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Effects of acute citalopram on the expression of conditioned freezing in naive versus chronic citalopram-treated rats
Progress in Neuro-Psychopharmacology & Biological Psychiatry 33 (2009) 113–117
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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 e v i e r. c o m / l o c a t e / p n p b p
Effects of acute citalopram on the expression of conditioned freezing in naive versus chronic citalopram-treated rats Shinji Hashimoto, Takeshi Inoue ⁎, Ihoko Muraki, Tsukasa Koyama Department of Psychiatry, Hokkaido University Graduate School of Medicine, North 15, West 7, Kita-ku, Sapporo 060-8638, Japan
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Article history: Received 3 June 2008 Received in revised form 26 October 2008 Accepted 27 October 2008 Available online 5 November 2008 Keywords: Citalopram Conditioned fear stress Freezing behavior Serotonin (5-hydroxytryptamine; 5-HT)
a b s t r a c t An acute challenge with selective serotonin (5-HT) reuptake inhibitors (SSRIs) reduces the conditioned freezing in rats, a model of anxiety. The increase in the 5-HT levels in the nerve terminal induced by SSRIs is closely related to its pharmacological effects. Clinically, SSRIs exert an anxiolytic effect after chronic treatment. The effects of repeated treatment with citalopram on conditioned freezing in rats were examined in the present study. Acute citalopram (10 mg/kg) reduced freezing at a short post-training interval (1 day) significantly. While the effect of citalopram (10 mg/kg) on freezing was diminished by prolonging the interval between conditioning and the exposure to conditioned fear stress, repeated citalopram (10 mg/kg) injection twice daily for 7 days restored the inhibitory effect of acute challenge of citalopram (10 mg/kg) on freezing. By prolonging the period between conditioning and exposure to conditioned fear stress, this model may have a more precise predictive validity of anxiety disorder as an animal model. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Recent clinical studies have indicated that various serotonin (5-hydroxytryptamine; 5-HT)-related agents, such as selective serotonin reuptake inhibitors (SSRIs; Eriksson and Humble, 1990; Boyer, 1995; Gorman, 1997; Zohar and Westenberg, 2000), 5-HT/ noradrenaline reuptake inhibitors (Nagata et al., 2003; Allgulander et al., 2004; Meoni et al., 2004; Tsukamoto et al., 2004), 5-HT1A receptor agonists (Kahn et al., 1988; Boyer and Feighner, 1993), 5-HT2 receptor antagonists (Kahn et al., 1988), monoamine oxidase inhibitors (Eriksson and Humble, 1990; Schneier et al., 1998) and 5-HT-precursor 5-hydroxyL-tryptophan (Kahn et al.,1988; Eriksson and Humble,1990; Blanco et al., 2002), are effective for the treatment of anxiety disorders. These findings support the hypothesis that hypofunction in the brain 5-HT neuron system is closely related to anxiety disorders. In rodents, exposure to an environment previously paired with inescapable electric foot shock, reliably elicits a response characterized by a period of crouching and complete immobility (Blanchard and Blanchard, 1969; Bolles, 1970; Bolles and Collier, 1976; Bouton and Bolles, 1980). This behavior is termed conditioned fear stress-induced freezing behavior (conditioned freezing). Conditioned fear stress is regarded as psychological stress without physical stimuli, and it has been proposed that freezing behavior could be used as an animal model of anxiety (Fanselow, 1984).
Abbreviations: ANOVA, analysis of variance; 5-HT, 5-hydroxytryptamine; SSRI, selective serotonin reuptake inhibitor; SDL, state-dependent learning. ⁎ Corresponding author. Tel.: +81 11 706 5160; fax: +81 11 706 5081. E-mail address: [email protected] (T. Inoue). 0278-5846/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2008.10.015
Conditioned freezing in rats is attenuated by an acute challenge of SSRIs, without affecting the motor activity nonspecifically (Hashimoto et al., 1996, 1997; Inoue et al., 1996b). These results are consistent with the clinical findings that SSRIs are effective for the treatment of anxiety disorders. Clinically, however, SSRIs exert an anxiolytic effect only after chronic treatment (Den Boer and Westenberg, 1988; Oehrberg et al., 1995). Partly consistent with this clinical finding, a recent study showed that chronic treatment with SSRI reduced the conditioned freezing more than acute treatment did (Li et al., 2001), thus suggesting that chronic SSRIs are more effective with a longinterval between fear conditioning and testing. In view of these findings, this study examined the effects of various intervals between fear conditioning and testing on the SSRI-induced inhibition of conditioned freezing, and effects of repeated treatment with the SSRI citalopram on conditioned freezing. Citalopram is a potent and highly selective inhibitor of 5-HT reuptake (Hyttel, 1982). In addition to its lack of effect on other monoamine reuptake mechanisms, citalopram has a very low affinity for the receptors of a variety of neurotransmitters (Hyttel, 1982).
2. Materials and methods 2.1. Animals and drug Male Sprague–Dawley rats were obtained from the Shizuoka Laboratory Animal Center (Shizuoka, Japan). They weighed between 250 and 320 g, were housed in groups of 4 and maintained in a 12 h light/dark (lights on at 7:00), temperature-controlled environment (22 ± 1 °C) with free access to food and water. The experiments began
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after a 1-week acclimatization period. The rats were tested between 8:30 and 11:30. Citalopram hydrobromide (citalopram, H. Lundbeck & Co. A/S, Denmark) was dissolved in 0.9% sterile saline and injected subcutaneously in a volume of 1 ml/kg. The dosage of citalopram (10 mg/kg) was chosen based on the previous in vivo microdialysis study (Hashimoto et al., 1999). From the inhibitory potency (ED50) of 5-HT reuptake of citalopram in human (3.4 mg/day in vivo) (Meyer et al., 2004) and rats (0.7 mg/kg s.c. in vivo) (Sánchez and Hytell, 1999), 10 mg/kg of citalopram in rats is estimated to be equivalent to 49 mg/ day in human (clinical daily doses of citalopram are 20–60 mg/day) (Trivedi et al., 2006).
10 mg/kg) and the other half were trained and tested in a different drug state (saline–citalopram 10 mg/kg or citalopram 10 mg/kg–saline). The rats were subjected to inescapable electric foot shock in a chamber with a grid floor, 60 min after citalopram (10 mg/kg) or saline administration. One day after the electric foot shock, they were again placed and observed for 10 min in the shock chamber, but no current was applied to the floor of the chamber. Citalopram (10 mg/kg) or saline was administered 60 min before the observation period. These procedures were approved by the Hokkaido University Graduate School of Medicine Animal Care and Use Committee and were in compliance with the Guide for the Care and Use of Laboratory Animals, Hokkaido University Graduate School of Medicine.
2.2. Conditioned freezing
2.4. Data analysis
For contextual fear conditioning, rats individually underwent inescapable electric foot shock for a total of 2.5 min in a shock chamber with a grid floor (30 × 24 × 30 cm, Med Associates Inc., USA) (Hashimoto et al., 1996). Five foot shocks (2.5 mA scrambled shock, each of 30 s duration) were delivered at intershock intervals of 30 s using an ENV-410 shock generator (Med Associates Inc., USA). One, 3, 7 or 11 days after the electric foot shock, the rats were again placed and observed for 10 min in the shock chamber, but no current was applied to the floor of the chamber. During the observation period, the duration of the freezing behavior was recorded as previously described (Hashimoto et al., 1996, 1997). Freezing was defined as the absence of all observable movement of the skeleton and the vibrissae, except those related to respiration. All other behavior was scored as activity. The animal was classified as either freezing or active according to its behavior throughout the entire 10-s period. The percentage score represented the number of 10-s periods during which the animal froze for the entire 10 s. The behavior was recorded on videotape and scored by 2 independent observers (one of whom was blind to the experimental grouping of the animals). The interscorer reliability by this method was very high (more than 0.95).
All the data are presented as the means ± S.E.M. of individual values of the rats from each group. Statistical analyses were performed with the two-way analysis of variance (ANOVA) followed by Duncan's test. 3. Results 3.1. Experiment I. Effect of the various intervals between conditioning and exposure to conditioned fear on acute inhibition of conditioned freezing behavior by citalopram The results of Experiment I are shown in Fig. 1B. Two-way ANOVA revealed no significant main effect of the interval or acute challenge of citalopram [Effect of interval, F(3,80) = 0.974, P = 0.4092; Effect of acute challenge of citalopram , F(1,80) = 0.516, P = 0.4746], but there was a
2.3. Experimental design Experiment I was designed to study the effects of various intervals between fear conditioning and exposure to conditioned fear. Simultaneously, the effects of acute challenge of citalopram on conditioned freezing were tested. The rats were subjected to inescapable electric foot shock in a chamber with a grid floor. One, 3, 7 or 11 days after the electric foot shock, they were again placed and observed for 10 min in the shock chamber, but no current was applied to the floor of the chamber. Citalopram (10 mg/kg) or saline was administered 60 min before the observation period (Fig. 1A). Experiment II was designed to study the effects of repeated treatment with citalopram on conditioned freezing. The rats were subjected to inescapable electric foot shock in a chamber with a grid floor and injected saline or citalopram (10 mg/kg) twice daily (9:00 and 18:00) from the next day of foot shock for 7 days. Three and a half days after the last treatment (11 days after foot shock), the rats were again placed in the shock chamber but no current was applied and the rats were observed for 10 min. An acute challenge dose of 10 mg/kg citalopram or saline was administered 60 min before the observation period (Fig. 2A). Experiment III was designed to study whether state-dependent learning (SDL) (Overton, 1974) was produced by citalopram. This SDL hypothesis postulates that acquisition of a task under a drug may require the same or similar drug state for recall. Accordingly, given that animals were trained in one state (drug-free) and tested in another state (drug), an impairment in the expression of conditioned freezing could potentially result from SDL. We examined an SDL effect in a 2 × 2 factorial design: Half of the animals were trained and tested in the same drug state (saline–saline or citalopram 10 mg/kg–citalopram
Fig. 1. Effect of acute citalopram challenge (s.c.) on the expression of conditioned fear after various intervals (1, 3, 7 and 11 days) after fear conditioning (foot shock). (A) Citalopram (10 mg/kg) or the vehicle (saline) was subcutaneously administered at 1 h before testing. The different shaped arrows indicate the timing of foot shock, conditioned fear stress (testing) and drug treatment (SC). (B) The mean percentages ± S.E.M. of freezing scored for a 10-min observation period are given. The behavior was sampled at 10-s intervals. Eight rats per group except for saline—1 day (24 rats) and saline—11 days (16 rats) groups. ⁎P b 0.05 vs saline—1 day, citalopram—3 days and citalopram—11 days groups.
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statistically marginal effect of interaction [F(3,80)= 2.653, P = 0.0542]. When the rats were exposed to conditioned fear stress 1, 3, 7 or 11 days after the electric foot shock, controls, which were treated with saline, exhibited stable freezing behavior (75–82% for 10-min observation period). When the rats were exposed to conditioned fear stress on the day after the electric foot shock, an acute challenge dose of 10-mg/kg citalopram attenuated conditioned freezing in comparison to the saline–1 day, citalopram–3 day and citalopram–11 day groups (Duncan, P b 0.05). An acute challenge dose of 10 mg/kg citalopram, however, did not attenuate the freezing behavior when the rats were exposed to conditioned fear stress 3, 7 or 11 days after the electric foot shock. 3.2. Experiment II. Effects of repeated treatment with citalopram on the conditioned freezing Fig. 2B shows the results of Experiment II. The inhibitory effect of an acute challenge dose of 10 mg/kg citalopram on conditioned freezing was restored by repeated treatment with 10 mg/kg citalopram twice daily for 7 days [two-way ANOVA; effect of acute challenge of citalopram, F(1,28) = 5.088, P b 0.05; interaction of repeated treatment with citalopram × acute challenge of citalopram, F(1,28) = 4.634, P b 0.05; effect of repeated treatment with citalopram, F(1,28) = 3.436, P = 0.074]. 3.3. Experiment III. Effects of citalopram on state-dependent learning in conditioned freezing Citalopram administration either before the training or the test attenuated conditioned freezing, consistent with the result of Fig. 1
Fig. 3. Effect of acute citalopram administration (s.c.) before fear conditioning (Preshock) and/or testing (PreCFS) on conditioned freezing. The mean percentages ± S.E.M. of freezing scored for a 10-min observation period are given. The behavior was sampled at 10-s intervals. Sixteen rats per group. ⁎⁎P b 0.01 vs saline–saline control group.
and our previous results (Inoue et al., 1996a). Furthermore, when citalopram was administered both before the training and before the test, conditioned freezing was also attenuated significantly (Fig. 3) [two-way ANOVA; effect of citalopram administration before training, F(1,60) = 10.783, P = 0.0017; effect of citalopram administration before test, F(1,60) = 5.365, P = 0.024; interaction of citalopram before training × citalopram before testing F(1,60) = 6.585, P = 0.0128]. 4. Discussion
Fig. 2. Effect of an acute citalopram challenge (s.c.) on the expression of conditioned fear after an interval of 11 days after fear conditioning (foot shock). (A) Citalopram (10 mg/kg) or the vehicle (saline) was subcutaneously administered at 1 h before testing on day 11. On day 0, foot shock was given to rats. On days 1–7, citalopram [10 mg/kg, CIT (10)] or the vehicle (saline, SAL) was subcutaneously injected to rats twice daily. The different shaped arrows indicate the timing of foot shock, conditioned fear stress (testing) and drug treatment [SAL or CIT (10)]. (B) The mean percentages ± S.E.M. of freezing scored for a 10-min observation period are given. The behavior was sampled at 10-s intervals. Eight rats per group. ⁎⁎P b 0.01 vs repeated saline–acute saline, repeated citalopram–acute saline and repeated saline–acute citalopram groups.
Clinically, SSRIs exert an anxiolytic effect after chronic treatment. Accordingly, the effects of repeated treatment with citalopram after fear conditioning were examined on conditioned freezing in the present study. The following results were obtained: while the inhibitory effect of an acute challenge of citalopram on conditioned freezing diminished by prolonging the interval between conditioning by foot shock and exposure to conditioned fear stress, the inhibitory effect of acute citalopram on freezing was restored by repeating the treatment with citalopram. In summary, when the interval between conditioning and testing is 1 day, acute citalopram is anxiolytic, but when the interval is 11 days, only repeated citalopram is anxiolytic. Therefore, a long interval between conditioning and testing may give the predictive validity as an animal model of human anxiety disorder to conditioned fear stress. Experiment I confirmed that the effect of conditioned fear lasts for many days (at least up to 11 days following foot shock), which is consistent with previous reports showing contextual conditioned fear stably appeared up to 100 days after conditioning (Anagnostaras et al., 2001). In this test, the intervals between conditioning and exposure to conditioned fear stress (testing) did not affect the duration, i.e. degree, of freezing behavior. The similar intensities of fear were observed throughout 1–11 days intervals between conditioning and testing. Therefore, intervals between conditioning and testing did not increase or decrease freezing. Nevertheless, the inhibitory effect of acute challenge of citalopram was weakened by prolonging the intervals between conditioning and testing from 1 day to 3–11 days. This diminishment of the acute SSRI effect on freezing has been suggested and hypothesized in previous reports showing that freezing was resistant to acute SSRI treatment in the setting of 2 weeks intervals between conditioning and testing; and either extremely high doses of SSRI were thus needed to significantly inhibit conditioned freezing (Li et al., 2001) or they still proved to be ineffective (Nishikawa et al., 2007). The present data directly confirmed the hypothesis of an interval–response relationship in acute anxiolytic effects of citalopram on conditioned fear.
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The reason why the acute anxiolytic effect of citalopram was diminished during long intervals after foot shock is not clear, but the memory consolidation process of fear acquisition may be related to this phenomenon. Several lines of evidence have suggested that recent memory after conditioning is less stable than remote memory: First, the lesions of the hippocampus in a short, but not long, interval (28 days or longer) after contextual fear conditioning disrupted the fear expression in subsequent tests (Anagnostaras et al., 2001), and, in a recent study, the reversible inactivation of the basolateral amygdala (0.25–48 h) and dorsal hippocampus (0.25–1.5 h) in a shorter interval after fear conditioning showed similar disruptive effects (Sacchetti et al., 1999). Second, after contextual fear conditioning, the inhibition of protein synthesis before reexposure to the shock chamber disrupted the subsequent expression of recent memory (1 and 3 weeks after conditioning) but not remote memory (8 weeks after conditioning), although the time course is different from the current results (Suzuki et al., 2004). Therefore, fear memory is unstable for a short period after fear conditioning, but this instability is time-limited and fear memory becomes more stable in long intervals after fear conditioning. The failure of acute SSRI to reduce freezing during long intervals after fear conditioning, as shown in this study, may therefore be explained by the stability or the degree of consolidation of fear memory, although the effect of SSRI on fear memory consolidation has not yet been reported and thus requires future research. In Experiment II, an acute challenge of citalopram significantly reduced freezing only in rats that had received citalopram pretreatment twice daily for 7 days. Clinically, SSRIs have an anxiolytic action after chronic treatment (Den Boer and Westenberg, 1988; Oehrberg et al., 1995). Therefore, by prolonging the period between conditioning and exposure to conditioned fear stress, the feasibility of establishing an animal model that has a more precise predictive validity of anxiety disorder was suggested. Since the half-life of citalopram is 3 h in rats (Hyttel et al., 1984) and the wash-out period in this experiment was 3.5 days, the possibility that the citalopram concentrations after acute citalopram challenge in the rat brain increased in rats with repeated treatment due to accumulation can thus be ruled out. Nevertheless, in future experiments, it is important to examine whether blood concentrations of citalopram after repeated treatment are relevant to its effect on conditioned freezing. Moreover, it may be important when repeated citalopram treatment begins after the footshock, if the effect of repeated citalopram on conditioned freezing is relevant to its effect on fear memory consolidation. Further experiments, in which repeated citalopram begins after fear memory consolidation, will be necessary. The excess extracellular 5-HT produced by 5-HT reuptake inhibitors in the cell body region (raphe nuclei) activates presynaptic 5-HT1A autoreceptors and slows down the firing rate of 5-HT neurons and their terminal release (Artigas, 1993). Previous in vivo microdialysis studies have shown that the effect of a low dose (1 mg/kg) of citalopram on extracellular 5-HT concentrations in the frontal cortex of rats was negligible, but it was markedly enhanced after repeated citalopram (10 mg/kg) injection for 14 days (Invernizzi et al., 1994). However, the effect of a higher dose of citalopram (5 mg/kg) on extracellular 5-HT concentrations was not enhanced after repeated citalopram (10 mg/kg) treatment (Gundlah et al., 1997; Hjorth and Auerbach, 1999). Taken together with these previous in vivo microdialysis studies, it seems unlikely that 10 mg/kg of citalopram increased the extracellular 5-HT levels in the brain of rats with repeated citalopram more than in the brain of rats with repeated saline, but to draw a conclusion from in vivo microdialysis studies, it will therefore be necessary to confirm the effect of repeated citalopram on extracellular 5-HT in rats exposed to conditioned fear. One might account for the effects of citalopram on the expression of conditioned freezing by a state-dependent learning (SDL) hypothesis (Overton, 1974). This SDL hypothesis postulates that acquisition of
a task under a drug may require the same or similar drug state for recall. However, the effects of citalopram before testing on conditioned freezing cannot be explained by SDL because citalopram administration both before conditioning and before testing also impaired conditioned freezing. If the impairments observed in this experiment were due to SDL, only animals that were trained and tested in different drug states would have been impaired. In conclusion, the acute anxiolytic effect of citalopram in conditioned fear was observed to diminish when the interval between fear conditioning and exposure to conditioned fear was prolonged up to 3– 11 days. This diminishment might be due to the change in the status of fear memory. Repeated citalopram pretreatment restored the anxiolytic effect of acute challenge of citalopram in conditioned fear. At present, this effect of repeated treatment cannot be explained by either increased extracellular 5-HT concentrations or drug accumulation. This model with a long interval between fear conditioning and exposure to conditioned fear may have a predictive validity of an animal model of human anxiety disorders. The clarification of the mechanism of anxiolytic action of repeated citalopram in this animal model may therefore lead to a new understanding of the pathophysiology of anxiety disorders and the mechanism of action of SSRIs. Acknowledgements I gratefully acknowledge helpful discussions with Daisuke Mochizuki and Tadashi Nagatani on several points in this paper. We would like to thank Asahi-kasei Pharma Co., Ltd. (Japan) for their support. References Allgulander C, Mangano R, Zhang J, Dahl AA, Lepola U, Sjodin I, et al. Efficacy of Venlafaxine ER in patients with social anxiety disorder: a double-blind, placebocontrolled, parallel-group comparison with paroxetine. Hum Psychopharmacol 2004;19:387–96. Artigas F. 5-HT and antidepressants: new views from microdialysis studies. Trends Pharmacol. Sci. 1993;14:262. Anagnostaras SG, Gale GD, Fanselow MS. Hippocampus and contextual fear conditioning: recent controversies and advances. Hippocampus 2001;11:8-17. Blanchard RJ, Blanchard DC. Crouching as an index of fear. J Comp Physiol Psychol 1969;67:370–5. Blanco C, Antia SX, Liebowitz MR. Pharmacotherapy of social anxiety disorder. Biol Psychiatry 2002;51:109–20. Bolles RC. Species-specific defense reactions and avoidance learning. Psychol Rev 1970;77:32–48. Bolles RC, Collier AC. The effect of predictive cues on freezing in rats. Anim Learn Behav 1976;4:6–8. Bouton ME, Bolles RC. Conditioned fear assessed by freezing and by the suppression of three different baselines. Anim Learn Behav 1980;8:429–34. Boyer WF. Serotonin uptake inhibitors are superior to imipramine and alprazolam in alleviating panic attacks: a meta analysis. Int Clin Psychopharmacol 1995;10:45–9. Boyer WF, Feighner JP. A placebo-controlled double-blind multicenter trial of two doses of ipsapirone versus diazepam in generalized anxiety disorder. Int Clin Psychopharmacol 1993;8:173–6. Den Boer JA, Westenberg HGM. Effect of serotonin and noradrenaline uptake inhibitor in panic disorder: a double-blind comparative study with fluvoxamine and maprotiline. Int Clin Psychopharmacol 1988;3:59–74. Eriksson E, Humble M. Serotonin in psychiatric pathophysiology: a review of data from experimental and clinical research. In: Pohl R, Gershon S, editors. Progress in Basic Clinical Pharmacology. The Biological Basis of Psychiatric TreatmentBasel: Karger; 1990. p. 66-119. Fanselow MS. What is conditioned fear? Trends Neurosci 1984;7:460–2. Gorman JM. The use of newer antidepressants for panic disorder. J Clin Psychiatry 1997;58(Suppl. 14):54–8. Gundlah C, Hjorth S, Auerbach SB. Autoreceptor antagonists enhance the effect of the reuptake inhibitor citalopram on extracellular 5-HT: this effect persists after repeated citalopram treatment. Neuropharmacology 1997;36:475–82. Hashimoto S, Inoue T, Koyama T. Serotonin reuptake inhibitors reduce conditioned fear stress-induced freezing behavior in rats. Psychopharmacology 1996;123:182–6. Hashimoto S, Inoue T, Koyama T. Effects of the co-administration of 5-HT1A receptor antagonists with an SSRI in conditioned fear stress-induced freezing behavior. Pharmacol Biochem Behav 1997;58:471–5. Hashimoto S, Inoue T, Koyama T. Effects of conditioned fear stress on serotonin neurotransmission and freezing behavior in rats. Eur J Pharmacol 1999;378:23–30. Hjorth S, Auerbach SB. Autoreceptors remain functional after prolonged treatment with a serotonin reuptake inhibitor. Brain Res 1999;835:224–8. Hyttel J. Citalopram: Pharmacological profile of a specific serotonin uptake inhibitor with antidepressant activity. Prog Neuropsychopharmacol Biol Psychiatry 1982;6:277–95.
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Effects of acute citalopram on the expression of conditioned freezing in naive versus chronic citalopram-treated rats Shinji Hashimoto, Takeshi Inoue ⁎, Ihoko Muraki, Tsukasa Koyama Department of Psychiatry, Hokkaido University Graduate School of Medicine, North 15, West 7, Kita-ku, Sapporo 060-8638, Japan
a r t i c l e
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Article history: Received 3 June 2008 Received in revised form 26 October 2008 Accepted 27 October 2008 Available online 5 November 2008 Keywords: Citalopram Conditioned fear stress Freezing behavior Serotonin (5-hydroxytryptamine; 5-HT)
a b s t r a c t An acute challenge with selective serotonin (5-HT) reuptake inhibitors (SSRIs) reduces the conditioned freezing in rats, a model of anxiety. The increase in the 5-HT levels in the nerve terminal induced by SSRIs is closely related to its pharmacological effects. Clinically, SSRIs exert an anxiolytic effect after chronic treatment. The effects of repeated treatment with citalopram on conditioned freezing in rats were examined in the present study. Acute citalopram (10 mg/kg) reduced freezing at a short post-training interval (1 day) significantly. While the effect of citalopram (10 mg/kg) on freezing was diminished by prolonging the interval between conditioning and the exposure to conditioned fear stress, repeated citalopram (10 mg/kg) injection twice daily for 7 days restored the inhibitory effect of acute challenge of citalopram (10 mg/kg) on freezing. By prolonging the period between conditioning and exposure to conditioned fear stress, this model may have a more precise predictive validity of anxiety disorder as an animal model. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Recent clinical studies have indicated that various serotonin (5-hydroxytryptamine; 5-HT)-related agents, such as selective serotonin reuptake inhibitors (SSRIs; Eriksson and Humble, 1990; Boyer, 1995; Gorman, 1997; Zohar and Westenberg, 2000), 5-HT/ noradrenaline reuptake inhibitors (Nagata et al., 2003; Allgulander et al., 2004; Meoni et al., 2004; Tsukamoto et al., 2004), 5-HT1A receptor agonists (Kahn et al., 1988; Boyer and Feighner, 1993), 5-HT2 receptor antagonists (Kahn et al., 1988), monoamine oxidase inhibitors (Eriksson and Humble, 1990; Schneier et al., 1998) and 5-HT-precursor 5-hydroxyL-tryptophan (Kahn et al.,1988; Eriksson and Humble,1990; Blanco et al., 2002), are effective for the treatment of anxiety disorders. These findings support the hypothesis that hypofunction in the brain 5-HT neuron system is closely related to anxiety disorders. In rodents, exposure to an environment previously paired with inescapable electric foot shock, reliably elicits a response characterized by a period of crouching and complete immobility (Blanchard and Blanchard, 1969; Bolles, 1970; Bolles and Collier, 1976; Bouton and Bolles, 1980). This behavior is termed conditioned fear stress-induced freezing behavior (conditioned freezing). Conditioned fear stress is regarded as psychological stress without physical stimuli, and it has been proposed that freezing behavior could be used as an animal model of anxiety (Fanselow, 1984).
Abbreviations: ANOVA, analysis of variance; 5-HT, 5-hydroxytryptamine; SSRI, selective serotonin reuptake inhibitor; SDL, state-dependent learning. ⁎ Corresponding author. Tel.: +81 11 706 5160; fax: +81 11 706 5081. E-mail address: [email protected] (T. Inoue). 0278-5846/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2008.10.015
Conditioned freezing in rats is attenuated by an acute challenge of SSRIs, without affecting the motor activity nonspecifically (Hashimoto et al., 1996, 1997; Inoue et al., 1996b). These results are consistent with the clinical findings that SSRIs are effective for the treatment of anxiety disorders. Clinically, however, SSRIs exert an anxiolytic effect only after chronic treatment (Den Boer and Westenberg, 1988; Oehrberg et al., 1995). Partly consistent with this clinical finding, a recent study showed that chronic treatment with SSRI reduced the conditioned freezing more than acute treatment did (Li et al., 2001), thus suggesting that chronic SSRIs are more effective with a longinterval between fear conditioning and testing. In view of these findings, this study examined the effects of various intervals between fear conditioning and testing on the SSRI-induced inhibition of conditioned freezing, and effects of repeated treatment with the SSRI citalopram on conditioned freezing. Citalopram is a potent and highly selective inhibitor of 5-HT reuptake (Hyttel, 1982). In addition to its lack of effect on other monoamine reuptake mechanisms, citalopram has a very low affinity for the receptors of a variety of neurotransmitters (Hyttel, 1982).
2. Materials and methods 2.1. Animals and drug Male Sprague–Dawley rats were obtained from the Shizuoka Laboratory Animal Center (Shizuoka, Japan). They weighed between 250 and 320 g, were housed in groups of 4 and maintained in a 12 h light/dark (lights on at 7:00), temperature-controlled environment (22 ± 1 °C) with free access to food and water. The experiments began
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after a 1-week acclimatization period. The rats were tested between 8:30 and 11:30. Citalopram hydrobromide (citalopram, H. Lundbeck & Co. A/S, Denmark) was dissolved in 0.9% sterile saline and injected subcutaneously in a volume of 1 ml/kg. The dosage of citalopram (10 mg/kg) was chosen based on the previous in vivo microdialysis study (Hashimoto et al., 1999). From the inhibitory potency (ED50) of 5-HT reuptake of citalopram in human (3.4 mg/day in vivo) (Meyer et al., 2004) and rats (0.7 mg/kg s.c. in vivo) (Sánchez and Hytell, 1999), 10 mg/kg of citalopram in rats is estimated to be equivalent to 49 mg/ day in human (clinical daily doses of citalopram are 20–60 mg/day) (Trivedi et al., 2006).
10 mg/kg) and the other half were trained and tested in a different drug state (saline–citalopram 10 mg/kg or citalopram 10 mg/kg–saline). The rats were subjected to inescapable electric foot shock in a chamber with a grid floor, 60 min after citalopram (10 mg/kg) or saline administration. One day after the electric foot shock, they were again placed and observed for 10 min in the shock chamber, but no current was applied to the floor of the chamber. Citalopram (10 mg/kg) or saline was administered 60 min before the observation period. These procedures were approved by the Hokkaido University Graduate School of Medicine Animal Care and Use Committee and were in compliance with the Guide for the Care and Use of Laboratory Animals, Hokkaido University Graduate School of Medicine.
2.2. Conditioned freezing
2.4. Data analysis
For contextual fear conditioning, rats individually underwent inescapable electric foot shock for a total of 2.5 min in a shock chamber with a grid floor (30 × 24 × 30 cm, Med Associates Inc., USA) (Hashimoto et al., 1996). Five foot shocks (2.5 mA scrambled shock, each of 30 s duration) were delivered at intershock intervals of 30 s using an ENV-410 shock generator (Med Associates Inc., USA). One, 3, 7 or 11 days after the electric foot shock, the rats were again placed and observed for 10 min in the shock chamber, but no current was applied to the floor of the chamber. During the observation period, the duration of the freezing behavior was recorded as previously described (Hashimoto et al., 1996, 1997). Freezing was defined as the absence of all observable movement of the skeleton and the vibrissae, except those related to respiration. All other behavior was scored as activity. The animal was classified as either freezing or active according to its behavior throughout the entire 10-s period. The percentage score represented the number of 10-s periods during which the animal froze for the entire 10 s. The behavior was recorded on videotape and scored by 2 independent observers (one of whom was blind to the experimental grouping of the animals). The interscorer reliability by this method was very high (more than 0.95).
All the data are presented as the means ± S.E.M. of individual values of the rats from each group. Statistical analyses were performed with the two-way analysis of variance (ANOVA) followed by Duncan's test. 3. Results 3.1. Experiment I. Effect of the various intervals between conditioning and exposure to conditioned fear on acute inhibition of conditioned freezing behavior by citalopram The results of Experiment I are shown in Fig. 1B. Two-way ANOVA revealed no significant main effect of the interval or acute challenge of citalopram [Effect of interval, F(3,80) = 0.974, P = 0.4092; Effect of acute challenge of citalopram , F(1,80) = 0.516, P = 0.4746], but there was a
2.3. Experimental design Experiment I was designed to study the effects of various intervals between fear conditioning and exposure to conditioned fear. Simultaneously, the effects of acute challenge of citalopram on conditioned freezing were tested. The rats were subjected to inescapable electric foot shock in a chamber with a grid floor. One, 3, 7 or 11 days after the electric foot shock, they were again placed and observed for 10 min in the shock chamber, but no current was applied to the floor of the chamber. Citalopram (10 mg/kg) or saline was administered 60 min before the observation period (Fig. 1A). Experiment II was designed to study the effects of repeated treatment with citalopram on conditioned freezing. The rats were subjected to inescapable electric foot shock in a chamber with a grid floor and injected saline or citalopram (10 mg/kg) twice daily (9:00 and 18:00) from the next day of foot shock for 7 days. Three and a half days after the last treatment (11 days after foot shock), the rats were again placed in the shock chamber but no current was applied and the rats were observed for 10 min. An acute challenge dose of 10 mg/kg citalopram or saline was administered 60 min before the observation period (Fig. 2A). Experiment III was designed to study whether state-dependent learning (SDL) (Overton, 1974) was produced by citalopram. This SDL hypothesis postulates that acquisition of a task under a drug may require the same or similar drug state for recall. Accordingly, given that animals were trained in one state (drug-free) and tested in another state (drug), an impairment in the expression of conditioned freezing could potentially result from SDL. We examined an SDL effect in a 2 × 2 factorial design: Half of the animals were trained and tested in the same drug state (saline–saline or citalopram 10 mg/kg–citalopram
Fig. 1. Effect of acute citalopram challenge (s.c.) on the expression of conditioned fear after various intervals (1, 3, 7 and 11 days) after fear conditioning (foot shock). (A) Citalopram (10 mg/kg) or the vehicle (saline) was subcutaneously administered at 1 h before testing. The different shaped arrows indicate the timing of foot shock, conditioned fear stress (testing) and drug treatment (SC). (B) The mean percentages ± S.E.M. of freezing scored for a 10-min observation period are given. The behavior was sampled at 10-s intervals. Eight rats per group except for saline—1 day (24 rats) and saline—11 days (16 rats) groups. ⁎P b 0.05 vs saline—1 day, citalopram—3 days and citalopram—11 days groups.
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statistically marginal effect of interaction [F(3,80)= 2.653, P = 0.0542]. When the rats were exposed to conditioned fear stress 1, 3, 7 or 11 days after the electric foot shock, controls, which were treated with saline, exhibited stable freezing behavior (75–82% for 10-min observation period). When the rats were exposed to conditioned fear stress on the day after the electric foot shock, an acute challenge dose of 10-mg/kg citalopram attenuated conditioned freezing in comparison to the saline–1 day, citalopram–3 day and citalopram–11 day groups (Duncan, P b 0.05). An acute challenge dose of 10 mg/kg citalopram, however, did not attenuate the freezing behavior when the rats were exposed to conditioned fear stress 3, 7 or 11 days after the electric foot shock. 3.2. Experiment II. Effects of repeated treatment with citalopram on the conditioned freezing Fig. 2B shows the results of Experiment II. The inhibitory effect of an acute challenge dose of 10 mg/kg citalopram on conditioned freezing was restored by repeated treatment with 10 mg/kg citalopram twice daily for 7 days [two-way ANOVA; effect of acute challenge of citalopram, F(1,28) = 5.088, P b 0.05; interaction of repeated treatment with citalopram × acute challenge of citalopram, F(1,28) = 4.634, P b 0.05; effect of repeated treatment with citalopram, F(1,28) = 3.436, P = 0.074]. 3.3. Experiment III. Effects of citalopram on state-dependent learning in conditioned freezing Citalopram administration either before the training or the test attenuated conditioned freezing, consistent with the result of Fig. 1
Fig. 3. Effect of acute citalopram administration (s.c.) before fear conditioning (Preshock) and/or testing (PreCFS) on conditioned freezing. The mean percentages ± S.E.M. of freezing scored for a 10-min observation period are given. The behavior was sampled at 10-s intervals. Sixteen rats per group. ⁎⁎P b 0.01 vs saline–saline control group.
and our previous results (Inoue et al., 1996a). Furthermore, when citalopram was administered both before the training and before the test, conditioned freezing was also attenuated significantly (Fig. 3) [two-way ANOVA; effect of citalopram administration before training, F(1,60) = 10.783, P = 0.0017; effect of citalopram administration before test, F(1,60) = 5.365, P = 0.024; interaction of citalopram before training × citalopram before testing F(1,60) = 6.585, P = 0.0128]. 4. Discussion
Fig. 2. Effect of an acute citalopram challenge (s.c.) on the expression of conditioned fear after an interval of 11 days after fear conditioning (foot shock). (A) Citalopram (10 mg/kg) or the vehicle (saline) was subcutaneously administered at 1 h before testing on day 11. On day 0, foot shock was given to rats. On days 1–7, citalopram [10 mg/kg, CIT (10)] or the vehicle (saline, SAL) was subcutaneously injected to rats twice daily. The different shaped arrows indicate the timing of foot shock, conditioned fear stress (testing) and drug treatment [SAL or CIT (10)]. (B) The mean percentages ± S.E.M. of freezing scored for a 10-min observation period are given. The behavior was sampled at 10-s intervals. Eight rats per group. ⁎⁎P b 0.01 vs repeated saline–acute saline, repeated citalopram–acute saline and repeated saline–acute citalopram groups.
Clinically, SSRIs exert an anxiolytic effect after chronic treatment. Accordingly, the effects of repeated treatment with citalopram after fear conditioning were examined on conditioned freezing in the present study. The following results were obtained: while the inhibitory effect of an acute challenge of citalopram on conditioned freezing diminished by prolonging the interval between conditioning by foot shock and exposure to conditioned fear stress, the inhibitory effect of acute citalopram on freezing was restored by repeating the treatment with citalopram. In summary, when the interval between conditioning and testing is 1 day, acute citalopram is anxiolytic, but when the interval is 11 days, only repeated citalopram is anxiolytic. Therefore, a long interval between conditioning and testing may give the predictive validity as an animal model of human anxiety disorder to conditioned fear stress. Experiment I confirmed that the effect of conditioned fear lasts for many days (at least up to 11 days following foot shock), which is consistent with previous reports showing contextual conditioned fear stably appeared up to 100 days after conditioning (Anagnostaras et al., 2001). In this test, the intervals between conditioning and exposure to conditioned fear stress (testing) did not affect the duration, i.e. degree, of freezing behavior. The similar intensities of fear were observed throughout 1–11 days intervals between conditioning and testing. Therefore, intervals between conditioning and testing did not increase or decrease freezing. Nevertheless, the inhibitory effect of acute challenge of citalopram was weakened by prolonging the intervals between conditioning and testing from 1 day to 3–11 days. This diminishment of the acute SSRI effect on freezing has been suggested and hypothesized in previous reports showing that freezing was resistant to acute SSRI treatment in the setting of 2 weeks intervals between conditioning and testing; and either extremely high doses of SSRI were thus needed to significantly inhibit conditioned freezing (Li et al., 2001) or they still proved to be ineffective (Nishikawa et al., 2007). The present data directly confirmed the hypothesis of an interval–response relationship in acute anxiolytic effects of citalopram on conditioned fear.
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The reason why the acute anxiolytic effect of citalopram was diminished during long intervals after foot shock is not clear, but the memory consolidation process of fear acquisition may be related to this phenomenon. Several lines of evidence have suggested that recent memory after conditioning is less stable than remote memory: First, the lesions of the hippocampus in a short, but not long, interval (28 days or longer) after contextual fear conditioning disrupted the fear expression in subsequent tests (Anagnostaras et al., 2001), and, in a recent study, the reversible inactivation of the basolateral amygdala (0.25–48 h) and dorsal hippocampus (0.25–1.5 h) in a shorter interval after fear conditioning showed similar disruptive effects (Sacchetti et al., 1999). Second, after contextual fear conditioning, the inhibition of protein synthesis before reexposure to the shock chamber disrupted the subsequent expression of recent memory (1 and 3 weeks after conditioning) but not remote memory (8 weeks after conditioning), although the time course is different from the current results (Suzuki et al., 2004). Therefore, fear memory is unstable for a short period after fear conditioning, but this instability is time-limited and fear memory becomes more stable in long intervals after fear conditioning. The failure of acute SSRI to reduce freezing during long intervals after fear conditioning, as shown in this study, may therefore be explained by the stability or the degree of consolidation of fear memory, although the effect of SSRI on fear memory consolidation has not yet been reported and thus requires future research. In Experiment II, an acute challenge of citalopram significantly reduced freezing only in rats that had received citalopram pretreatment twice daily for 7 days. Clinically, SSRIs have an anxiolytic action after chronic treatment (Den Boer and Westenberg, 1988; Oehrberg et al., 1995). Therefore, by prolonging the period between conditioning and exposure to conditioned fear stress, the feasibility of establishing an animal model that has a more precise predictive validity of anxiety disorder was suggested. Since the half-life of citalopram is 3 h in rats (Hyttel et al., 1984) and the wash-out period in this experiment was 3.5 days, the possibility that the citalopram concentrations after acute citalopram challenge in the rat brain increased in rats with repeated treatment due to accumulation can thus be ruled out. Nevertheless, in future experiments, it is important to examine whether blood concentrations of citalopram after repeated treatment are relevant to its effect on conditioned freezing. Moreover, it may be important when repeated citalopram treatment begins after the footshock, if the effect of repeated citalopram on conditioned freezing is relevant to its effect on fear memory consolidation. Further experiments, in which repeated citalopram begins after fear memory consolidation, will be necessary. The excess extracellular 5-HT produced by 5-HT reuptake inhibitors in the cell body region (raphe nuclei) activates presynaptic 5-HT1A autoreceptors and slows down the firing rate of 5-HT neurons and their terminal release (Artigas, 1993). Previous in vivo microdialysis studies have shown that the effect of a low dose (1 mg/kg) of citalopram on extracellular 5-HT concentrations in the frontal cortex of rats was negligible, but it was markedly enhanced after repeated citalopram (10 mg/kg) injection for 14 days (Invernizzi et al., 1994). However, the effect of a higher dose of citalopram (5 mg/kg) on extracellular 5-HT concentrations was not enhanced after repeated citalopram (10 mg/kg) treatment (Gundlah et al., 1997; Hjorth and Auerbach, 1999). Taken together with these previous in vivo microdialysis studies, it seems unlikely that 10 mg/kg of citalopram increased the extracellular 5-HT levels in the brain of rats with repeated citalopram more than in the brain of rats with repeated saline, but to draw a conclusion from in vivo microdialysis studies, it will therefore be necessary to confirm the effect of repeated citalopram on extracellular 5-HT in rats exposed to conditioned fear. One might account for the effects of citalopram on the expression of conditioned freezing by a state-dependent learning (SDL) hypothesis (Overton, 1974). This SDL hypothesis postulates that acquisition of
a task under a drug may require the same or similar drug state for recall. However, the effects of citalopram before testing on conditioned freezing cannot be explained by SDL because citalopram administration both before conditioning and before testing also impaired conditioned freezing. If the impairments observed in this experiment were due to SDL, only animals that were trained and tested in different drug states would have been impaired. In conclusion, the acute anxiolytic effect of citalopram in conditioned fear was observed to diminish when the interval between fear conditioning and exposure to conditioned fear was prolonged up to 3– 11 days. This diminishment might be due to the change in the status of fear memory. Repeated citalopram pretreatment restored the anxiolytic effect of acute challenge of citalopram in conditioned fear. At present, this effect of repeated treatment cannot be explained by either increased extracellular 5-HT concentrations or drug accumulation. This model with a long interval between fear conditioning and exposure to conditioned fear may have a predictive validity of an animal model of human anxiety disorders. The clarification of the mechanism of anxiolytic action of repeated citalopram in this animal model may therefore lead to a new understanding of the pathophysiology of anxiety disorders and the mechanism of action of SSRIs. Acknowledgements I gratefully acknowledge helpful discussions with Daisuke Mochizuki and Tadashi Nagatani on several points in this paper. We would like to thank Asahi-kasei Pharma Co., Ltd. (Japan) for their support. References Allgulander C, Mangano R, Zhang J, Dahl AA, Lepola U, Sjodin I, et al. Efficacy of Venlafaxine ER in patients with social anxiety disorder: a double-blind, placebocontrolled, parallel-group comparison with paroxetine. Hum Psychopharmacol 2004;19:387–96. Artigas F. 5-HT and antidepressants: new views from microdialysis studies. Trends Pharmacol. Sci. 1993;14:262. Anagnostaras SG, Gale GD, Fanselow MS. Hippocampus and contextual fear conditioning: recent controversies and advances. Hippocampus 2001;11:8-17. Blanchard RJ, Blanchard DC. Crouching as an index of fear. J Comp Physiol Psychol 1969;67:370–5. Blanco C, Antia SX, Liebowitz MR. Pharmacotherapy of social anxiety disorder. Biol Psychiatry 2002;51:109–20. Bolles RC. Species-specific defense reactions and avoidance learning. Psychol Rev 1970;77:32–48. Bolles RC, Collier AC. The effect of predictive cues on freezing in rats. Anim Learn Behav 1976;4:6–8. Bouton ME, Bolles RC. Conditioned fear assessed by freezing and by the suppression of three different baselines. Anim Learn Behav 1980;8:429–34. Boyer WF. Serotonin uptake inhibitors are superior to imipramine and alprazolam in alleviating panic attacks: a meta analysis. Int Clin Psychopharmacol 1995;10:45–9. Boyer WF, Feighner JP. A placebo-controlled double-blind multicenter trial of two doses of ipsapirone versus diazepam in generalized anxiety disorder. Int Clin Psychopharmacol 1993;8:173–6. Den Boer JA, Westenberg HGM. Effect of serotonin and noradrenaline uptake inhibitor in panic disorder: a double-blind comparative study with fluvoxamine and maprotiline. Int Clin Psychopharmacol 1988;3:59–74. Eriksson E, Humble M. Serotonin in psychiatric pathophysiology: a review of data from experimental and clinical research. In: Pohl R, Gershon S, editors. Progress in Basic Clinical Pharmacology. The Biological Basis of Psychiatric TreatmentBasel: Karger; 1990. p. 66-119. Fanselow MS. What is conditioned fear? Trends Neurosci 1984;7:460–2. Gorman JM. The use of newer antidepressants for panic disorder. J Clin Psychiatry 1997;58(Suppl. 14):54–8. Gundlah C, Hjorth S, Auerbach SB. Autoreceptor antagonists enhance the effect of the reuptake inhibitor citalopram on extracellular 5-HT: this effect persists after repeated citalopram treatment. Neuropharmacology 1997;36:475–82. Hashimoto S, Inoue T, Koyama T. Serotonin reuptake inhibitors reduce conditioned fear stress-induced freezing behavior in rats. Psychopharmacology 1996;123:182–6. Hashimoto S, Inoue T, Koyama T. Effects of the co-administration of 5-HT1A receptor antagonists with an SSRI in conditioned fear stress-induced freezing behavior. Pharmacol Biochem Behav 1997;58:471–5. Hashimoto S, Inoue T, Koyama T. Effects of conditioned fear stress on serotonin neurotransmission and freezing behavior in rats. Eur J Pharmacol 1999;378:23–30. Hjorth S, Auerbach SB. Autoreceptors remain functional after prolonged treatment with a serotonin reuptake inhibitor. Brain Res 1999;835:224–8. Hyttel J. Citalopram: Pharmacological profile of a specific serotonin uptake inhibitor with antidepressant activity. Prog Neuropsychopharmacol Biol Psychiatry 1982;6:277–95.
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