; Helmstetter and Bellgowan, 1994; Muller et al., 1997). The hippocampus is also known to be involved in contextual modulation of fear extinction including spontaneous recovery, reinstatement and renewal (Ji and Maren, 2007). While the hippocampus makes unique contributions to memory, it has also long been associated with sensorimotor processes, i.e. innate processes involving control of motor responses to sensory stimuli (Bast and Feldon, 2003). Taken together, our results suggest that the influences of contextual fear conditioning and extinction to the hippocampus might appear to reflect sensorimotor gating systems as alterations of PPI. On the other hand, the prefrontal cortex (PFC) is
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known to play an important role in fear extinction and project directly to a number of subcortical brain structures, including amygdala and hypothalamus, exerting inhibitory influences (Fuster, 1997). Stimulation of the PFC inhibits discharges of amygdaloid neurons (Quirk et al., 2003; Rosenkranz and Grace, 2001, 2002) and reduces conditioned fear responses of the animal (Milad and Quirk, 2002; Milad et al., 2004) in the process of fear extinction. Although our results suggested that the inhibitory effect of the PFC on the amygdala in fear extinction might not affect sensorimotor gating system, further researches about brain structures for association between PPI and fear conditioning will be necessary. There are many studies that investigated each mechanism of PPI or fear conditioning, respectively, but the relation between PPI and fear conditioning remains unclear. In the near future, we plan to examine the molecular connections between contextual fear conditioning and sensorimotor gating systems. 5. Conclusions In summary, the current study showed that contextual fear conditioning in mice might enhance prepulse inhibition for the auditory startle reflex, and maintain even after fear extinction. These results suggested that sensorimotor gating system may be associated with contextual fear conditioning and context effects of extinction including spontaneous recovery, reinstatement and renewal. Acknowledgments This research was supported by the Grant-in-Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology, Japan. References Anagnostars SG, Maren S, Fanselow MS. Temporally graded retrograde amnesia of contextual fear after hippocampal damage in rats: within-subjects examination. J. Neurosci. 1999;19:1106–14. Bast T, Feldon J. Hippocampal modulation of sensorimotor processes. Prog Neurobiol 2003;70(4):319–45 July. Braff DL, Geyer MA, Light GA, Sprock J, Perry W, et al. Impact of prepulse characteristics on the detection of sensorimotor gating deficits in schizophrenia. Schizophr Res 2001;49:171–8. Campeau S, Hayward MD, Hope BT, Rosen JB, Nestler EJ, Davis M. Induction of the c-fos proto-oncogene in the rat amygdala during unconditioned and conditioned fear. Brain Res 1991;565:349–52. Curzon P, Rustay NR, Browman KE. Cued and contextual fear conditioning for rodents. Methods of behavior analysis in neuroscience. In: Buccafusco Jerry J, editor. New Frontiers in Neuroscience, Second EditionTaylor & Francis Group; 2009. LLC PP.33-35. Davis M. Pharmacological and anatomical analysis of fear conditioning using the fearpotentiated startle paradigm. Behav Neurosci. 1986;100:814–24. Davis M. The role of the amygdala in fear-potentiated startle: implications for animal models of anxiety. Trends Pharmacol Sci 1992;13:35–41. Davis M, Falls WA, Campeau S, Kim M. Fear-potentiated startle: a neural and pharmacological analysis. Behav Brain Res. 1993;58:175–98. Du Y, Li J, Wu X, Li L. Precedence-effect-induced enhancement of prepulse inhibition in socially reared but not isolation-reared rats. Cogn Affect Behav Neurosci 2009;9:44–58. Fuster JM. The Prefrontal Cortex. Philadelphia: Lippincott-Raven; 1997. Geyer MA, Krebs-Thomson K, Braff DL, Swerdlow NR. Pharmacological studies of prepulse inhibition models of sensorimotor gating deficits in schizophrenia: a decade in review. Psychopharmacology (Berl). 2001;156:117–54. Geyer MA, Mcllwain KL, Paylor R. Mouse genetic models for prepulse inhibition: an early review. Mol Psychiatry 2002;7:1039–53. Greenwald MK, Bradley MM, Cuthbert BN, Lang PJ. Startle potentiation: shock sensitization, aversive learning, and affective picture modulation. Behav Neurosci 1998;112:1069–79. Grillon C, Davis M. Fear-potentiated startle conditioning in humans: explicit and contextual cue conditioning following paired versus unpaired training. Psychophysiology 1997a;34:451–8. Grillon C, Morgan CA, Southwick SM, Davis M, Charney DS. Baseline startle amplitude and prepulse inhibition in Vietnam veterans with posttraumatic stress disorder. Psychiatry Res 1996;64:169–78. Grillon C, Davis M. Effects of stress and shock anticipation on prepulse inhibition of the startle reflex. Psychophysiology 1997b;34:511–7.
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