Research reportAdrenal-dependent diurnal modulation of conditioned fear extinction learning
Introduction
The ability of organisms to form associations between innately dangerous events (unconditioned stimuli or US) and surrounding cues (conditioned stimulus or CS) serves an adaptive function – it permits organisms to better escape or avoid dangerous situations. Normally, conditioned fear responses diminish with repeated exposure to the CS in the absence of the US due to the acquisition of new competing conditioned fear extinction associations [1]. However, in many individuals with anxiety-related psychiatric disorders the expression of conditioned fear persists and becomes pathological [2], [3]. Patients suffering from these maladies, as well as subjects in an animal model of post traumatic stress disorder (PTSD), show deficits in fear extinction learning [4], [5]. Given the debilitating and persistent nature of PTSD, research leading to a better understanding of the underlying neural processes that regulate conditioned fear and extinction expression may lead to improved treatment strategies.
Conditioned fear is an aversive learning process that is highly sensitive to environmental conditions such as context and degree of stress [6]. Time of day has also been found to modulate various aspects of conditioned fear expression and extinction in rodent and human studies [7], [8], [9], [10]. Glucocorticoid hormones (CORT, cortisol in humans and corticosterone in rats) are a well-known transducer of the biological effects of environmental stressors [11] and time of day [12], [13], [14]. There has been some examination of CORT-dependent modulation of auditory cue and contextual conditioned fear acquisition, memory, and extinction but virtually no examination of CORT effects on auditory cue conditioned fear extinction learning (reviewed in Rodrigues et al. [15]). Furthermore, no prior studies have examined whether CORT regulates various aspects of conditioned fear and extinction expression in a diurnal fashion. Subregions of the amygdala are central to conditioned fear and extinction learning, and the medial prefrontal cortex (mPFC) and hippocampus contribute critical modulation of these processes [16], [17], [18]. Neuroplasticity in the mPFC and hippocampus is sensitive to circadian modulation [8], [19], and a number of related cognitive functions exhibit diurnal performance patterns [20], [21], [22].
The goals of our study were to determine if there was a time of day difference in conditioned fear and/or extinction learning in rats (experiment 1), and to assess whether the presence of adrenal hormones was necessary for any observed time of day differences (experiment 2). Emotionally driven memories are strongly modulated by the presence and/or timing of acute CORT administration [23]. Therefore, diurnal variations in conditioned fear learning and/or extinction learning may be modulated by the presence or absence of endogenous CORT. For this study we assessed conditioned fear to a discrete auditory cue as well as extinction learning when it was presented in a context separate from the initial cue-shock pairing in order to assess the diurnal and adrenal-dependent effects on cue specific conditioned fear learning and extinction [24].
Section snippets
Animals
For experiments 1 (n = 6, N = 12) and 2 (n = 6, N = 24) male Sprague Dawley rats (250–280 g; Harlan Laboratories, Indianapolis, IN) were housed two per cage (polycarbonate tubs, 47 cm × 23 cm × 20 cm) and given food (Teklad Rodent Diet 8640; Harlan) and water ad libitum. For experiment 2, rats were given food and either tap water (Sham rats) or 0.9% saline (adrenalectomized, ADX rats) ad libitum. Rats were housed in two separate rooms within a suite of rooms that included the behavioral testing rooms. Each
Session 1 (day 1) conditioned fear acquisition
There was no effect of time of day on conditioned fear acquisition freezing behavior (Fig. 2A). Rats trained at either ZT4 or ZT16 exhibited minimal freezing behavior before and during the tone and displayed high levels of freezing during the 1 min post-shock observation period.
Session 2 (day 2) expression of conditioned fear and extinction
Rats tested at either ZT4 or ZT16 exhibited comparable levels of freezing throughout the conditioned fear recall/extinction session (Fig. 2B). Both groups of rats froze to a similar extent during block one trials,
Discussion
Our experiments revealed an adrenal-dependent time of day difference in auditory cue conditioned fear extinction learning. In contrast, neither adrenal status nor time of day influenced the acquisition of conditioned fear. Specifically, 24 h recall of auditory cue conditioned fear was comparable in adrenal-intact and ADX rats trained and tested at ZT16 (active phase) or ZT4 (inactive phase). However, adrenal-intact rats, but not ADX rats, trained and tested at ZT16 displayed reduced freezing
Authors’ contribution
The roles of the contributing authors are as follows: Elizabeth R Woodruff: experiment design and conceptualization, experiment execution, data analysis, data interpretation, and manuscript writing and editing; Benjamin N Greenwood: experiment design and conceptualization, data interpretation, and manuscript writing and editing; Lauren E Chun: experiment design and conceptualization, experiment execution, data analysis, data interpretation, and manuscript writing and editing; Sara Fardi:
Acknowledgments
The authors would also like to thank Professor Jerry Rudy for his expert advice and edits of this manuscript.
This work was supported by grant NIH MH075968. The NIMH and NIH had no further role in the study design; in the collection, analysis and interpretation of the data; in the writing of the report; or in the decision to submit the paper for publication.
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