Title: The hippocampus integrates context and shock into a configural memory in contextual fear conditioning Author names and affiliation: Shih-Dar Chang1 and K. C. Liang1, 2, 3 1

Department of Psychology,

2

Graduate Institute for Brain and Mind Science,

3

Neurobiology and Cognitive Science Center

National Taiwan University, Taipei, Taiwan 10617 Running title: dorsal hippocampus and configural fear memory Number of pages: 34 Number of figures: 6 Number of tables: 0 Corresponding author: K. C. Liang Tel: 886-2-3366-3084 Fax: 886-2-2362-9909 E-mail: [email protected] Grant sponsor: Ministry of Science and Technology, Taiwan, ROC; Grant Number: NSC 98-2410-H002-024-MY3, NSC 101-2410-H-002-082-MY3 Ministry of Education, Research Projects for Promoting University Excellence, Taiwan, ROC; Grant Number: 10R80918/101R8921 Key words: direct pattern completion, mediated pattern completion, amygdala, conditioned freezing, rats

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/hipo.22679 This article is protected by copyright. All rights reserved.

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Abstract Contextual fear conditioning involves forming a representation for the context and associating it with a shock, which were attributed by the prevailing view to functions of the hippocampus and amygdala respectively. Yet our recent evidence suggested that both processes require integrity of the dorsal hippocampus (DH). In view of the DH involvement in uniting multiple stimuli into a configuration, this study examined whether the DH would integrate context and shock into a shocked-context representation. Male Wistar rats were trained on a two-phase training paradigm of contextual fear conditioning. They explored a novel context on the first day to acquire a contextual representation, and received a shock in that context on the second day to form the context-shock memory. Tests of conditioned freezing given on the following days revealed two properties of configural memory—direct and mediated pattern completion: First, the contextual fear memory was retrieved in a novel context by a cue embedded in the configural set—a shock that did not elicit significant freezing on its own. Second, freezing was also elicited in a novel context by a transportation chamber that was not directly paired with the shock but could activate the fear memory inferentially. The effects were specific to the cue and not due to context generalization. Infusion of lidocaine into the DH, but not the amygdala, immediately after context-shock training impaired conditioned freezing elicited through either type of pattern completion. Our data suggest that the DH in contextual fear conditioning associates context and shock in parallel with the amygdala by incorporating the shock into an otherwise neutral context representation and turning it into a shocked-context representation.

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1. Introduction

The hippocampus is proposed to serve memory by integrating multiple sources of information into a configuration (Eichenbaum, 1999; Phelps et al., 1994; Sutherland and Rudy, 1989). Over the years, this function has been widely investigated in animals by using contextual fear conditioning in which an otherwise neutral context is paired with an aversive shock (Fanselow, 2000; Rudy and O'Reilly, 1999). As a context encompasses stimuli of various modalities, a dominant theory proposed that all context cues are first encoded into a representation by the hippocampus, and then this representation can associate with the shock in another brain region, say, the amygdala (Fanselow and Poulos, 2005; Rudy, 2009). Under this scenario, the hippocampus is viewed as a cognitive structure processing only contextual information and the amygdala adds an emotional tone to the otherwise neutral representation of context. However, the exact nature of hippocampal involvement in associative fear learning has been debated (Fanselow and Dong, 2010; Nalloor et al., 2012). In a two-phase paradigm of contextual fear conditioning that allows temporal detachment of context-shock learning from context coding, we have shown that inactivation of the dorsal hippocampus (DH) shortly after either phase of training impaired conditioned freezing in the test, suggesting involvement of the DH in both context binding and context-shock association (Chang et al., 2008), a notion consistent with activation of different immediate early genes or unit ensembles in the DH by these two learning processes (Lee, 2010; Moita et al., 2004). Anatomically, the hippocampus receives inputs from multiple brain areas (Amaral and Lavenex, 2007) including those activated by nociception and/or arousal, such as the cingulate cortex, locus coeruleus, ventral tegmental area, and raphe nuclei (Pickel et al., 1974; StormMathisen, 1977; Swanson and Hartman, 1975; White et al., 1990). The hippocampus also

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possesses abundant receptors of hormones released by stress (McEwen and Wallach, 1973). With all these inputs the hippocampus receives information not only from the external world but also from the internal milieu (Hsiao and Isaacson, 1971; Kennedy and Shapiro, 2004; Smith and Mizumori, 2006). In contextual fear conditioning, however, the so-called context representation formed in the hippocampus was usually referred to those external stimuli in the training environment (Bannerman et al., 2006; Nadel, 2008; Rudy, 2009). Yet in addition to these physical stimuli offered by the external context (Moita et al., 2004), the unconditioned stimulus (US) shock that elicits pain and arousal may activate somatic and visceral inputs to the hippocampus. There is no a priori reason to exclude all the shock-initiated information from the input domain of the DH in binding to the context cues of other sensory modalities to form a configural representation. In contrast to the Pavlovian association subserved by the amygdala, memory depending on the hippocampus is conceived to flexibly and adaptively express in new circumstances other than that of original learning (Eichenbaum et al., 1990; Phelps et al., 1994; Squire, 1992). One flexibility in such congfigural memory is referred to as pattern completion that relies on reciprocal connections between the hippocampus and cortex (O'Reilly and McClelland, 1994; Teyler and Discenna, 1986). Two kinds of pattern completion have been proposed based on behavioral observation. First, a memory trace can be reactivated fully by presenting part of its original stimulus configuration as a cue (O'Reilly and McClelland, 1994; Squire, 1992), which is noted as direct pattern completion. Second, the memory can also be retrieved by stimuli that are not contained in the original memory episode but shared common elements with it (Bunsey and Eichenbaum, 1996; Eichenbaum, 1999; Eichenbaum et al., 1999), which is noted as mediated pattern completion. Either property should be apparent in retrieving a configural memory that is hippocampus-dependent.

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To evaluate if the DH indeed binds context and shock into a configural representation of fear memory, this study tested the two aforementioned corollary features of hippocampusdependent configural memory in contextual fear conditioning and assessed roles of the DH in formation or consolidation of such memory.

2. Materials and Methods

2.1. Subjects Male Wistar rats obtained from Animal Breeding Center of National Taiwan University and BioLASCO Taiwan Co., Taipei, Taiwan were used in this study. Upon arrival, rats were individually housed and had free access to food and water. The vivarium was maintained on a 12 hr light/dark cycle (lights on at 9:00 am) with ambient temperature at 22-25 °C. Animals were subjected to stereotaxic brain surgery when they weighed 350-400 g. Experiments were always run in the light phase. All procedures were conducted according to Guidelines for the Care and Use of Laboratory Animals in Taiwan and were approved by Institutional Animal Care and Use Committee of National Taiwan University.

2.2 Surgery For cannula implantation, animals were anesthetized with sodium pentobarbital (ip, 45 mg/kg); atropine sulfate (ip, 0.4 mg/kg) was also given to reduce respiratory congestion. Two 23-gauge guide cannulae were bilaterally implanted into the DH (AP -3.8 mm, ML ±2.5 mm, DV -2.2 mm) or basolateral amygdala (BLA) (AP -2.7 mm, ML ±5 mm, DV -6.5 mm) under stereotaxic guidance (Kopf Instrument, Tujunga, California) from a position above the target regions. The cannulae were affixed onto the skull by three anchored jewelry screws with dental cement. A stainless stylet was inserted into the cannula to prevent contamination. Rats

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were allowed to recover from surgery for at least 7 days. Before the formal experiment, they were handled 1 min per day for 5 days. At the last day of handling, rats bearing cannulae were acclimated to the drug administration procedure by inserting the infusion needle into the cannula without infusing any liquid.

2.3. Apparatus Two identical boxes (30 × 24 × 24 cm; MED Associates Inc.), designated as Context A, were individually enclosed in sound/light attenuation chambers (60 × 40 × 58 cm) and used as the conditioning context for all experiments. The floor of Context A was composed of 18 grid bars (4 mm in diameter) with each spaced 16 mm apart from center to center and connected to a programmable shocker (Model VT 05448; MED Associates Inc.). A 10-W incandescent light bulb was mounted on the center of left wall in each sound/light attenuation chamber for illumination and a fan was located at the back wall for ventilation and generation of masking white noise. To provide a specific odor, the box was cleansed with 75% alcohol after each time of use. Another box designated as Context B bearing different contextual cues was used in some tests. It was a chamber (35 × 35 × 35 cm) with a trapezoid-shape interior created by black Plexiglas. The chamber floor consisted of 24-bar rods with a 10-degree slant. The rods were 3.5 mm in diameter and spaced 14 mm apart from center to center. In some experiments the floor of Context B was connected to the shocker for shock delivery. Illumination was from a 60-W house light on the ceiling of the room. The chamber was wiped with 2% acetic acid prior to each trial to provide a distinct odor. A preliminary experiment found negligible fear generalization between Context A and B if rats were shocked in one context and tested in the other (see supporting information Fig. S1).

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2.4. Behavioral Procedure The latent learning paradigm of contextual fear conditioning To temporally detach context-shock learning from context encoding, the present study adopted the latent learning paradigm of contextual fear conditioning. This paradigm trained rats in two successive daily sessions as previously described (Chang et al., 2008; Chang and Liang, 2012). Briefly, in the Context session, rats were carried individually from their home cages to Context A and allowed to freely explore the context for 2 min in order to create the contextual representation. Twenty-four hours later, in the Context-Shock session, rats were placed back to Context A for 20 s and received a footshock (2 s) with no signal at the end of it. The shock intensity was 0.75 mA for the intact or DH implanted animals. For the amygdala implanted animals the shock was 1.5 mA in the direct pattern completion experiment and 0.75 mA for 3 times in the mediated pattern completion experiment such that the freezing scores of these animals would be comparable to those of the hippocampus implanted animals if tested in the training context according to our preliminary results. The 20-s pre-shock interval in the context was chosen based on the previous findings that this interval was sufficient for animals to reactivate an already-formed contextual representation but not so to establish a new one (Fanselow, 1990; Kiernan and Westbrook, 1993). Some experiments included an immediate shock group in which the rat received the shock immediately after entering the context. This group served as a shock control group because the rats had little time to retrieve the context representation from memory of the last day, thus the context-shock association could not be formed in these animals (Fanselow, 1990; Kiernan and Westbrook, 1993). Evaluation of the direct pattern completion To evaluate the effect of direct pattern completion, one day after context-shock training in the cue-reminder test, upon entering Context B all animals received an immediate shock (0.75 mA, 2 s) as a reminder cue, which had been shown not to elicit freezing on its own

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(Fanselow, 1986), and were then tested for 6 min. Four days later, in the context-reminder test, all rats returned to Context A for another 6-min test. According to the principle of direct pattern completion, presenting either the shock or context should activate the memory, if these two stimuli have been integrated into a fear configuration (Fig. S2a). In order to minimize the interference of shock-elicited activity burst with the freezing behavior (Fanselow, 1982) and to make the data comparable for the two tests, the final analysis excluded the first-minute data of either test. Evaluation of the mediated pattern completion To evaluate the effect of mediated pattern completion, the latent learning paradigm was slightly modified based on the previous studies (Chang et al., 2008; Rudy and O'Reilly, 2001). Briefly, in the Context session a rat was transported from its home cage to Context A by a transportation Chamber T carried on a cart, this procedure required about 20 s. Chamber T was a white translucent plastic cage (45 × 25 × 15 cm) filled with sawdust on the floor and covered with plywood on the top. Once entering the context, the rat was allowed to freely explore Context A for 2 min at the first stay and then was carried back by Chamber T to its home cage and remained there for 40 s before the next transportation. This procedure was repeated for four extra times, but each time the rat stayed in Context A for only 40 s. This training protocol aimed to associate Chamber T with the full representation of Context A and thus turn it into a reminding cue for the trained context representation (Rudy and O'Reilly, 2001). In the Context-Shock session given 24 hrs later, animals were individually carried in their home cage to Context A and received the designated footshock 20 s after entry. After the shock training, rats were removed immediately from the context and received the posttraining treatment if necessary before returning to their home cages. It is important to note that Chamber T never appeared in this phase. Thus, animals retrieved their context memory

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by the 20-s re-exposure to the training context for learning the context-shock association (Fanselow, 1990). Further, in this session the representation contained only Context A and the shock, which was different from that formed in the Context session containing Chamber T and Context A. Yet both shared the common elements of Context A (Fig. S2b). In the cue-reminder test given one day after the shock training, animals were taken from their home cage and transported individually in Chamber T (20 s) to Context B to assess freezing behavior for 6 minutes. This procedure would allow Chamber T, acting as a retrieval cue, to activate first the representation of Context A that would in turn activate the configural representation of fear association, if the latter indeed exists. Four days after the cue-reminder test, all rats were carried individually by the experimenter to Context A and tested for another 6 minutes in the context-reminder test.

2.5. Drug infusion Lidocaine hydrochloride (Sigma, St. Louis, USA) was dissolved into phosphatebuffered saline (pH = 7.4). The concentration of lidocaine was 4% (W/V), which was shown to effectively inactivate the target regions (Malpeli, 1999; Martin, 1991). The infusion was accomplished by inserting a 30-gauge infusion needle into the guide cannula with its tip protruding beyond the cannula 1 mm for the DH infusion, and 2 mm for the amygdala infusion. Lidocaine or vehicle was infused simultaneously into both sides of the DH or BLA immediately after context-shock training. A total volume of 0.5 µl was infused into each side at a rate of 0.5 µl/min by a syringe pump (CMA/100, Carnegie Medicin, Stockholm, Sweden). After infusion, the needle stayed in the cannula for 1 min before being withdrawn.

2.6. Data analysis The behavior during the testing sessions was videotaped for 6 min. Two independent

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raters blind to treatments evaluated every 4 s whether the sampled behavior was freezing or not. Freezing was defined as absence of any movement except for respiration (Fanselow, 1982). The inter-rater reliability was over .95 across all experiments. The percentage of freezing in all behavior samples was calculated as a measurement of fear and subjected to data analysis. Analysis of variance (ANOVA) was applied to detect the general difference among groups. Post-hoc analyses for individual comparisons were conducted according to the adjusted procedures.

2.7. Histology At the conclusion of each experiment, the implanted rats were anesthetized with an overdose of anesthetics and perfused through the heart with saline followed by 10% formalin. The brain was removed and stored in formalin solution with 20% sucrose for at least 7 days, then 40-µm coronal sections were taken by a cryostat. The frozen sections throughout the cannula tract were mounted on slides and stained with thionin. Only animals with both cannulae correctly hitting the DH or BLA were accepted. Figure 1 shows the distribution of cannula tips in a sample of the experimental animals included in the final data analysis.

Insert Figure 1 about here

3. Results

3.1. Direct Pattern Completion in Contextual Fear Conditioning To evaluate whether context and shock are integrated into a configural representation of fear memory that can be activated through direct pattern completion by a shock, rats were trained and tested with the latent learning paradigm described in the Methods section. In the Context-Shock session animals were divided randomly into three groups: The No-Shock

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group (n = 12) stayed in Context A for 20 s without shock, the Imm group (n = 10) received an immediate shock upon entering Context A (< 1 s), and the Delay group (n = 10) received a shock 20 s after entering the context. Figure 2a shows the experiment protocol schematically. Figure 2b shows the mean freezing scores of the three groups in the two test sessions. A mixed design two-way ANOVA, with training treatments (No-Shock, Imm and Delay) as a between-subject variable and test sessions (cue-reminder and context-reminder) as a withinsubject variable. Results revealed a significant main effect of training treatments (F(2, 29) = 24.91; p < .01). However, neither the main effect of test sessions (F(1, 29) < 1) nor the twofactor interaction (F(2, 29) = 2.32; p > .05) was statistically significant. Planned comparisons with one-way ANOVA followed by the Tukey HSD post-hoc analysis showed that the Delay group had significantly higher freezing than the No-Shock or Imm group in both test sessions (p < .05). The data indicate that only the Delay group learned the contextual fear conditioning and this memory was readily activated by the training context or by a shock cue presented in a novel context.

Insert Figure 2 about here

3.2. Mediated Pattern Completion in Contextual Fear Conditioning To test if contextual fear memory can be activated by mediated pattern completion, two groups of naïve rats were trained with the protocol diagramed in Figure 3a. In the ContextShock session, they received either an immediate (Imm group; n = 5) or delay shock (Delay group; n = 6) upon entering the context. All animals were subsequently tested as described in the Methods section. Figure 3b shows the mean freezing scores of the two groups in the two tests. A mixed design two-way ANOVA with training treatments (immediate or delay shock) as a betweensubject variable and test sessions (cue-reminder and context-reminder tests) as a within-

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subject variable revealed a significant main effect for training methods (F(1, 9) = 12.26; p < .01) and test sessions (F(1, 9) = 13.32; p < .01). No interaction effect between two variables was found (F(1, 9) = 3.07; p > .05). Planned comparisons with two one-way ANOVAs revealed that the Delay group showed statistically more freezing than the Imm group in both tests (p < .01). These data indicate that the Delay group learned significantly better fear conditioning than the Imm group and this memory could be readily activated in the original training context or in a novel context by an intermediate cue linked to the fear representation.

Insert Figure 3 about here

3.3. Freezing in the cue-reminder test was related to the cues and not due to fear generalization To further evaluate whether freezing shown in the cue-reminder tests was indeed elicited by the specific cue, animals were subjected to the same training procedure as the delay group in experiment 3.2. In the Test 1 session, rats were randomly assigned to one of the three conditions: With no cue (the Non group; n = 8), an immediate shock (the Direct group; n = 8) or transportation by Chamber T (the Mediated group; n = 8) as a reminder just before testing their freezing behavior in the novel context (Context B). Four days later, all animals received the context reminder test in the training context (Fig. 4a). Figure 4b shows the mean freezing scores of three groups in the two test sessions. A mixed design two-way ANOVA with groups (Non, Direct and Mediated groups) as a betweensubject variable and test sessions (cue-reminder and context-reminder tests) as a withinsubject variable revealed significant main effects for groups (F(2, 21) = 13.46; p < .01) and test sessions (F(1,21) = 22.98; p < .01). Most importantly, the interaction between two factors was significant (F(2, 21) = 4.83; p < .05). Post-hoc comparisons with the Tukey HSD test revealed

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that the Non group displayed significantly less freezing than the Direct and Mediated groups (both p < .01) in the cue-reminder test session, but no significant difference was found among the three in the context-reminder test session. When freezing of the corresponding group was compared between Test 1 and Test 2, the Non group displayed less freezing in Test 1 than in Test 2 (p < .01), but the Direct and Mediated groups had compatible freezing in Test 1 and Test 2. The data suggest that the freezing behavior in the novel context was indeed elicited by the specific cue applied immediately before the test and not due to fear generalization.

Insert Figure 4 about here

3.4. Differential Effects of DH and BLA Suppression in Direct Pattern Completion Given the above results that a shock could be a cue to remind contextual fear memory in a novel context, we further tested whether temporary inactivation of the DH shortly after the Context-Shock session would disrupt formation of a configural representation of fear memory and thus rendered the reminding shock unable to elicit conditioned freezing. Two groups of DH-implanted animals were subjected to the training procedure identical to that of the Delay group in experiment 3.1 and received intra-DH infusion of vehicle (Veh; n = 12) or lidocaine (Lido; n = 11) immediately after the Context-Shock session (Fig. 5a). The mean freezing scores in the tests are shown in Figure 5b. A mixed design ANOVA, with drug treatments (vehicle or lidocaine) as a between-subject variable and test sessions (cue-reminder and context-reminder) as a within-subject variable, showed a significant main effect of drug treatments (F(1, 21) = 17.83; p < .01), indicating that lidocaine infusion after the training impaired freezing in both tests. Neither the main effect of test sessions (F(1, 21) < 1) nor the interaction between the two factors (F(1, 21) < 1) was statistically significant. According to the view that the amygdala, particularly the BLA, is involved in direct

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context-shock association in contextual fear conditioning (Fanselow and LeDoux, 1999; Maren and Hobin, 2007; Phillips and LeDoux, 1992), one would expect that inactivating the BLA shortly after the Context-Shock session should impair freezing elicited by the training context but not that by a reminder shock in a novel context, as the latter effect relies on the hippocampus-dependent configural memory. To test this notion, two groups of rats bearing indwelling cannulae in the BLA were trained and tested with the procedure of Figure 5a. After shock training, vehicle (Veh; n = 13) or lidocaine (Lido; n = 13) was infused into the BLA. The mean freezing scores in the two tests are shown in Figure 5c. Perturbing the BLA with cannulae implantation rendered rather moderate fear memory in the cue-reminder test; however, the context-shock learning was still substantial as freezing behavior expressed in the test phase was significantly higher than that in the Context session (dependent t-tests; Veh group: 18.97% ± 6.21% vs. 1.02% ± 0.58%, t(12) = 2.91, p < .01; Lido group: 11.49% ± 1.83% vs.1.54% ± 0.72%, t(12) = 5.22, p < .01). A mixed design two-way ANOVA with drug treatments as a between-subject variable and test sessions as a withinsubject variable showed no main effect of drug treatments (F(1, 24) = 3.16; p > .05) but a significant main effect of test sessions (F(1, 24) = 20; p < .01). Most importantly, there was a significant interaction between the two factors (F(1, 24) = 7; p < .05). Post-hoc comparisons with the Tukey HSD test revealed that the Veh group displayed significantly more freezing than the Lido group in context-reminder test (p < .01) but not in the cue-reminder test.

Insert Figure 5 about here

3.5. Differential Effects of DH and BLA Suppression in Mediated Pattern Completion Based on the findings that conditioned freezing can be activated by cues indirectly linked to the representation of fear memory, we further tested whether intra-DH infusion of

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lidocaine immediately after the Context-Shock session would impair retrieval of configural memory by a mediated cue. Two groups of animals were subjected to the training procedure as the Delay group in experiment 3.2. After the Context-Shock session, rats received either vehicle (Veh; n = 17) or lidocaine (Lido; n = 17) infused into the DH (Fig. 6a). Results showed that animals receiving lidocaine expressed less freezing in both tests (Fig. 6b). A mixed design two-way ANOVA, with drug treatments (vehicle or lidocaine) as a betweensubject variable and test sessions (cue-reminder and context-reminder tests) as a withinsubject variable, showed significant main effects for drug treatments (F(1, 32) = 9.21; p < .01) and test sessions (F(1, 32) = 33.13; p < .01). The interaction effect between two variables did not reach statistical significance (F(1, 32) = 3.65; p > .05). To evaluate whether the mediated pattern completion effect would also involve the BLA, two groups of animals bearing BLA cannulae were trained with the same procedure except for three footshocks being administered. Animals received intra-BLA infusion of either vehicle (n =13) or lidocaine (n =11) shortly after the final shock. The mean freezing scores in the two tests are shown in Figure 6c. A mixed design two-way ANOVA, with drug treatments as a between-subject variable and test sessions as a within-subject variable, revealed significant main effects of drug treatments (F(1, 22) = 10.31; p < .01) and test sessions (F(1, 22) = 63.86; p < .01), as well as significant interaction between the two variables (F(1, 22) = 17.81; p < .01). Post-hoc analysis with the Tukey HSD revealed that the Veh group displayed significantly more freezing than the Lido group in the context-reminder test (p < .01), but no difference was detected in the cue-reminder test. While both groups with BLA infusion in the cue-reminder test had moderate performance, the freezing scores in the test phase were still significantly higher than those in the Context session (dependent t-tests; Veh group: 18.71% ± 2.08% vs. 1.02% ± 0.44%, t(12) = 8.9, p < .01; Lido group: 17.38% ± 5.24% vs. 1.51% ± 0.69% t(10) = 5.55, p < .01), suggesting substantial learning in both groups and no floor effect

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in the comparison between them for the cue-reminder test.

Insert Figure 6 about here

4. Discussion

The present results, to the best of our knowledge, provide the first piece of empirical evidence demonstrating direct and mediated pattern completion in retrieval of contextual fear memory. Such findings suggest that memory of context-shock pairing can be represented in the hippocampus as a kind of configural memory. This view was further supported by the findings that temporary inactivation of the DH, but not BLA, shortly after the Context-Shock session impaired consolidation of the configural fear memory and compromised conditioned freezing elicited by direct or mediated pattern completion in the memory tests. The first experiment demonstrated that memory of the context-shock association can be activated by a shock given immediately prior to the test in a novel context, which by itself did not elicit freezing (Fanselow, 1986; Fanselow, 1990), consistent with an interpretation of direct pattern completion in retrieving the hippocampus-dependent memory. It has been proposed that the unconditioned responses (UR) of a shock are biphasic and freezing is a secondary UR with a longer response latency (Wagner and Brandon, 1989). This view raised a possibility that the freezing observed after the reminder shock might have been an UR of the shock rather than a conditioned response (CR) related to the configural memory of a context paired with shock. However, this interpretation failed to account for results of the Imm or No-Shock group in experiment 3.1 that rats acquiring little or no context-shock association expressed much lower freezing to the reminding shock in the cue-reminder test (Fig. 2). This finding supports the view that in contextual fear conditioning, freezing is not a direct UR to

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the shock but instead a CR relying on fear conditioning (Fanselow, 1982; Fanselow, 1986). The low freezing score in the Imm group also ruled out a possibility that the higher freezing in the Delay group of experiment 3.1 was mainly due to shock sensitization induced by the first shock given on the day before, as the Imm group also received the same stimulus 24 hrs before the reminding shock in the test. The notion that context and shock can be integrated into a single configuration is further supported by the present findings that the fear memory can also be activated in a new context by a reminder cue that never appeared along with the shock during training but was somehow linked to it indirectly (Fig. 3). This mediated retrieval property is viewed as another character of configural memory (Eichenbaum et al., 1999). This property found in experiment 3.2 is incompatible with the explanation that conditioned freezing observed in the novel context could have been a UR, as no shock was administered in this case. It is possible that rats in experiment 3.2 might first associate Chamber T to Context A and then Contex A to the shock. Thus, the freezing response elicited by Chamber T during the cue-reminder test might still rely on the simple association process of sensory preconditioning. While such a possibility cannot be entirely ruled out, however, it is important to note that Chamber T and Context A in our training protocol were presented sequentially for a limited period of time without any temporal overlap at all. Previous evidence has shown that sensory preconditioning is hard to occur between two non-overlapping sensory stimuli (Kamin, 1961; Prewitt, 1967). Experiment 3.3 demonstrated direct and mediated pattern completion in retrieving the context-shock representation with the same training procedure. It showed that the freezing behavior observed in a novel context was indeed due to the specific cue stimulus (the shock or transportation chamber in experiment 3.1 or 3.2, respectively) incorporated into the configural memory, as freezing became negligible when such cue was omitted in the new Context B (Fig. 4). The low freezing score observed in the Non group of experiment 3.3 also suggests little or

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no generalization between Context A and B, congruent with the supporting information in Fig. S1. A fear generalization effect between the two adopted contexts thus cannot account for our data. Inactivation of the DH but not BLA after Context-Shock training clearly compromised retention in the pattern completion test, suggesting that the process uniting the context and the shock into a configural representation of fear memory relies on the DH rather than the BLA. However, it is noted that freezing scores of rats bearing amygdala cannulae were relatively low in the pattern completion test even under a more intense training shock. Implantation of cannula or electrode into a brain target as ventral as the amygdala may cause neural damages contributing to this modest retention, as former studies have shown that in an avoidance task amygdala-implanted controls had retention much poorer than the non-implanted controls (Gold et al., 1978; Liang et al., 1982). One might argue that the low control performance in cue-reminder tests might have rendered a floor effect to mask a potential amygdala influence. However, our data did show substantial acquisition of the context-shock association in the amygdala-implanted rats as their freezing after the shock training in the test was much higher than that in exploring a novel context before the shock training, suggesting a possibility of expressing deficits induced by the amygdala treatment and thus rendering the floor-effect interpretation less plausible. A role of the DH in integrating context and shock appears to differ from the prevailing view that in contextual fear conditioning the hippocampus is involved in coding stable stimuli into a context memory but not associating it with a shock (Nadel, 2008; Phillips and LeDoux, 1994; Rudy, 2009; Zelikowsky et al., 2014). Such a view was mainly based on manipulative effects or neural correlates detected in the DH for the context or context-shock learning task and also on attribution of the effect or correlate found in both kinds of tasks to a common part of the two—context representation. However, a former study showed that while expression

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of Arc in the DH increased in both context learning and context-shock association, amygdala suppression blocked the effect only in the latter case (Huff et al., 2006), implying that the Arc expression in the two cases relied on different mechanisms. Further, our previous study on a two-phase contextual fear conditioning task found that forming a context representation and associating it to the shock differentially involved GABAA and muscarinic activity in the DH (Chang and Liang, 2012). Such data support that the DH has a role in context-shock learning in addition to context representation, which is consistent with other studies showing that some DH genes were activated only in learning a new context or in updating the context by shock (Lee, 2010), while others were activated specifically by context exposure plus presentation of shock but not by either event alone (Hall et al., 2000; von Hertzen and Giese, 2005). Our study did not exclude the role of the amygdala in context-shock association of fear conditioning. Indeed, the results that intra-BLA infusion of lidocaine impaired conditioned freezing in the original training context are consistent with the view that the amygdala is critical for contextual fear conditioning (Helmstetter and Bellgowan, 1994; Kim et al., 1993; Phillips and LeDoux, 1992). Yet BLA suppression after context-shock training did not affect expression of fear memory in the direct or mediated pattern completion test. The differential effects of BLA and DH suppression after context-shock training suggest that the two areas may code the experience through different mechanisms: The amygdala accomplishes fear conditioning by treating context and shock as independent events and linking them by simple Pavlovian association to elicit various emotion responses through S-R circuitry (Davis, 1992; Fanselow and Poulos, 2005; Phillips and LeDoux, 1992). Conversely, the DH integrates all sensory inputs from the two events into a whole to form a combinatorial S-S representation, as first proposed by Tolman (1948) and then followed by others (Eichenbaum et al., 1999; Nadel et al., 1985). This view is in parallel with findings in human fear conditioning that participants acquired both conditioned autonomic responses to a CS and declarative facts of

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the CS-US relationship (Cacciaglia et al., 2014), amygdala lesions impaired the former and hippocampal lesions impaired the latter (Bechara et al., 1995; Clark and Squire, 1998; LaBar et al., 1995). If both the DH and amygdala in parallel support context-shock association, removing a configural component of this memory by inactivation of the DH should leave what subserved by the amygdala in operation. Rats with the DH inactivated after shock training should have shown more freezing when tested in the training context than in a novel context reminded by a cue, as simple association in the amygdala was available in the former case. However, our data showed that rats receiving intra-DH infusion of lidocaine had comparably low freezing in both cue- and context-reminder tests for either direct or mediated pattern completion (Fig. 5 and 6). One possible interpretation is that the DH may modulate the BLA to alter a simple association between context and shock (Calandreau et al., 2006; Maren and Fanselow, 1995; Maren and Hobin, 2007; Mello et al., 1992). Thus, the DH manipulation may not only disrupt the configural fear memory formed in the DH but somehow weaken the Pavlovian association in the BLA. Alternatively, in view of the influence of amygdala stimulation on hippocampal long-term potentiation (Frey et al., 2001; Ikegaya et al., 1996), the memory effects formerly ascribed to the amygdala could be due to amygdala modulation of memory processing in the DH that receives amygdala inputs (Pikkarainen et al., 1999). Suppressing the target site DH would compromise not only memory processing in situ but also that from a modulating site and thus yield similar memory deficits in both the cue- and context-reminder tests. Direct evidence discriminating these two possibilities remains to be found. In summary, this study provides the first piece of supportive evidence for configural learning of context and shock in fear conditioning. These results modify and extend the previous view that such conditioning only involves simple association within the amygdala (Fanselow and LeDoux, 1999; Fanselow and Poulos, 2005) by showing that the hippocampus

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can achieve a similar goal by combinatorial learning in forming a configural memory, which can be retrieved by pattern completion either in a direct or mediated fashion. These findings, taken together with the previous ones, suggest that the brain may code the same learning task in multiple ways that engage different neural circuits in parallel (McDonald and White, 1993; Squire, 1986; Squire and Dede, 2015)—each subserving the same function by different means. Such brain degeneracy (Edelman and Gally, 2001; Tononi et al., 1999) may not only afford graceful memory degradation after local brain damages but also allow the organism to code an experience in various ways and apply it adaptively to an ever-changing world. The exact neural circuit of how a configural representation of context with shock activates fear reaction remains to be elucidated. In addition to connection with the amygdala, the hippocampus also projects to other brain regions implicated in anxiety and fear, e.g. the bed nucleus of the stria terminalis (Staff et al., 2000; Walaas and Fonnum, 1980), stimulating the DH with drug could alter emotional responses (Engin and Treit, 2007). In light of the common features between rat’s configural learning and human declarative memory (Phelps et al., 1994), further pursue of this issue may eventually lead to disclosure of neural mechanisms underlying memory of pathological fear in humans.

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Acknowledgement

This study was supported by grants NSC 98-2410-H-002-024-MY3, NSC 101-2410-H-002082-MY3 from the Ministry of Science and Technology as well as Research Projects for Promoting University Excellence 10R80918/101R8921 from Ministry of Education, Taiwan, ROC.

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Figure Legends

Figure 1. Anatomical placement of infusion needle tips in a sample of animals that received vehicle or lidocaine infusion in the (a) dorsal hippocampus and (b) basolateral amygdala as shown on the section plates of brain atlas adapted from Paxinos and Watson (1986).

Figure 2. Presentation of a reminder shock elicited freezing only in animals acquiring the conditioned fear response. (a) A schematic illustration of the experimental procedure. During the Context-Shock session, animals in the No-Shock group were placed into Context A for 20 s without any shock presentation. The Imm group received the shock immediately upon entering Context A. The Delay group received a shock 20 s after being placed into Context A. One day after shock training, animals were placed into Context B and received an immediate shock (IMM) as a reminder cue to activate the context fear memory and tested for 6 min (cuereminder). Four days later, rats were tested again in Context A for additional 6 min (contextreminder). (b) Percentage of freezing (mean ± SEM) during 2-6 minutes of two test sessions. *p < .05; **p < .01 compared with the Delay group.

Figure 3. Activation of configural fear memory by mediated pattern completion. (a) A schematic illustration of the experimental procedure. During the Context session, a specific Chamber T was used to transport animals from their home cage (H) to Context A. During the Context-Shock session, rats were transported by their home cage to Context A and received either an immediate (< 1s; Imm group) or 20-s delayed shock (Delay group) after entering the context. During the test phase, animals were transported by Chamber T to Context B in the cue-reminder test or by the experimenter (EXPer) to Context A in the context-reminder test for assessing freezing behavior. (b) Percentage of freezing (mean ± SEM) in the two test

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sessions. **p < .01 between the Delay and Imm groups.

Figure 4. Freezing was specific to the cue in the cue-reminder test. (a) A schematic illustration of the experimental procedure. Animals were subjected to the training procedure of the mediated pattern completion paradigm. In the cue-reminder test session administered in the novel Context B, animals in the Non group did not receive any retrieval cue before the test while animals in the Direct group received an immediate shock and animals in Mediated group received chamber transportation as a reminder cue. Four days later, all rats were tested again in Context A for additional 6 min (context-reminder). T: transportation chamber; H: home cage; EXPer: experimenter. (b) Percentage of freezing (mean ± SEM) during 2-6 minutes of the two test sessions. **p < .01 compared with Non group.

##

p < .01 compared

with the corresponding performance during the cue-reminder test.

Figure 5. Effects of lidocaine infused into the DH or BLA on fear memory activated by direct pattern completion. (a) A schematic illustration of the experimental procedure. (b) Freezing behavior (mean ± SEM) of animals receiving intral-DH infusion of vehicle (Veh) or lidocaine (Lido) on the cue-reminder and context-reminder tests. (c) Freezing behavior (mean ± SEM) of animals receiving intra-BLA drug infusion on the two test sessions. *p < .05; **p < .01 compared with its related control.

Figure 6. Effects of lidocaine infused into the DH or BLA on mediated pattern completion. (a) A schematic illustration of the experimental procedure. (b) Freezing behavior (mean ± SEM) of animals receiving intral-DH infusion of vehicle (Veh) or lidocaine (Lido) on the two tests. (c) Freezing behavior (mean ± SEM) of animals receiving intra-BLA infusion of drug on the two tests. *p < .05; **p < .01 compared with its related control.

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The hippocampus integrates context and shock into a configural memory in contextual fear conditioning.

Contextual fear conditioning involves forming a representation for the context and associating it with a shock, which were attributed by the prevailin...
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