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International Journal of Neuropsychopharmacology logoLink to International Journal of Neuropsychopharmacology
. 2025 Sep 15;28(9):pyaf065. doi: 10.1093/ijnp/pyaf065

Acquisition of trace fear conditioning without functional nucleus reuniens did not require dorsal or ventral hippocampus NMDA receptor activation in male Long-Evans rats

Ru-Hsuan Liu 1, Chun-hui Chang 2,3,✉
PMCID: PMC12477607  PMID: 40971798

Abstract

Importance

The nucleus reuniens (RE) is a crucial component that interconnects the medial prefrontal cortex (mPFC) and hippocampus (HPC), completing the HPC-dependent circuit underlying the regulation of trace fear. We previously demonstrated that RE inactivation during acquisition impaired the encoding of trace fear, while RE inactivation during both the acquisition and retrieval led to heightened trace fear throughout the test session, raising questions about the involvement of HPC-independent circuit in trace fear acquisition without functional RE.

Objective

To investigate whether rats without functional RE throughout the entire behavioral sessions can acquire trace fear using an HPC-independent circuit.

Design

A balanced factorial design was used to assess the role of the dorsal hippocampus (DH) or ventral hippocampus (VH) in trace fear acquisition with or without functional RE.

Setting

The study was conducted in a controlled laboratory environment.

Participants

Adult male Long-Evans rats were used as experimental subjects.

Interventions

Consecutive intracranial micro-infusions of either GABAA receptor agonist “Muscimol” or vehicle targeted the RE during both trace fear acquisition and retrieval. Micro-infusions of the N-methyl-D-aspartate (NMDA) receptor antagonist “DL-2-amino-5-phosphonovaleric acid” or saline targeted the DH or VH during trace fear acquisition.

Main Outcomes

Fear level of respective groups was measured.

Measures

Freezing was quantified as immobility during baseline and conditioned stimulus during trace fear acquisition and retrieval.

Results

Control animals required proper DH or VH NMDA receptor activation for the acquisition of trace fear. Rats without functional RE still acquired trace fear, but independent of DH or VH NMDA receptor activation, suggesting the reliance of an HPC-independent circuit during fear encoding.

Conclusions and Relevance

These findings highlighted potential alternative neural pathways that may support and compensate trace fear acquisition under pathological conditions.

Significance Statement

Impaired fear regulation resulted in psychiatric disorders like panic disorder and anxiety. Pavlovian trace fear conditioning using male Long-Evans rats as the subjects models human emotional learning. Although hippocampus (HPC)–medial prefrontal cortex (mPFC) interactions facilitate trace fear acquisition, the role of thalamic nucleus reuniens (RE), which connects these regions, is unclear. In this study, we found that in terms of within-session fear expression during acquisition, dorsal hippocampus (DH) N-methyl-D-aspartate (NMDA) receptor blockade impaired fear response only in RE-intact rats, whereas ventral hippocampus (VH) blockade impaired the response regardless of the RE functionality. Critically, rats without functional RE still acquired trace fear memory independent of DH or VH NMDA receptor activation, indicating the recruitment of alternative HPC-independent pathways. These findings highlight possible compensatory pathways engaged following RE dysfunction, providing insight into how the brain adapts under pathological conditions.

Keywords: trace fear conditioning, nucleus reuniens, hippocampus

INTRODUCTION

In trace fear conditioning, individuals learn to associate a neutral conditioned stimulus (CS) with an aversive unconditioned stimulus (US) separated by a “trace” interval,1–3 resulting in the elicitation of conditioned response when only the CS is presented.4,5 Subjects usually need several trials to acquire trace fear, and higher-order brain regions are engaged, such as the hippocampus (HPC) and the medial prefrontal cortex (mPFC).6,7 Earlier animal studies have shown that lesions of the HPC3,8–10 and the mPFC11–13 impaired trace conditioning. In addition, blockade of NMDA receptors with DL-2-amino-5-phosphonovaleric acid (APV) prevented long-term potentiation in the HPC and disrupted the acquisition of trace conditioning.14–16 Evidence from human neuroimaging studies also indicated the involvement of both the mPFC and the HPC in fear acquisition, and HPC activation in trace conditioning supported its role in bridging the temporal gap between the 2 stimuli.17,18 Among patients, trace conditioning deficits have been reported in some psychiatric conditions, such as panic disorder, anxiety-like disorders, and amnesia.19–22

Anatomically, although the hippocampal CA1 and subiculum (SUB) have dense direct projections to the mPFC, there is no direct return projection from the mPFC to the HPC. The nucleus reuniens (RE), a midline thalamic nucleus, serves as an important relay in orchestrating the communication between the mPFC and the HPC through its respective reciprocal connections to the mPFC, the SUB, and the ventral CA1.23–29 Given the importance of the RE in mPFC-HPC interaction, we previously examined the role of the RE in trace fear conditioning and demonstrated that RE inactivation during the acquisition phase resulted in lower cued fear during retrieval, whereas RE inactivation during both the acquisition and the retrieval phases of trace conditioning resulted in heightened cued fear during test.30 In the follow-up study, we further reported that for rats underwent trace conditioning without functional RE throughout the entire behavioral sessions, these animals showed heightened contextual fear to the conditioning context that did not decline much with the passage of time, which generalized to the other context, and fear to tones reoccurred when the tones were presented.31 These behavioral phenomena suggested that for rats without functional RE, they may have acquired contextual and cued trace fear using HPC-independent circuit, resulted in behavioral expression that was not well-controlled.32–34 Indeed, a previous study revealed that the RE supported HPC-dependent contextual fear acquisition, whereas contextual memories acquired without functional RE were insensitive to hippocampal NMDA receptor inactivation.35

Based on the above results, we used a behavioral pharmacological approach to examine whether rats without a functional RE throughout the entire behavioral sessions acquired trace fear using an HPC-independent circuit. Because of the anatomical and functional differences between the dorsal (DH) and ventral (VH) parts of the HPC,36–38 we selectively blocked NMDA receptor activation in the DH (experiment 1) or the VH (experiment 2) during the acquisition phase and hypothesized that animals without functional RE would acquire trace fear, but RE-intact animals would not. Specifically, for RE-intact animals, DH/VH NMDA receptor blockade was expected to impair fear acquisition, leading to reduced freezing during the tone test. If our hypotheses held true, for RE inactivation animals, DH/VH NMDA receptor blockade was not expected to impair fear acquisition, and therefore their freezing levels was expected to remain high throughout the tone test, suggesting an HPC-independent fear encoding strategy was adapted.

METHODS

Subjects

A total of 174 male Long-Evans rats (200–300 g, 6–7 weeks old upon arrival) obtained from National Center for Biomodels (NCB, Taiwan) were used. The animals were under standard care as previously described39 and all procedures were conducted during the light cycle (9 am to 6 pm) at National Yang Ming Chiao Tung University Animal Center with approval from both the National Tsing Hua University and the National Yang Ming Chiao Tung University, Institutional Animal Care and Use Committees (IACUC).

Surgery

After arrival, the animals were handled daily for 1 week. At the time of the surgical procedures, the rats were at the age of 7–8 weeks old. The animals were anesthetized with ketamine (100 mg/kg, i.p.,) and xylazine (10 mg/kg, i.p.). Core body temperature was maintained at 37°C by a temperature-controlled heating pad (CWE), while the head was fixed by the stereotaxic instrument (Stoelting, IL, USA). For experiment 1, three 26-gauge stainless steel guide cannulae were implanted to target the RE (single; relative to bregma: anterior–posterior [AP] −1.1 mm, medial–lateral [ML] +2.3 mm, dorsal–ventral [DV] −6.7 mm, 20° angle to the vertical axis, 27° angle to the coronal plane) and the DH (bilateral; relative to bregma: [AP] −3.8 mm, [ML] ±2.7 mm, and [DV] −2.2 mm). For experiment 2, 3 guide cannulae were implanted to target the RE and the VH (bilateral; relative to bregma: [AP] −5.2 mm, [ML] ±5 mm, and [DV] −6.6 mm). To complete the surgery, 3 additional anchor screws were mounted, and then the headstage was fixed in place with dental acrylic. Standard post-surgery care was administered as described before.39

Behavioral Apparatus

Four identical conditioning chambers (Med-Associates) were used. Context A and context B were generated to provide 2 distinct context settings as described before.39

Behavioral Procedures

Trace Fear Conditioning

On day 1, auditory trace fear conditioning was conducted in context A. The conditioning session consisted of 10 pairings of a tone (CS; 20 s, 85 dB, 2 kHz) and a footshock (US; 2 s, 1.0 mA) with a 30-s trace interval. The first tone was presented after a 3-min baseline (BL) and subsequent trials were separated by a 240-s inter-trial interval (ITI). NoCOND rats received tones only but no footshocks. On day 2, the test session was performed in context B to minimize contextual fear. Following a 3-min BL, all animals received 10 discrete tones (20 s) separated by a 60-s ITI (Figure 1A).

Figure 1.

Figure 1

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Experimental design. (A) In experiment 1, all rats underwent a 2-d behavioral procedure. Trace fear conditioning was conducted in context A on day 1 and tested in context B on day 2. Animals received either VEH or MUS into the RE immediately before both the conditioning and the test sessions, and either SAL or APV into the DH immediately before conditioning session. (B) In experiment 2, the 2-d behavioral procedure, the same as described in experiment 1, was done, with either SAL or APV infused into the VH immediately before conditioning session. On day 3, animals from the NoCOND groups underwent the EPM/marble burying test and shock sensitivity test. For control experiments, the drug history of the animals during conditioning, as well as the order of EPM and marble burying test were counterbalanced, with shock sensitivity test always done the last. APV, DL-2-amino-5-phosphonovaleric acid; DH, dorsal hippocampus; EPM, elevated plus maze; MUS, muscimol; RE, nucleus reuniens; SAL, saline; VEH, vehicle; VH, ventral hippocampus.

For experiment 2, NoCOND rats further underwent 3 control experiments on day 3 after the trace fear conditioning procedures (Figure 1B). These experiments were aimed to clarify whether RE inactivation per se affected the innate anxiety state or the shock sensitivity of the animals.

Elevated Plus Maze (EPM)

One day before the test, animals were habituated to the behavioral room in their home cages for 2 h. On test day, each animal was placed onto the center of the maze facing a closed arm with video recorded for 5 min. The percentage of time spent in the open arms (time in open arms/total time in open and closed arms) and the percentage of entries into the open arms (entries into open arms/total entries into open and closed arms) were measured.

Marble Burying Test

The animals were placed individually in a 45 × 21 × 20 cm cage box filled with 5 cm depth of bedding for 10 min. Eighteen marbles were put on the bedding in a 3 × 6 pattern. At the end, the rat was removed from the cage and the number of marbles buried by at least two-third was recorded.

Shock Sensitivity Test

Each animal was placed in the same conditioning chamber used in experiments 1 and 2, but under a novel context setting distinct from contexts A and B. During test, each animal received a series of footshocks (2 s duration, 10-s ITI) started at 0.05 mA and increasing by 0.05 mA increments until the animal exhibited reactions such as freezing, flinching, jumping, and sonically vocalizing. The minimal magnitude of electrical current to induce any of the behavioral response was recorded.

Drug Infusions

One day before the behavioral experiment, the animals were familiarized with the sound of the micro-infusion pump for 2 min. For trace fear conditioning, immediately before both the conditioning (on day 1) and the test (on day 2), GABAA receptor agonist “Muscimol” (MUS; 0.1 μg/μL) or vehicle (VEH; 0.9% saline) was infused into the RE (REMUS or REVEH). Immediately before the conditioning (on day 1), competitive NMDA receptor antagonist “APV” (APV; 10 μg/μL) or saline (SAL) was also infused into the DH (experiment 1; DHAPV or DHSAL) or the VH (experiment 2; VHAPV or VHSAL). For NoCOND rats, the RE was infused with MUS or VEH (REMUS or REVEH), while the DH or the VH was infused with SAL (NoCONDSAL). For the control experiments (on day 3 of experiment 2), the RE was infused with MUS or VEH (1 infusion only), followed by elevated plus maze (EPM)/marble burying test and shock sensitivity test. Respective drugs were delivered at a rate of 0.25 μL/min for 2 min (0.5 μL total per site), followed by an extra 30 s for drug diffusion, using an injector (33-gauge) extended 1.0 mm beyond the guide cannula implanted during the surgery. The dosage of MUS and APV was chosen based on earlier studies.14,15,40,41

Histology

Standard Nissl staining procedures were applied as described before42 upon completion of the behavioral procedures to confirm the injection sites.

Statistics

Freezing behavior, characterized by a rigid posture with the absence of movement except for respiration, served as the behavioral readout to evaluate fear response.4,43 In our study, freezing level was designated as “immobility” because the NoCOND rats never received the footshocks. Immobility was defined as consecutively observed movements below the motion threshold for 1s and was measured continuously during the trace conditioning procedures by Video Freeze (Med Associates). The percentage of total observations in which immobility occurred at BL and during CSs was calculated. These values were analyzed using 3-way repeated measure analysis of variance (RM ANOVA) with between-subject factors of “DRUG” (REVEH and REMUS) and “GROUP” (experiment 1, NoCONDSAL, DHSAL, and DHAPV; experiment 2, NoCONDSAL, VHSAL, and VHAPV), and within-subject factor of “Trial.” Because we specifically aimed to examine whether the rats without functional RE acquired cued fear using HPC-independent circuit, planned comparisons of behavioral performance during the test session on day 2 were further analyzed for respective DRUG conditions using 2-way RM ANOVA with between-subject factor of “GROUP” and within-subject factor of “Trial.” Differences were considered significant at P < .05 and marginal at .05 ≤ P < .1. Both significant and marginal effects were reported, and Student–Newman–Keuls (S-N-K) post hoc tests were performed after significant/marginal F ratios were obtained. For all ANOVAs, effect sizes were reported as partial eta squared (η2p). All control experiments were analyzed using Student’s t-test. Data were calculated using SPSS (IBM) and presented as means ± SEMs.

RESULTS

Experiment 1

To evaluate the role of DH NMDA receptor in trace fear acquisition between animals with or without functional RE, the RE was inactivated during both the conditioning and test sessions, and NMDA receptor activity in the DH was blocked prior to conditioning.

A total of 90 animals were used. Thirty-three rats were excluded due to cannula misplacements (n = 21), inadvertent lesions caused by contamination of guide cannulae (n = 9), incorrect infusion volume (n = 2), and infusion of APV into RE (n = 1). The final group sizes are as follows: REVEH-NoCONDSAL (n = 10), REVEH-DHSAL (n = 10), REVEH-DHAPV (n = 10), REMUS-NoCONDSAL (n = 7), REMUS-DHSAL (n = 10), and REMUS-DHAPV (n = 10). The representative images of Nissl-stained coronal section for injection sites in the RE and the DH are shown in Figure 2A, and the placements of injector tips for all animals included in analyses are summarized in Figure 2B.

Figure 2.

Figure 2

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Cannula placements in the RE and the DH. (A) Examples of cannula placements in the RE (left) and the DH (right). (B) Injection sites for all the subjects included in data analyses across 4 different levels along the anterior–posterior axis in the RE (left) and the DH (right). Numbers indicated the coronal levels relative to bregma. DH, dorsal hippocampus; RE, nucleus reuniens.

On day 1, the animals underwent trace fear conditioning. The upper and middle left panels of Figure 3 show immobility levels of the animals during the BL and each of the 10 tones during conditioning, and the lower left panel summarized the overall immobility level across the BL and tones for respective groups during conditioning. There were significant main effects of “DRUG” [F (1, 51) = 6.36, P = .02, Inline graphic = 0.11], “GROUP” [F (2, 51) = 9.25, P < .001, Inline graphic = 0.27], and “Trial” [F (10, 510) = 73.88, P < .001, Inline graphic = 0.59], significant 2-way interactions between “DRUG” and “GROUP” [F (2, 51) = 4.00, P = .02, Inline graphic = 0.14] and “GROUP” and “Trial” [F (20, 510) = 3.57, P < .001, Inline graphic = 0.12], a marginal 2-way interaction between “DRUG” and “Trial” [F (10, 510) = 1.84, P = .052, Inline graphic = 0.04], and a significant 3-way interaction among “DRUG”, “GROUP,” and “Trial” [F (20, 510) = 4.15, P < .001, Inline graphic = 0.14]. The overall immobility levels of REMUS groups were significantly higher than those of REVEH groups and there was a significant difference between REVEH-NoCONDSAL and REMUS-NoCONDSAL groups (P < .05) (lower left panel of Figure 3). For RE-intact animals (REVEH), post hoc comparisons among “GROUP” (lower left panel of Figure 3) suggested that immobility levels were significantly different among groups, with respective levels highest in the REVEH-DHSAL group, intermediate in the REVEH-DHAPV group, and lowest in the REVEH-NoCONDSAL group (all Ps < .05), indicating that the overall within-session fear expression was slightly impaired when the DH NMDA receptor activity was blocked. Post hoc comparisons by trials (upper left panel of Figure 3) suggested that immobility levels of REVEH-DHSAL group were significantly higher than REVEH-NoCONDSAL from trial #3 to #10, and also significantly higher than REVEH-DHAPV group in trials #3 and #4 (all Ps < .05), indicating that within-session fear expression at early trials was impaired when the DH NMDA receptor activity was blocked. For RE inactivation animals (REMUS), there were no statistical differences among 3 groups (lower left panel of Figure 3). Post hoc comparisons by trials (middle left panel of Figure 3) also revealed that there were no statistical differences among 3 groups in any trials, suggesting that REMUS-DHAPV group was insensitive to DH NMDA receptor inactivation, and REMUS-NoCONDSAL group showed a nonspecific increase in immobility levels.

Figure 3.

Figure 3

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Acquisition of trace fear conditioning in rats without functional RE did not require DH NMDA receptor activation. The upper and middle panels showed the immobility levels of the RE-intact animals (upper panels) and the RE inactivation animals (middle panels) during the BL and each of the 10 tones (CS) during the conditioning and test sessions, and the lower panels summarized the overall immobility level across the BL and tones for respective groups. For RE-intact animals, DH NMDA receptor blockade resulted in a slower increase in immobility level during acquisition and a lower freezing response to the tone during early retrieval (upper panels). For RE inactivation animals, these animals displayed a rapid increase in immobility level during acquisition and a high level of fear expression during test session regardless of DH NMDA receptor blockade during conditioning (middle panels). Moreover, REMUS groups exhibited an overall higher immobility level compared to the REVEH groups during both the conditioning and the test sessions. BL, baseline; CS, conditioned stimulus; DH, dorsal hippocampus; RE, nucleus reuniens. *, P < .05. a, P < .05 between REVEH-NoCONDSAL and REMUS-NoCONDSAL groups.

On day 2, all animals were tested for their fear to tones. The upper and middle right panels of Figure 3 showed immobility levels of the animals during the BL and each of the 10 tones during test, and the lower right panel summarized the overall immobility level across the BL and tones for respective groups during test. There were significant main effects of “DRUG” [F (1, 51) = 7.44, P = .01, Inline graphic = 0.13], “GROUP” [F (2, 51) = 5.34, P = .01, Inline graphic = 0.17], and “Trial” [F (10, 510) = 7.88, P < .001, Inline graphic = 0.13], a significant 2-way interaction between “GROUP” and “Trial” [F (20, 510) = 1.67, P = .04, Inline graphic = 0.06], but no statistical differences in other interactions. We further analyzed REMUS and REVEH groups, respectively, to evaluate whether animals without functional RE throughout the behavioral sessions acquired cued fear using HPC-independent circuit. For RE-intact animals (REVEH), there were significant main effects of “GROUP” [F (2, 27) = 4.34, P = .02, Inline graphic = 0.24] and “Trial” [F (10, 270) = 2.76, P = .003, Inline graphic = 0.09], and a marginal effect of 2-way interaction between “GROUP” and “Trial” [F (20, 270) = 1.59, P = .054, Inline graphic = 0.11]. Post hoc comparisons among “GROUP” (lower right panel of Figure 3) suggested that the immobility level of REVEH-DHSAL group was significantly higher than that of REVEH-NoCONDSAL group (P < .05). Post hoc comparisons by trials (upper right panel of Figure 3) revealed that immobility levels of REVEH-DHSAL group were significantly higher than REVEH-NoCONDSAL in trials #1, #2, #4, and #6, and also significantly higher than REVEH-DHAPV group in trial #1 (all Ps < .05), suggesting that in control animals (REVEH groups), acquisition to cued fear was impaired if the DH NMDA receptor activation was blocked during conditioning by showing a lower fear response during early retrieval. Post hoc comparisons by drugs (upper right panel of Figure 3) further revealed that immobility levels of REVEH-NoCONDSAL group showed no difference between BL and any other trials (all Ps > .05), while immobility levels of REVEH-DHSAL group showed sustained increase during tones and reached significant difference compared to BL in trials #1, #2, #4, #6, #9, and #10 (all Ps < .05). Importantly, immobility levels of REVEH-DHAPV group showed initial increase to tones and reached significant difference compared to BL from trial #1 to #4 (all Ps < .05), but dropped quickly toward later trials, with no statistical difference from trial #5 to #10 (all Ps > .05), suggesting that in control animals (REVEH groups), they did not learn well if the DH NMDA receptor activation was blocked during conditioning by showing faster within-session extinction during test. For RE inactivation animals (REMUS), there was a significant main effect of “Trial” [F (10, 240) = 6.52, P < .001, Inline graphic = 0.21], a marginal effect of “GROUP” [F (2, 24) = 3.34, P = .052, Inline graphic = 0.22], but no statistical difference in interaction between the 2. Post hoc comparisons among “GROUP” (lower right panel of Figure 3) suggested that immobility of the REMUS-DHAPV group was significantly higher than the REMUS-NoCONDSAL group (P < .05), but not others, suggesting that without functional RE throughout the behavioral sessions, these animals were insensitive to DH NMDA receptor inactivation during conditioning and acquired cued fear.

Experiment 2

To evaluate the role of VH NMDA receptor in trace fear acquisition between animals with or without functional RE, the RE was inactivated during both the conditioning and test sessions, and NMDA receptor activity in the VH was blocked prior to conditioning.

A total of 84 animals were used. Thirty rats were excluded due to cannula misplacements (n = 16), inadvertent lesions caused by contamination of guide cannulae (n = 5), death during surgery (n = 7), injury due to self-scratch after surgery (n = 1), and injury due to operation error during the test session (n = 1). The final group sizes are as following: REVEH-NoCONDSAL (n = 8), REVEH-VHSAL (n = 11), REVEH-VHAPV (n = 9), REMUS-NoCONDSAL (n = 9), REMUS-VHSAL (n = 8), and REMUS-VHAPV (n = 9). The representative images of Nissl-stained coronal section for injection sites in the RE and VH are shown in Figure 4A and the placements of injector tips for all animals included in the analyses are summarized in Figure 4B.

Figure 4.

Figure 4

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Cannula placements in the RE and the VH. (A) Examples of cannula placements in the RE (left) and the VH (right). (B) Injection sites for all the subjects included in data analyses across 4 different levels along the anterior–posterior axis in the RE (left) and the VH (right). Numbers indicated the coronal levels relative to bregma. RE, nucleus reuniens; VH, ventral hippocampus.

On day 1, the animals underwent trace fear conditioning. The upper and middle left panels of Figure 5 showed immobility levels of the animals during the BL and each of the 10 tones during conditioning, and the lower left panel summarized the overall immobility level across the BL and tones for respective groups during conditioning. There were significant main effects of “DRUG” [F (1, 48) = 15.29, P < .001, Inline graphic = 0.24], “GROUP” [F (2, 48) = 10.41, P < .001, Inline graphic = 0.30], and “Trial” [F (10,480) = 62.42, P < .001, Inline graphic = 0.57], and a significant 2-way interaction between “GROUP” and “Trial” [F (20, 480) = 3.80, P < .001, Inline graphic = 0.14]. The overall immobility levels of REMUS groups were significantly higher than those of REVEH groups. Post hoc comparisons among groups revealed that the immobility of VHSAL group was significantly higher than those of NoCONDSAL and VHAPV groups (both Ps < .05), with no statistical difference between the latter 2 (P > .05). The results suggested that regardless of whether the animals had functional RE, immobility levels increased rapidly in VHSAL group but slower in VHAPV group, indicating impaired within-session fear expression at early trials when VH NMDA receptor activity was blocked.

Figure 5.

Figure 5

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Acquisition of trace fear conditioning in rats without functional RE did not require VH NMDA receptor activation. The upper and middle panels showed the immobility levels of the RE-intact animals (upper panels) and the RE inactivation animals (middle panels) during the BL and each of the 10 tones (CS) during the conditioning and test sessions, and the lower panels summarized the overall immobility level across the BL and tones for respective groups. VH NMDA receptor blockade resulted in a slower increase in immobility level during acquisition in both RE-intact (upper left panel) and RE inactivation (middle left panel) animals. During test session, RE-intact animals (upper right panel) showed lower freezing response to the tone if conditioned under VH NMDA receptor blockade. However, RE inactivation animals (middle right panel) showed a high level of fear expression regardless of VH NMDA receptor blockade during conditioning. BL, baseline; CS, conditioned stimulus; RE, nucleus reuniens; VH, ventral hippocampus. *, P < .05.

On day 2, all animals were tested for their fear to tones. The upper and middle right panels of Figure 5 showed immobility levels of the animals during the BL and each of the 10 tones during test, and the lower right panel summarized the overall immobility level across the BL and tones for respective groups during test. There were significant main effects of “DRUG” [F (1, 48) = 25.56, P < .001, Inline graphic = 0.35], “GROUP” [F (2, 48) = 18.61, P < .001, Inline graphic = 0.44], and “Trial” [F (10, 480) = 10.59, P < .001, Inline graphic = 0.18], and a significant 2-way interaction between “GROUP” and “Trial” [F (20, 480) = 1.80, P = .02, Inline graphic = 0.07]. We further analyzed REMUS and REVEH groups, respectively, to evaluate whether animals without functional RE throughout the behavioral sessions acquired cued fear using HPC-independent circuit. For RE-intact animals (REVEH), there were significant main effects of “GROUP” [F (2, 25) = 7.28, P = .003, Inline graphic = 0.37] and “Trial” [F (10, 250) = 5.98, P < .001, Inline graphic = 0.19], but no interaction between the 2. Post hoc comparisons among groups (lower right panel of Figure 5) revealed that immobility levels of REVEH-VHSAL group were significantly higher than those of REVEH-NoCONDSAL and REVEH-VHAPV groups (both Ps < .05), with no statistical difference between the latter 2 (P > .05), indicating a learning deficit in REVEH-VHAPV group. For RE inactivation animals (REMUS), there were significant main effects of “GROUP” [F (2, 23) = 13.28, P < .001, Inline graphic = 0.54] and “Trial” [F (10, 230) = 5.26, P < .001, Inline graphic = 0.19], but no interaction between the 2. Post hoc comparisons among groups (lower right panel of Figure 5) revealed that there was no statistical difference in immobility levels between REMUS-VHSAL and REMUS-VHAPV groups (P > .05), which were significantly higher than that of REMUS-NoCONDSAL group (both Ps < .05), suggesting that without functional RE throughout the behavioral sessions, these animals were insensitive to VH NMDA receptor inactivation during test and acquired cued fear.

Because the overall immobility levels of REMUS groups were higher than that of RESAL groups during both the conditioning and test sessions in experiments 1 and 2, we further evaluated that under RE inactivation, whether there were differences in locomotor activity, innate anxiety state, or threshold to shocks compared to RE-intact animals in experiment 2. To address this question, the locomotor activity and sleeping state of REVEH-NoCONDSAL and REMUS-NoCONDSAL groups during the BL and the 10 interstimulus intervals (ISIs) in conditioning session were analyzed, and 3 control experiments were conducted using these 2 groups of animals after the conclusion of trace conditioning and retrieval. Elevated plus maze and marble burying test were incorporated to assess innate anxiety state, while shock sensitivity test was performed to evaluate the threshold to shocks. The drug history of the animals during conditioning, as well as the order of EPM and marble burying test, were counterbalanced. Shock sensitivity test was always done at the last.

The results of locomotor activity (Figure 6A) showed that both groups initially exhibited high locomotor activity; however, subsequent locomotor activity level declined more rapidly under RE inactivation. There were significant main effects of “DRUG” [F (1, 15) = 25.25, P < .001, Inline graphic = 0.63] and “Trial” [F (10, 150) = 10.22, P < .001, Inline graphic = 0.41], and a significant 2-way interaction between “DRUG” and “Trial” [F (10, 150) = 3.48, P < .001, Inline graphic = 0.19]. Post hoc comparisons by trials revealed that the locomotor activity levels of REVEH-NoCONDSAL group were significantly higher than REMUS-NoCONDSAL group during the BL, and in all trials except for trial #5 (all Ps < .05). We further quantified the time the animals spent in sleep, and there was an increase in RE inactivation animals as the trials progressed. The results of sleeping states (Figure 6B) showed that there was a significant main effect of “Trial” [F (10, 150) = 3.21, P = .001, Inline graphic = 0.18] and a marginal effect of “DRUG” [F (1, 15) = 3.83, P = .07, Inline graphic = 0.20]. The time the animals spent in sleep was marginally higher in the REMUS-NoCONDSAL group than the REVEH-NoCONDSAL group. Under RE inactivation, there was no difference in the threshold to shocks that elicited any motor reactions [t (15) = 0.44, P = .67] (Figure 6C). There was a trend of decrease in innate anxiety as RE inactivation rats spent more time in the open arms in EPM [t (15) = −2.06, P = .06] (Figure 6D, right). However, there were no differences in the percentage of entries into the open arms in EPM [t (15) = −1.28, P = .22] (Figure 6D, left), or the number of buried marbles in the marble burying test [t (15) = 0.81, P = .43] (Figure 6E).

Figure 6.

Figure 6

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The general higher immobility levels under RE inactivation during conditioning and test could be partially attributed to the lower locomotor activity, but not changes in shock sensitivity or innate anxiety state. The locomotor activity and sleeping state of REVEH-NoCONDSAL and REMUS-NoCONDSAL groups during the BL and the 10 ISIs in conditioning session were analyzed. Three control experiments were conducted using these 2 groups of animals after the conclusion of trace conditioning and retrieval. The drug history of the animals during conditioning, as well as the order of the EPM and marble burying test, was counterbalanced. (A) The locomotor activity levels of REVEH-NoCONDSAL group were significantly higher than REMUS-NoCONDSAL group. (B) The time the animals spent in sleep was marginally higher in the REMUS-NoCONDSAL group than the REVEH-NoCONDSAL group. No statistical differences were found between the control and the RE inactivation group in (C) the magnitude of the footshock eliciting any reaction in shock sensitivity test, (D) the percentage of entries into open arms in EPM (left), or (E) the number of buried marbles in the marble burying test. RE inactivation group showed a trend of spending more time in the open arms compared to the control group (right of D; +, .05 ≤ P < .1). BL, baseline; EPM, elevated plus maze; ISIs, interstimulus intervals; RE, nucleus reuniens; VH, ventral hippocampus.

DISCUSSION

In the present study, we observed that within-session fear expression during acquisition was delayed under DH NMDA receptor blockade only in the RE-intact animals, but was delayed under VH NMDA receptor blockade regardless the rats had functional RE or not. For RE-intact animals, acquisition of trace fear was impaired under DH or VH NMDA receptor blockade. For animals without functional RE, they were still capable to acquire trace fear and were insensitive to DH or VH NMDA receptor blockade, suggesting that these animals likely acquired trace fear using HPC-independent circuit. Finally, the overall higher immobility in animals without functional RE throughout the behavioral sessions was partially attributed to the decreased locomotor activity, but not changes in their shock sensitivity or innate anxiety state.

For RE-intact animals, we replicated previous findings that pre-conditioning infusions of APV into either the DH or VH led to a deficit of trace fear acquisition.14,15,44 Furthermore, within-session fear expression was impaired under VH NMDA receptor blockade during the conditioning session, as reported in an earlier study.15 Impairment in within-session fear expression under DH NMDA receptor blockade, on the other hand, has not been reported before.14,15 For animals without functional RE throughout the entire behavioral sessions, those receiving infusions of SAL into the DH or VH exhibited a rapid increase in fear expression to tones during conditioning, along with heightened immobility levels throughout the entire test session, consistent with our previous work30 that these animals were capable to acquire trace fear.

Compared to DH NMDA receptor blockade, VH NMDA receptor blockade had a more pronounced effect on trace fear acquisition. DH NMDA receptor blockade affected within-session fear expression during conditioning and led to a decrease in fear response only at the first trial during test in RE-intact animals. In contrast, VH NMDA receptor blockade affected within-session fear expression regardless of the presence of functional RE and resulted in significantly lower fear response during test in RE-intact animals. Anatomical projections of the DH and VH may have contributed to their differential involvement in fear expression and acquisition. The mPFC receives dense projection from the VH, while direct projection from the DH (dorsal SUB subregion) is sparse.23,26 Whereas the DH receives sensory information derived from the sensory cortex via the entorhinal cortex (ERC), a process required for spatial learning, the VH has direct reciprocal connections with the amygdala, as well as strong connections with the bed nucleus of the stria terminalis, hypothalamus, and the nucleus accumbens, suggesting a more significant role of the VH in modulation of fear.36,45–47

In previous report,31 the REMUS animals tended to show generalization of contextual fear, which was resilient to extinction even after prolonged context exposure. The animals in this study replicated this phenomenon. For RE inactivation animals in experiment 1, there was an increase in immobility level during the BL in the test session, which did not reach statistical difference among groups. In experiment 2, there was a significant main effect among groups during the BL in the test session. Post hoc comparisons revealed that immobility of the REMUS-VHSAL group was significantly higher than that of the REMUS-NoCONDSAL group, and the level of the REMUS-VHAPV was in between the REMUS-VHSAL group and REMUS-NoCONDSAL groups, suggesting that VH NMDA receptor inactivation might have alleviated the context fear generalization in REMUS animals. We noticed that overall immobility level of the REMUS-DHSAL control group (around 60%) in experiment 1 was lower than that of the REMUS-VHSAL control group (around 85%) in experiment 2, which may have resulted in their lack of statistical difference in immobility level during BL. We have no good explanation of these basal differences across experiments, which could be a result due to different cohort of animals or slight difference in housing/manipulation conditions. Nonetheless, the high generalized contextual fear during BL made it harder to differentiate the contribution of cue-induced fear in RE inactivation animals.

We noticed that for animals without functional RE, there was a general up-shift in immobility level. We ruled out certain factors in that we did not detect an increase in shock sensitivity or innate anxiety under RE inactivation compared to controls. However, these animals indeed showed a faster decline in locomotor activity, and spent more time in sleep, with sleep occurring earlier. One possible explanation is that RE inactivation interfered with the regulation of wakefulness and sleep state, making the animals more likely to fall asleep. In addition, RE inactivation may reduce the interest of the rats in exploring a novel environment, leading to less exploratory behavior and a further increase of the time in immobility. Nevertheless, although RE inactivation resulted in a general increase in immobility, such general increase did not contradict our key findings that under RE inactivation (REMUS), the APV groups still exhibited significantly higher freezing levels compared to their respective NoCOND groups during test in both experiments 1 and 2.

Previous studies have shown that the RE supported HPC-dependent encoding of precise contextual and trace memories.35,48 In trace procedures, learning becomes more complicated due to the temporal interval between the CS and US. The formation of temporal associative memory is dependent on both the mPFC and HPC, implicating that the mPFC-RE-HPC loop supports higher-order functions.26,48 Interestingly, we previously reported that animals without functional RE were still able to acquire trace fear.30 In this study, we further demonstrated that their learning of trace fear did not rely on DH or VH NMDA receptor activation, suggesting that these animals acquired trace fear using HPC-independent circuit. The reciprocal connections between the mPFC and amygdala,23,28 as well as evidence from lesion studies,49 pharmacological studies,50,51 and genetics studies,52 suggested an important role of the amygdala in trace fear memory formation. Therefore, it is possible that direct mPFC–amygdala interaction supported trace fear conditioning when the RE is off-line. Moreover, previous reports have shown that the retrosplenial cortex (RSC), which has connections with the HPC, thalamus, mPFC, and amygdala,53,54 plays a crucial role in trace fear acquisition and retrieval.55,56 Other than the RSC, perirhinal cortex, ERC, and paraventricular nucleus of the thalamus also have direct reciprocal connections with the mPFC,23,57 which could potentially serve as alternative hubs to support trace fear acquisition, These regions can also relay information to the amygdala, completing a potential HPC-independent circuit.45,52,58–60

One of the limitations of our study is the use of the only male subjects, which may be a concern due to increasing evidence that highlights differences in the neural mechanisms of emotional learning between males and females. Indeed, hormonal cycles may cause males and females to show different behaviors in fear regulation. Therefore, future work that includes both male and female rats is critical for assessing potential sex differences.

Dysfunction of fear control may lead to anxiety-related disorders.17,61–63 Using the trace fear conditioning paradigm, fear control that requires high-order brain circuits can be further explored.18,64,65 Overall, the present data revealed that the encoding of trace fear without functional RE did not rely on DH or VH NMDA receptor activation, likely through the HPC-independent circuit, which sheds light on research into potential alternative brain circuits in compensation of the function under pathological conditions.

Acknowledgments

We thank the animal care team in Laboratory Animal Center of National Yang Ming Chiao Tung University for their support in animal housing.

Contributor Information

Ru-Hsuan Liu, Institute of Systems Neuroscience, National Tsing Hua University, Hsinchu 30013, Taiwan.

Chun-hui Chang, Institute of Systems Neuroscience, National Tsing Hua University, Hsinchu 30013, Taiwan; Brain Research Center, National Tsing Hua University, Hsinchu 30013, Taiwan.

Author Contributions

Ru-Hsuan Liu (Formal analysis [equal], Investigation [lead], Visualization, Writing—original draft [equal]), Chun-hui Chang (Conceptualization, Funding acquisition, Methodology, Resources, Writing—review & editing [lead]).

Funding

This work was supported by the National Science and Technology Council (NSTC) of Taiwan under research project “110-2326-B-007-001-MY3” to Chun-hui Chang and by the Brain Research Center of National Tsing Hua University under the Higher Education Sprout Project funded by the Ministry of Education in Taiwan.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

Data Availability

The data underlying this article will be shared upon reasonable request to the corresponding author.

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