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Systems Reconsolidation Reveals a Selective Role for the Anterior Cingulate Cortex in Generalized Contextual Fear Memory Expression

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Systems Reconsolidation Reveals a Selective Role for the Anterior Cingulate Cortex in Generalized Contextual Fear Memory Expression Neuropsychopharmacology (2015) 40, 480–487 & 2015 American College of Neuropsychopharmacology. All rights reserved 0893-133X/15 www.neuropsychopharmacology.org Systems Reconsolidation Reveals a Selective Role for the Anterior Cingulate Cortex in Generalized Contextual Fear Memory Expression ,1,2 1 ,1 Einar O¨ Einarsson* , Jennifer Pors and Karim Nader* 1 Department of Psychology, McGill University, Montreal, QC, Canada After acquisition, hippocampus-dependent memories undergo a systems consolidation process, during which they become independent of the hippocampus and dependent on the anterior cingulate cortex (ACC) for memory expression. However, consolidated remote memories can become transiently hippocampus-dependent again following memory reactivation. How this systems reconsolidation affects the role of the ACC in remote memory expression is not known. Using contextual fear conditioning, we show that the expression of 30-day-old remote memory can transiently be supported by either the ACC or the dorsal hippocampus following memory reactivation, and that the ACC specifically mediates expression of remote generalized contextual fear memory. We found that suppression of neural activity in the ACC with the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) impaired the expression of remote, but not recent, contextual fear memory. Fear expression was not affected by this treatment if preceded by memory reactivation 6 h earlier, nor was it affected by suppression of neural activity in the dorsal hippocampus with the GABA-receptor agonist muscimol. However, simultaneous targeting of both the ACC and the dorsal hippocampus 6 h after memory reactivation disrupted contextual fear memory expression. Second, we observed that expression of a 30-day-old generalized contextual fear memory in a novel context was not affected by memory reactivation 6 h earlier. However, intra-ACC CNQX infusion before testing impaired contextual fear expression in the novel context, but not the original training context. Together, these data suggest that although the dorsal hippocampus may be recruited during systems reconsolidation, the ACC remains necessary for the expression of generalized contextual fear memory. Neuropsychopharmacology (2015) 40, 480–487; doi:10.1038/npp.2014.197; published online 3 September 2014 INTRODUCTION the memory to function independently of the hippocampus (Frankland and Bontempi, 2005). In support of this view, Lesions or pharmacological inactivation of the hippocam- imaging studies have revealed sequential activity in area pus preferentially impact recently acquired contextual fear CA1 in the hippocampus and ACC for recent and remote memory (Anagnostaras et al, 1999; de Oliveira Alvares et al, time points after learning, respectively, in terms of 2012; Goshen et al, 2011; Kitamura et al, 2009; Ward et al, immediate-early gene expression (Frankland et al, 2004), 1999; Wiltgen et al, 2010; Winocur et al, 2009). Conversely, and dendritic spine growth (Vetere et al, 2011). This transient disruption of the anterior cingulate cortex (ACC) transition has been suggested to reflect systems consolida- impairs the expression of remote, but not recent memory tion, during which post-training hippocampal replay (Frankland et al, 2004; Goshen et al, 2011). Such findings of episodic or detailed memory leads to memory trace have led to the idea that the hippocampus serves a critical reactivation in the cortex, promoting the development of a role in integrating information from a distributed more schematic cortical representation (McClelland et al, cortical network representing various features of a memory, 1995; Winocur et al, 2010). and that over time, the medial prefrontal cortex (including Recent studies suggest that retrieval can make remote the ACC) gradually takes over this integrative role, allowing hippocampus-independent contextual fear memory again dependent on the hippocampus for a brief time (Debiec *Correspondence: Dr E O¨ Einarsson, INSERM U862, Neurocentre et al, 2002; Winocur et al, 2009). Such renewed dependence Magendie, 146 Rue Le´o Saignat, Bordeaux cedex 33077, France, Tel: on the hippocampus after memory reactivation is called +33 557 573719, Fax: +33 557 573669, systems reconsolidation. Specifically, these studies showed E-mail: [email protected] or Dr K Nader, Department of that electrolytic lesions of the dorsal hippocampus impaired Psychology, McGill University, 1205 Dr. Penfield Avenue, Montreal, QC, Canada H3A 1B1. E-mail: [email protected] remote memory only when applied after memory reactiva- 2Current address: INSERM U862, Neurocentre Magendie, 146 Rue tion. It remains unclear, however, whether remote memory Le´o Saignat, Bordeaux cedex 33077, France reactivation also affects the role of the ACC. For example, it Received 30 March 2014; revised 28 July 2014; accepted 31 July 2014; is not known whether the ACC critically supports the accepted article preview online 5 August 2014 remote memory expression after reactivation, or if the The anterior cingulate and generalized memory EO¨ Einarsson et al 481 reactivated remote memory only requires the hippocampus, metabolize rapidly within a few hours after cortical infu- as is the case for recent memory. sions (Attwell et al, 1999; Honore et al, 1988). Other drugs, To examine how remote memory reactivation affects the such as muscimol, are known to suppress neuronal activity brain structures supporting contextual fear memory for longer periods. In one experiment involving the dorsal expression, we tested whether pharmacological suppression hippocampus, we used muscimol for suppressing neuronal of neural activity in the ACC and/or dorsal hippocampus activity during test 2 as the drug’s effects and diffusion have disrupts expression of remote contextual fear memory been extensively characterized for that structure. A previous following memory reactivation. First, we found that after study (Corcoran et al, 2005) targeting the dorsal hippo- remote memory reactivation, suppression of neural activity campus, using the same dose as in this study with in the ACC impaired memory expression when tested 24 h, autoradiographic analysis of the spread of [3 H]muscimol, but not 6 h, later. Second, we found that only simultaneous found that the drug did not bind to other subcortical suppression of neural activity in the ACC and the dorsal structures, although it did diffuse to more dorsal cortical hippocampus impaired memory expression 6 h after regions. To further test the functional effects of possible memory reactivation, suggesting that memory expression dorsal diffusion from infusion to the dorsal hippocampus, can be supported by either structure alone. Third, we found we included a group receiving muscimol infusion into that pre-test suppression of neural activity in the ACC 6 h cortical regions dorsal to the dorsal hippocampus. after memory reactivation impaired fear expression in Drugs were infused via an infusion pump at a rate of a novel context, but did not affect memory expression in 0.25 ml/min. CNQX disodium salt (Tocris, Ellisville, MO) the training context, suggesting that the ACC specifically was dissolved in nanopure water and infused at 2.5 mg/ mediates generalized fear memory expression. 0.25 ml per side. Muscimol was dissolved in artificial cerebrospinal fluid and injected at 0.5 mg/0.5 ml per side. Representative photomicrographs of infusion sites marked MATERIALS AND METHODS with methylene-blue solution in the ACC or the dorsal General Behavioral Procedures hippocampus are depicted in Supplementary Figure 1. Subjects. Male Sprague–Dawley rats (Charles River, Saint- Constant, PQ) weighing at least 250 g at the start of training Apparatus. The training context consisted of rodent con- were used in experiments. Animals were housed individu- ditioning cages with clear Plexiglas walls and a metal grid ally and maintained on a 12/12 h light/dark cycle (lights on floor (Coulbourn Instruments) that was enclosed within a at 0700 hours) with food and water provided ad libitum. sound-attenuating chamber in a well-lit room. The cages The rats were handled daily on 5 consecutive days before were dimly lit with a single house light, had a fan on for training for B2 min. All procedures were in accordance background noise, and were scented with diluted vanilla. with the Canadian Council on Animal Care Guide, and were The novel context was in a dark room and consisted of a approved by the McGill University Animal Care and Use rodent conditioning cage (Med Associates, St Albans, VT) Committee. with a striped Plexiglas wall front, aluminum sides, clear Plexiglas top, curved gray plastic back, and plastic floor covered with bedding. The cage was enclosed in a sound- Surgery and histology. Under ketamine (55 mg/kg), attenuating chamber with blinking white LED light above xylazine (3.33 mg/kg), and domitor (27 mg/kg) anesthesia, the cage. 26-G stainless steel cannulae were implanted bilaterally into the ACC (injector coordinates: AP 2.6 mm relative to bregma; ML±0.7 mm; DV À 1.6 mm to dura), the Behavioral procedures. The day before conditioning, rats dorsal hippocampus (AP À 3.6 mm relative to bregma; ML were habituated to the conditioning chamber (training ±3.1 mm; DV À 3.1 mm to dura, and À 1.6 mm to dura for context) for 5 min. For conditioning, rats received eight the control group receiving infusions dorsally to the dorsal unsignaled foot-shocks (1.5 mA, 1 s) at 62 s intervals 2 min hippocampus). Rats were given a week to recover. after being placed in the context and then remained there In experiments testing recent memory (3 days post- for additional 30 s. For all tests, freezing (defined as the conditioning), rats were operated on before conditioning. complete absence of movement, except that of respiration) In experiments testing remote memory, surgery was was scored with an instantaneous time-sampling procedure, performed between conditioning and testing. Guide cannu- in which each animal was observed as either freezing or not lae were fixed to the skull with dental cement and stainless every 5 s. All test and memory reactivation sessions were steel screws. At the end of the experiment, animals were 3 min long.
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