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. 2022 Aug;29(8):216–222. doi: 10.1101/lm.053611.122

Nucleus reuniens inactivation does not impair consolidation or reconsolidation of fear extinction

Krithika Vasudevan 1, Karthik R Ramanathan 1, Valerie Vierkant 1, Stephen Maren 1
PMCID: PMC9374271  PMID: 35902273

Abstract

Recent data reveal that the thalamic nucleus reuniens (RE) has a critical role in the extinction of conditioned fear. Muscimol (MUS) infusions into the RE impair within-session extinction of conditioned freezing and result in poor long-term extinction memories in rats. Although this suggests that RE inactivation impairs extinction learning, it is also possible that it is involved in the consolidation of extinction memories. To examine this possibility, we examined the effects of RE inactivation on the consolidation and reconsolidation of fear extinction in male and female rats. Twenty-four hours after auditory fear conditioning, rats underwent an extinction procedure (45 CS-alone trials) in a novel context and were infused with saline (SAL) or MUS within minutes of the final extinction trial. Twenty-four hours later, conditioned freezing to the extinguished CS was assessed in the extinction context. Postextinction inactivation of the RE did not affect extinction retrieval. In a second experiment, rats underwent extinction training and, 24 h later, were presented with a single CS to reactivate the extinction memory; rats were infused with SAL or MUS immediately after the reactivation session. Pharmacological inactivation of the RE did not affect conditioned freezing measured in a drug-free retrieval test the following day. Importantly, we found in a subsequent test that MUS infusions immediately before retrieval testing increased conditioned freezing and impaired extinction retrieval, as we have previously reported. These results indicate that although RE inactivation impairs the expression of extinction, it does not impair either the consolidation or reconsolidation of extinction memories. We conclude that the RE may have a critical role in suppressing context-inappropriate fear memories in the extinction context.

New memories, once acquired, must undergo consolidation to become stabilized in long-term memory (McGaugh 2000; Frankland and Bontempi 2005). During this consolidation period, memories are labile and can be disrupted by a variety of manipulations (Davis and Squire 1984; Haubrich and Nader 2018). Interestingly, long-term memories are also sensitive to disruption after the retrieval. The sensitivity of long-term memories to disruption suggests that, upon retrieval or reactivation, these memories become destabilized and must undergo a reconsolidation process to be maintained (Nader et al. 2000; Alberini 2005). The fact that retrieval increases the lability of long-term memory has considerable clinical relevance, because reconsolidation update procedures may have efficacy in treating phobias and posttraumatic stress disorder (Agren et al. 2012; Beckers and Kindt 2017; Phelps and Hofmann 2019; Ressler et al. 2021).

Another intervention for patients with stressor- and trauma-related disorders focuses on suppressing pathological fear memories using behavioral interventions. These procedures, which include prolonged exposure therapy, take advantage of extinction learning (Maren 2011; Milad and Quirk 2012; Craske et al. 2018). During extinction-based therapies, cues (or thoughts) associated with trauma are repeatedly experienced in a safe setting. This procedure yields a new extinction memory that competes with the original fear memory for expression in behavior (Pavlov 1927; Konorski 1967; Bouton et al. 2006, 2021). However, extinction memories are not as robust as their respective fear memories. Extinguished fear can relapse under a variety of conditions, including a change in context (renewal), a reminder of the aversive event (reinstatement), or the passage of time (spontaneous recovery) (Rescorla 2004; Goode and Maren 2019; Bouton et al. 2021). Therefore, understanding the ways in which extinction memories are stabilized may prove particularly useful in clinical interventions for stress- and trauma-related disorders.

Decades of work have revealed brain circuits involved in extinction learning and memory (Myers and Davis 2002; Quirk and Mueller 2008; Orsini and Maren 2012; Maren et al. 2013). This work has revealed a critical role for neural activity in the hippocampus (HPC) (Corcoran and Maren 2001; Corcoran et al. 2005; Tronson et al. 2009; Marek et al. 2018; Lacagnina et al. 2019), amygdala (Falls et al. 1992; Sotres-Bayon et al. 2007; Laurent et al. 2008; Zimmerman and Maren 2010; Trouche et al. 2013; Davis et al. 2017), and medial prefrontal cortex (mPFC) (Quirk et al. 2006; Sotres-Bayon et al. 2009; Peters et al. 2010; Do-Monte et al. 2015; Giustino and Maren 2015; Marek et al. 2018). Importantly, it has recently been shown that the thalamic nucleus reuniens (RE), a brain area that interconnects with the HPC and mPFC (Vertes et al. 2007; Cassel et al. 2013; Cassel et al. 2021), is critical for both the specificity of contextual memory (Xu and Südhof 2013; Ramanathan et al. 2018b) and extinction learning (Ramanathan et al. 2018a; Ramanathan and Maren 2019). For example, we found that infusion of the GABAA receptor agonist, muscimol, into the RE prior to extinction training resulted in severe impairments in within-session extinction learning and a lasting impairment in extinction retrieval (Ramanathan et al. 2018a; Ramanathan and Maren 2019). Moreover, chemogenetic inhibition of mPFC projections to RE reproduced these deficits in extinction learning and retrieval (Ramanathan et al. 2018a).

These results suggest that neuronal activity in the RE is essential for extinction learning, though it is also possible that the pharmacological manipulations of RE and its afferents also disrupted the consolidation of extinction memory after training. Consistent with this possibility, it has been reported that muscimol inactivation of the RE immediately after contextual fear conditioning can influence the consolidation and reconsolidation of contextual fear memories (Troyner et al. 2018; Troyner and Bertoglio 2021). Extinction and reconsolidation have often been characterized as opposing processes that result from memory retrieval (Pedreira and Maldonado 2003; Suzuki et al. 2004; Merlo et al. 2014). Brief (single) exposure to the CS favors the recruitment of reconsolidation, whereas many CS exposures generate extinction. Additionally, extinction memories are susceptible to disruption after retrieval, suggesting that they also become labile upon retrieval and undergo reconsolidation (Vianna et al. 2001; Rossato et al. 2010). Therefore, in this study, we examined the role of the RE in the consolidation and reconsolidation of extinction memory using Pavlovian fear conditioning and extinction procedures in rats and pharmacological inhibition of the RE during consolidation and reconsolidation windows.

Results

RE inactivation does not impair consolidation of extinction

To investigate the role of the RE in the consolidation of extinction, we inactivated the RE (Fig. 1A,B) with muscimol immediately after the extinction training session. The behavioral procedure for the experiment is schematized in Figure 2A. As shown in Figure 2B, rats exhibited low levels of baseline freezing prior to the first conditioning trial, but freezing behavior increased across the conditioning session (repeated measures ANOVA, main effect of trial, F(5,28) = 50.446, P < 0.001) and there were no differences in the groups assigned to receive SAL or MUS after extinction training. To reduce context fear to the conditioning context and limit generalization to the extinction context, rats were returned to the conditioning context the following day and underwent a 15-min context exposure session (data not shown). Rats exhibited a decrease in context freezing across the 15 min (F(5,28) = 42.169, P < 0.001). The following day, rats underwent extinction training (45 CS-alone trials) conducted in a novel context (context B). Rats in each group demonstrated robust freezing to the first CS presentation and exhibited a within-session decrease in freezing (repeated measures ANOVA, main effect of trial, F = 20.365, P < 0.001). No group differences were observed during extinction. Either immediately after extinction (IMM) or after a 6-h delay (DEL), rats received intra-RE microinfusions of SAL or MUS.

Figure 1.

Figure 1.

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(A) Representative thionin-stained coronal section showing cannula placement in RE. The inset shows cannula placement with anatomical labels overlaid. (B,C) Cannula injector tip locations for experiment 1 (B) and experiment 2 (C). Cannula placements were mapped onto rat brain atlas templates (Swanson 2018). Open circles indicate injector tips for MUS rats, and filled circles indicate injector tips for SAL rats.

Figure 2.

Figure 2.

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(A) Schematic for the behavioral procedures for experiment 1. Illustrations are adapted from Marek et al. (2018). (B) Conditioning: Percentage of freezing during the 3-min baseline and 1-min ISI following each of five CS–US pairings. Extinction: Percentage of freezing averaged across a 3-min baseline and nine five-trial blocks (30-sec ISIs). Testing: Average freezing across five CS test trials for rats infused with either saline (SAL; filled circles and squares) or muscimol (MUS; open circles and squares) either 10 min (SAL, n = 8; MUS, n = 7) or 6 h (SAL, n = 8; MUS, n = 7) after extinction. All data are means ± SEM. P < 0.05, P < 0.01, one-way factorial and repeated measures ANOVA.

Twenty-four hours after extinction, rats were returned to context A to assess renewal of extinguished fear in the conditioning context; this was followed by an extinction retrieval test the following day. We ran the renewal test because earlier work suggested that RE inactivation might affect the specificity of contextual memory (Xu and Südhof 2013; Ramanathan et al. 2018b; Troyner et al. 2018). If rats overgeneralized the memory of the extinction context, then they may exhibit less renewal of fear in the conditioning context. As shown in Figure 2B, average freezing across the five-trial retrieval tests was similar in VEH- and MUS-treated rats in both the renewal and extinction retrieval contexts. Nonetheless, rats exhibited robust renewal, exhibiting significantly greater CS-elicited freezing in the conditioning context compared with the extinction context. There were no differences between rats receiving intracranial infusions either immediately or 6 h after extinction. Importantly, average pre-CS freezing was low and there were no group or context differences in this measure (data not shown). These observations were confirmed in an ANOVA that showed a significant main effect of test context (F(1,28) = 45.11, P < 0.0001). There was neither a significant main effect of either postextinction infusion interval (F(1,28) = 0.005, P = 0.9461) or drug condition (F(1,28) = 0.003, P > 0.9602) nor a significant interaction between these factors (F(1,28) = 0.077, P = 0.7831). This reveals that postextinction inactivation of the RE does not impair the consolidation of extinction memories.

RE inactivation does not impair reconsolidation of extinction

The previous experiment indicates that the consolidation of extinction memory is not impaired by pharmacological inhibition of RE activity. However, it remains possible that RE activity, which we have previously shown to be essential for extinction retrieval (Ramanathan et al. 2018a; Ramanathan and Maren 2019), may be involved in the reconsolidation of extinction memories. Indeed, it has been reported previously that the RE is involved in the reconsolidation of contextual fear memories (Troyner and Bertoglio 2021). To investigate this possibility, we examined the effect of muscimol infusion in the RE immediately after the reactivation of an extinguished memory. The behavioral schematic for the experiment is shown in Figure 3A. Both conditioning (F(1,10) < 1.386, P = 0.2663) and extinction (F(1,10) = 0.139, P = 0.7173) proceeded similarly in animals designated to receive SAL or MUS after the reactivation session. The day after extinction, rats were returned to the extinction context for a reactivation procedure in which a single CS-only trial was delivered. One minute after CS reactivation, the rats were removed from the chambers and received intra-RE infusions of either SAL or MUS. The following day, the rats were returned to the extinction context and presented with five CS-alone trials to assess extinction retrieval. As shown in Figure 3B, rats receiving either SAL or MUS infusions exhibited similar levels of freezing, which were comparable with those in the previous experiment. This suggests that the RE is not necessary for the reconsolidation of extinction memory.

Figure 3.

Figure 3.

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(A) Schematic for behavioral design for experiment 2. (B) Conditioning: Percentage of freezing during a 3-min baseline and 1-min ISI for each trial. Extinction: Average freezing during a 3-min baseline, the first five-CS trial block, and the last five-CS trial block. Reactivation: Freezing averaged during the 1-min period after a single CS. The shaded bar indicates the timing of muscimol (MUS; open circles, n = 8) or saline (SAL; filled circles, n = 6) infusions immediately after reactivation. Retrieval, off drug: Average freezing across five CS test trials. (C) Average freezing for 3-min baseline and 30-sec ISI for the drug-free retrieval test. All data are means ± SEM. P < 0.05, P < 0.01, one-way factorial and repeated measures ANOVA.

To confirm that muscimol inactivation of the RE was effective, we infused the RE with either SAL or MUS prior to an extinction retrieval test. We have previously reported robust impairments in extinction retrieval with this procedure (Ramanathan et al. 2018a; Ramanathan and Maren 2019). As shown in Figure 3C, MUS infusions into the RE produced a robust increase in conditioned freezing to the extinguished CS. This was confirmed in a repeated measures ANOVA, which revealed a significant main effect of drug (F(1,10) = 6.635, P = 0.0276); there was no main effect of trial (F(1,10) = 1.372, P = 0.2610), though there was a significant drug × trial interaction when comparing baseline freezing with the freezing across the first five tones (F(1,10) = 7.176, P = 0.0231). These results reveal that although pharmacological inactivation of the RE produces robust impairments in extinction retrieval, it has no effect on the consolidation or reconsolidation of extinction memories.

Discussion

We have previously reported that muscimol inactivation of RE impairs the acquisition and expression of extinction after auditory fear conditioning in rats. It is possible that the effects of RE inactivation on extinction learning are mediated by enduring effects of muscimol that influence the consolidation of the extinction memory. However, the present results reveal that postextinction (or postreactivation) inactivation of the RE has no effect on conditioned freezing behavior in subsequent retrieval tests. The absence of a deficit in extinction retrieval or renewal was not due to a failure of muscimol to regulate memory processes mediated by the RE. Indeed, we showed that MUS inactivation of the RE produced robust impairments in extinction retrieval when infused immediately before the test session. Hence, although the RE is necessary for the successful encoding and retrieval of extinction memory, it is not involved in either the consolidation or reconsolidation of that extinction memory. Local inhibition of RE neurons with muscimol immediately after extinction training or after reactivation of the extinction memory did not prevent successful retrieval of the extinction memory in subsequent tests. The present study therefore suggests a distinction between the role of the RE in the encoding and retrieval of extinction memories on the one hand and the consolidation and reconsolidation of those memories on the other.

These findings are consistent with previous research suggesting that the RE is selectively involved in memory retrieval and encoding, but not in “offline” learning processes such as consolidation (Mei et al. 2018). However, it has been reported that posttraining RE inactivation can influence the consolidation of contextual fear memories under some conditions (Troyner et al. 2018). Specifically, postconditioning RE muscimol infusions reduced contextual discrimination in later retention tests. This effect was manifest as similar levels of freezing in SAL- and MUS-infused rats tested in the conditioning context, but greater levels of freezing in MUS-infused rats tested in a novel context. Additional experiments showed that postconditioning RE inactivation increased incubation of the contextual fear memory over a 21-d retention interval and increased its resistance to extinction. This work suggests that postconditioning RE inactivation might reduce the specificity of the contextual fear memory, which is consistent with earlier work (Xu and Südhof 2013; Ramanathan et al. 2018b). If the RE functions similarly in the consolidation of extinction, then RE inactivation after extinction might be expected to reduce renewal of extinguished fear in a novel context; that is, if the inhibitory properties of the extinction context are more likely to generalize to a novel context, it would lead to less renewal of fear (which occurs when an extinguished CS is encountered in a novel context). However, we found that postextinction RE inactivation did not affect conditioned freezing to the extinguished CS in either the conditioning context where rats exhibited renewal or in the extinction context where rats suppressed freezing behavior. These results indicate that RE inactivation after extinction training does not affect the consolidation of extinction memory.

The storage of memories requires protein synthesis, and protein synthesis inhibitors such as anisomycin impair both the consolidation and reconsolidation of fear memories (Davis and Squire 1984; Haubrich and Nader 2018). These processes are also disrupted by muscimol, which is sufficient to disrupt both the consolidation and reconsolidation of fear memories (Wilensky et al. 2000; Troyner and Bertoglio 2020). We used muscimol rather than anisomycin in the present experiments to assess the possibility that impairments in extinction learning with pretraining infusions (Ramanathan et al. 2018a) result from RE inhibition that persists into the consolidation period. The present results reveal that the impairments in extinction learning that we previously observed with pretraining muscimol infusions are not the result of consolidation impairments. Importantly, muscimol inactivation of the RE produced dramatic deficits in extinction retrieval when infused prior to the retrieval test, as we have previously reported; these effects are not due to state-dependent effects (Ramanathan et al. 2018a; Ramanathan and Maren 2019). Nonetheless, it remains possible that posttraining protein synthesis inhibition in the RE would result in impairments in the consolidation or reconsolidation of fear extinction. Further experiments are required to examine this possibility.

Considerable work has explored the conditions that render reactivated memories labile and sensitive to manipulations that interfere with reconsolidation. For example, retrieval/reactivation conditions that are associated with “surprise” (prediction error) are particularly effective in destabilizing memories and rendering them sensitive to drugs that impair reconsolidation (Sevenster et al. 2012; Beckers and Kindt 2017; Sinclair and Barense 2019). It is noteworthy that the reactivation procedure that we used after extinction training involved a nonreinforced CS presentation (i.e., another extinction trial). This would not be expected to result in a prediction error. Paradoxically, reactivating the extinction memory by presenting the CS along with an unpaired US or a novel CS or in a novel context (reactivation procedures that would result in a prediction error) might have resulted in a more effective destabilization of the extinction memory. The absence of prediction error during our reactivation procedure may have limited the sensitivity of the retrieved extinction memory to reconsolidation inhibition.

The results of the present study reveal that the RE is necessary for the acquisition and expression of extinction memories but not for the consolidation or reconsolidation of those memories. This parallels work showing that muscimol infusions into the basolateral amygdala impair the acquisition and expression of fear memory (Helmstetter and Bellgowan 1994; Wilensky et al. 2000) but not the consolidation of those memories (Wilensky et al. 2000). Although the RE does not appear to be involved in the posttraining stabilization of new or reactivated extinction memories, it does contribute to the stabilization of contextual fear memories. Further research is necessary to elucidate the specific conditions under which the RE is recruited in the consolidation or reconsolidation of memory.

Materials and Methods

Subjects

Adult male and female rats (200–224 g; Long-Evans Blue Spruce; Envigo) were used for the experiments. Rats were individually housed on a 14/10-h light/dark cycle and had food and water access ad libitum. All experiments were performed during the light cycle. The rats were handled for 1 min every day for 3 d before surgery and 5 d after surgery to habituate them to the experimenters. All experimental procedures were performed in accordance with the protocols approved by the Texas A&M University Animal Care and Use Committee.

Surgery

For the microinfusion experiments, rats were anesthetized with isoflurane (5% for induction and ∼2% for maintenance) and fixed with ear bars into a stereotaxic instrument (Kopf Instruments). An incision was made in the scalp, the head was leveled (A/P <0.1 mm between bregma and lambda), and bregma was identified. Coordinates for craniotomy were marked on the skull. Small holes were drilled into the skull to affix three jeweler's screws to the skull and to target a single midline guide cannula (26 gauge, 9 mm; P1 Tech) above the RE. The cannula was implanted at a 10° angle on the midline (A/P: −2.10 mm, M/L +1.25 mm, D/V: −7.20 mm from skull; coordinates measured from bregma). Each cannula was affixed to the skull with dental cement, and a stainless steel dummy cannula (30 gauge, 10 mm; P1 Tech) was inserted into the guide cannula. Rats were allowed to recover for 7 d after surgery before behavioral testing.

Drug delivery

For RE microinfusions, rats were transported in squads of four to eight animals from the vivarium to an infusion room using white 5-gal buckets (one rat per bucket). Dummy cannulae were removed from the implanted guide cannulae, and stainless steel injectors (33 gauge, 10 mm; P1 Tech) connected to polyethylene tubing were inserted into the guide cannulae for intracranial infusions. Tubing connected the injectors to 10-µL Hamilton syringes, which were mounted on an infusion pump (KD Scientific). Infusions were monitored by movement of an air bubble separating the drug or saline solutions from distilled water within the tubing. Muscimol (MUS; Sigma) was diluted in sterile saline to a concentration of 0.1 µg/µL. For the infusions, muscimol or saline solution was infused intracranially into the RE at a rate of 0.1 µL/min for 3 min (0.3 µL total); the injectors remained in the animal for 2 min after the infusion to allow for diffusion. After infusions, clean dummies were secured to the guide cannulae.

Behavioral apparatus and contexts

Sixteen identical rodent conditioning chambers (30 cm × 24 cm × 21 cm; Med Associates) housed in sound-attenuating cabinets that were used for behavioral testing. Chambers consisted of two aluminum side walls, Plexiglas ceilings and rear walls, and a hinged Plexiglas door. Floors consisted of 19 stainless steel rods that were wired to a shock source and solid-state grid scrambler (Med Associates) for delivery of footshocks. A speaker mounted outside the grating in one of the aluminum walls was used to deliver auditory stimuli. Ventilation fans, house lights, and removeable Plexiglas floors inside the chambers were used to distinguish between contexts. Each chamber rested on a load-cell platform that transduced chamber displacement in response to each rat's motor activity; load-cell voltages were amplified, digitized, and acquired online via Threshold Activity software (Med Associates) as previously described (Maren 1998, 2001). Load-cell voltages were digitized at 5 Hz, resulting in an assessment of locomotor activity every 200 msec. Freezing behavior (a conditioned fear response) was quantified by the number of observations for each rat below a freezing threshold (load-cell voltage ≤10). Rats were classified as freezing if the rat was immobile for at least 1 sec. Behavioral procedures were conducted in two distinct contexts that were constructed by adjusting stimuli in the conditioning chamber, sound-attenuating cabinet, and testing room. For context A, a 15-W white house light within each chamber was illuminated and the room was illuminated with red overhead fluorescent lights; ventilation fans (65 dB) in the sound-attenuating cabinets were turned on and the cabinet doors were left open. The chambers were cleaned with a 1% ammonium hydroxide solution, and a small volume of this solution was placed in the waste pan under each grid floor. Rats were transported from the vivarium to the test room in white plastic boxes without bedding. For context B, black Plexiglas floors covered the grid floor (no shocks were administered in context B), house lights were off, and the room was illuminated with white overhead fluorescent lights. The sound-attenuating cabinet doors were closed, and the boxes were cleaned with a 3% acetic acid solution. Rats were transported from the vivarium to the chambers in black plastic boxes with fresh bedding.

Behavioral procedures

Experiment 1: postextinction muscimol infusions into RE

Approximately 1 wk after cannula surgeries, rats underwent fear conditioning (context A), conditioning context exposure (to limit generalization of fear to the extinction context), extinction training (context B), and retrieval testing (context B). Fear conditioning consisted of five tones (CS: 2 kHz, 80 dB, 10 sec), each paired with a footshock (US: 1 mA, 2 sec), with 70-sec intertrial intervals (ITIs). Prior to extinction, rats were returned to the conditioning context (35.5 min) to extinguish conditioned freezing to the conditioning context and limit generalization of fear to the extinction context. The day after exposure, the rats were placed in a novel context (context B), where they experienced a 3-min stimulus-free pretrial period followed by 45 CS-alone presentations with 40-sec intertrial intervals and a 3-min posttrial period. After extinction, rats were transported either directly to the infusion room or returned to the colony and then were given MUS or SAL microinfusions into the RE either 10 min after extinction (immediate condition) or 6 h after extinction (delayed condition). To assess the context specificity of the extinction memory, the animals were returned to the conditioning context (context A) and presented with the extinguished CS for a renewal test. After a 10-min pretrial period, the animals received five CS-alone presentations (40-sec ITIs), and a 3-min posttrial period. Twenty-four hours later, the rats were returned to context B, where they received five CS-alone presentations (40-sec ITIs) 10 min after placement in the chambers. This test assessed extinction retrieval in the extinction context.

Experiment 2: postreactivation muscimol infusions into the RE

Behavioral testing began ∼1 wk after cannula surgeries. Fear conditioning and conditioning context exposure were conducted as previously described; however, context A extinction was reduced to 15 min insofar as this seemed to be sufficient time to extinguish fear to context A. The next 2 d, rats underwent extinction as previously described. Twenty-four hours after extinction, the extinction memory was reactivated by returning rats to context B and presenting a single CS after a 3-min baseline period; rats remained in the chambers 1 min after the CS. Immediately after this session, rats were transported to the infusion room, where they received microinfusions of intra-RE MUS or SAL. The following day, extinction retrieval was assessed by returning rats to context B, where they received five CS presentations (70-sec ITIs) 3 min after placement in the chambers; the rats remained in the chambers 1 min after the last CS. One day later, we assessed whether RE inactivation would affect the expression of extinguished fear as we have previously reported (Ramanathan et al. 2018a; Ramanathan and Maren 2019). Rats were given intra-RE microinfusions of MUS or SAL (each rat received the same drug treatment as the one administered after the reactivation session) prior to an extinction retrieval test in context B.

Histology

Upon completion of the experiment, rats were overdosed with 0.5 mL of 100 mg/mL sodium pentobarbital (Fatal-Plus) i.p. and perfused transcardially with physiological saline followed by 10% formalin. Brains were extracted and stored for 16–18 h at 4°C in 10% formalin, after which they were transferred to a 30% sucrose solution for a minimum of 5 d. Brains were then sectioned using a cryostat (Leica Microsystems) at −20°C and stained with thionin to verify cannula placements (Fig. 1).

Data analysis

There were no sex differences in any of the analyses, and male and female rats were therefore collapsed. Exclusions criteria included off-target placement of the cannula and unusually high freezing that persisted into extinction retrieval (≥2.5 SD above mean freezing). Two rats were excluded from experiment 1 for off-target cannula placement. One rat was excluded from experiment 2 on account of unusually high freezing, and another was excluded for off-target cannula placement. The group sizes for each experiment were as follows: experiment 1: SAL n = 16 and MUS n = 14 and experiment 2: SAL n = 6 and MUS n = 8. All freezing data represent the percentage of freezing during each trial, which consisted of a 10-sec CS and ISIs (60 sec during conditioning and 30 sec during extinction). For the extinction sessions, the trial-by-trial data were averaged to yield nine five-trial blocks. All data were analyzed using analysis of variance (ANOVA), and post-hoc comparisons in the form of Fisher's protected least significant difference (PLSD) tests were performed after a significant overall F ratio in the ANOVAs. All the data are represented as means ± SEM.

Acknowledgments

This study was supported by National Institutes of Health grants R01MH065961 and R01MH117852.

Footnotes

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