Fear extinction learning can be impaired or enhanced by modulation of the CRF system in the basolateral nucleus of the amygdala

doi:10.1016/j.bbr.2014.06.021

Behavioural Brain Research

Volume 271, 1 September 2014, Pages 234-239

https://doi.org/10.1016/j.bbr.2014.06.021 Get rights and content

Highlights

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Fear extinction memory is impaired by increases in endogenous CRF in the BLA.
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Fear extinction memory is impaired by intra-BLA infusions of CRF.
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Fear extinction memory is enhanced by intra-BLA infusions of a CRF receptor antagonist.

Abstract

The neuropeptide corticotropin-releasing factor (CRF) is released during periods of anxiety and modulates learning and memory formation. One region with particularly dense concentrations of CRF receptors is the basolateral nucleus of the amygdala (BLA), a critical structure for both Pavlovian fear conditioning and fear extinction. While CRF has the potential to modify amygdala-dependent learning, its effect on fear extinction has not yet been assessed. In the present study, we examined the modulatory role of CRF on within-session extinction and fear extinction consolidation. Intra-BLA infusions of the CRF binding protein ligand inhibitor CRF_(6–33) which increases endogenous levels of free CRF, or intra-BLA infusions of exogenous CRF made prior to fear extinction learning did not affect either fear expression or within-session extinction learning. However, when these animals were tested twenty-four hours later, drug free, they showed impairments in extinction memory. Conversely, intra-BLA infusions of the CRF receptor antagonist α-helical CRF_(9–41) enhanced memory of fear extinction. These results suggest that increased CRF levels within the BLA at the time of fear extinction learning actively impair the consolidation of long-term fear extinction.

Introduction

Human subjects with anxiety disorders exhibit abnormalities in how they acquire and/or extinguish conditioned fear responses [1], [2]. Understanding how anxiety and conditioned fear interact at the neuronal level may thus yield fundamental insights into the causes of anxiety disorders and provide a foundation for clinical investigation. Corticotropin-releasing factor (CRF) is a key neuropeptide for initiating behavioral, endocrine and autonomic responses to stress [3], [4], [5]. It is released by neurons in the paraventricular nucleus of the hypothalamus to regulate pituitary ACTH secretion, thus triggering a key component of the hypothalamic-pituitary-adrenal (HPA) axis [6]. Growing evidence suggests that the CRF system plays a significant role in the regulation of anxiety [7], [8]. For example, chronic hyperactivation of the CRF system has been linked to several anxiety disorders and depression [9], [10]. Levels of CRF are enhanced in patients with post-traumatic stress disorder (PTSD) [11], while single nucleotide polymorphisms in the CRF gene have been associated with childhood risk factors for panic disorders [12].

Traditionally, stress-related behavior was thought to be mediated solely by activation of the HPA axis. However, growing evidence suggests that the extrahypohalamic CRF system plays a significant role in the regulation of anxiety. Both the basolateral nucleus of the amygdala (BLA) and central nucleus of the amygdala (CE) are rich in CRF immunoreactive cell bodies, terminals and receptors [13], [14], [15]. Acute stress elevates extracellular CRF levels in the amygdala [16], [17]. A large number of studies have implicated the amygdala as a key player in mediating anxiety responses [18], [19], [20]. Moreover, altered amygdala function has been implicated in several anxiety disorders such as generalized anxiety disorder as well as in PTSD [21], [22], [23]. While the amygdala is only one of a constellation of structures involved in mediating anxiety [24], it plays a crucial and clearly defined role is Pavlovian fear conditioning and extinction.

In classical fear conditioning, an initially neutral stimulus, such as a tone (conditioned stimulus; CS) is paired with a noxious stimulus such as a brief electrical footshock (unconditioned stimulus; US). Afterwards, when presented alone, the CS elicits responses in the animal characteristic of fear [25]. Extinction of conditioned fear is a form of new learning in that the CS is repeatedly presented alone so that it ceases to elicit a fear response [26], [27]. The original fear memory is inhibited, but not erased, as extinction actively suppresses fear responses in a context-dependent fashion [27]. Evidence suggests that the BLA is a critical site of plasticity for fear conditioning and is also required for the acquisition and storage of extinction memory [28], [29], [30].

A number of studies suggest that CRF may modulate both amygdala-dependent and amygdala-independent learning. CRF antagonists infused into the BLA disrupt contextual fear conditioning [31] and impair memory formation of an inhibitory avoidance task [32], suggesting that in general CRF may enhance learning. Moderate increases in CRF also enhance performance in a spatial learning task, visual discrimination paradigm and an inhibitory avoidance task [32], [33]. However, a recent report suggests that infusions of CRF into the BLA might impair fear conditioning [34]. The effects of CRF or CRF antagonists on fear extinction learning have not been assessed. However, levels of CRF are enhanced in PTSD patients [35] and PTSD patients exhibit deficits in their ability to extinguish learned fear [36], [37], [38], suggesting that CRF in the amygdala might impair fear extinction learning as well.

Here we manipulated the CRF system within the BLA in several different ways prior to fear extinction learning to test the involvement of this neuropeptide on the extinction of fear memories. We took advantage of the fact that endogenous CRF binds to the high affinity binding protein (CRF-BP), a membrane-associated protein which sequesters and inhibits CRF [39]. Administration of CRF-BP ligand inhibitors displace CRF which is then free to act at available CRF receptors [40]. This increase in endogenous CRF is thought to be comparable to administration of a low concentration of exogenous CRF and has been shown to enhance performance in several learning tasks [32], [33], [40]. In this study, we evaluated the effects of intra-BLA infusions of (1) a CRF-BP ligand inhibitor, (2) CRF itself and (3) a CRF receptor antagonist each administered prior to fear extinction learning. Our results suggest that increased levels of CRF within the BLA inhibit the formation of long-term memory of fear extinction.

Section snippets

Subjects

Adult male Sprague Dawley rats (Charles River Laboratories; 250–325 g) were housed individually with ad libitum access to food and water and maintained on a 12 h light/dark cycle. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by Columbia University's Animal Care and Use Committee.

Surgery

Rats were anesthetized with a mixture of isoflurane and oxygen and mounted in a stereotaxic apparatus. Betadine was

Results

We first asked whether increasing endogenous levels of CRF in the BLA by inhibiting CRF-BP affects fear extinction. Rats were habituated to the training context on day 1 and conditioned with 3 tone-shock pairings on day 2 (Fig. 2A; each CS was a 5 kHz 30 s tone co-terminating with a footshock US = 0.5 mA, 1 s). Freezing to each tone was quantified (Fig. 2B). A repeated measures ANOVA across all 3 tones found no significant difference between groups (F_(2,16) = 0.86; p = 0.44), ensuring that there were no

Discussion

In this study we evaluated whether modulating the CRF system within the BLA affects fear extinction. Our data suggest that increasing the concentration of CRF within in the BLA impairs the consolidation of long-term fear extinction memories.

While this is the first study to examine the effects of CRF on fear extinction, it has been previously demonstrated that CRF enhances fear learning in the BLA. CRF antagonists infused into the BLA disrupt contextual fear conditioning [31] and impair memory

Acknowledgements

This work was supported by the National Institute of Mental Health Grant R15-MH-095032.

References (61)

C. Grillon
Startle reactivity and anxiety disorders: Aversive conditioning, context, and neurobiology
Biol Psychiatry
(2002)
M.R. Milad et al.
Presence and acquired origin of reduced recall for fear extinction in PTSD: results of a twin study
J Psychiatr Res
(2008)
G.F. Koob et al.
A role for corticotropin releasing factor and urocortin in behavioral responses to stressors
Brain Res
(1999)
A.J. Dunn et al.
Physiological and behavioral responses to corticotropin- releasing factor administration: is CRF a mediator of anxiety or stress responses?
Brain Res Brain Res Rev
(1990)
M. Heilig et al.
Corticotropin-releasing factor and neuropeptide Y: role in emotional integration
Trends Neurosci
(1994)
T.L. Bale
Sensitivity to stress: dysregulation of CRF pathways and disease development
Horm Behav
(2005)
F.J. Sautter et al.
Corticotropin-releasing factor in posttraumatic stress disorder (PTSD) with secondary psychotic symptoms, nonpsychotic PTSD, and healthy control subjects
Biol Psychiatry
(2003)
J.W. Smoller et al.
The corticotropin-releasing hormone gene and behavioral inhibition in children at risk for panic disorder
Biol Psychiatry
(2005)
J.M. Reul et al.
Corticotropin-releasing factor receptors 1 and 2 in anxiety and depression
Curr Opin Pharmacol
(2002)
A. Jaferi et al.
Mu-opioid and corticotropin-releasing-factor receptors show largely postsynaptic co-expression, and separate presynaptic distributions, in the mouse central amygdala and bed nucleus of the stria terminalis
Neuroscience
(2009)

R. Adolphs

Neural systems for recognizing emotion

Curr Opin Neurobiol

(2002)

S.L. Rauch et al.

Exaggerated amygdala response to masked facial stimuli in posttraumatic stress disorder: a functional MRI study

Biol Psychiatry

(2000)

M.D. De Bellis et al.

A pilot study of amygdala volumes in pediatric generalized anxiety disorder

Biol Psychiatry

(2000)

K.M. Myers et al.

Behavioral and neural analysis of extinction

Neuron

(2002)

D.T. Hubbard et al.

Activation of basolateral amygdala corticotropin-releasing factor 1 receptors modulates the consolidation of contextual fear

Neuroscience

(2007)

S.C. Heinrichs et al.

Enhancement of performance in multiple learning tasks by corticotropin- releasing factor-binding protein ligand inhibitors

Peptides

(1997)

K. Isogawa et al.

Contrasting effects of pretraining, posttraining and pretesting infusions of corticotropin-releasing factor into the lateral amygdala: attenuation of fear memory formation but facilitation of its expression

Biol Psychiatry

(2013)

V.B. Risbrough et al.

Role of corticotropin releasing factor in anxiety disorders: a translational research perspective

Horm Behav

(2006)

T. Peri et al.

Psychophysiologic assessment of aversive conditioning in posttraumatic stress disorder

Biol Psychiatry

(2000)

A.F. Donatti et al.

Activation of corticotropin-releasing factor receptors from the basolateral or central amygdala increases the tonic immobility response in guinea pigs: an innate fear behavior

Behav Brain Res

(2011)

T.J. Sajdyk et al.

Role of corticotropin-releasing factor and urocortin within the basolateral amygdala of rats in anxiety and panic responses

Behav Brain Res

(1999)

M.S. Cratty et al.

Prenatal stress increase corticotropin-releasing factor (CRF) content and release in rat amygdala minces

Brain Res

(1995)

C.J. Cook

Stress induces CRF release in the paraventricular nucleus, and both CRF and GABA release in the amygdala

Physiol Behav

(2004)

T. Blank et al.

The corticotropin-releasing factor receptor 1 antagonist CP-154,526 reverses stress-induced learning deficits in mice

Behav Bran Res

(2003)

K. Uryu et al.

Fine structure and possible origins of nerve fibers with corticotropin-releasing factor-like immunoreactivity in the rat central amygdaloid nucleus

Brain Res

(1992)

A. Pitkanen et al.

Organization of intra-amygdaloid circuitries in the rat: an emerging framework for understanding functions of the amygdala

Trends Neurosci

(1997)

C. Rivier et al.

Modulation of stress-induced ACTH release by corticotropin-releasing factor, catecholamines and vasopressin

Nature

(1983)

M.R. Brown et al.

Corticotropin-releasing factor: actions on the sympathetic nervous system and metabolism

Endocrinology

(1982)

A.V. Turnbull et al.

Corticotropin-releasing factor (CRF) and endocrine responses to stress: CRF receptors, binding protein, and related peptides

Proc Soc Exp Biol Med

(1997)

S.C. Heinrichs et al.

Corticotropin-releasing factor in brain: a role in activation, arousal, and affect regulation

J Pharmacol Exp Ther

(2004)

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Moreover, the detrimental effects of BLA-CRF were suggested to depend on CRF signaling at the CRFR1 receptor since antagonizing its actions via an intra-BLA injection of the CRFR1 receptor antagonist α-helical CRF(9 −4 1) prior to extinction learning improved later extinction recall (Abiri et al., 2014). Thus, increased CRF levels in the amygdala at the time of extinction learning seem to strongly impede successful fear extinction (Abiri et al., 2014; Gafford et al., 2012). Since CRF release is strongly enhanced by stressful and threatening situations, it is reasonable to assume that CRF has a prominent role in mediating the IED, which can be caused by intense fear acquisition paradigms that evoke heightened arousal during immediate fear extinction learning (Fitzgerald et al., 2015; Maren & Chang, 2006).
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Research reportFear extinction learning can be impaired or enhanced by modulation of the CRF system in the basolateral nucleus of the amygdala

Highlights

Abstract

Introduction

Section snippets

Subjects

Surgery

Results

Discussion

Acknowledgements

Biol Psychiatry

J Psychiatr Res

Brain Res

Brain Res Brain Res Rev

Trends Neurosci

Horm Behav

Biol Psychiatry

Biol Psychiatry

Curr Opin Pharmacol

Neuroscience

Curr Opin Neurobiol

Biol Psychiatry

Biol Psychiatry

Neuron

Neuroscience

Peptides

Biol Psychiatry

Horm Behav

Biol Psychiatry

Behav Brain Res

Behav Brain Res

Brain Res

Physiol Behav

Behav Bran Res

Brain Res

Trends Neurosci

Modulation of stress-induced ACTH release by corticotropin-releasing factor, catecholamines and vasopressin

Nature

Corticotropin-releasing factor: actions on the sympathetic nervous system and metabolism

Endocrinology

Corticotropin-releasing factor (CRF) and endocrine responses to stress: CRF receptors, binding protein, and related peptides

Proc Soc Exp Biol Med

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Research report
Fear extinction learning can be impaired or enhanced by modulation of the CRF system in the basolateral nucleus of the amygdala