Roles of hippocampal GABAA and muscarinic receptors in consolidation of context memory and context–shock association in contextual fear conditioning: A double dissociation study

Neurobiology of Learning and Memory 98 (2012) 17–24

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Roles of hippocampal GABAA and muscarinic receptors in consolidation of context memory and context–shock association in contextual fear conditioning: A double dissociation study Shih-Dar Chang a, K.C. Liang a,b,⇑ a b

Department of Psychology, National Taiwan University, Taipei, Taiwan, ROC Neurobiology and Cognitive Science Center, National Taiwan University, Taipei, Taiwan, ROC

a r t i c l e

i n f o

Article history: Received 9 December 2011 Revised 6 April 2012 Accepted 12 April 2012 Available online 21 April 2012 Keywords: Latent learning Conditioned freezing Configural memory Neural plasticity Rat

a b s t r a c t Contextual fear conditioning involves forming a context representation and associating it to a shock, both of which involved the dorsal hippocampus (DH) according to our recent findings. This study tested further whether the two processes may rely on different neurotransmitter systems in the DH. Male Wistar rats with cannula implanted into the DH were subjected to a two-phase training paradigm of contextual fear conditioning to separate context learning from context–shock association in two consecutive days. Immediately after each training phase, different groups of rats received bilateral intra-DH infusion of the GABAA agonist muscimol, 5HT1A agonist 8-OH-DPAT, NMDA antagonist APV or muscarinic antagonist scopolamine at various doses. On the third day, freezing behavior was tested in the conditioning context. Results showed that intra-DH infusion of muscimol impaired conditioned freezing only if it was given after context learning. In contrast, scopolamine impaired conditioned freezing only if it was given after context–shock training. Posttraining infusion of 8-OH-DPAT or APV had no effect on conditioned freezing when the drug was given at either phase. These results showed double dissociation for the hippocampal GABAergic and cholinergic systems in memory consolidation of contextual fear conditioning: forming context memory required deactivation of the GABAA receptors, while forming context–shock memory involved activation of the muscarinic receptors. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction The hippocampus is implicated in learning and memory functions, particularly for coding the relationship among stimuli (Eichenbaum, 1999; Squire, 1992; Sutherland & Rudy, 1989). Contextual fear conditioning is often employed to study such a function as the context being associated with the shock is composed of multiple stimuli (Fanselow, 2000; Rudy, 2009). In this task, a typical training procedure is to place a rat into a novel chamber and a few minutes later a footshock is administered; freezing is measured subsequently in the same context as an index of fear memory (Fanselow, 1990; Kiernan & Westbrook, 1993). Lesions or temporary inactivation of the dorsal hippocampus (DH) before or shortly after training impaired performance in a retention test, suggesting a role of the DH in learning and memory for this task (Esclassan, Coutureau, Di Scala, & Marchand, 2009; Gale, Anagnostaras, & Fanselow, 2001). As similar DH lesions did not

⇑ Corresponding author at: Department of Psychology, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 10617, Taiwan, ROC. Fax: +886 2 2362 9909. E-mail address: [email protected] (K.C. Liang). 1074-7427/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nlm.2012.04.004

affect acquisition and/or retention in cue conditioning (Kim & Fanselow, 1992; Phillips & LeDoux, 1992), most researchers proposed that the DH is involved in context representation, but not in shock association that is achieved in the amygdala (Fanselow & Poulos, 2005; Maren & Fanselow, 1995; Rudy, Barrientos, & O’Reilly, 2002). However, in a training paradigm separating context coding and shock association into two phases (Fanselow, 1990; Rudy & O’Reilly, 2001) some recent studies have shown that blocking the DH shortly after either phase of training impaired conditioned freezing (Chang, Chen, & Liang, 2008; Lee, 2010), suggesting involvement of the DH in both processes of learning. This suggestion is corroborated by further evidence that memory formation of context coding and context–shock association activated within the hippocampus different sorts of immediate early genes (Hall, Thomas, & Everitt, 2000; Huff et al., 2006; von Hertzen & Giese, 2005), neuronal responses (Berry & Seager, 2001; Berry & Thompson, 1978; Moita, Rosis, Zhou, LeDoux, & Blair, 2004) or synaptic modifications (Sacchetti et al., 2001, 2002). These findings, taken altogether, raise a question of whether these two types of learning may be mediated by different processes in the DH, such as distinct neurotransmitter systems, and thus can be doubly dissociated.

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Among the various neurotransmitter systems in the DH, roles of muscarinic, GABAA, 5HT1A and NMDA receptors in learning and memory have been well documented (Buzsaki & Chrobak, 1995; Farr, Flood, & Morley, 2000; Meneses & Hong, 1997; Morris, Davis, & Butcher, 1990; Tinsley, Quinn, & Fanselow, 2004). Previous studies using the typical contextual fear conditioning task have shown that conditioned freezing to a context was compromised by intra-DH infusion of GABAA agonists (Bast, Zhang, & Feldon, 2001), 5-HT1A agonists (Stiedl, Misane, Spiess, & Ogren, 2000), NMDA antagonists (Quinn, Loya, Ma, & Fanselow, 2005; Schenberg & Oliveira, 2008) or muscarinic antagonists (Gale et al., 2001; Wallenstein & Vago, 2001) given before or after training. However, as typical training trials in contextual fear conditioning engage both context representation and context–shock association, these findings failed to dissociate the drug effect on each of the two processes. In addition, those studies adopting a pretraining drug regimen could not clearly rule out the confounding of the influence on performance from that on learning and memory processing per se. To pursue whether memory formation for the two types of learning in contextual fear conditioning involves different neurotransmitters in the DH, this study adopted a two-phase training paradigm of contextual fear conditioning in which context coding and context–shock association were separated in two learning phases and examined effects of intra-DH infusion of the GABAA agonist muscimol, 5HT1A agonist 8-OH-DPAT, NMDA antagonist APV or muscarinic antagonist scopolamine immediately after either learning phase. Our results revealed that hippocampal GABAA and muscarinic receptors were differentially involved in the memory consolidation processes of context coding and context–shock association. 2. Materials and methods 2.1. Subjects Male Wistar rats were obtained from the Animal Center of National Taiwan University, Taipei, Taiwan. They were individually housed with free access to food and water, and were maintained on a 12/12 h light–dark cycle (lights on from 9:00 am to 9:00 pm) with 22–25 °C ambient temperature and 60–70% relative humidity. The animal care followed Guidelines for Animal Research of Agriculture Council, Taiwan, ROC. All experiments were carried out in the light phase and were conducted in accordance with the protocol approved and monitored by the Institutional Animal Care and Use Committee of National Taiwan University. 2.2. Surgery Rats received brain surgery for cannula implantation after their body weight reaching 350 g. They were treated with atropine sulfate (0.4 mg/kg) to prevent respiratory congestion and were anesthetized with sodium pentobarbital (45 mg/kg ip). The deeply anesthetized rat was mounted onto a stereotaxic instrument (Model 900, David Korpf Instrument, Tijuna, USA) and a midline incision was made on the scalp to expose the skull. Two 23-gauge stainless steel guide cannulae were implanted bilaterally into the DH (AP 3.8 mm, ML ±2.5 mm, DV 2.2 mm). Cannulae were affixed on the skull with dental cement anchored by three screws. To prevent contamination, a stainless stylet was inserted into each cannula after the surgery. Rats were allowed to recuperate in their home cage for at least 7 days until complete recovery. Before the behavioral task, rats were handled 1 min per day for 5 days, and 1 day before the experiment all rats were acclimated to the infusion procedure except no liquid actually dispensed.

2.3. Apparatus Fear conditioning for all experiments was carried out in two identical training boxes (30  24  24 cm; MED Associates Inc.) used as the conditioned context. Each of them was individually housed in a wooden chamber for attenuation of sound and light (60  40  58 cm). Electric shock was delivered through the bargrid floor of the box connected to a programmable shocker (Model VT 05448; MED Associates Inc.). A 10-W incandescent light bulb was mounted on the chamber wall for illumination. A fan was located at the back of the chamber for ventilation and generation of a masking white noise. Each box was cleansed with 75% alcohol before each use. 2.4. Behavioral procedure The two-phase training paradigm of contextual fear conditioning was composed of two training sessions in two successive days as previously described (Chang et al., 2008). In the Context session on the first day, rats were carried individually from their home cages to the conditioning context for an exposure of 2 min and then returned to their home cages. In the Context–Shock session on the next day, rats returned to the same context and 20 s after its entering a single footshock (2 s, 0.75 mA) was presented. Immediately after receiving the shock, rats were removed from the context and returned to its home cage. Based on the previous findings (Chang et al., 2008; Fanselow, 1990; Kiernan & Westbrook, 1993), 20 s of re-exposure was enough for the animal to reactivate the existing memory of a pre-exposed context but not enough to encode a new context representation. Thus such a session allowed merely association of shock to an already-formed context representation. Consequently, the present paradigm separated context learning from context–shock association and permitted clean examination of the drug effect on each of the two learning processes. Twenty-four hours later, rats were carried individually to the conditioning context for a retention test. Freezing behavior, defined as absence of any body movement except for respiration (Fanselow, 1982), was sampled every 4 s for 6 min during the Test session. Two independent raters blind to the type of posttraining treatments evaluated whether the sampled behavior was freezing or not. The inter-rater reliability was over .95 across experiments. 2.5. Drug infusion This study adopted a posttraining infusion regimen such that any effect created could be attributed clearly to influences on memory processing per se (McGaugh, 2000). Different doses of the muscarinic receptor antagonist scopolamine (Sigma, St. Louis, MO, USA, 5–50 lg), the GABAA receptor agonist muscimol (Sigma, St. Louis, MO, USA, 0.2–1.25 lg), the NMDA receptor antagonist APV (DLAPV; Tocris, Bristol, UK, 5 lg), or the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; Tocris, Bristol, UK; 2 or 4 lg) was dissolved into 0.5 ll phosphate-buffered saline (pH = 7.4). The doses were chosen based on the previous findings (Chang et al., 2008; Egashira et al., 2006; Mao & Robinson, 1998; Wallenstein & Vago, 2001). Infusion was carried out by a 30gauge infusion needle that was extended beyond the tip of the guide cannula for 1 mm. A total volume of 0.5 ll was simultaneously dispensed to each side of the DH at a rate of 0.5 ll/min by a syringe pump (CMA/100, Carnegie Medicin, Stockholm, Sweden). In view of the data that 1 ll of lidocaine, muscimol or DAPV inactivated an area in a tear-drop shape within 1 mm of the infusion needle tip (Martin, 1991; Steele & Morris, 1999), a volume of 0.5 ll used in this study would presumably encroach on an even smaller area in the target region without much invasion into the

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adjacent non-target tissue. After infusion, the needle stayed in the guide cannula for 1 min before being withdrawn. In some experiments animals were run on several batches due to capacity limitation of the laboratory; rats infused with vehicle were included in each batch of running. Preliminary analyses of the first-batch data allowed estimation of the subject number needed in following batches to detect an effect. This strategy contributed in part to the unequal subject numbers in various experiments. The data of different batches were combined only if the performance of vehicle-infused rats across various batches of running did not significantly differ and could be pooled together to form a collapsed control group of vehicle animals. Because the vehicle rats were run in every batch, this often rendered a greater number of subjects in the controls. 2.6. Histology At the completion of each experiment, animals 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 a formalin solution with 20% sucrose for at least 7 days. Coronal sections (40 lm) were taken throughout the cannula tract with a cryostat at 20 °C and every third section was mounted on a slide. Sections were stained with thionin and examined under a light microscope. Rats with both cannulae tips located correctly in the DH were included in the data analysis. Exclusion of subjects bearing misplaced cannulae also contributed to the variation in subject numbers in different groups. The distribution of cannula tips in a sample of animals receiving vehicle or various drug treatments is shown in Fig. 1. 3. Results 3.1. Posttraining intra-DH infusion of muscimol impaired formation of context memory To evaluate the role of DH GABAA receptors in memory processing of contextual cues, rats received intra-DH infusion of vehicle (n = 22) or muscimol in 0.2 lg (n = 9), 0.5 lg (n = 14) or 1.25 lg (n = 12) immediately after the Context session. Mean freezing scores of the four groups at the retention test shown in Fig. 2A indicate that muscimol caused a dose-dependent deficit. A one-way ANOVA revealed a significant difference among the groups (F(3, 53) = 3.34; p < .05). Post-hoc analyses with the Dunnett test revealed that the 1.25 lg group displayed less conditioned freezing than the vehicle group (p < .01). To examine the role of the DH GABAA receptor in context–shock learning, additional three groups of rats were trained on the task. Immediately after the Context–Shock session, they were infused with vehicle (n = 17) or muscimol at a dose of 0.5 lg (n = 14) or 1.25 lg (n = 10). Mean freezing scores of the three groups are shown in Fig. 2B. A one-way ANOVA revealed no significant difference among three groups (F(2, 38) < 1), suggesting that infusion of muscimol immediately after the Context–Shock session had no apparent effect.

Fig. 1. A schematic diagram showing distribution of infusion needle tips in the dorsal hippocampus on the brain plates adapted from Paxinos and Watson (1986) for a sample of animals receiving vehicle or various drug treatments.

freezing scores of the three groups during testing are shown in Fig. 3A. A one-way ANOVA revealed no significant difference amount the groups (F(2, 24) < 1). Two other groups of rats infused with vehicle (n = 6) or 4 lg 8-OH-DPAT (n = 9) into the DH immediately after the Context–Shock session. Fig. 3B shows the mean freezing score of each group in the retention test. While rats infused with 4 lg 8-OH-DPAT appeared to have a lower mean freezing score, an independent t-test revealed that it did not significantly differ from that of the vehicle group (t(13) = 1.1; p = 0.29). These findings suggested that 8-OH-DPAT administered after either the Context or Context–Shock session had a negligible effect on conditioned freezing.

3.2. Posttraining intra-DH infusion of 8-OH-DPAT did not affect formation of context memory or context–shock memory

3.3. Posttraining intra-DH infusion of APV did not impair formation of context memory or context–shock memory

To test the role of 5-HT1A receptors in the two memory processes of contextual fear conditioning, posttraining intra-DH infusion of 8-OH-DPAT was administered immediately after the Context or Context–Shock session. Three groups of rats received intra-DH infusion of vehicle (n = 12) or 8-OH-DPAT at a dose of 2 lg (n = 8) or 4 lg (n = 7) immediately after the Context session. Mean

This experiment examined the role of NMDA receptors in memory formation for each type of learning in contextual fear conditioning. Two groups of rats received either vehicle (n = 6) or 5 lg APV (n = 8) infused into the DH immediately after the Context session and two additional groups received intra-DH infusion of vehicle (n = 18) or 5 lg APV (n = 16) immediately after the

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Fig. 2. A schematic illustration of the experimental procedure and the effects of intra-DH infusion of muscimol at different doses on contextual fear conditioning. Scores in the groups indicate the percentage of freezing during the 6-min testing phase (mean ± SEM). The number of subjects in each group is shown at the bottom of each bar. (A) Effects of intra-DH infusion of muscimol after the Context session. ⁄⁄p < .01 compared with the vehicle group. (B) Lack of effect of intra-DH infusion of muscimol after the Context–Shock session.

Fig. 3. A schematic illustration of the experimental procedure and lack of effects of intra-DH infusion of 8-OH-DPAT at different doses on contextual fear conditioning. Scores in the groups indicate the percentage of freezing during the 6-min testing phase (mean ± SEM). The number of subjects in each group is shown at the bottom of each bar. No effect was found for intra-DH infusion of 8-OH-DPAT given after either the Context session (A) or the Context–Shock session (B) in comparison with the correspondent vehicle group.

Context–Shock session. The dose was chosen because it effectively impaired context–shock learning if given prior to shock training in a similar training paradigm (Chang et al., 2008). Mean freezing scores in the retention test for the groups receiving infusion after the Context or Context–Shock session are shown, respectively, in Fig. 4A and B. Independent t-tests revealed no significant difference between the two groups receiving vehicle and drug infusion either after the Context session (t(12) = 0.37; p = 0.37) or after the shock session (t(32) = 0.26; p = 0.8). Thus, posttraining infusion of APV into the DH immediately after either type of learning had no effect on conditioned freezing in contextual fear conditioning. 3.4. Posttraining intra-DH infusion of scopolamine impaired formation of context–shock memory To test for the role of hippocampal muscarinic receptors in the two memory processes, scopolamine was infused into the DH immediately after the Context session or Context–Shock session. Results from some of the above experiments hinted that performance of rats having vehicle after the Context session was not comparable to that of rats having vehicle after the Context–Shock

session. To better control for possible influences of infusion at different training phases on freezing, seven groups of rats were used in this experiment and they all received infusion at both training sessions. A vehicle group (n = 13) received vehicle after the Context and Context–Shock sessions; three other groups received scopolamine at a dose of 5 lg (n = 8), 25 lg (n = 10) or 50 lg (n = 6) after the Context session but they all received vehicle after the Context– Shock session; the remaining three groups received vehicle after the Context session but they were treated with scopolamine at the dose of 5 lg (n = 8), 25 lg (n = 10) or 50 lg (n = 10) after the Context–Shock session. The mean freezing scores of various groups at the retention test are shown in Fig. 5A. Scopolamine induced a deficit of conditioned freezing during the Test session only if it was infused immediately after the Context–Shock session but had no effect if it was given after the Context session. The data were analyzed by two oneway ANOVAs with rats receiving the drug after the Context or Context–Shock session compared separately with the vehicle group. Results indicated that the three groups receiving scopolamine following the Context session did not differ from the vehicle group (F(3, 33) < 1) but the three groups receiving scopolamine after the

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Fig. 4. A schematic illustration of the experimental procedure and lack of effects of intra-DH infusion of APV on contextual fear conditioning. Scores in the groups indicate the percentage of freezing during the 6-min testing phase (mean ± SEM). The number of subjects in each group is shown at the bottom of each bar. APV (5 lg) infused into the DH after (A) the Context-session or (B) the Context–Shock session did not affect conditioned freezing.

Fig. 5. A schematic illustration of the experimental procedure and the effects of intra-DH infusion of scopolamine in contextual fear conditioning at different doses and time points. Scores in the groups indicate the percentage of freezing during the 6-min testing phase (mean ± SEM). The number of subjects in each group is shown at the bottom of each bar. (A) Effects of intra-DH vehicle or scopolamine infusion after the Context (post-C) or the Context–Shock session (post-C/S). (B) Effects of intra-DH scopolamine (50 lg) after the Context–Shock session at different time points. ⁄p < .05, ⁄⁄p < .01 compared with the vehicle group.

Context–Shock session significantly differed from the vehicle group (F(3, 37) = 3.34; p < .05). Post-hoc analyses with the Dunnett test revealed that the group receiving 50 lg of scopolamine after the Context–Shock session displayed less freezing than the vehicle group (p < .05). To investigate the time-dependent effect of the drug, three pairs of two groups received vehicle or the effective dose of scopolamine (50 lg) into the DH immediately (n = 10 for each group), 6 h (n = 8 for each group) or 12 h (n = 9 for each group) after the Context– Shock session. As the three groups receiving vehicle infusion at different times after training did not differ from each other (F(2, 24) < 1), their data were collapsed into a single control group (n = 27) to increase the power of the test. Mean freezing scores of the four groups at the retention test shown in Fig. 5B reveals that intra-DH infusion of scopolamine caused a time-dependent deficit. A one-way ANOVA revealed a significant difference among the groups (F(3, 50) = 3.57; p < .05). Post-hoc analyses with the Dunnett test indicated that scopolamine impaired retention only if it was given immediately after the shock (p < .01) but had no effect if given 6 h or 12 h after the shock (p > .05). 4. Discussion This study aimed to explore the neural bases within the DH underlying the formation of context memory and context–shock

memory in contextual fear conditioning. Results showed that posttraining infusion of the GABAA agonist muscimol and the muscarinic antagonist scopolamine into the DH respectively affected memory formation for the context representation and the context–shock association. These results not only replicated our former findings (Chang et al., 2008) that the DH was involved in both of these two processes in contextual fear conditioning but also extended those findings by showing a double dissociation for the neurochemical substrates underlying them. The observed effects in our study can be neatly attributed to alterations in memory formation for the two learning processes as the effects were induced by posttraining treatments and were time-dependent (McGaugh, 2000). In the first experiment, muscimol impaired contextual fear conditioning only if it was given after the Context session. This result is in line with previous findings showing that intra-DH infusion of interleukin-1 beta, a treatment facilitating the hippocampal GABA transmission (He et al., 2010), impaired contextual fear conditioning if the drug was given immediately after the Context session of a similar paradigm (Barrientos et al., 2002). These data suggest that normally GABAA receptors in the DH were deactivated during the context-coding period. In agreement with this view, previous studies also showed that intra-DH administration of muscimol impaired tasks that rely on contextual information, such as Morris water maze (Diergaarde, Schoffelmeer, & De Vries, 2008) and

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expression of an extinguished fear response (Corcoran & Maren, 2001). Our results, however, are incongruent with a previous study reporting that intra-DH infusion of muscimol (0.5 lg/0.5 ll) after the Context session of a similar task had no effect on conditioned freezing (Matus-Amat, Higgins, Barrientos, & Rudy, 2004). It should be noted that the only dose of muscimol tested in that study was ineffective according to our results, despite it was effective if given before the session (Matus-Amat et al., 2004). The differential susceptibility of acquisition and memory formation processes to the GABA suppression effect may account for the discrepancy of results. As muscimol silences the target areas by increasing inhibition on neurons bearing GABAA receptors, the effect of muscimol should be due to a specific GABAA influence on the DH. While the possibility that the observed effect was an indirect consequence of GABAA activation remains viable, the effect is very unlikely due to general suppression over the DH, as certain sodium channel blockers that silence all neuronal activities in the DH caused even greater interference than muscimol (Bast et al., 2001). It has been shown that microinfusion of 5-HT1A agonists into the DH affected memory processing in several tasks (Ben Mamou, Gamache, & Nader, 2006; Farr et al., 2000) including contextual fear conditioning (Stiedl, Misane, et al., 2000). However, according to our observation, 8-OH-DPAT given after context learning had no apparent effect on context memory, even at a dose up to 8 lg (data not shown). The seemingly lower performance of the post-shock 8OH-DPAT group still did not differ significantly from a control group in which all rats having vehicle at either phase were pooled together to increase the test power (t(25) = 1.58, p = 0.13). This study thus failed to find a strong effect of 8-OH-DPAT on memory formation for the two types of learning in contextual fear conditioning. Our results were incongruent with the previous findings that intra-DH infusion of this drug prior to a typical training paradigm of contextual fear conditioning impaired conditioned freezing in mice (Stiedl, Misane, et al., 2000). While the species difference might be a factor underlying this discrepancy, the effect reported by Stiedl and colleagues could be due to drug influence on processes other than memory formation as 8-OH-DPAT or other 5HT1A agonists, such as buspirone, given peripherally or into the DH altered animals’ nociception (Carli, Tranchina, & Samanin, 1992). Thus, the exact role of DH 5-HT1A receptors in memory processes of contextual fear conditioning should be pursued further in the future. Using the latent learning procedure previous studies showed that intra-DH infusion of APV prior to the Context session or Context–Shock session of this task impaired conditioned freezing (Chang et al., 2008; Matus-Amat, Higgins, Sprunger, Wright-Hardesty, & Rudy, 2007). Based on the findings that posttraining intraDH infusion of NMDA antagonists impaired retention in the water maze and inhibitory avoidance tasks (Liang, Hon, Tyan, & Liao, 1994; Packard & Teather, 1997; Roesler et al., 2005) and post-tetanic administration of APV impaired development of a late-phase LTP (Gong et al., 2011), we probed the role of NMDA receptors in memory formation of either process and found that APV infused after either training phrase failed to have an effect. The results are consistent with the findings that intra-DH infusion of APV before a typical training trial in contextual fear conditioning impaired conditioned freezing but the same treatment given after the training trial did not (Schenberg & Oliveira, 2008; Stiedl, Birkenfeld, Palve, & Spiess, 2000). With substantial number of subjects in each group, rats receiving post-shock APV had low freezing but did not differ from the controls that also showed low freezing. Whether APV, in other doses or under other conditions, exerts a mild effect on context–shock association that was concealed by the unexpected low control performance in this experiment should be pursued in the future. The present data, taken together with the

previous ones from this and other laboratories, may nonetheless be interpreted as that the DH NMDA receptors are less critical for consolidating than for encoding fear memory. Intra-DH infusion of scopolamine caused a dose- and timedependent memory deficit in conditioned freezing only if the drug was given after the Context–Shock session. Such findings were in line with the previous evidence that intra-DH infusion of scopolamine either before or after the training session of a typical contextual fear conditioning task impaired fear memory (Gale et al., 2001; Wallenstein & Vago, 2001). Most importantly, our results confirmed a critical role of the DH, via acting on its muscarinic receptors, in modulating context–shock memory of contextual fear conditioning. This effect could not be attributed to interference with processing shock information per se as intra-DH infusion of scopolamine did not affect fear conditioning to a cue (Rogers & Kesner, 2004; Wallenstein & Vago, 2001). However, our results appear to be inconsistent with the recent findings that intra-DH infusion of oxotremorine improved memory for the context rather than that for the shock in a modified inhibitory avoidance task separating the apparatus acclimation and shock administration in 2 days (Malin & McGaugh, 2006). This incongruence might be due to differences in the task nature and the type of behavior assessed (avoidance tendency versus freezing) as studies have shown that different measures of contextual fear engaged different neural substrates (Antoniadis & McDonald, 2000; Maren, 2007). Further, the oxotremorine findings by Malin and McGaugh (2006) assert that stimulation of the DH muscarinic receptors is sufficient for improving context memory but not for improving context–shock memory; our data on the other hand suggest that activation of muscarinic receptors in the DH is necessary to form context–shock memory and blocking these receptors simply renders failure of it. It remains to be speculated on how the two neurotransmission systems accomplish their functions. During the Context session, multiple cues in a context need to be integrated into a unified representation (Fanselow, 1990, 1999; Rudy & O’Reilly, 1999). Such learning might rely on the rhythmic oscillation in membrane potentials of hippocampal neurons that sets up a dynamic state allowing relevant neurons to fire together for binding sensory inputs from the cortex (Bland & Oddie, 2001; Buzsaki & Chrobak, 1995; Eichenbaum, Dudchenko, Wood, Shapiro, & Tanila, 1999). The intrinsic GABA network within the DH contributes to such rhythmic activities, which are further subjected to intricate modulation of extrinsic GABA input acting on the GABAA receptors (Ylinen et al., 1995). Indiscriminate activation of the GABAA receptors by drug infusion during the period of context coding might thus impair formation of context memory by disrupting such rhythmic oscillation. The effect of intra-DH scopolamine may be related to modification of a pre-existing neutral context memory into an emotional one (Lee, 2010; Chang & Liang, 2010, Soc. Neurosci. Abstr.). According to a model proposed by Hasselmo (1999, 2006), any mismatch between the stored memory and the current sensory input may cause release of acetylcholine in the hippocampus, which would facilitate new information processing and modification of the existent memory (Hasselmo, 1999, 2006; also see Lorincz & Buzsaki, 2000). In line with this view, after lesions of septal cholinergic neurons that project mainly to the hippocampus, the DH place cells could be normally activated to form a new context representation, yet they were unable to remap their place fields if the context changed (Ikonen, McMahan, Gallagher, Eichenbaum, & Tanila, 2002; Leutgeb & Mizumori, 1999). However, the present results are unable to rule out a possibility that intra-DH infusion of scopolamine may modulate neural plasticity in the amygdala, such as down regulating the extracellular signal-regulated kinase (ERK) and thus impair conditioned freezing according to some previous

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