Neurobiology of Learning and Memory 110 (2014) 64–71

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Entorhinal cortex contribution to contextual fear conditioning extinction and reconsolidation in rats Elisabetta Baldi, Corrado Bucherelli ⇑ Dipartimento di Medicina, Sperimentale e Clinica, Sezione di Fisiologia, Università degli Studi di Firenze, Viale G.B. Morgagni 63, I-50134 Firenze, Italy

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Article history: Received 11 October 2013 Revised 23 January 2014 Accepted 11 February 2014 Available online 22 February 2014 Keywords: Conditioned freezing Memory consolidation TTX inactivation Amygdala Hippocampus

a b s t r a c t During contextual fear conditioning a rat learns a temporal contiguity association between the exposition to a previously neutral context (CS) and an aversive unconditioned stimulus (US) as a footshock. This condition determines in the rat the freezing reaction during the subsequent re-exposition to the context. Potentially the re-exposition without US presentation initiates two opposing and competing processes: reconsolidation and extinction. Reconsolidation process re-stabilizes and strengthens the original memory and it is initiated by a brief re-exposure to context. Instead the extinction process leads to the decrease of the expression of the original memory and it is triggered by prolonged re-exposure to the context. Here we analyzed the entorhinal cortex (ENT) participation in contextual fear conditioning reconsolidation and extinction. The rats were trained in contextual fear conditioning and 24 h later they were subjected either to a brief (2 min) reactivation session or to a prolonged (120 min) re-exposition to context to induce extinction of the contextual fear memory. Immediately after the reactivation or the extinction session, the animals were submitted to bilateral ENT TTX inactivation. Memory retention was assessed as conditioned freezing duration measured 72 h after TTX administration. The results showed that ENT inactivation both after reactivation and extinction session was followed by contextual freezing retention impairment. Thus, the present findings point out that ENT is involved in contextual fear memory reconsolidation and extinction. This neural structure might be part of parallel circuits underlying two phases of contextual fear memory processing. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction In rodents contextual fear conditioning is a paradigm useful to study emotional learning and memory (Anagnostaras, Gale, & Fanselow, 2001; LeDoux, 2000; Maren, 2001). This form of learning involves the association of an otherwise neutral context with an aversive stimulus, e.g. an electrical footshock (the unconditioned stimulus, US). After training, the context alone elicits a conditioned fear response such as freezing behavior, i.e. the suppression of all somatic movements, with the exception of respiration, behaving as a conditioned stimulus (CS) (Fanselow, 1980; LeDoux, Sakaguchi, & Reis, 1983; Sacchetti, Ambrogi Lorenzini, Baldi, Tassoni, & Bucherelli, 1999a). This form of memory can be easily maintained for a long time (LeDoux, 2000; Maren, 2001). Long-term memory is generated through a process known as consolidation. According to the classical theory of memory consolidation, through this process the newly formed mnemonic trace, initially sensitive to disruption by several treatments ⇑ Corresponding author. Fax: +39 55 4379506. E-mail address: corrado.bucherelli@unifi.it (C. Bucherelli). http://dx.doi.org/10.1016/j.nlm.2014.02.004 1074-7427/Ó 2014 Elsevier Inc. All rights reserved.

(e.g. electroconvulsive shock, intracerebral or systemic pharmacological treatments), becomes stable over time (Dudai, 1996; McGaugh, 2000). Thus, once stabilized the engram remains insensitive to disruption. However, results have shown that after reaching a stable state, memory becomes transiently sensitive to disruption if it is reactivated (for instance by retrieval trial) (Bucherelli & Tassoni, 1992a; Judge & Quartermain, 1982; Misanin, Miller, & Lewis, 1968; Nader, Schafe, & LeDoux, 2000). In many instances the same treatments that disrupt consolidation are effective in disrupting a reactivated memory (Alberini, 2005; Dudai, 2004; Nader, 2003; Sara, 2000). The process by which a reactivated memory becomes again stable and insensitive to disruption has been termed reconsolidation (Alberini, 2005; Dudai, 2006; Nader, 2003; Tronson & Taylor, 2007). To induce contextual fear conditioning memory trace reactivation it is sufficient to expose the experimental subject to the training context (CS) in the absence of aversive US (footshock) (Nader, 2003; Tronson & Taylor, 2007). This type of trial can also be considered as an extinction trial. The mnemonic trace extinction results in the decrease of the conditioned fear response evoked by the context when the context no longer predicts footshock for the animal (Baldi & Bucherelli, 2010;

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Myers & Davis, 2007; Quirk & Mueller, 2008). This suggested that memory retrieval is a dynamic process that may potentially initiate two competing processes: reconsolidation and extinction (De la Fuente, Frendenthal, & Romano, 2011; Mamiya et al., 2009; Rossato, Bevilaqua, Izquierdo, Medina, & Cammarota, 2010; Suzuki et al., 2004). In fact, reconsolidation stabilizes and extinction weakens the expression of the original memory. An important determinant of subsequent engram processing following the retrieval is the temporal duration of re-exposure to the CS (context) without the exposition to the US: brief reactivation sessions lead to memory reconsolidation, whereas longer reexposition sessions lead to memory extinction (Barak & Hamida, 2012; de la Fuente et al., 2011; Debiec, LeDoux, & Nader, 2002; Eisenberg, Kobilo, Berman, & Dudai, 2003; Lee, Milton, & Everitt, 2006; Pedreira & Maldonado, 2003; Suzuki et al., 2004). Current understanding of the neural basis of fear reconsolidation and extinction is much poorer compared with the acquisition/consolidation of conditioned fear. The current knowledge indicates that these different mnemonic phases are characterized by both distinctive and coincident features regarding anatomical and molecular requirements (Alberini, 2005; Berman & Dudai, 2001; Bucherelli, Baldi, Mariottini, Passani, & Blandina, 2006; Chen et al., 2005; Izquierdo et al., 2006; Lee, Everitt, & Thomas, 2004; Lin, Yeh, Lu, & Gean, 2003; Szapiro, Vianna, McGaugh, Medina, & Izquierdo, 2003; Vianna, Szapiro, McGaugh, Medina, & Izquierdo, 2001). Understanding the mechanisms of fear memory reconsolidation and extinction may have clinical relevance in treatment of human anxiety disorders such as post-traumatic stress disorder. Indeed, reconsolidation and extinction procedures may be used to reduce the expression of fear memory (Alberini, 2005; Auber, Tedesco, Jones, Monfils, & Chiamulera, 2013; Davis, Myers, Chhatwal, & Ressler, 2006; Hartley & Phelps, 2010; Monfils, Cowansage, Klann, & LeDoux, 2009; Nader, 2003; Parsons & Ressler, 2013; Quirk et al., 2010; Rao-Ruiz et al., 2011; Rossato et al., 2010; Schiller et al., 2010). Thus, the identification of both neural circuits underlying the reconsolidation and extinction processes and pharmacological agents that impair reconsolidation or potentiate extinction appears to be crucial. Experimental results have shown that the basolateral amygdala (BLA) and hippocampus are involved in contextual fear conditioning consolidation (Anagnostaras et al., 2001; Kim & Fanselow, 1992; McGaugh, 2000; Sacchetti et al., 1999a), reactivation/reconsolidation (Baldi, Mariottini, & Bucherelli, 2008; Bucherelli et al., 2006; Debiec et al., 2002; Lee et al., 2004; Mamiya et al., 2009) and extinction (Baldi & Bucherelli, 2010; Fisher, Sananbenesi, Schrick, Spiess, & Radulovic, 2004; Fisher et al., 2007; Myers & Davis, 2007; Quirk & Mueller, 2008; Sananbenesi et al., 2007; Vianna et al., 2001). These neural structures have extensive reciprocal connections with the entorhinal cortex (ENT) (Amaral & Witter, 1989; McDonald & Mascagni, 1997; Pitkanen, Pikkarainen, Nurminen, & Ylinem, 2000; Swanson & Cowan, 1977; Witter, Wouterlood, Naber, & Van Haeften, 2000). It has been suggested an interplay between the BLA and ENT in the regulation of memory consolidation (Majak & Pitkanen, 2003; Roesler, Roozendaal, & McGaugh, 2002). Moreover, the ENT has a pivotal role in processing information that is critical to hippocampal functioning (Eichenbaum, Otto, & Cohen, 1994; Maren & Fanselow, 1997). Hence, it appears interesting to analyse whether this neural site is involved in the same phases of contextual fear memory in which BLA and hippocampus play a role. Our previous results have shown that ENT is involved in contextual fear consolidation in the rat. Post-acquisition bilateral ENT tetrodotoxin (TTX) inactivation up to 1.5 h after training results in retention deficit of contextual freezing (Baldi, Liuzzo, & Bucherelli, 2013). Regarding reconsolidation, there are few and contrasting data that do not provide direct

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evidence for a role of this neural site in contextual fear conditioning reconsolidation. In fact, in rats trained in an inhibitory avoidance task, the infusion of anisomycin (a protein synthesis inhibitor) into ENT performed 15 min before or 3 h after memory reactivation, did not affect subsequent memory retention (Cammarota, Bevilaqua, Medina, & Izquierdo, 2004). On the other hand, it has been shown that reconsolidation of long-term recognition memory is associated with BDNF and Egr-1 mRNA expression and hyperphosphorylation of ERK in the ENT (Kelly, Laroche, & Davis, 2003; Romero-Granados, Fontan-Lozano, Delgado-Garcia, & Carrion, 2010), whereas protein synthesis in the ENT does not seem necessary for reconsolidation of this type of memory (Lima et al., 2009). Finally up to now an active role of the ENT in memory extinction was only observed in tasks other than classical contextual fear conditioning. Lesions in the lateral entorhinal cortex increased resistance to extinction of an operant-conditioning task in mice (Gauthier, Destrade, & Soumiren-Mourat, 1983), and immediately post-extinction intra-ENT infusions of a NMDA antagonist, protein synthesis or CaMKII inhibitors impaired inhibitory avoidance extinction (Bevilaqua et al., 2006), that was associated with significant c-Fos expression in this neural site (Huang, Shyu, Hsiao, Chen, & He, 2013). Because of the lack of direct evidence for ENT role in contextual fear memory reconsolidation and extinction, the aim of the present work was to inactivate the rat ENT by the stereotaxic administration of the depressor of neuron excitability tetrodotoxin (TTX) for studying ENT involvement in these memorization processes. The inactivation was bilaterally performed either immediately after trace reactivation or immediately after extinction training of contextual fear conditioning. In this way it has been possible to clarify the involvement of this brain site in these two memorization phases.

2. Materials and methods 2.1. Animals Seventy-day old male albino Wistar rats (average body weight 290 g) (Harlan, Italy) were used. The animals were individually housed in stainless steel cages in a room with a natural light–dark cycle and constant temperature of 20 ± 1 °C. The rats had free access to food and water throughout the experiment. All animal care and experimental procedures were conducted in accordance with Italian legislation and the official regulations of the European Communities Council on use of laboratory animals (Directive of 24 November 1986; 86/609/EEC).

2.2. Behavioral procedures 2.2.1. Apparatus As in previous experiments a basic Skinner box module (Modular Operant Cage, Coulbourn Instruments Inc.) was used to induce fear conditioning (Sacchetti, Ambrogi Lorenzini, Baldi, Tassoni, & Bucherelli, 1999b; Sacchetti et al., 1999a). Box dimensions were 29  31  26 cm. The top and two opposite sides were made of aluminum panels, the other two sides of transparent plastic and the floor of stainless steel rods connected to a shock delivery apparatus (Grid Floor Shocker, Coulbourn Instruments Inc., Model E13-08). The apparatus was connected to a stimulus programming device (Scatola di comando Arco 2340 – Ugo Basile) in order to predetermine number, duration and rate of US delivery. The apparatus was placed in an acoustically insulated room (3.5  1.8  2.1 (h) m), kept at a constant temperature of 20 ± 1 °C. Illumination inside the room was 60 lux.

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2.2.2. Conditioning On Day 1 the rat was gently taken manually from the home cage, placed in a bucket and carried from the housing room to the soundproof room, where it was placed inside the conditioning apparatus and left undisturbed for 3 min. After this time, seven electrical foot-shocks (1.0 mA, 1 s) at 30-s intervals were delivered. The rat was left undisturbed for 2 min after the end of the stimulation sequence. During first 3 min and last 2 min periods freezing duration was measured. Rats were brought back to the home cage immediately thereafter. 2.2.3. Extinction To induce extinction of contextual fear conditioning, 22 h after conditioning, the rats were placed in the conditioning apparatus and left inside for 120 min without receiving any footshocks. They were then returned to the home cage (Fig. 1). 2.2.4. Reactivation To induce reactivation, rats were again placed in the conditioning apparatus 24 h after the training trial. Each rat was left for 2 min within the apparatus without delivering footshocks. Rats were brought back to the home cage immediately thereafter (Fig. 1). 2.2.5. Contextual fear conditioning retention trial To measure contextual freezing 4 days (24 h + 72 h) after conditioning, the animals were again placed inside the conditioning apparatus and left there for 6 min without receiving foot-shocks. After that time they were returned to the home cage (Fig. 1). The rat’s behavior was recorded by means of a closed circuit TV system. Freezing (immobility) was defined as the complete absence of somatic motility except for respiratory movements (LeDoux et al., 1983). Measurements were performed by means of a stop-watch by personnel who did not know to which experimental group each animal belonged. Total accumulated freezing time (i.e. total seconds spent freezing during each period) was measured. 2.2.6. Surgery and drug administration Functional inactivation of ENT was induced by the stereotaxic administration of 5 ng TTX (Sigma, Italy) dissolved in 0.5 ll saline, into sites with the following coordinates: (the target was on the median zone), antero-posterior (AP) = 6.8, lateral (L) = ±5.0, and ventral (V) = 7.7 according to the atlas by Paxinos and Watson (1986) (see Fig. 2). It was estimated that after TTX administration (5 ng in 0.5 ll) inactivation effects were circumscribed within a tissue volume less than 2 mm in diameter tendentially centered more ventrally than rostrally respect the end of the cannula (Fig. 2) (see also Freund, Manns, & Rose, 2010). At least 20 min

Fig. 2. Extension of TTX functional inactivation of ENT area estimated on the basis of injection sites (., end of some needle tracks) and on known characteristics of TTX diffusion (for explanation see Section 2).

were necessary to obtain maximal neural inactivation lasting for no less than 120 min and disappearing within 24–36 h (Baldi, Mariottini, & Bucherelli, 2007b; Freund et al., 2010; Zhuravin & Bures, 1991). These assessments are based on previous reports

Fig. 1. Schematic representation of experimental paradigm to induce contextual fear conditioning extinction or reconsolidation and subsequent retention testing.

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both by our laboratory and others. For instance, a group of rats receiving unilateral injection of TTX (8 ng in 0.8 ll) in the substantia nigra, exhibited rotational deambulation lasting between 2 and 4 h and disappeared after 12–24 h, whereas rats injected 1.5 mm more laterally did not exhibit any motor alteration (Baldi et al., 2007b). Zhuravin and Bures (1991) described the extent and duration of the blockade induced by a TTX dosage of 10 ng in 1 ll saline. These authors monitored pupillary diameter in anesthetized rats at various intervals after injecting TTX 2, 1.5, 1, 0 mm lateral from the Edinger-Westphal nucleus. The resulting mydriasis indicated that, for such a dosage, the mean blockade radius was 1 mm. The maximum effect was obtained 30–120 min after injection, to decay exponentially to control level over the subsequent 24 h. Other authors have shown that TTX (10 ng/1 ll) administration into the dorsal hippocampus is followed by a functional inactivation measuring about 2.5 mm across its longitudinal axis (Quiroz et al., 2003), and that the effective spread in the cerebral cortex of raccoons is 2 mm (Boehnke & Rasmusson, 2001). TTX was injected under general anesthesia (Natriumpentobarbital, 50 mg/kg, i.p.). This procedure induces relatively minor trauma due to the single needle penetration instead of permanent-cannulating procedure, and is employed exclusively during the post-training phase (Ambrogi Lorenzini, Baldi, Bucherelli, Sacchetti, & Tassoni, 1997). It has been shown that the general anesthesia necessary to perform the manipulations employed in the present as in previous experiments in our laboratory, does not interfere with memory trace processing. When compared to control, unanesthetized rats, experimental subjects exhibit memory notwithstanding being anesthetized after training (Ambrogi Lorenzini et al., 1997; Bucherelli & Tassoni, 1992b; Ivanova & Bures, 1990; Sacchetti et al., 1999a; Tassoni, Bucherelli, & Bures, 1992). Inactivation was performed in rats placed in a stereotaxic apparatus. The injection needle (outside diameter 0.3 mm), connected with a short piece of polyethylene tubing to a Hamilton syringe, was fixed in the electrode holder of the stereotaxic apparatus and lowered into the target structure; 0.5 ll of the solution was injected over a 1–2 min period, and the needle was left in place for another 1 min before being slowly withdrawn. 2.2.7. Experimental groups A total of 104 rats was employed, randomly divided into 9 groups. Three of these underwent extinction procedure (22 h after conditioning). Immediately thereafter (i.e. 24 h after conditioning), TTX (E-T) or saline (E-S) was bilaterally injected into the ENT. One group was not operated as control (E). Three groups of rats underwent reactivation procedure 24 h after conditioning. Immediately after, TTX (R-T) or saline (R-S) were bilaterally injected into the ENT. One group was not operated as control (R). The other 3 groups did not undergo reactivation or extinction. 24 h after conditioning two groups were respectively TTX (C-T) or saline (C-S) bilaterally injected into the ENT. One group was not operated as control (C). Eight animals were excluded so that 96 animals comprised 9 groups with 10–12 animals each.

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were excluded from data processing because of inappropriate placements of injection needle. 3. Results 3.1. Spontaneous and conditioned behavior during conditioning (training) During the pre-footshock 3 min exploration period of the acquisition training session, rats of all 9 groups exhibited a homogeneous level of immobility ranging between 9% and 14% of total exposure time. One-way ANOVA showed that there were no significant differences between groups [F(8, 87) = 0,62, N.S.]. A very long freezing duration was exhibited by the rats of all groups during the 2-min post-shock period in the conditioning chamber. The mean freezing duration of the 9 groups ranged between 79.2% and 85.7% of total time. One-way ANOVA showed that there were no significant differences between groups [F(8, 87) = 0.41, N.S.], i.e. the freezing response was homogeneous in all groups. 3.2. Effectiveness of extinction but not of reactivation procedures per se on fear conditioning retention During retention test there were evidently different freezing durations between the 3 non-operated groups, i.e. the group that had undergone the extinction procedure (E), the reactivation (R) or no extinction no reactivation one (C) (Fig. 3). Freezing duration of E group was about one third of those of the other two groups (R and C). One-way ANOVA showed statistically significant differences between groups [F(2, 28) = 21.10, P < 0.001]. Newman Keuls post hoc test showed significant differences between the E group and both C and R groups (P < 0.05 in both instances). On the other hand there were no significant differences between C and R groups. 3.3. Post-extinction and post-reactivation ENT inactivation effects At the retention test, the E-T group showed much longer freezing duration compared to the extinguished saline-injected one (E-S) (Fig. 4). In addition, Fig. 4 shows that the E-S group froze significantly less than either C-S or C-T groups, displaying comparable amounts of freezing to those that did not undergo the

2.3. Statistical analysis Between groups comparison one-way ANOVA and post hoc Newman–Keuls’multiple comparisons test between group pairs were used. 2.3.1. Morphology At the end of the experiments, injected sites (ENT) were histologically verified. Rats were deeply anesthetized and intracardially perfused with saline, followed by 4% formaldehyde. Brains were cut at 40 lm using a freezing microtome and injection needle tracks were identified in Nissl-stained serial sections. Eight rats

Fig. 3. Effects of 2 min (reactivation R) or 2 h (extinction E) or re-exposure to the conditioning context on contextual fear conditioning retention. C not extinction-not reactivation control group. Means ± SEM are shown. P < 0.05.

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Fig. 4. Effects on contextual fear conditioning extinction and reconsolidation of bilateral administration of TTX (T) or saline (S) into ENT. E extinguished groups, R reactivated groups, C not extinguished, not reactivated control groups. Means ± SEM are shown. P < 0.05.

extinction procedure. One-way ANOVA revealed statistically significant differences between E-S, E-T, C-S and C-T groups [F(3, 40) = 9.82, P < 0.001]. Newman Keuls post hoc test showed significant differences between E-T and E-S-groups (P < 0.05) and between E-S and C-S-groups (P < 0.05). There were not significant differences between C-S and C-T groups. At the retention test, the R-T group showed shorter freezing duration compared to the reactivated saline-injected one (R-S) (Fig. 4). Fig. 4 shows that the R-S group exhibited freezing duration comparable to that exhibited by the not-reactivated, saline-injected C-S group and by the no-reactivated, TTX-injected C-T group. One-way ANOVA revealed statistically significant differences between R-S, R-T, C-S and C-T groups [F(3, 39) = 11.32, P < 0.001]. Newman Keuls post hoc test showed significant differences between R-T and R-S (P < 0.05). On the other hand there were not statistically significant differences between R-S and C-S.

4. Discussion In the present experiments both ENT post-extinction and postreactivation inactivation results showed that ENT is involved in both extinction and reconsolidation of contextual fear conditioning. The protocol used showed to be adequate to induce conditioning, extinction and reconsolidation. Contextual fear response appears to be harder to learn than that to a discrete CS (Fanselow, 1990; Rudy & Morledge, 1994; Sacchetti et al., 1999b). The conditioning protocol used, already used in previous work, allowed the formation of a strong contextual memory trace that a long exposure times to the CS (context) without shocks extinguished (Ambrogi Lorenzini, Baldi, Bucherelli, & Tassoni, 1993; Bucherelli & Tassoni, 1992a; Hikind & Maroun, 2008). Present results were consensual with data in the literature showing that the duration of re-exposure is a key variable that controls subsequent behavior: brief re-exposures to the context seem to trigger memory reconsolidation, whereas prolonged re-exposures to the context lead to extinction (Barak & Hamida, 2012; de la Fuente et al., 2011; Debiec et al., 2002; Eisenberg et al., 2003; Lee et al., 2006; Pedreira & Maldonado, 2003; Suzuki et al., 2004). Freezing measured during animal testing is a conditioned (learned) response. Indeed, the experimental subjects exhibited only short-lasting immobility when placed in the conditioning apparatus prior to the training session (Corodimas & LeDoux, 1995; Fanselow, 1990; Sacchetti et al., 2001, 1999b). Immediately after conditioning, the freezing behavior of the 9 groups of experimental animals was

homogeneous in response to shocks. At retention testing, performed 4 days after conditioning, the control group (C) which did not undergo extinction exhibited a good contextual fear conditioning freezing response whereas 120 min context re-exposed group, 22 h after conditioning training (group E), showed a significant decrease of the contextual freezing response. The reactivation procedure (2 min re-exposition to context without footshocks) did not modify freezing levels at the retention test. Consequently, this last experimental procedure did not induce partial extinction nor enhances freezing levels at the retention testing. Thus, the possible effects due to ENT functional inactivation performed immediately after re-exposure to the context were to be ascribed to interference on reconsolidation process. In the present study we used an inactivating agent, TTX, which depresses neuronal excitability, instead of the agents like protein synthesis inhibitors. In fact, previous literature has shown that these agents do not always appear to be sure markers for studying a neural site involvement in memorization processes. For instance, the object recognition memory reconsolidation was associated with an increase in the phosphorylation of ERK and in BDNF and EGR-1 expression in ENT (Kelly et al., 2003; Romero-Granados et al., 2010), although did not require new protein synthesis in this brain site (Lima et al., 2009). Consensually, when local administrations of protein synthesis inhibitors were employed to examine ENT involvement in passive avoidance memory reconsolidation, this brain site does not appear to affect this mnemonic phase (Cammarota et al., 2004). As stated in the Section 2, concerning the temporal and spatial extent of local inactivation by TTX, it can be stated that in the present experiment TTX inactivation effects (maximal lasting between 20–30 and 120 min after injection) were circumscribed within a tissue volume less than 2 mm in diameter centered on medial–lateral ENT without affecting other bordering regions (Fig. 2). It is important to notice that to study the role of a neural site in a specific phase of memorization process (in this case reconsolidation and extinction) such phase is to be induced when the neural structure examined is no longer involved in the post-training consolidation. Our previous experiments have shown that unlike other neural sites (such as BLA, nucleus basalis magnocellularis or substantia nigra) (Baldi, Mariottini, & Bucherelli, 2007a; Baldi et al., 2007b; Sacchetti et al., 1999a) the ENT is no longer involved in the consolidation of contextual mnemonic trace 24 h after conditioning trial (Baldi et al., 2013). This finding is also confirmed in the present experiments as ENT inactivation performed 24 h after conditioning training does not induce any mnemonic retention impairment. In fact, as reported in Fig. 4, C-T group displayed freezing levels comparable to those of C-S control group. Retention testing was always performed 72 h after TTX administration, i.e. when no residual TTX effects were present (Zhuravin & Bures, 1991). The absence of interference with normal function both during acquisition and retention trial excludes not only state-dependent effects, but also interference with sensory perception or with motor control functions. Our paradigm excludes the hypothesis that experimental manipulations interfered with sensory perception and/or motor performance control (Maren & Fanselow, 1997). Therefore, our results can be fully explained with the effects of the experimental procedures on memory processing. In general, the most widely accepted view is that extinction is a form of new learning, a new inhibitory association between the CS and US (i.e. a CS-no US association) and consists of several phases: acquisition, consolidation and retrieval (Myers & Davis, 2007; Pape & Pare, 2010; Quirk & Mueller, 2008). The present results show that ENT plays a role in consolidation of the contextual fear extinction. The experimental procedure used here (post-extinction training local inactivation) allows the study targeted of this phase. At retention test the rats of E-T group showed longer freezing duration

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than E-S control group, indicating an impaired extinction of contextual fear response. These data are consensual with reports indicating an ENT involvement in inhibitory avoidance extinction. The authors have found that intra-ENT infusions of AP5 (a NMDA antagonist) or protein synthesis inhibitor anisomycin or CaMKII inhibitor KN-93, but not MEK1/2 inhibitor PD98059, immediately following the first of four daily extinction exposures produced an extinction impairment (Bevilaqua et al., 2006). In addition, inhibitory avoidance extinction procedure was followed by a significant enhanced c-fos expression in the ENT (Huang et al., 2013). It is thought that contextual fear extinction relies on a complex network of neural structures, mainly involving the amygdala, the medial prefrontal cortex (mPFC) and the hippocampus: (i) the amygdala is the site of acquisition and storage of the extinction memory, (ii) the mPFC, in particular its infralimbic area (IL), mediates the consolidation of extinction, and (iii) the hippocampus plays a role in the context-dependence of extinction. It is believed that during fear extinction the IL inhibits fear expression mainly through its projections to the amygdala. Indeed, IL inputs may synapse on ‘‘extinction neurons’’ within the basal nuclei of the amygdala, which fire selectively to an extinguished CS (Herry et al., 2008). Extinction neurons may then influence amygdala central nucleus (CeA) activity, the main output nucleus of the amygdala sending direct projections to brain structures controlling conditioned fear responses. Moreover between the BLA and CeA there are clusters of GABAergic interneurons, the intercalated cell masses (ITC) neurons, that inhibit the CeA output (Royer, Martina, & Pare, 1999). Following extinction training ITC neurons are driven by IL projections inhibiting the output of CeA and providing a mechanism of extinction (Amano, Amur, Goswani, & Pare, 2012; Amano, Unal, & Pare, 2010; Ehrlich et al., 2009; Likhtik, Popa, Apergis-Schoute, Fidacaro, & Pare, 2008; Pape & Pare, 2010; Pare & Duvarci, 2012; Quirk & Mueller, 2008). The hippocampus may contribute to the fear extinction sending contextual information to both amygdala and IL. There are robust reciprocal connections between the amygdala and the hippocampus (Canteras & Swanson, 1992; Pitkanen et al., 2000) and these connections seem to be involved in extinction learning and in the context-specific retrieval of extinction. On the other hand, the IL receives also inputs from the hippocampus (Hoover & Vertes, 2007) and recent evidence suggests that hippocampal projections to the mPFC elicit synaptic changes that may be responsible for the extinction consolidation (Orsini & Maren, 2012; Pape & Pare, 2010; Peters, Dieppa-Perea, Melendez, & Quirk, 2010; Quirk & Mueller, 2008). In this scenario the ENT might constitute an important component of neuronal network underlying contextual fear extinction. In particular the ENT is positioned in such a way to represent the link between mPFC and amygdala on the one hand and between mPFC and the hippocampus on the other. In fact, the rat ENT sends and receives projections from the PFC (Courtin, Bienvenu, Einarsson, & Herry, 2013; Hyman, Van Hoesen, & Damasio, 1990). Moreover, McDonald and Mascagni (1997) have established that the ENT is a source of significant inputs to the amygdala, to both BLA and CeA and also to the ITC neurons. The inactivation of the ENT immediately after extinction-training would prevent activation of the ENT-amygdala projections preventing the activation of the amygdaloid inhibitory circuits and thereby hindering extinction consolidation of contextual fear. Alternatively, or additionally, the effects observed may be due to blockade of amygdala-ENT projections. In fact, ENT receives heavy direct projections from the BLA (Pitkanen et al., 2000). On the other hand, it has been showed that the amygdala modulates the consolidation of long-term memory by influencing other brain regions involved in this memorization phase (McGaugh, McIntyre, & Power, 2002). As extinction is a new learning, it should be susceptible to post-training modulation by BLA (Boccia, Blake, Baratti, & McGaugh, 2009) and the ENT may play

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a downstream role in this modulation. As recalled above, the ENT provides the major gateway for transmission of information between the hippocampus and cortex being a critical relay for the contextual ones (Hyman et al., 1990; Maren & Fanselow, 1997). Because TTX inactivates not only somata but also passing fibers, TTX injection centered on ENT could temporarily disconnect the hippocampus, a site critically involved in the contextual representation, from cortical regions. In this contention it remains to clarify if the presently described contextual fear extinction impairment could be due to the functional disconnection of the hippocampus from neocortex or to an ENT role as autonomous site of polymodal sensory input elaboration confirming the inhibitory avoidance extinction results interpretation (Bevilaqua et al., 2006). The present results show that the ENT is involved also in contextual fear reconsolidation. Indeed, rats undergoing a brief reactivation session (2 min) of contextual memory followed by ENT TTX inactivation exhibit low levels of freezing response at the retention testing compared to saline-injected controls (Fig. 4). Thus, this finding points out the ENT as a part of a neural network involved in the reactivation/reconsolidation of contextual fear memory after retrieval. These results are in agreement with those obtained in recognition memory reconsolidation associated with an increase in the phosphorylation of ERK and in BDNF and EGR-1 expression in ENT (Kelly et al., 2003; Romero-Granados et al., 2010), even if this process does not require new protein synthesis in this neural site (Lima et al., 2009). This last result confirms other findings obtained in inhibitory avoidance where the injection of anisomycin into ENT 15 min before or 3 h after a pure reactivation session, unable by itself to induce extinction, did not produce effects on subsequent retention (Cammarota et al., 2004). As stated above, reconsolidation and extinction are two opposing and competing processes, where only extinction involves a new learning which competes with the original one (de la Fuente et al., 2011; Mamiya et al., 2009; Quirk & Mueller, 2008; Rossato et al., 2010; Suzuki et al., 2004). The present findings show that ENT, besides to be involved in contextual fear consolidation, participates both in reconsolidation and extinction consolidation of contextual fear conditioning. The two distinct processes might take place in distinct connections between ENT and critical structures for contextual fear conditioning, such as amygdala and hippocampus. Indeed, observations obtained by electrophysiological recordings and immunohistochemistry methods have identified distinct amygdaloid (‘‘fear neurons’’ and ‘‘extinction neurons’’) and hippocampal (‘‘cFos+ cells’’ and ‘‘pERK+ cells’’) neurons activated during conditioning and extinction of fear (Herry et al., 2008; Tronson et al., 2009). Specifically, amygdaloid fear neurons and hippocampal cFos+ cells might be connected with ENT projections that are activated during contextual fear reconsolidation, whereas amygdaloid extinction neurons and hippocampal pERK+ cells might be connected with ENT projections activated during extinction of this fear response. On the other hand, reconsolidation and extinction might interact and some authors have begun to evaluate the possible interactions of the two processes. In fact, several studies have shown that extinction performed during the ‘‘reconsolidation window’’ enhanced effects of extinction training session preventing the re-expression of emotional memory both in animals and humans and it has been proposed that this strategy may be used to treat fear-related disorders (Monfils et al., 2009; Rao-Ruiz et al., 2011; Rossato et al., 2010; Schiller et al., 2010). On this point, the amygdala has been showed to be a locus for the interaction between the reconsolidation and extinction processes at molecular level. In fact, amygdalar CREB (cAMP-response element-binding protein) activation is differentially regulated in these two memory phases because it shows a different time course from the beginning of the re-exposure to the context between reconsolidation and extinction: during extinction CREB activation is delayed compared

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with reconsolidation (Mamiya et al., 2009). The ENT might also be identified as a possible locus for this interaction. In summary, together previous and present work show that ENT is involved both in contextual fear conditioning consolidation, extinction and reconsolidation. In particular the present findings represent the first report of an ENT involvement in both reconsolidation and extinction consolidation of contextual fear conditioning. Consequently, ENT should be considered together with the amygdala and hippocampus as a site crucial for contextual fear conditioning memory. Acknowledgments We thank Dr. Maria Beatrice Passani for comments on a previous draft of this manuscript and A. Aiazzi, S. Cammarata, C. Pregno, and A. Vannucchi for their technical assistance. References Alberini, C. M. (2005). Mechanisms of memory stabilization: Are consolidation and reconsolidation similar or distinct processes? Trends in Neurosciences, 28, 51–56. 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