Neuropsychopharmacology (2009) 34, 2574–2584 & 2009 Nature Publishing Group All rights reserved 0893-133X/09 $32.00 www.neuropsychopharmacology.org Pharmacological Discrimination of Extinction and Reconsolidation of Contextual Fear Memory by a Potentiator of AMPA Receptors Daisuke Yamada1,2, Ko Zushida1,2, Keiji Wada1,2 and Masayuki Sekiguchi*,1,2 1 Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, 2 Tokyo, Japan; CREST, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan Conditioned fear memory, once formed through fear conditioning, is modulated by reexposure of individuals to a conditioned stimulus. The reexposure reactivates the fear memory, which induces reconsolidation of the memory first, and then extinction of the fear response. Both attenuating the former and facilitating the latter are effective in reducing the fear response, and these findings are potentially translatable to the enhancement of exposure therapy for complex anxiety disorders. Currently, there is no drug that is established to modulate either reconsolidation or extinction selectively, which are thought to be independent processes. Here, we report that an extinction-facilitating AMPA potentiator, 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide (PEPA), does not act on the reconsolidation of fear memory formed by contextual fear conditioning in mice. The freezing rates observed in contextually conditioned mice following short reexposure (3 min) to the context were not influenced by intraperitoneal or intra-amygdala administration of PEPA. The same short reexposure to the context enhanced freezing responses in mice that were similarly administered D-cycloserine (DCS), a drug that facilitates both extinction and reconsolidation, and this enhancement of freezing responses in mice intraperitoneally administered DCS was abolished by propranolol, a drug that suppresses reconsolidation. At the same doses used in the short reexposure experiments, PEPA and DCS facilitated extinction of the fear response induced by long reexposure to the context and suppressed reinstatement of the conditioned fear memory. PEPA and DCS did not affect reextinction. These results suggest that PEPA acts on extinction of contextual fear memory without having detectable influences on its reconsolidation. Neuropsychopharmacology (2009) 34, 2574–2584; doi:10.1038/npp.2009.86; published online 12 August 2009 Keywords: fear memory; reconsolidation; extinction; PEPA; D-cycloserine; exposure therapy INTRODUCTION nucleus of the amygdala (BLA) is mainly involved in reconsolidation (Nader et al, 2000; Duvarci and Nader, Conditioned fear memory, once formed through Pavlovian 2004; Baldi et al, 2008), and that the amygdala, hippocam- fear conditioning, is modulated by reexposure of indivi- pus, and medial prefrontal cortex (mPFC) form a circuit duals to a conditioned stimulus (CS). The reexposure necessary for extinction in rodents (for review, see reactivates the fear memory, which induces reconsolidation Corcoran and Quirk, 2007; Sotres-Bayon et al, 2004; Quirk of the memory first, and then extinction of the fear response and Mueller, 2008). (Myers and Davis, 2002; Ouyang and Thomas, 2005; Suzuki Both attenuating reconsolidation and facilitating extinc- et al, 2004). Reconsolidation is a process that results in a tion are effective to reduce the emergence of a fear response persistent or greater fear response once consolidated (Diergaarde et al, 2008; Davis et al, 2006). From a clinical memory is destabilized and reconstructed into a more point of view, this strategy has attracted attention as a stable state by short exposure to a CS (Sara, 2000; Nader method to enhance exposure therapy for complex anxiety et al, 2000). Extinction is a process by which a new disorders such as phobias, post-traumatic stress disorder inhibitory association is formed following a longer exposure (PTSD), and obsessive-compulsive disorder. Based on this to a CS, resulting in a reduced fear response (Myers and strategy, a partial agonist for NMDA receptors, D-cycloser- Davis, 2002). Previous studies suggest that the basolateral ine (DCS), a highly studied extinction-facilitating drug, was applied to humans in a clinical trial to enhance exposure *Correspondence: Dr M Sekiguchi, Department of Degenerative therapy for acrophobia, social anxiety disorder, and Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, obsessive-compulsive disorder, and was confirmed to Tokyo 187-8502, Japan, Tel: + 81 42 346 1715, Fax: + 81 423 46 enhance exposure therapy (Ressler et al, 2004; Hofmann 1745, E-mail: [email protected] or [email protected] et al, 2006; Kushner et al, 2007; Guastella et al, 2008; Received 2 February 2009; revised 2 July 2009; accepted 2 July 2009 Vervliet, 2008; Norberg et al, 2008). A fear extinction-selective drug D Yamada et al 2575 Reconsolidation and extinction are thought to be environment of our institute’s animal center for at least independent processes. However, because common mole- 1 week. The mice were 10- to 12-week old at the beginning cular mechanisms, such as protein synthesis and activation of experiments. The mice were housed four or five per cage of NMDA receptors, b-adrenergic receptors, protein kinase under controlled conditions of temperature (25±1 1C) and A, and mitogen-activated protein kinases, are involved in lighting (12 h light/dark cycle) and provided with food and these two processes (for review, see Tronson and Taylor, water ad libitum. All experiments were conducted in strict 2007; Myers and Davis, 2007), drugs that act on extinction accordance with the regulations of the National Institute of may also influence reconsolidation. Therefore, at present, to Neuroscience (Japan) for animal experiments. manipulate either of the two processes with systemic pharmacological reagents, strict control of the duration of Apparatus reexposure is required to optimize the conditions for each process (Suzuki et al, 2004). Specifically, with long All behavioral experiments were conducted in the same reexposures systemic DCS attenuates fear responses by conditioning chamber (20 Â 20 cm bottom, 35 cm high; facilitating extinction, but with shorter reexposures it Muromachi Kikai, Tokyo, Japan) inside a sound-attenuating enhances fear responses by facilitating reconsolidation box. The chamber was made of white polyvinyl chloride, (Walker et al, 2002; Ledgerwood et al, 2003; Lee et al, exception for the grid floor, which was made of stainless 2006). Conversely, with short reexposures systemic propra- steel bars (0.5 cm in diameter, spaced 1.0 cm apart). The nolol (PROP) attenuates fear responses by suppressing rods were wired to a shock generator/scrambler (SGS-002; reconsolidation, but with long reexposures it enhances fear Muromachi Kikai) for the delivery of electrical footshocks. responses by reducing extinction (Ouyang and Thomas, A digital video camera connected to a VHS video recorder 2005; Debiec and LeDoux, 2004; Mueller et al, 2008). was mounted above the chambers to permit video scoring of Therefore, seeking a compound that selectively acts on the animal’s behavior. The illumination on the floor of the reconsolidation or extinction is potentially important for box was 200 lux. two reasons: (1) to provide a pharmacological demonstra- tion that reconsolidation and extinction are independent Drugs processes; and (2) to facilitate development of a therapeutic drug that enhances exposure therapy without the undesired For intraperitoneal administration, PEPA (Sigma, St Louis, side effect of fear augmentation. For the second aspect, a MO; or kindly supplied by Nihon Suisan, Tokyo, Japan) and compound that could be administered systemically would DCS (Sigma) were dissolved (3 g/l) into vehicle consisting be desirable. of 0.45% NaCl and 33% (2-hydroxypropyl)-b-cyclodextrin Previously, we reported (Zushida et al, 2007) that by shaking vigorously more than 3 h (in case of PEPA). intraperitoneal or intra-mPFC administration of the AMPA (±)-Propranolol hydrochloride (Sigma) was dissolved in potentiator, 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro- saline (1 g/l). These drug solutions were administered into phenoxyacetamide (PEPA), facilitates extinction learning of mice at a dose of 30 mg/kg body weight (volume 10 ml/kg), contextual fear in mice, and that this facilitation is mainly except for PROP, which was injected at a dose of 10 mg/kg. due to activation of the mPFC because (1) the potentiating The timings of injections varied among experiments and are actions of PEPA on the excitatory synaptic response described in the Results section. For intra-amygdala recorded in BLA pyramidal neurons and hippocampal administration, PEPA and DCS were dissolved (2.5 g/l) into CA1 pyramidal neurons were weaker than those on the vehicle (17% (2-hydroxypropyl)-b-cyclodextrin solution). synaptic response recorded in mPFC pyramidal neurons, and (2) intra-amygdala administration of PEPA did not Behavioral Procedures significantly facilitate extinction learning. These weaker electrophysiological and behavioral actions of PEPA on the Grouping. Before each experiment, the mice used in the BLA prompted us to hypothesize that PEPA may not act on experiment were randomly divided into two (Figure 1a–c reconsolidation of fear memory, because the BLA is mainly and g–i) or three groups (Figure 1d–f; Figures 2–5). The involved