Elsevier

Behavioural Brain Research

Volume 237, 15 January 2013, Pages 23-31
Behavioural Brain Research

Research report
Neural substrates of fear conditioning, extinction, and spontaneous recovery in passive avoidance learning: A c-fos study in rats

https://doi.org/10.1016/j.bbr.2012.09.024Get rights and content

Abstract

Extinguishing fear conditioning and preventing the return of fear are the goal in the treatment of anxiety disorders. However, the neural substrates that mediate fear conditioning, extinction, and spontaneous recovery (i.e., the return of fear) remain uncertain. We utilized the aversive passive avoidance learning paradigm and Fos-like immunoreactivity to elucidate this issue. Exception for naïve rats that did not receive any treatment served as the control group, the other rats were subjected to three sessions of context/footshock (0.5 mA, 2 s) pairings followed by 12 extinction sessions (context-no footshock). After the last extinction test, these rats were assigned to one of three groups reflecting the number of resting days before the test session (context-no footshock): Day 8, Day 9, and Day 10 groups. Only the Day 10 group exhibited spontaneous recovery during the test session. Fos-like immunoreactivity associated with fear conditioning was seen in the amygdala and cingulate cortex area 1 (Cg1). The extinction of fear was seen to be related to Cg1, cingulate cortex area 2 (Cg2), piriform cortex (Pir), and entorhinal cortex (Ect). Spontaneous recovery was seen to be related to amygdala, Pir, and Ect. The present findings indicate that the brain substrates of fear acquisition, extinction and spontaneous recovery have different ensembles of brain activations. These differences suggest that different brain targets may be considered for fear extinction and for avoiding the return of fear in anxiety disorders.

Highlights

► Fear conditioning was extinguished when encountering a context without footshock. ► Spontaneous recovery only occurred in Day 10 following extinction session. ► Conditioning, extinction and spontaneous recovery have distinct brain activations. ► The results provide clinical implications in human anxiety disorders.

Introduction

Anxiety disorders include panic disorder, posttraumatic stress disorder, obsessive–compulsive disorder, and some specific phobias [1]. Growing evidence has shown that those symptoms are formed through Pavlovian conditioning, in which the conditioned stimulus (CS) is a neutral stimulus or context and the unconditioned stimulus (US) is a traumatic or aversive event [2], [3], [4]. Extinguishing fear and preventing its return is the goal of the treatment of anxiety disorders [5], [6], [7]. Aversive Pavlovian conditioning paradigm has been used in an animal model to examine this issue [7], [8], [9]. In an aversive learning task, animals learn to avoid entering a dark compartment (CS) in a light/dark avoidance apparatus after encountering a footshock (US) in the dark compartment. The latency to move into the dark compartment is a measure of fear conditioning [10]. Moreover, fear diminishes when the CS is repeatedly presented without the US, demonstrating extinction [11]. Following extinction for a length of time, fear may recover; this is called spontaneous recovery [12] and reflecting the return of fear [13]. The time interval following extinction is a crucial variable for spontaneous recovery to occur [14], [15].

Studies that investigated the brain mechanisms underlying fear conditioning, extinction, and spontaneous recovery have yielded rather inconsistent results. For example, the amygdala has been shown to be involved in fear conditioning [16], but recent investigations have dissociated the basolateral amygdala [17], [18] and lateral amygdala [19] to manifest its different functions in the fear conditioning. Moreover, it has been shown that the cerebellum might regulate the consolidation of fear memory [20]. Some studies that have explored the neural substrates of extinction indicated that the basolateral, but not central nucleus of the amygdala mediates the extinction of conditioned taste aversion [21] and conditioned fear [22], [23]. Additionally, a recent review reported that the prefrontal cortex (PFC)–amygdala interaction may underlie the extinction of conditioned fear [24]. Compared to the study of neural substrates of fear conditioning and extinction, study of neural substrates of spontaneous recovery is quite scarce. One study has demonstrated that the ventromedial PFC is necessary for the recovery of fear [25].

This study is designed to directly compare the neural substrates of fear conditioning during acquisition phase, during extinction phase and during spontaneous recovery phase. The past research studied the neural substrates of each phase of fear conditioning, but to our knowledge this is the first one to look into all three phases within one experiment. Taken together, the present research has two goals: (a) to determine which time point would induce acquisition, extinction, and spontaneous recovery of fear conditioning. (b) According to the results of the critical time points, the study explores which brain areas activate each of the acquisition, extinction, and spontaneous recovery phases. We utilized the Fos-like immunohistochemical staining and chose some typical brain areas to involvements of Pavlovian fear conditioning [i.e., amyagala, cingulate cortext area 1 (Cg1), cingulate cortex area 2 (Cg2), and prelimbic cortex (PrL)] and some non-relevant brain areas with fear conditioning [i.e., nucleus accumbens (NAc), orbital cortex (Oct), piriform cortex (Pir), and entorhinal cortex (Ect)] to examine the involvement of specific neural substrates on each of three phases.

Section snippets

Animals

Fifty-four male Sprague-Dawley rats were purchased from BioLASCO Taiwan Co., Ltd (Yi-Lan County, Taiwan). All of the rats weighed 250–300 g at the beginning of the experiment. They were individually housed in plastic cages with sawdust bedding in a colony room with temperature maintained at 20 ± 2 °C and a 12 h/12 h light/dark cycle (light on 7:00 AM–7:00 PM) with food and water available ad libitum. All of the experiments were performed in accordance with the guidelines of the Academia Sinica

Acquisition, extinction, and spontaneous recovery of conditioned fear

We measured the latency to enter the dark compartment as the fear response. During the acquisition phase, a minor footshock (US) was presented in the dark compartment in three trials in one session, followed by four extinction sessions and one test session (three trials per session) in which no shock was present in the dark compartment (Fig. 1B). A 3 × 3 mixed two-way ANOVA revealed no effect of group (SP/Day 8, SP/Day 9, and SP/Day 10) on latency (F2,27 = 0.75, p > 0.05), a significant effect of

Discussion

The present results show that fear increases during the acquisition session when CS and US are paired repeatedly, decreases during the extinction session when CS alone is presented repeatedly. Spontaneous recovery (returning of fear responses) occurs following a period of 10 days of inactive time, but not before. Fos-like activation was observed in the amygdala and Cg1 during the acquisition session. The brain areas involved in the extinction process include the Cg1, Cg2, Pir, and Ect. The

Conclusions

Conditioned fear as indicated in increased latency to avoid the place where aversive event had occurred increased gradually during acquisition sessions. It decreased gradually during extinction sessions. Some 10 days following extinction fear returned as shown in spontaneous recovery sessions. Fos-like immunohistochemical staining indicated that c-fos overexpression occurred in the amygdala and Cg1 during the acquisition session. The brain areas activated during extinction included the Cg1,

Acknowledgements

Research was supported by grants NSC 100-2410-H-431-003 and NSC 99-2410-H-431-013 from the National Science Council to ACWH and grant NSC 96-2320-B-001-017-MY3 from the National Science Council to BCS. We thank Mr. Dong-Wei Lu and Ms. Kuan-Mei Lu for their help counting Fos-like-immunoreactive nuclei. This study was performed at the Institution of Biomedical Science, Academia Sinica, Taipei, Taiwan.

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