Individuals exhibit differences in their propensity to undergo fear conditioning.
BDNF level in the BLA determines the magnitude of conditioned fear response.
Increased adrenergic activity promotes the acquisition of conditioned fear.
Results
We demonstrate that C57BL/6 inbred mouse strain subjected to chronic social defeat training (CSDT) can be separated into susceptible and resistant subpopulations that display different levels of fear responses in an auditory fear conditioning paradigm. Susceptible mice had significantly more c-Fos protein expression in neurons of the basolateral amygdala (BLA) following CSDT and showed exaggerated conditioned fear responses, while there were no significant differences between groups in innate anxiety- and depressive-like behaviors. Through the use of conditional brain-derived neurotrophic factor (BDNF) knockout strategies, we show that elevated BLA BDNF level following fear conditioning training is a key mediator contributing to determine the levels of conditioned fear responses. Our combined behavioral, autonomic, and neuroendocrine approaches reveal that relative to susceptible mice, resistant mice had a much faster recovery from conditioned stimuli-induced cardiovascular and corticosterone responses. Systemic administration of norepinephrine reuptake inhibitor atomoxetine increased c-Fos protein expression in BLA neurons following fear conditioning training and promoted the expression of conditioned fear in resistant mice. Conversely, administration of -adrenergic receptor antagonist propranolol reduced fear conditioning training-induced c-Fos protein expression in BLA neurons and reduced conditioned fear responses in susceptible mice. These findings reveal a novel role for the BDNF signaling within the BLA in mediating individual differences in autonomic, neuroendocrine and behavioral reactivity to fear conditioning.
Discussion
The adaptive responses of the organism to stressful environmental challenges, including both behavioral and neuroendocrine adjustments, are crucial for maintaining physiological homeostasis, whereas a pathologically excessive response may result in the development of psychopathology. Fortunately, the vast majority of stress-exposed individuals can overcome crisis and maintain normal neuropsychological functioning. The present study provides novel evidence for the existence of individual differences in the acquisition and expression of conditioned fear.
We have shown that C57BL/6 mice subjected to CSDT can be separated into susceptible and resistant subpopulations on the basis of a measure of social avoidance, and that susceptible mice displayed higher levels of conditioned fear responses than did resistant mice. We have identified new functions for BLA BDNF signaling in mediating individual differences in autonomic, neuroendocrine and behavioral responses to auditory fear conditioning. Furthermore, pharmacological manipulations of adrenergic activity successfully normalize individual differences in conditioned fear responses.
We propose that inherent differences in reactivity to CSDT may help to predict which individuals may develop a higher level of conditioned fear response. We recognize that increase in BDNF protein levels within the BLA may serve as useful biomarker to further our mechanistic understanding of inherent differences in autonomic, neuroendocrine and behavioral reactivity to fear conditioning. Therapeutic interventions aimed at reducing BDNF secretion or blocking the BDNF signaling within the BLA may provide beneficial effects in preventing or decreasing the development of fear-related anxiety disorders. (The full article has been included in the Appendix).
This is the first study to demonstrate inherent differences in reactivity to CSDT may help to predict which individuals may develop a higher level of conditioned fear response.
National Cheng Kung University
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Brain-derived neurotrophic factor in the amygdala mediates susceptibility to fear conditioning
Dylan Choua,b,Chiung-Chun Huanga and Kuei-Sen Hsua,b,*
aDepartment of Pharmacology1, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan
bInstitute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan
(43 pages with 6 figures)
*Corresponding author: Kuei-Sen Hsu, Ph.D.,
Department of Pharmacology, College of Medicine, National Cheng Kung University, No. 1, University Rd., Tainan 701, Taiwan.
Tel: 886-6-2353535 ext. 5498; Fax: 886-6-2749296 E-mail:[email protected]
*Manuscript
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Appendix
Abstract
Fear conditioning in animals has been used extensively to model clinical anxiety disorders. While individual animals exhibit marked differences in their propensity to undergo fear conditioning, the physiologically relevant mediators have not yet been fully characterized. Here, we demonstrate that C57BL/6 inbred mouse strain subjected to chronic social defeat training (CSDT) can be separated into susceptible and resistant subpopulations that display different levels of fear responses in an auditory fear conditioning paradigm. Susceptible mice had significantly more c-Fos protein expression in neurons of the basolateral amygdala (BLA) following CSDT and showed exaggerated conditioned fear responses, while there were no significant differences between groups in innate anxiety- and depressive-like behaviors.
Through the use of conditional brain-derived neurotrophic factor (BDNF) knockout strategies, we show that elevated BLA BDNF level following fear conditioning training is a key mediator contributing to determine the levels of conditioned fear responses. Our combined behavioral, autonomic, and neuroendocrine approaches reveal that relative to susceptible mice, resistant mice had a much faster recovery from conditioned stimuli-induced cardiovascular and corticosterone responses.
Systemic administration of norepinephrine reuptake inhibitor atomoxetine increased c-Fos protein expression in BLA neurons following fear conditioning training and promoted the expression of conditioned fear in resistant mice. Conversely, administration of -adrenergic receptor antagonist propranolol reduced fear conditioning training-induced c-Fos protein expression in BLA neurons and reduced conditioned fear responses in susceptible mice. These findings reveal a novel role for the BDNF signaling within the BLA in mediating individual differences in autonomic, neuroendocrine and behavioral reactivity to fear conditioning.
Keywords: Fear conditioning; Social defeat stress; BDNF; Anxiety; Amygdala
Abbreviations: BDNF, brain-derived neurotrophic factor; BLA, basolateral amygdala;
CaMK, calmodulin-kinase II; CR, conditioned response; CS, conditioned stimulus;
CSDT, chronic social defeat training; EGFP, enhanced green fluorescent protein;
FORT, familiar odor recognition test; FST, forced swimming test; LV, lentivirus; MAP, mean arterial pressure; NeuN, neuronal nuclei; NORT, novel object recognition test;
OPRT, object placement recognition test; PCR, polymerase chain reaction; SIT, social interaction test; SMP, significant motion pixels; SPT, sucrose preference test; TBS, Tris-HCl buffer solution; TST, tail suspension test; US, unconditioned stimulus;
VEGF, vascular endothelial growth factor; WT, wild type.
Highlight
Individuals exhibit differences in their propensity to undergo fear conditioning.
BDNF level in the BLA determines the magnitude of conditioned fear response.
Increased adrenergic activity promotes the acquisition of conditioned fear.
Introduction
Experiencing stressful life events increase the risk of later developing anxiety and depressive disorders. While everyone experiences stressful events, there is considerable heterogeneity in responses (Kessler et al., 2005; Feder et al., 2009;
Russo et al., 2012). One of the most critical questions is why some individuals are able to overcome crisis whereas others experiencing the same stressors develop severe psychopathology. Understanding the neurobiological mechanisms of individual differences in risk for psychiatric disorders and developing more cost-effective treatments are of vital importance.
Fear conditioning paradigm has been used extensively as an experimental model to investigate the neurobiological basis of fear and anxiety (Mineka and Zinbarg, 2006). In the classical paradigm, a neutral conditioned stimulus (CS), usually an auditory tone, is paired with an aversive unconditioned stimulus (US), such as a footshock. After repeated CS-US pairings, an association is formed such that presentation of the CS alone elicits the same set of behavioral and autonomic responses (conditioned responses; CR) formerly produced by the US (LeDoux, 2000;
Maren, 2001). Although many brain areas have been implicated in fear conditioning, the amygdala is thought to be the main control center for the acquisition and expression of auditory fear conditioning (Goosens and Maren, 2001; Gale et al., 2004;
Rodrigues et al., 2004; Phelps and LeDoux, 2005). To date, the vast majority of fear conditioning research has focused on determining the neural mechanisms underlying typical responding in idealized average individuals. However, there is increasing awareness that considerable individual differences exist in the acquisition and expression of conditioned fear among species even among individuals from the same strain. For instance, judging on the measures of freezing behavior and ultrasonic vocalization, previous work has shown that Wistar rats, although identical in stock,
sex, age, and housing conditions, can differ considerably in their reactivity to auditory fear conditioning (Borta et al., 2006). However, little is known about molecular mediators in determining an individual's susceptibility to fear conditioning.
Using the measure of social interaction, Nestler and his colleagues have provided compelling experimental evidence that the inbred population of C57BL/6 mice subjected to chronic social defeat stress can be separated into susceptible and unsusceptible subpopulations based on the interaction ratio obtained from the social interaction test (SIT) (Berton et al., 2006; Krishnan et al., 2007; Vialou et al., 2010).
Since mice display distinct behavioral and physiological phenotypes following social defeated stress, the question arises as to whether similar paradigm could help to identify individual’s susceptibility to fear conditioning. Here, we take advantage of this dichotomy in behavioral outcomes after a chronic social defeat training (CSDT) in male C57BL/6 mice to study the molecular basis of individual differences in the acquisition and expression of conditioned fear. We found that mice that display high reactivity to CSDT exhibit significantly more freezing behavior in the conditioned fear test. Furthermore, the BDNF signaling within the BLA is associated with individual differences in conditioned fear responses.
Materials and methods
Animals
Adult male C57BL/6 (8-12 weeks old), homozygous brain-derived neurotrophic factor (BDNF)-floxed (Bdnftm3Jae/J), and calmodulin-kinase II (CamK)-Cre transgenic mice were originally obtained from The Jackson Laboratory and bred within our animal facility. Bdnftm3Jae/J mice were crossed to CamK-Cre mice to generate BDNF conditional knockout mice in the C57BL/6 genetic background. Mice were genotyped by a polymerase chain reaction (PCR)-based method using genomic DNA isolated from tail samples. Mice were housed in groups of three to four in a humidity- and temperature-controlled (25 1 °C) vivarium on a 12 h light/dark cycle (lights on 06:00-18:00 h) with access to food and water ad libitum and were acclimated in the animal research facility for at least one week prior to use in behavioral experiments. All behavioral procedures were performed during the light cycle between 10:00 and 15:00 h. All animal procedures described were executed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of National Cheng Kung University. All efforts were made to minimize the number of animals used and their suffering.
Chronic social defeat training
C57BL/6 mice were subjected to a 5 min social defeat training twice per day for 10 consecutive days as previously described (Berton et al., 2006; 2007; Krishnan et al., 2007) with some modifications. During each defeat episode, C57BL/6 mouse was introduced into the home cage of an unfamiliar and aggressive CD1 retired breeder mouse (4-6 months old; Charles River Laboratories) for physical encounter. The 5 min training session was divided into two consecutive periods. During the initial
period (20 sec, time counted starting from the first contact), C57BL/6 mouse was allowed for direct physical interaction with the CD1 mouse. During the period 2 (280 sec), C57BL/6 mouse was placed in the protective cage inside the CD1 mouse home cage, allowing olfactory, visual, and auditory communication but preventing tactile contact. Following CSDT, C57BL/6 mice were then placed back into their home cage and were housed individually. Control mice were housed in equivalent cage with members of the same strain. SIT was carried out 24 h after the last training day. For the SIT, we measured the time spent in the interaction zone during the first (target absent) and second (target present) trials. Social approach-avoidance behaviors were recorded using a digital video camera and analyzed with the Ethovision tracking system (Nodus). The interaction ratio was calculated as (time spent in the interaction zone in the presence of target)/(time spent in the interaction zone in the absence of target). Mice with ratio < 1 were labeled as susceptible subpopulation, and those with ratio 1 were labeled as resistant subpopulation.
Auditory fear conditioning
Mice were transferred to fear conditioning chamber (15.9 × 14.0 × 12.7 cm;
ENV-307A, MED Associates) that was equipped with a shock floor and placed into a ventilated sound-attenuating isolation cubicle. Following a 3-min acclimation period, mice received three presentations of an auditory conditioning stimulus (CS; 90 dB sound at 2 kHz for 20 sec) paired with an aversive footshock unconditioned stimulus (US; 0.6 mA for 2 sec), separated by 30 sec interval. The CS-induced CR was measured 1 h, 24 h, or 7 d after the last conditioning trial. The CR was scored as the total time of the mouse spent in freezing during a 3-min test session. The extinction trials were performed at 24 h intervals for two consecutive days and consisted of 10 trials of 20 sec re-exposure to the conditioning tone cue without presentation of
footshock. Twenty-four hours later, mice were returned to a novel chamber to test for recall of extinction learning with additional tone-alone trials. The behavior data were analyzed by differential subtraction of two consecutive images captured at 7.5 Hz to calculate the significant motion pixels (SMP). A freezing behavior was defined as the value of SMP < 20 at any indicated time point.
Odor avoidance test
An odor avoidance test was used to evaluate the innate fear. Mice were initially habituated to the testing chamber (42 × 42 × 42 cm) for 3 days (10 min/day) in a low illumination (approximately 10 Lux) condition room. In the test session, mice were exposed to no or three odors in sequence (no odor, cat urine, 5 % acetic acid and 0.1
% osmanthus), separated by 10 min inter-trial intervals. Odors were mixed into sand in 10 cm plastic cups. On each test trial, one odor was exposed to 5 min. After each trial, the apparatus was thoroughly cleaned with ethanol solution (10% v/v).
The behavior of the animals was video recorded using a digital video camera and scoring was performed with the behavioral tracking system Ethovision (Nodus).
Fear scores were calculated as the time spent in the odor interaction zone (12 × 12 cm).
Tail suspension test
Mice were tested on a modified version of the tail suspension test (TST) described by Steru et al. (1985). At the day of testing, experimental mice were transferred to the experiment room and allowed to acclimatize for 3 h. Mice were individually suspended by the tail to a horizontal ring-stand bar (distance from floor = 25 cm) using adhesive tape wrapped around its tail (1 cm from tip). Typically, mice displayed multiple escape-oriented behaviors interspersed with bouts of immobility as
the session progressed. A 3-min test session was used that was recorded by a digital video camera positioned in front of the TST apparatus. The total time of immobility for individual animal was calculated. Mice were considered immobile when they hung passively and motionless. Because of a propensity of C57BL/6 mice to climb up their tails during the testing session (Huang et al., 2012), mice that climbed their tail or fell off the hanger were excluded from the final analysis (less than 20% of the total population).
Sucrose preference test
Sucrose preference test (SPT) was performed as described previously (Huang et al., 2012). Briefly, mice were first habituated to consume water in the two-bottle-choice paradigm for 3 days. At the beginning of the experiment, mice were housed individually in the test chamber identical to their home cage and water was deprived for one hour to increase drinking behavior. In the test session, mice were provided access to two bottles with 1% sucrose solution and water, respectively, for 3 h. The sucrose preference score was determined dividing the volume of sucrose consumed by the total liquid consumption.
Forced swimming test
Forced swimming test (FST) was used as previously described (Porsolt et al., 1977). Mice were individually placed in Plexiglas cylinder (30 cm in diameter and 40 cm deep) containing 20 cm water (24 ± 1 °C) and were video recorded for 5 min.
Active (swimming, climbing and struggling) or passive (immobility) behaviors were scored with the behavioral tracking system Ethovision (Noldus). After the forced swim session, mice were towel dried thoroughly and then returned to their home cages. The water was changed after each mouse.
Novel object recognition test
Novel object recognition test (NORT) was performed as described previously (Bevins and Besheer, 2006). Briefly, mice were initially habituated to the empty chamber (42 × 42 × 42 cm) by allowing them to freely explore for 10 min each day for 3 days. After 24 h, mice were rehabituated to the empty chamber for 1 min and then returned to their home cage while two identical objects were placed at the rear left and right corners of the chamber. Afterward, mice were placed in the chamber and were allowed to explore two identical objects for 10 min. One hour after training, object recognition was tested by substituting a novel object for a familiar training object. Mice were placed in the testing chamber for 5 min and the time spent in exploring each object was recorded using a digital video camera and scoring was performed with the behavioral tracking system Ethovision (Noldus). To analyze cognitive performance, a discrimination index was calculated as the time exploring the novel object, expressed as the ratio of the total time spent exploring both novel and familiar objects.
Object placement recognition test
Object placement recognition test (OPRT) was performed as described previously (Yang et al., 2012). Briefly, mice were initially habituated to the empty chamber (42 × 42 × 42 cm) by allowing them to freely explore for 10 min each day for 3 days. After 24 h, mice were rehabituated to the empty chamber for 1 min and then returned to their home cage while two identical objects were placed at two diagonal corners of the chamber. Afterward, mice were placed in the chamber and were allowed to explore two identical objects for 10 min. In the test phase, one familiar object was repositioned, and the other familiar object was placed in the same location as during the training trial. One hour after training, mice were placed in the
testing chamber for 5 min and the time spent in exploring each object was recorded using a digital video camera and scoring was performed with the behavioral tracking system Ethovision (Noldus). To analyze cognitive performance, a discrimination index was calculated for each rat as the following formula: (time spent on the object in novel location)/ (time spent on the object in novel location + time spent on the object in familiar location).
Familiar odor recognition test
Familiar odor recognition test (FORT) was conducted as described previously (Farovik et al., 2011) with some modifications. Mice were initially starved for 24 h before experiments and were then trained to dig for reward (one chocolate pellet) buried in a cup filled with unscented sand. Once mice had learned to retrieve the reward by digging in the cup, they were allowed to an odor recognition test in which each trial consisted of a sample and a test phase. In the sample phase, mice were presented with a cup filled with sand and scented with one distinct odor. After 5 min delay, two cups, scented with a different odor (one old and one new), were presented in consecutive fashion and in a random order and chocolate pellet was buried in the cup of familiar odor that mice could obtain by digging in the sand for correct old response. Trials that required the same response did not occur more than three times in a row during the test phase. For new trial, a response was defined as mice moving the sand with the forepaw. Once mice reached the criterion of 80% correct over two consecutive sessions (each session consisted of 10 trials), each session consisted of a serial three sample odors, then a 5-min delay, then 3 old and 3 new odors in a random
Familiar odor recognition test (FORT) was conducted as described previously (Farovik et al., 2011) with some modifications. Mice were initially starved for 24 h before experiments and were then trained to dig for reward (one chocolate pellet) buried in a cup filled with unscented sand. Once mice had learned to retrieve the reward by digging in the cup, they were allowed to an odor recognition test in which each trial consisted of a sample and a test phase. In the sample phase, mice were presented with a cup filled with sand and scented with one distinct odor. After 5 min delay, two cups, scented with a different odor (one old and one new), were presented in consecutive fashion and in a random order and chocolate pellet was buried in the cup of familiar odor that mice could obtain by digging in the sand for correct old response. Trials that required the same response did not occur more than three times in a row during the test phase. For new trial, a response was defined as mice moving the sand with the forepaw. Once mice reached the criterion of 80% correct over two consecutive sessions (each session consisted of 10 trials), each session consisted of a serial three sample odors, then a 5-min delay, then 3 old and 3 new odors in a random