Immediate pre-learning stress enhances baseline startle response and fear acquisition in a fear-potentiated startle paradigm

Behavioural Brain Research 371 (2019) 111980

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Immediate pre-learning stress enhances baseline startle response and fear acquisition in a fear-potentiated startle paradigm

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Mackenzie R. Riggenbacha, Jordan N. Weisera, Brianne E. Mosleya, Jennifer J. Hipskinda, Leighton E. Wiremana, Kelsey L. Hessa, Tessa J. Duffya, Julie K. Handela, MacKenzie G. Kaschalka, Kassidy E. Reneaua, Boyd R. Rorabaughb, Seth D. Norrholmc,d, Tanja Jovanovicd, ⁎ Phillip R. Zoladza, a

Department of Psychology, Sociology, & Criminal Justice, Ohio Northern University, Ada, OH, 45810, USA Department of Pharmaceutical & Biomedical Sciences, Raabe College of Pharmacy, Ohio Northern University, Ada, OH, 45810, USA Mental Health Service Line, Atlanta VA Medical Center, Decatur, GA, USA d Department of Psychiatry and Behavioral Sciences, Emory University, Atlanta, GA, USA b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Stress Memory Cortisol Fear conditioning Extinction

Extensive work has shown that stress time-dependently influences hippocampus-dependent learning and memory. In particular, stress that is administered immediately before learning enhances long-term memory, while stress that is temporally separated from learning impairs long-term memory. We have extended these findings by examining the impact of immediate, pre-learning stress on an amygdala-dependent fear conditioning task. One hundred and forty-one healthy participants underwent a stress (socially evaluated cold pressor test) or control manipulation immediately before completing differential fear conditioning in a fear-potentiated startle paradigm. Participants then completed extinction and extinction memory testing sessions 24 and 48 h later, respectively. Stress administered immediately before acquisition increased baseline startle responses and enhanced fear learning, as evidenced by greater fear-potentiated startle to the CS + . Although no group differences were observed during extinction training on Day 2, stressed participants exhibited evidence of impaired extinction processes on Day 3, an effect that was driven by group differences in acquisition. Importantly, stressed participants’ cortisol responses to the stressor on Day 1 were positively associated with CS discrimination on Days 2 and 3. These findings suggest that stress immediately before fear conditioning strengthens fear memory formation and produces a more enduring fear memory, perhaps via corticosteroid activity. Such a paradigm could be useful for understanding factors that influence traumatic memory formation.

1. Introduction Stress can enhance, impair, or have no effect on learning and memory [1–3]. These differential effects are partly mediated by stress exerting different effects on different stages of learning and memory. Indeed, stress administered after learning enhances long-term memory [e.g., 4]; stress administered before retrieval impairs long-term memory [e.g., 5]; and, stress administered prior to learning can enhance or impair long-term memory [e.g., 6]. All of these effects have been associated with an interaction between stress-related neurochemicals, such as norepinephrine and corticosteroids, in the amygdala, hippocampus, and prefrontal cortex (PFC) [7,8]. Regardless of the stage of learning and memory affected by stress, the timing of the stressor appears critical. Post-learning stress must ⁎

occur close ( < 30 min) to the learning experience to enhance longterm memory [9], because the stress-induced increase in norepinephrine and corticosteroids must converge in time and space with brain regions activated by the learning experience [10]. In contrast, pre-retrieval stress must be temporally separated (e.g., > 20–30 min) from retrieval to impair long-term memory [11,12], because these effects depend on delayed, stress-induced increases in corticosteroids. The effects of pre-learning stress on long-term memory are more complex and appear to be biphasic in nature. When stress is administered shortly before learning, long-term memory is generally enhanced [1,6,13–17]. When stress is administered more than 20–30 min before learning, long-term memory is generally impaired [6,13,18,19] (there is some evidence that these effects are sex-dependent [18,20]). The time-dependent effects of pre-learning stress on long-term memory

Corresponding author at: Ohio Northern University, Department of Psychology, Sociology, & Criminal Justice, 525 S. Main St. Hill 013, Ada, OH, 45810, USA. E-mail address: [email protected] (P.R. Zoladz).

https://doi.org/10.1016/j.bbr.2019.111980 Received 13 February 2019; Received in revised form 29 April 2019; Accepted 25 May 2019 Available online 27 May 2019 0166-4328/ © 2019 Elsevier B.V. All rights reserved.

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with the strength of their fear conditioning during acquisition. In contrast, when the stress was administered 50 min prior to fear conditioning, participants’ cortisol responses to the stressor were negatively associated with fear learning. In general, these findings support the idea that stress administered temporally proximal to learning enhances long-term fear memory via autonomic mechanisms (e.g., norepinephrine), while stress that is temporally separated from learning tends to impair fear memory formation via stress-induced increases in corticosteroids. Antov and colleagues were the first to show that pre-learning stress time-dependently influences fear conditioning. However, in their study, the fear memory was assessed and extinguished in the same day as acquisition, thus preventing the assessment of a long-term fear memory. The investigators also did not measure extinction memory. Furthermore, most studies examining the influence of stress or stress hormones on fear conditioning have measured skin conductance responses (SCRs) to quantify fear [e.g., [33,34,40–44]], and, unlike fearpotentiated startle [45,46], SCRs are not highly coupled with amygdala activity. Thus, the purpose of the present study was to extend upon the findings of Antov and colleagues by examining how stress administered immediately before learning would affect fear acquisition, extinction, and extinction memory in a fear-potentiated startle paradigm. Because only one study has examined the effects of stress administered immediately before fear learning, we chose to focus on this time point. We hypothesized that stress would enhance fear acquisition and delay longterm extinction, effects that would be associated with autonomic and/ or corticosteroid activity. We also predicted that any stress-induced alterations of fear learning might be sex-dependent.

have been attributed to a biphasic influence of stress-induced amygdala activity on synaptic plasticity in cognitive brain regions, particularly the hippocampus, as well as different temporal profiles of stress-related neurochemical release [1–3,8,21,22]. Specifically, stress administered shortly before learning enhances long-term memory via a rapid increase of excitatory neurochemicals, such as norepinephrine, and slowly rising corticosteroids exerting excitatory influences on synaptic plasticity. In contrast, stress that is temporally separated from learning impairs longterm memory as a result of rising corticosteroid levels exerting delayed, inhibitory influences on synaptic plasticity. Although stress can enhance or impair learning and memory, the cognitive responses to stress are adaptive. When experiencing stress, our attention is directed to stress-related stimuli to help us remember the stressful event and what occurs around it [3,22,23]. This enhanced memory for stress-related information aids in survival by facilitating our responses to similar situations that we experience later in life. Nevertheless, the adaptive nature of the stress response can have inadvertent consequences. It can result in powerful and intrusive traumatic memories that underlie psychological disorders like post-traumatic stress disorder (PTSD), and it can even impair learning and memory, particularly for information unrelated to the stressor. Because stress-related alterations of cognition are characteristic of everyday life and associated with some psychological disorders, developing a better understanding of stress-memory interactions has great scientific and clinical value. Individuals with PTSD typically display intense memories of the traumatic event [24] and have difficulty extinguishing these memories [25–27]. One explanation for the development and maintenance of such debilitating memories is the presence of abnormal fear conditioning processes in these individuals. Indeed, several studies have reported enhanced fear conditioning acquisition and impaired fear extinction in PTSD patients [28–32]. Given that stress is inherent in traumatic experiences, examining the effects of stress on fear conditioning processes provides a useful model to better understand the formation and maintenance of traumatic memories. However, such studies, especially in humans, are extremely limited, and those that have been conducted have yielded inconsistent, sex-dependent results. For instance, Merz et al. [33] found that stress administered 40 min before fear conditioning selectively impaired differential fear conditioning in males, while having no effect on females; moreover, the cortisol levels of males in response to stress were negatively associated with the strength of their fear conditioning. On the other hand, Jackson et al. [34] reported that stress administered 1 h before fear conditioning enhanced differential fear conditioning in males, but impaired it in females. Interestingly, in this study, the cortisol levels of males in response to stress were positively associated with the strength of their fear conditioning. These inconsistent outcomes, at least in females, could be explained by the failure to account for the influence of sex hormones on fear conditioning. Indeed, low estradiol levels have been associated with greater fear responses during extinction, greater intrusive memories, and impaired extinction retention [35–38], and some work has shown that the effects of pre-learning stress on extinction recall depend on estradiol levels in females [39]. Most research examining the time-dependent effects of pre-learning stress on long-term memory have assessed the effects of stress on nonemotional, hippocampus-dependent tasks, such as word list learning. However, theoretical approaches to the time-dependent effects of prelearning stress on long-term memory have also speculated that there is a biphasic effect of stress on amygdala functioning [1]. We are aware of only one study that has evaluated the time-dependent effects of stress on an amygdala-dependent task, such as cued fear conditioning. Antov et al. [40] examined the influence of stress administered 10 or 50 min prior to a differential fear conditioning paradigm on acquisition and extinction. These investigators found that stress administered 10 min before fear conditioning led to delayed extinction; moreover, participants’ autonomic responses to the stressor were positively associated

2. Material and methods 2.1. Participants The data presented in this manuscript represent a subset of data from a larger study examining the influences of stress, sex, and multiple genetic variants on fear-potentiated startle. One hundred and forty-one healthy undergraduate students (68 males, 73 naturally cycling females; age: M = 19.36, SD = 1.40) from Ohio Northern University volunteered to participate in the experiment. Individuals were excluded from participating if they met any of the following conditions: diagnosis of Raynaud’s or peripheral vascular disease; presence of skin diseases, such as psoriasis, eczema, or scleroderma; history of syncope or vasovagal response to stress; history of any heart condition or cardiovascular issues (e.g., high blood pressure); history of severe head injury; current treatment with psychotropic medications, narcotics, betablockers, steroids, or any other medication that was deemed to significantly affect central nervous or endocrine system function; mental or substance use disorder; regular tobacco use; regular use of recreational drugs; regular nightshift work; auditory disorder; hearing impairment. Participants were asked to refrain from drinking alcohol or exercising extensively for 24 h prior to the experimental sessions and to refrain from eating or drinking anything but water for 2 h prior to the experimental sessions. The inclusion of naturally cycling females in the data analyses was a post hoc decision made by the experimenters, given the influence of contraception on emotional memory [e.g., [47–49]]. All experimental procedures were approved by the Institutional Review Board at Ohio Northern University, carried out in accordance with the Declaration of Helsinki, and undertaken with the understanding and written consent of each participant. Participants were awarded class credit and $20 cash upon completion of the study.

2.2. Experimental procedures All experimental procedures took place between 1000 and 1700 h. 2

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potentiated startle as the primary dependent measure and consisted of three phases: Day 1 – fear acquisition, Day 2 – fear extinction, and Day 3 – extinction memory testing. Each of these phases was separated by approximately 24 h.

2.2.1. Socially evaluated cold pressor test (SECPT) Following completion of a short demographics survey and the collection of baseline physiological measures (described below), participants were asked to submerge their dominant hand in a bath of water for 3 min. Participants who had been randomly assigned to the stress condition (N = 67; 39 males, 28 females) placed their hand in a bath of ice cold (0–2 °C) water, while participants who had been randomly assigned to the control condition (N = 74; 29 males, 45 females) placed their hand in a bath of warm (35–37 °C) water. The water was maintained at the appropriate temperature by a circulating water bath (ColeParmer; Vernon Hills, IL). To maximize the stress response during the SECPT, participants were encouraged to keep their hand in the water bath for the entire 3-min period. However, if a participant found the water bath too painful, he or she was allowed to remove his or her hand from the water and continue with the experiment. Fourteen participants from the stress condition removed their hand from the water prior to 3 min elapsing (M = 156.48 s, SD = 50.38), and all participants from the control condition kept their hand in the water for the entire 3-min period. A social evaluative component was also added to the cold pressor manipulation. Participants in the stress condition were misleadingly informed that they were being videotaped during the procedure for subsequent evaluation of their facial expressions, and throughout the water bath manipulation, they were asked to keep their eyes on a camera that was located on the wall of the laboratory. Previous work has shown that the SECPT results in increased autonomic nervous system activity and increased salivary cortisol levels [50]. Moreover, the SECPT produces greater increases in salivary cortisol levels than the standard cold pressor test that does not include a social evaluative component.

2.2.3.1. Stimuli. The startle probe was a 40-ms, 108-dB burst of broadband noise with near instantaneous rise time, delivered binaurally through headphones. The conditioned stimuli (CSs), which were two geometric shapes, were presented on a white background via a computer monitor (via SuperLab software; Cedrus Corporation, San Pedro, CA) that was situated in front of participants. The CS + was a blue square (9.5 x 9.5 cm), and the CS- was a purple triangle (11 x 9.5 cm). The unconditioned stimulus (US) was a 250-ms, 140p.s.i. airblast directed at the larynx. This US has been used in several previous studies and consistently produces robust fear-potentiated startle [e.g., [28,51–57]]. On CS + trials, the startle probe was presented 6 s after CS onset, followed 500 ms later by the US; the CS + terminated 500 ms following US onset. On CS- trials, the startle probe was presented 6 s after CS onset, without any US presentation; the CS- terminated 250 ms following the startle probe. On noise alone (NA) trials, the startle probe was presented alone as participants stared at a white background on the computer monitor; NA trials were the length of the startle probe (i.e., 40-ms). 2.2.3.2. Startle response measurement. The eyeblink component of the acoustic startle response was measured by electromyographic (EMG) recordings of the right orbicularis oculi muscle. Ag/AgCl electrodes (5mm) filled with electrolyte gel were positioned 1 cm below the pupil of the right eye, 1 cm below the lateral canthus, and behind the right ear over the mastoid (ground). Impedance levels were less than 6 kΩ for each participant. Startle response data were acquired using the Acqknowledge data acquisition and analysis software (Biopac Systems, Inc., Aero Camino, CA) and the EMG module of the Biopac MP150 system (Biopac Systems, Inc., Aero Camino, CA). The EMG signal was sampled at a frequency of 1 kHz.

2.2.2. Subjective and objective stress response measures 2.2.2.1. Subjective pain and stress ratings. Participants rated the painfulness and stressfulness of the water bath at 1-min intervals on 11-point scales ranging from 0 to 10, with 0 indicating a complete lack of pain or stress and 10 indicating unbearable pain or stress.

2.2.3.3. US expectancy measurement. A three-button response keypad (SuperLab software, Cedrus Corporation, San Pedro, CA) was used during each fear-potentiated startle session to collect trial by trial ratings of US expectancies. During each CS presentation, participants pressed one of three buttons: an “AIR” key when they expected the CS to be followed by an airblast, a “NO AIR” key when they did not expect the CS to be followed by an airblast, and a “?” key when they were uncertain of what to expect. For the purposes of data analysis, participant responses of “AIR” were scored as +1, responses of “?” were scored as 0, and responses of “NO AIR” were scored as -1 [28,51–57].

2.2.2.2. Cardiovascular analysis. Heart rate (HR) was measured continuously for approximately 1 min before the water bath until its completion via a BioNomadix pulse transducer (Biopac Systems, Inc.; Goleta, CA) placed on the ring finger of participants’ non-dominant hand. The pulse transducer was connected to the PPG module of the MP150 Biopac hardware. Average baseline HR (average of 1 min before water bath) and water bath HR (average of water bath) were calculated for statistical analysis. 2.2.2.3. Cortisol analysis. On Day 1, saliva samples were collected from participants immediately before and 25 min after the water bath to analyze salivary cortisol concentrations. On Day 2, saliva samples were collected from participants immediately before and after fear extinction (see below) to analyze salivary cortisol levels. Saliva samples were collected in a Salivette saliva collection device (Sarstedt, Inc., Newton, NC). The samples were stored at −20 °C until being thawed and extracted by low-speed centrifugation. Salivary cortisol levels were then determined by an investigator blind to the conditions of the participants via enzyme immunoassay (EIA; Cayman Chemical Co., Ann Arbor, MI) according to the manufacturer’s protocol. The assay has a range of 6.6-4,000 pg/ml and a sensitivity of 35 pg/ml. The intra-assay coefficient of variation was 3.05%, and the inter-assay coefficient of variation was 17.96%.

2.2.3.4. Days 1–3 (fear acquisition, extinction, extinction memory). Immediately following exposure to the water bath on Day 1 (Section 2.2.1), participants completed fear acquisition training. This phase began with three NA trials, followed by a habituation segment that consisted of four CS+, CS-, and NA trials. Importantly, no CS during the habituation segment was reinforced with an airblast US. An ensuing conditioning phase consisted of three blocks with four trials of each type (CS+, CS-, NA) for a total of 12 trials per block and 36 total trials. During the conditioning phase, the CS + was always followed by the airblast US, resulting in a 100% reinforcement rate. On Days 2 and 3 of the experiment, participants underwent fear extinction and extinction memory testing, respectively. Each of these phases began with three NA trials. Then, participants were exposed to four blocks with four trials of each type (CS+, CS-, NA) for a total of 12 trials per block and 48 total trials. None of the CS presentations during these phases were reinforced with an airblast US. A fixed trial order was used for all participants, with the only restriction being that there were 4 trials of each trial type (i.e., CS+, CS-,

2.2.3. Differential fear conditioning paradigm The differential fear conditioning paradigm used in the present study followed that which has been studied extensively in previous work [e.g., [28,51–57]], but with a modified timeline. Specifically, unlike previous work with this paradigm, each fear-potentiated startle session was separated by a period of 24 h. The paradigm included fear3

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Fig. 1. The top part of the figure provides a schematic illustration of the fear-potentiated startle paradigm (a). Fear acquisition, fear extinction, and extinction memory testing were separated by 24 h, and each began with 3 exposures to the startle probe alone [noise alone (NA) trials]. Fear acquisition included a habituation phase (during which no stimulus was followed by the aversive US) and a conditioning phase (3 blocks of trials during which the CS + was always reinforced by the aversive US airblast). Fear extinction and extinction memory testing consisted of 4 blocks of NA, CS+, and CS- trials, during which no stimulus was followed by the aversive US. The lower part of the figure provides a representative breakout diagram of the conditioned stimuli [reinforced conditioned stimulus (CS+) and nonreinforced conditioned stimulus (CS-)] trial types during the conditioning phase of fear acquisition (b). Within each block, participants were exposed to 12 trials of varying trial types. The timelines for CS + and CS- stimulus exposure, relative to the startle probe and US, are depicted under these trial types.

potentiated startle relative to each participant’s baseline startle response (i.e., NA trials) and is supported by work evidencing their superiority to standardized difference scores and percent change scores [59]. Because of the variable nature of the startle response, difference scores were calculated for each trial type within each block (i.e., average of 4 trials of each trial type) in order to obtain a more accurate representation of fear-potentiated startle within each block [60–62]. The trial-by-trial raw startle data and US expectancies can be found in the supplementary material. Separate mixed-model ANOVAs were used to analyze baseline startle responses (i.e., responses to the first 3 NA trials), fear-potentiated startle, and US expectancies on Days 1–3, with stress and sex serving as the between-subjects factors and, for the analyses of fearpotentiated startle and US expectancies, stimulus (CS+, CS-) and trial block (4 levels for each phase) serving as the within-subjects factors. Late acquisition was defined as block 4 of acquisition on Day 1, when discrimination learning was at maximum, and late extinction was defined as block 4 of extinction on Day 2 and provided a measure of extinction success. The first blocks of extinction on Day 2 and extinction memory testing on Day 3 were considered measures of fear and extinction memory, respectively. Initial analyses of startle responses during extinction and extinction memory testing were followed up by additional analyses in which participants’ startle responses during late acquisition were included as a covariate to control for group differences in fear learning. To conclude the analyses of fear-potentiated startle and US expectancies, we employed mixed-model ANOVAs to analyze each set of data across all three days of testing. We performed exploratory bivariate correlations to determine whether participants’ physiological responses to the acute stress predicted their fear-potentiated startle responses to the CS + during

NA) presented during each block of 12 trials. The initial preparation of the fixed trial order involved randomizing the order of trial type within each block. The intertrial intervals were randomized to be between 9 and 22 s in duration. Fig. 1 depicts the timeline, stimuli, and trial block composition that made up each experimental session. 2.2.3.5. Startle data preprocessing. Acknowledge data acquisition files were imported into the MindWare EMG analysis program (MindWare Technologies, Ltd., Gahanna, OH), which was used to filter and rectify the EMG signals that occurred between 20–200 ms following presentation of each startle probe. The EMG signal was amplified by a gain of 2000 and filtered with low- and high-frequency cutoffs at 28 and 500 Hz, respectively. A 60-Hz notch filter was also applied. The resulting data were then exported for analysis. The peak EMG signal 20–200 ms after presentation of the startle probe was used as a measure of the acoustic startle response. EMG responses were excluded from data analysis only if instrument or human error occurred and the signal was not acquired. 2.3. Statistical analyses Mixed-model ANOVAs were used to analyze subjective pain/stress ratings of the water bath manipulation, HR, and cortisol concentrations, with stress and sex serving as the between-subjects factors and time point of measurement serving as the within-subjects factor. Similar to previous work employing the fear-potentiated startle paradigm [e.g., [52–55,58]], we quantified fear-potentiated startle by computing a difference score for the EMG recordings [(startle magnitude to the CS + or CS- in each block) – (startle magnitude to the NA trials in each block)]. The use of raw difference scores allows one to calculate fear4

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participants (effect of time point: F(1,137) = 29.03, p < 0.001, η2p = 0.18; Stress x Time Point interaction: F(1,137) = 51.55, p < 0.001, η2p = 0.27; Fig. 2b). No other main effects or interactions were significant (all F < 1.32, all p > 0.25). No significant main effects or interactions were observed for the analysis of Day 2 salivary cortisol concentrations (all F < 0.87, all p > 0.35).

acquisition, extinction, and extinction memory testing. To perform such correlations, we calculated difference scores for salivary cortisol (Day 1 post-stress – Day 1 pre-stress) and HR (water bath HR – baseline HR). We also converted fear-potentiated startle scores during each session into single CS discrimination scores (raw startle responses to CS+ – raw startle responses to CS-) to use as the dependent measures. Alpha was set at .05 for all analyses, and Bonferroni-corrected post hoc tests were employed when the omnibus F indicated the presence of a significant effect. If the assumption of sphericity was violated, Greenhouse-Geisser corrections were employed, with reduced degrees of freedom reported in the analyses.

3.2. Fear-potentiated startle 3.2.1. Day 1 – fear acquisition Baseline startle responses to the first three NA trials during acquisition were significantly greater in stressed participants, relative to nonstressed participants, F(1,131) = 7.28, p < 0.01, η2p = 0.05 (Fig. 3a). Females (M = 192.21 μV; SEM = 13.53) also exhibited significantly greater baseline startle responses than males (M = 147.40 μV; SEM = 14.08), F(1,131) = 5.27, p < 0.05, η2p = 0.04. The Stress x Sex interaction was not significant, F(1,131) = 0.02, p > 0.88. The analysis of fear acquisition revealed that, during the nonreinforced habituation segment, participants exhibited statistically equivalent startle responses following presentation of the CS + and CS-. In the first block of reinforced acquisition trials (ACQ 1), participants exhibited significantly greater fear-potentiated startle responses to the CS- than to the CS + . In the final two blocks of reinforced acquisition trials (ACQ 2 and 3), participants exhibited significantly greater fearpotentiated startle responses to the CS + than to the CS-, demonstrating successful differential fear conditioning (effect of stimulus: F (1,116) = 44.95, p < 0.001, η2p = 0.28; effect of trial block: F (2.81,348) = 19.71, p < 0.001, η2p = 0.15; Stimulus x Trial Block interaction: F(3,348) = 41.87, p < 0.001, η2p = 0.27; Fig. 3b). Importantly, stressed participants exhibited significantly greater fear-potentiated startle responses to the CS + during acquisition, relative to non-stressed participants (effect of stress: F(1,116) = 4.32, p < 0.05, η2p = 0.04; Stress x Stimulus interaction: F(1,118) = 4.86, p < 0.05, η2p = 0.04). No other main effects or interactions were significant (all F < 1.35, all p > 0.26). The analysis of late acquisition revealed that, by the end of training, stressed participants exhibited significantly greater CS discrimination than non-stressed participants (effect of stimulus: F(1,125) = 98.12, p < 0.001, η2p = 0.44; Stress x Stimulus interaction: F(1,125) = 4.50, p < 0.05, η2p = 0.04). In addition, females exhibited significantly greater CS discrimination than males (Sex x Stimulus interaction: F (1,125) = 6.70, p < 0.05, η2p = 0.05; Fig. 4a). No other main effects or interactions were significant (all F < 0.62, all p > 0.43).

3. Results 3.1. Subjective and objective stress response measures 3.1.1. Subjective pain and stress ratings Stressed participants rated the water bath as significantly more painful (effect of stress: F(1,137) = 944.17, p < 0.001, η2p = 0.87) than non-stressed participants. The pain (effect of time point: F (1.54,274) = 17.27, p < 0.001, η2p = 0.11; Stress x Time Point interaction: F(1.54,274) = 12.52, p < 0.001, η2p = 0.08) ratings from stressed participants significantly increased throughout the water bath manipulation. There was also a statistical trend for the effect of sex, F (1,137) = 3.74, p = 0.055, suggesting that females (M = 3.96; SEM = 0.16) exhibited greater pain ratings than males (M = 3.52; SEM = 0.17), overall. No other main effects or interaction were significant (all F < 2.42, all p > 0.12). Stressed participants rated the water bath as significantly more stressful (effect of stress: F(1,137) = 357.19, p < 0.001, η2p = 0.72) than non-stressed participants. Stress ratings significantly increased for all participants throughout the water bath manipulation (effect of time point: F(1.42,274) = 3.61, p < 0.05, η2p = 0.03). The Stress x Time Point interaction was only a statistical trend, F(1.42,274) = 3.10, p = 0.064, suggesting that stress ratings from stressed participants increased throughout the water bath manipulation. No other main effects or interactions were significant (all F < 1.50, all p > 0.22). 3.1.2. Heart rate Stressed participants exhibited significantly greater HR during the water bath, relative to non-stressed participants (effect of time point: F (1,128) = 46.58, p < 0.001, η2p = 0.27; Stress x Time Point interaction, F(1,128) = 14.13, p < 0.001, η2p = 0.10; Fig. 2a). No other main effects or interactions were significant (all F < 2.19, all p > 0.14).

3.2.2. Day 2 – extinction Baseline startle responses to the first three NA trials on Day 2 were significantly greater in females (M = 179.19 μV; SEM = 13.75) than in males (M = 122.66 μV; SEM = 13.79), F(1,135) = 8.43, p < 0.01,

3.1.3. Cortisol On Day 1, stressed participants exhibited significantly greater salivary cortisol levels following the water bath, relative to non-stressed

Fig. 2. Stressed participants exhibited greater HR than non-stressed participants during the water bath manipulation (a). Stressed participants also exhibited greater salivary cortisol levels than non-stressed participants following the water bath manipulation (b). Data are presented as means ± SEM. * p < 0.001 relative to no stress.

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Fig. 3. Stressed participants exhibited greater startle responses to the startle probe alone (NA = noise alone) than non-stressed participants during fear acquisition (a). During the CS habituation (CS HAB) phase of fear acquisition, participants did not respond differentially to the CS + or CS- (b). As the acquisition trials progressed, participants exhibited greater startle responses to the CS + than to the CS-, demonstrating successful differential fear conditioning. Across the entire acquisition phase and during the final block of acquisition trials (ACQ 3), stressed participants displayed greater startle responses to the CS + than did non-stressed participants. During fear extinction, participants exhibited greater startle responses to the CS + than to the CS- (c). Participant startle responses to the CS + decreased across trials but were still greater than startle responses to the CS- by the end of extinction. During extinction memory testing, stressed participants exhibited greater startle responses to the CS- than non-stressed participants during Blocks 1 and 2 (d). Stressed participants also exhibited greater startle responses to the CS + than non-stressed participants during Blocks 2, 3, and 4. Unlike nonstressed participants, stressed participants continued to exhibit greater startle responses to the CS + than to the CS- at the end of the session. Data are presented as means ± SEM. * p < 0.01 relative to no stress.

η2p = 0.06. No other main effects or interactions were significant (all F < 0.67, all p > 0.41). The analysis of the first block of extinction trials on Day 2, which provides an assessment of long-term fear retention, revealed that participants exhibited significantly greater fear-potentiated startle responses to the CS + than to the CS- (effect of stimulus: F (1,135) = 10.35, p < 0.01, η2p = 0.07). No other main effects or interactions were significant (all F < 2.80, all p > 0.09), indicating that

pre-learning stress had no significant impact on long-term fear retention. The analysis of the entire extinction session on Day 2 revealed a significant effect of stimulus, F(1,127) = 83.71, p < 0.001, η2p = 0.40, revealing that participants exhibited significantly greater fear-potentiated startle responses to the CS + than to the CS- (Fig. 3c). Participants’ fear-potentiated startle responses to the CS + and CS- significantly decreased across trial blocks (effect of trial block: F Fig. 4. Female participants exhibited greater CS discrimination than males by the final block of fear acquisition (a). Analyses of the first block of fear extinction revealed that stress increased overall startle responses in males, while decreasing them in females (b). Analyses of the final block of fear extinction revealed that female participants displayed greater startle responses to the CS + than did males (c). During extinction memory testing , females exhibited greater startle responses to the CS + than did males, particularly during Blocks 2 and 4. Data are presented as means ± SEM. * p < 0.01 relative to CS-; β = p < 0.05 relative to males.

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(2.58,381) = 8.96, p < 0.001, η2p = 0.07). There was also a significant Sex x Stimulus interaction, F(1,127) = 20.67, p < 0.001, η2p = 0.14, indicating that females exhibited significantly greater fear-potentiated startle responses to the CS + throughout extinction than did males. No other main effects or interactions were significant (all F < 2.59, all p > 0.06). The analysis of the last block of extinction on Day 2, which provides an assessment of extinction success, revealed that participants still exhibited significantly greater fear-potentiated startle responses to the CS + than to the CS- (effect of stimulus: F(1,129) = 25.96, p < 0.001, η2p = 0.17). Interestingly, females displayed significantly greater fearpotentiated startle responses to the CS + than did males (Sex x Stimulus interaction: F(1,129) = 4.48, p < 0.05, η2p = 0.03; Fig. 4c). No other main effects or interactions were significant (all F < 3.44, all p > 0.06), Including fear-potentiated startle responses from late acquisition as a covariate did not influence the significant effect of stimulus observed for the first block of extinction on Day 2. However, it did reveal a significant Stress x Sex interaction, F(1,124) = 3.95, p < 0.05, η2p = 0.03, indicating that while fear-potentiated startle responses to both the CS + and CS- increased in stressed males, they decreased in stressed females (Fig. 4b). Including fear-potentiated startle responses from late acquisition as a covariate did not influence the effects observed for the entire extinction session.

revealed that participants still exhibited significantly greater fear-potentiated startle responses to the CS + than to the CS- (effect of stimulus: F(1,129) = 25.96, p < 0.001, η2p = 0.17). Interestingly, females displayed significantly greater fear-potentiated startle responses to the CS + than did males (Sex x Stimulus interaction: F (1,129) = 4.48, p < 0.05, η2p = 0.03). No other main effects or interactions were significant (all F < 3.44, all p > 0.06), indicating that pre-learning stress had no significant impact on long-term fear retention. Including fear-potentiated startle responses from late acquisition as a covariate eliminated the significant effect of sex observed during the first block of extinction memory testing on Day 3. Including fear-potentiated startle responses from late acquisition as a covariate with It also eliminated the significant Sex x Stimulus x Trial Block and Stress x Stimulus x Trial Block interactions. However, stressed participants still exhibited significantly greater fear-potentiated startle responses to the CS + and CS- than did non-stressed participants, F(1,113) = 7.56, p < 0.01, η2p = 0.06. 3.2.4. Effects across days The analysis of fear-potentiated startle across all three phases revealed a significant Stimulus x Day interaction, F(1.88,202) = 7.94, p < 0.001, η2p = 0.07, indicating that participants exhibited significantly greater fear-potentiated startle responses to the CS + during acquisition and extinction on Days 1 and 2 than they did during extinction memory testing on Day 3. The analyses also revealed that females displayed significantly greater fear-potentiated startle responses to the CS + than did males, particularly during extinction and extinction memory testing on Days 2 and 3 (Sex x Stimulus interaction: F (1,101) = 10.15, p < 0.01, η2p = 0.09; Sex x Stimulus x Day interaction: F(1.88,202) = 3.41, p < 0.05, η2p = 0.03). Finally, the analyses indicated that stressed participants exhibited significantly greater fearpotentiated startle responses to the CS + and CS-, as well as greater CS discrimination, than non-stressed participants across all three days (effect of stress: F(1,101) = 8.37, p < 0.01, η2p = 0.08; Stress x Stimulus interaction: F(1,101) = 4.68, p < 0.05, η2p = 0.04).

3.2.3. Day 3 – extinction memory testing Baseline startle responses to the first three NA trials on Day 3 were significantly greater in females (M = 149.82 μV; SEM = 13.62) than in males (M = 106.02 μV; SEM = 13.60), F(1,134) = 5.18, p < 0.05, η2p = 0.04. No other main effects or interactions were significant (all F < 2.25, all p > 0.13). The analysis of the first block of extinction memory testing on Day 3, which provides a measure of long-term extinction memory, indicated that females exhibited significantly greater fear-potentiated startle responses to the CS + and CS- than did males (effect of sex: F (1,132) = 5.02, p < 0.05, η2p = 0.04). No other main effects or interactions were significant (all F < 2.02, all p > 0.15). The analysis of the entire extinction memory testing on Day 3 revealed a significant effect of stimulus, F(1,120) = 22.24, p < 0.001, η2p = 0.16, indicating that participants exhibited significantly greater startle fear-potentiated responses to the CS + than to the CS-. Participants’ fear-potentiated startle responses to the CS + and CSsignificantly decreased across trial blocks (effect of trial block: F (2.83,360) = 15.32, p < 0.001, η2p = 0.11). Females exhibited significantly greater fear-potentiated startle responses to the CS + than did males, particularly during blocks 2 and 4 of the session (effect of sex: F(1,120) = 7.55, p < 0.01, η2p = 0.06; Sex x Stimulus x Block interaction: F(2.72,360) = 2.78, p < 0.05, η2p = 0.02; Fig. 4d). Stressed participants exhibited significantly greater fear-potentiated startle responses to the CS + and CS- than did non-stressed participants, F(1,120) = 8.96, p < 0.01, η2p = 0.07. This effect was largely driven by a more gradual reduction of fear-potentiated startle responses in stressed participants across trial blocks. Indeed, relative to non-stressed participants, stressed participants displayed significantly greater fearpotentiated startle responses to the CS + in Blocks 2 and 3 and significantly greater fear-potentiated startle responses to the CS- in Blocks 1 and 2. Although non-stressed participants exhibited significantly greater fear-potentiated startle responses to the CS + than to the CSduring Blocks 1 and 2, this difference was not significant in Blocks 3 and 4. On the other hand, stressed participants exhibited significantly greater fear-potentiated startle responses to the CS + than to the CSduring Blocks 2, 3, and 4, suggesting a hindrance in fully extinguishing the CS+-US association (Stress x Stimulus x Trial Block interaction: F (2.72,360) = 2.73, p < 0.05, η2p = 0.02; Fig. 3d). No other main effects or interactions were significant (all F < 1.37, all p > 0.25). The analysis of the last block of extinction memory testing on Day 3

3.3. US expectancies 3.3.1. Day 1 – fear acquisition During acquisition, US expectancy ratings for the CS + were significantly greater than US expectancy ratings for the CS-, and this difference significantly increased as acquisition progressed (effect of stimulus: F(1,121) = 642.89, p < 0.001, η2p = 0.84; Stimulus x Trial Block interaction: F(2.18,363) = 246.94, p < 0.001, η2p = 0.67; Fig. 5a). Stress selectively impacted US expectancy ratings in males. Relative to non-stressed males, stressed males exhibited significantly lower US expectancy ratings for the CS + and significantly higher US expectancy ratings for the CS- (Stress x Stimulus interaction: F (1,121) = 4.73, p < 0.05, η2p = 0.04; Stress x Stimulus x Trial Block interaction: F(1,121) = 6.84, p < 0.01, η2p = 0.05). No other main effects or interactions were significant (all F < 1.16, all p > 0.32). 3.3.2. Day 2 – extinction During fear extinction on Day 2, US expectancy ratings for the CS + were significantly greater than US expectancy ratings for the CS-; this difference significantly decreased as trials progressed (effect of stimulus: F(1,124) = 108.27, p < 0.001, η2p = 0.47; effect of trial block: (2.06,372) = 27.51, p < 0.001, η2p = 0.18; Stimulus x Trial Block interaction: F(2.29,372) = 8.83, p < 0.001, η2p = 0.07; Fig. 5b). Stressed participants exhibited a significantly slower extinction of US expectancies for the CS + than non-stressed participants, particularly during Blocks 3 and 4 (Stress x Stimulus x Trial Block interaction: F (2.29,372) = 2.88, p < 0.05, η2p = 0.02). No other main effects or interactions were significant (all F < 2.15, all p > 0.11). 7

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Fig. 5. US expectancy ratings for the CS + were greater than US expectancy ratings for the CS- during fear acquisition; this difference increased as the trials progressed (a). During extinction and extinction memory testing , US expectancy ratings for the CS + were greater than US expectancy ratings for the CS-; these differences decreased across trials (b, c). Data are presented as means ± SEM. * p < 0.001 main effect of CS + relative to CS-.

3.3.3. Day 3 – extinction memory testing During extinction memory testing on Day 3, US expectancy ratings for the CS + were significantly greater than US expectancy ratings for the CS-; this difference significantly decreased as trials progressed (effect of stimulus: F(1,125) = 30.71, p < 0.001, η2p = 0.20; effect of trial block: F(1.24,375) = 26.01, p < 0.001, η2p = 0.17; Stimulus x Trial Block interaction: F(2.67,375) = 14.62, p < 0.001, η2p = 0.11; Fig. 5c). No other main effects or interactions were significant (all F < 2.63, all p > 0.10).

significantly increased and decreased, respectively, throughout learning. Importantly, stress administered immediately before acquisition significantly increased baseline startle responses and significantly enhanced fear-potentiated startle to the CS + during acquisition. There was no significant effect of stress on fear-potentiated startle responses to the CS + during extinction on Day 2, but stressed participants did exhibit a significantly slower reduction of their US expectancy ratings for the CS+, relative to non-stressed participants. During extinction memory testing on Day 3, stressed participants exhibited significantly greater fear-potentiated startle responses to the CS + than did nonstressed participants, which appeared to be driven by the stress-induced enhancement of acquisition on Day 1. Finally, the change in Day 1 salivary cortisol levels in stressed participants was associated with their CS discrimination during extinction on Day 2 and extinction memory testing on Day 3, with greater cortisol responses to the stressor predicting greater CS discrimination. These findings, consistent with previous research [40], support the idea that stress shortly before learning enhances the formation and endurance of fear memories, which may be associated with stress-induced changes in corticosteroid activity. Because stress-related disorders, such as PTSD, are associated with abnormalities in fear conditioning and extinction, this experimental design could be useful for examining factors that influence the development and maintenance of such disorders.

3.3.4. Effects across days The analysis of US expectancies across all three phases revealed a significant Stimulus x Day interaction, F(2.67,375) = 14.62, p < 0.001, η2p = 0.11, indicating that US expectancy ratings for the CS + and CS- significantly decreased across days, and the ratings for the CS + showed a significantly greater decrease. 3.4. Correlations We discovered that, in stressed participants, the magnitude of their change in salivary cortisol levels on Day 1 was positively associated with their CS discrimination during extinction on Day 2, r(66) = 0.27, p < 0.05, and extinction memory testing on Day 3, r(66) = 0.37, p < 0.01 (Fig. 6). No other significant relationships were observed in stressed participants, and no relationships between cortisol or HR and startle responses were significant in non-stressed participants (all absolute r < 0.08, all p > 0.57).

4.1. Time-dependent effects of stress Our laboratory, as well as others, have found that hippocampusdependent memory is enhanced when learning occurs in close proximity to the stressor but impaired when learning and stress are temporally separated [1,6,13–19]. These time-dependent effects of stress on learning have been attributed to the timing of physiological responses to stress. Diamond and colleagues explained that, initially, stress causes an increase in excitatory neurotransmitters (e.g., norepinephrine), which activate memory-involved brain areas such as the amygdala and hippocampus. Synaptic plasticity is facilitated by this increased neurochemical release, resulting in an enhancement of memory formation. Working in tandem with this early phase of arousal is the endogenous

4. Discussion The purpose of the present study was to investigate the effects of immediate, pre-learning stress on fear conditioning, extinction, and extinction memory. Overall, participants successfully learned the differential fear conditioning task, as they demonstrated significantly greater fear-potentiated startle to the CS + than to the CS- by the end of acquisition. Participants also exhibited cognitive awareness of the CS +-US contingency; their US expectancy ratings for the CS + and CS8

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Fig. 6. In stressed participants, the change in cortisol levels following the water bath manipulation on the day of fear acquisition was positively associated with their magnitude of CS discrimination during extinction (a) and extinction memory testing (b).

participants did not appear to completely extinguish the CS+-US association, as they still exhibited greater fear-potentiated startle to the CS + than to the CS- during the last block of trials. The lack of a prelearning stress effect on the extinction of fear-potentiated startle differs from the findings of Antov et al. [40] and could be the result of methodological differences between the two studies. For instance, Antov and colleagues examined extinction training on the same day as fear acquisition, whereas, here, a period of 24 h separated acquisition from extinction. Although we did not observe a group effect of stress on the extinction of fear-potentiated startle to the CS+, we did observe a significant positive correlation between stressed participants’ cortisol responses to the stressor on Day 1 and their CS discrimination during the Day 2 extinction session. Stressed participants exhibiting greater cortisol responses to the stressor on Day 1 displayed greater fear-potentiated startle to the CS+, relative to the CS-, throughout the Day 2 session. We also observed a significantly slower reduction of US expectancy ratings for the CS + in stressed participants on Day 2. Thus, our findings provide some evidence that immediate pre-learning stress diminished extinction in participants. During extinction memory testing on Day 3, stressed participants displayed significantly greater fear-potentiated startle to the CS- than non-stressed participants during the first two blocks of trials, suggesting enhanced generalization of fear in stressed participants. We also found that stressed participants exhibited significantly greater fear-potentiated startle to the CS + during the second and third blocks of trials of extinction memory testing. By the end of the Day 3 session, stressed participants, unlike non-stressed participants, still exhibited significantly greater fear-potentiated startle to the CS + than to the CS-. These effects appeared to be driven by the enhanced fear acquisition observed in stressed participants on Day 1, as including acquisition CS discrimination as a covariate in these analyses eliminated such effects. Nevertheless, the effects still suggest that immediate pre-learning stress produces a stronger fear memory that is less susceptible to subsequent extinction. Also, similar to the Day 2 extinction session, there was a significant positive correlation between stressed participants’ cortisol responses to the stressor on Day 1 and their CS discrimination during the Day 3 extinction memory testing. What is interesting is that the significant effects of pre-learning stress on extinction processes did not manifest until 48 h after learning. Future work should examine how pre-learning stress, perhaps through a sensitization process, might impact fear memory and fear extinction after even longer delays. Our results suggest that, in addition to noradrenergic activity [40,70,71], corticosteroid-induced effects on fear acquisition might also influence one’s ability to extinguish a learned fear association. In stressed participants, the cortisol response to the stressor on Day 1 was positively correlated with CS discrimination and startle responses to the CS + on Days 2 and 3, indicating that those who exhibited greater cortisol responses to the stress continued to demonstrate greater fear-

hormone cortisol. Directly following a stressor, rapid, nongenomic effects of cortisol intensify the effects of norepinephrine and help store newly formed memories by further promoting synaptic plasticity [63–65]. However, as time passes, corticosteroid activity raises the threshold of excitation in cognitive brain regions, particularly the hippocampus, hindering synaptic plasticity and making the formation of new memories more difficult. Therefore, when learning is separated from the initial stressor, the memory formation is impaired, suggesting that the temporal dynamics of the effects of stress on memory formation are due, in part, to time-dependent corticosteroid activity. We have extended on this theoretical framework by demonstrating that, in addition to hippocampus-dependent learning, stress administered immediately before an amygdala-dependent task enhances fear acquisition. Stress exposure also increased baseline startle responses during the acquisition phase, which is consistent with some [e.g., [66,67]], but not all [e.g. [68,69],], previous studies. Importantly, the observed stress-induced enhancement of fear acquisition cannot be explained by the alterations of baseline startle, given that fear-potentiated startle was calculated by correcting for each participant’s responses to NA trials. Most previous work examining the impact of stress or stress hormones on fear conditioning has measured SCRs to quantify fear [e.g., [33,34,40–44]]. Using fear-potentiated startle as a measure of fear in the present study provides a distinct advantage over the use of SCRs because, unlike SCRs, fear-potentiated startle is directly coupled with amygdala activity and provides a more direct measure of fear [45,46]. Still, the present study is not the first to extend the time-dependent effects of stress on learning to a fear conditioning paradigm. Antov et al. [40] also examined the effects of immediate pre-learning stress on fear conditioning, but these investigators did not observe an overall effect of stress on fear acquisition. Instead, they observed a positive association between participants’ autonomic responses to the stressor and the strength of their fear conditioning during acquisition. They also found that participants who were stressed just prior to fear learning exhibited delayed extinction relative to non-stressed participants. Similar to these findings, Soeter and Kindt [70,71] found that the administration of yohimbine, an α2-receptor antagonist that increases noradrenergic activity, 30 min prior to fear conditioning led to delayed extinction, broader fear generalization, and a greater recovery of fear (as measured by reinstatement and reacquisition). Collectively, these findings suggest that stress or arousal around the time of fear conditioning might strengthen the formation of a fear memory via autonomic, noradrenergic mechanisms. Although pre-learning stress enhanced fear acquisition on Day 1, it did not influence long-term fear memory, as evidenced by the lack of a stress effect on the first block of Day 2 extinction trials. We also did not observe an effect of stress on extinction success, which was measured by analyzing the last block of trials on Day 2. In fact, overall, 9

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traumatic event that precipitates the onset of PTSD. For instance, in Dutch soldiers, Lommen et al. [80] found that pre-deployment extinction performance negatively correlated with the severity of PTSD symptoms after deployment. In other words, impaired extinction abilities in Dutch soldiers before being deployed to Afghanistan predicted more PTSD symptoms in the soldiers when they returned home. Investigating the effects of stress on fear conditioning and extinction processes could thus lend insight into susceptibility factors for traumatic memory formation.

potentiated startle to the CS + during extinction and extinction memory testing. One explanation for this finding is that corticosteroids released as a result of stress strengthened memory formation on Day 1. Indeed, the administration of cortisol prior to learning has been shown to selectively enhance the recall of emotionally arousing images [72,73], and extensive preclinical work has shown that an interaction between glucocorticoids and norepinephrine around the time of learning enhances the consolidation of emotional memories [7]. In addition, investigators have reported that cortisol administration enhances the strength of fear acquisition or delays extinction in humans [33,42–44]; however, these effects have been selectively observed in females. These same studies actually reported that cortisol administration impaired fear acquisition in males. In contrast to these findings, Jackson et al. [34] found that stress administered 1 h before fear conditioning enhanced fear learning in men and impaired fear learning in women. Despite these inconsistencies, it is possible that corticosteroid activity strengthened the formation and/or consolidation of the fear memory, making it more resistant to subsequent extinction processes. Further examination of the effects of stress-related corticosteroid increases on fear acquisition and extinction would be useful to clarify the exact relationship between cortisol and fear memory maintenance.

Role of the funding source The research reported in this publication was supported by the National Institute of Mental Health of the National Institutes of Health under award number R15MH104836. The National Institutes of Health had no further role in the study design; in the collection, analysis, and interpretation of the data; in the writing of the manuscript; or in the decision to submit the manuscript for publication. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. References

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Extensive work has shown that stress exerts sex-dependent effects on learning and memory. Indeed, multiple studies have reported significant effects of stress on learning and memory in males, while observing no effects or opposite effects in females [e.g., [18,20,34,41,74–77]]. Past work specifically examining the effects of stress on fear conditioning and extinction has been no different. In these studies, some work has shown that pre-learning stress enhances fear learning in males and impairs fear learning in females [34]; other work has reported the exact opposite effects [33]. Research has also shown that stress-related cortisol levels correlate positively with fear acquisition in males but not females [41,78]. In the present study, we did not observe any sex-dependent effects of stress on fear learning. However, females did exhibit significantly greater baseline startle than males on each day of testing. Furthermore, relative to males, females displayed significantly greater fear-potentiated startle to the CS + throughout the Day 2 and Day 3 extinction and extinction memory sessions. Previous work has shown that circulating levels of estradiol correlate with fear, especially during extinction, in females. Specifically, low levels of estradiol have been associated with delayed extinction and impaired extinction memory [35–38]. Research has also shown that, during extinction, females with low levels of estradiol exhibit reduced activity of the ventromedial PFC, a brain area that is important for extinction processes [38]. It is possible that many of the females in the present study had lower levels of circulating estradiol, which led to greater fearrelated behaviors during the Day 2 and 3 sessions. However, given that we did not assess sex hormone levels in participants, this is a hypothesis that requires additional testing. 4.3. Conclusion and translational relevance The present study extends the time-dependent effects of stress on declarative learning to a fear conditioning paradigm. We found that stress immediately before learning enhanced the acquisition of conditioned fear and delayed extinction of the fear memory. The formation of more enduring fear memories in a fear conditioning paradigm could serve as an experimental model of traumatic memories that underlie the development of PTSD. Indeed, fear conditioning studies have demonstrated that PTSD patients exhibit enhanced fear acquisition and impaired extinction [28,29,79]. While it is possible that the extinction deficit observed in PTSD patients could be a result of physiological sensitization to stress (Grillon & Morgan, 1966), there is evidence to suggest that impaired extinction abilities are present before the 10

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