Edited by: Brittany L. Smith, University of Cincinnati, United States
Reviewed by: Evelin Cotella, Cincinnati Children’s Hospital Medical Center, United States; Michael Totty, Texas A&M University, United States
This article was submitted to Behavioral Endocrinology, a section of the journal Frontiers in Behavioral Neuroscience
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Fear memory retrieval is relevant to psychiatric disorders such as post-traumatic stress disorder (PTSD). One of the hallmark symptoms of PTSD is the repeated retrieval and re-experiencing of the initial fear memory even long after the traumatic event has occurred. Women are nearly twice as likely to develop PTSD following a trauma than men, thus sex differences in the retrieval of fear memories is highly relevant for understanding the development and maintenance of PTSD. In the current study, we aimed to examine sex differences in the retrieval and extinction of either recent or remote fear memories. To do so, we conditioned male and female rats either 1 day (recent) or 28 days (remote) prior to testing retrieval and extinction. While there was no effect of sex or retention interval on initial retrieval, we found that remotely conditioned females exhibited higher rates of freezing than remotely conditioned males in later retrieval/extinction sessions, suggesting a sex difference in the retrieval and/or extinction of remote, but not recent, fear memories. Overall, these results are the first to demonstrate a sex difference in the extinction of remote fear memory, and this may contribute to the differential expression of fear-related disorders like PTSD in men and women.
The ability to form and later retrieve fear memories is highly adaptive. Fear memories promote survival by guiding behavioral responses to avoid potential threats in the future. However, fear memory processes can also contribute to the ontology and maintenance of psychiatric disorders. For example, one of the hallmark diagnostic criteria for post-traumatic stress disorder (PTSD) is chronic re-experiencing of the memory of the traumatic event, even long after the event occurred (
Fear memories are often studied in rodents using contextual fear conditioning. In this procedure, rats or mice are placed in the conditioning apparatus and receive mild foot-shock(s). Re-exposure to the conditioning apparatus, or context, elicits conditioned fear responses (e.g.,
In addition to potential differences in the expression of contextual fear, there is some evidence that generalization of contextual fear to a novel context is influenced by sex. For instance,
In addition to contextual cues, fear responses can also be elicited by discrete cues that were present in the environment during the aversive event. For example, in Pavlovian fear conditioning, discrete cues (e.g., tones, lights) gain the ability to elicit fear responses through pairings with mild-foot shock. Although there are some exceptions, a general pattern in the literature is that that males and females exhibit relatively similar conditioned fear to discrete cues during retrieval tests [
The purpose of the present study was to compare male and female rats in the retrieval of cued fear conditioning acquired either recently or remotely. We chose to examine differences between recent and remote memories because it is broadly acknowledged that as memories age, their neurobiological correlates undergo significant reorganization (e.g., systems consolidation;
The subjects were 63 (31 male, 32 female) experimentally naïve Long-Evans rats (Envigo Laboratories, Indianapolis, IN, USA) 75–90 days old upon arrival. Rats were allowed 1 week to acclimate to the vivarium while housed in pairs. On the first day of behavioral procedures rats were then individually housed with plastic tunnels for enrichment in 12 × 7.5 × 7.5 in plastic caging for the remainder of the experiment. Rats were assigned to one of four groups: Male Remote (
Behavioral procedures occurred in 16 conditioning chambers (Med Associates, Inc., St. Albans, VT, ENV-007; 24 cm W × 30.5 cm L × 29 cm H), which were modified to create 4 sets of distinct “contexts.” All chambers had the following common features. Each chamber was housed in a sound-attenuating cabinet (Med Associates, ENV-017M; 66 cm W × 56 cm L × 56 cm H) outfitted with an exhaust fan to provide airflow and background noise (68 dB). All 16 chambers were outfitted with a food cup, recessed in the center of the front wall, a retracted lever (Med Associates, ENV-112CM), located on the right of the front wall, and an inactive nose-poke aperture (2 cm in diameter) located 3 cm above the food cup. All chambers also had a panel light (Med Associates, ENV-221M) on the right front wall (16 cm above the grid floor), a house light (Med Associates, ENV-215M) centered on the back wall 24 cm above the grid floor, and a speaker (Med Associates, ENV-224AM) located 20 cm above and to the right of the food cup. Only the house light was illuminated throughout the experiment. The speaker was used to deliver a 2000 Hz tone for 10 s (the conditioned stimulus, CS), and the grid floor was used to deliver a 1.0-mA, 1.0-s shock (the unconditioned stimulus, US). Security cameras were mounted to the wall outside each sound-attenuating cabinet, and an 8-cm hole in the chamber wall allowed for video recording from the wall opposite the door.
Sets of four chambers were modified to create four different contexts. For the first distinct context (“Bedding” context), the ceiling and side walls were clear acrylic plastic, the front and back walls were brushed aluminum, and the grid floor was stainless-steel rods (5 mm in diameter) spaced 1.5 cm apart (center-to-center). In addition, approximately 6 oz of woodchip bedding was placed in the tray below the grid floor. For the second set of boxes (“Anise” context), the ceiling and door were covered with laminated black and white checkerboard paper with 3.5 cm black and white squares, and three panels on the back wall were covered in black electrical tape to provide a distinct visual feature. The grid floor was staggered, such that every other bar was on a different plane offset by 0.5 cm, and the tray below the grid floor was painted black. Approximately 5 mL of 10% Anise extract (McCormick, Baltimore, MD, USA) was placed in a plastic dish on the floor directly outside the chamber (inside the cabinet) to the right of the chamber door at the beginning of every session to serve as a distinct olfactory cue.
For the third set of boxes (“Vicks” context), the ceiling and door were covered with wallpaper made from laminated gray construction paper. There was an additional panel light (which remained off) and retracted lever on the left side of the front wall. The floor consisted of alternating stainless-steel rods with different diameters (0.48 and 1.27 cm), spaced 1.6 cm apart from center to center, and the tray beneath the floor was painted gray. Prior to each session approximately 0.5 mL of Vicks VapoRub ointment (Vicks, Cincinnati, OH, USA) was placed in the plastic dish outside the door to chamber. For the fourth set of boxes (“Coconut” context), the ceiling and door were covered with rows of blue dots (3 cm in diameter) that were spaced approximately 1.75 cm apart. There was also an additional panel light (off) and retracted lever on the left side of the front wall. In these chambers the floor consisted of stainless steel rods (5 mm in diameter) arranged such that there was a slight arch in the floor between front and back wall: the highest rod at the center was approximately 1 cm higher than the two rods at either end of the grid floor. A small dish of 10% coconut extract (McCormick, Baltimore, MD, USA) was also placed on the floor to the right of the chamber door.
In the current experiment, each group of rats experienced two contexts, counterbalanced as Context A and B. Half of the rats experienced the Bedding and Anise boxes (counterbalanced as Context A and B) and the other half of the rats experienced the Vicks and Coconut boxes (counterbalanced as Context A and B). Assignment to a particular pair of contexts was counterbalanced across sex and retention interval. Thus, half of the rats in each of the four behavioral conditions (i.e., Male Remote, Male Recent, Female Remote, Female Recent) were trained in the Bedding/Anise pair, and the other half in the Vicks/Coconut pair.
All behavioral procedures were conducted between 8:00 am and 2:00 pm, and the timing of procedures was kept consistent for each group.
All rats received a single day of auditory fear conditioning in Context A. Each session consisted of 3 presentations of the CS, a 10-s tone, which terminated with the onset of the US, a 1-mA, 1-s shock. The first trial began 3 min after rats were placed in the chambers. The time between shock and the next CS presentation was 64 s. Subjects remained in the chambers for 90 s after the last trial before being returned to their home-cages. Half of the rats remained in their home cages for a 28-day retention interval. The other half received a 24-h retention interval. As shown in
Experimental timeline. All groups received a single session of auditory fear conditioning in Context A, followed by a 28-day retention interval (Remote Groups) or a 1-day retention interval (Recent Groups) before being tested for context fear retrieval in Contexts A and B (order counterbalanced). On the subsequent 2 days, all groups received 2 daily sessions of Context B Re-exposure, for a total of 4 sessions. Next, all groups received 3 daily sessions of tone retrieval/extinction in Context B for 2 days, for a total of 6 sessions.
Following either a 1- or 28-day retention interval (see
Over the course of the next 2 days, rats were exposed to Context B alone for four 20-min sessions (see
Tone retrieval was tested in Context B. Each session consisted of 30 presentations of the tone with no shocks presented (64 s ITI). The first trial began 3 min after rats were placed in the chamber, and rats were removed from the chamber following the last CS presentation. For two consecutive days (see
In order to monitor the estrous cycle, vaginal smears were collected from all female rats (both Recent and Remote groups) for 4 days prior and 4 days after conditioning for the Remote group, and again starting 4 days prior to conditioning for the Recent group, continuing through the end of the experiment. Smears were collected by inserting a cotton swab dampened with distilled water less than a centimeter into the vaginal canal (to avoid inducing pseudopregnancy) and rolling the tip against the vaginal wall. Samples were then transferred to dry glass slides. All samples were taken each day between 11:45 am and 1:30 pm. Following the end of the experiment, all slides were stained using 0.1% Crystal Violet stain (e.g.,
Freezing was the main dependent measure, defined as total motor immobility except for breathing (
Automated scoring of freezing was conducted using the following method: video streams were acquired in near-infrared (720P resolution, 29.97 frames per second) by Anpviz IPCameras (model IPC-B850W) mounted in each chamber. Streams were delivered over a dedicated ethernet network and captured by a computer running ffmpeg. Recordings were subsequently scored by first computing the absolute difference in pixel intensity at every pixel on each pair of subsequent frames. A per-frame activity measure was produced by averaging this difference over all pixels. Inspection of the distribution of (log10-transformed) activity scores revealed a clear bimodal distribution of activity, with the mode of the lowest scores reflecting video noise and mode of the higher scores reflecting rat movement. These distributions varied almost solely by chamber/camera. Presumptive freezing was therefore defined as occurring, on a per-chamber basis, when the activity score fell below the value visually marking the beginning of the rat-movement related portion of the distribution. Activity scores were then averaged in 1 s bins, and only 1 s bins that fell below the threshold were defined to represent freezing [approximating procedures used by the Fanselow laboratory, e.g.,
In addition to freezing behavior, this is some evidence that female rates engage in other, escape-like responses (e.g., “darting,” flight;
While the estrous cycle was monitored throughout the duration of the experiment, adequate analyses based on phase were not possible given that sample sizes for individual phases and/or low vs. high hormone phases were too underpowered (e.g., during tone retrieval/extinction sessions 1–3, only 5 out of the 31 females were in the proestrous phase) to reliably detect a statistical influence of estrous phase.
Mean percent freezing during conditioning is presented in
Mean percent freezing (±SEM) during baseline periods for each group during conditioning and tone extinction/retrieval sessions.
Conditioning |
Tone extinction/retrieval |
||||||
Group | 1 | 2 | 3 | 4 | 5 | 6 | |
Male remote | 1.03 (0.39) | 3.28 (1.09) | 15.23 (3.66) | 32.72 (7.27) | 44.00 (6.92) | 32.92 (9.03) | 29.03 (6.38) |
Male recent | 3.46 (1.26) | 4.10 (1.00) | 27.87 (8.10) | 31.49 (7.22) | 57.54 (8.96) | 25.54 (6.49) | 29.03 (7.07) |
Female remote | 0.29 (0.21) | 10.76 (4.19) | 22.91 (5.23) | 45.74 (8.36) | 32.00 (5.73) | 30.67 (6.38) | 37.74 (6.87) |
Female recent | 1.83 (0.56) | 8.41 (3.48) | 25.05 (5.96) | 28.00 (5.28) | 33.23 (6.68) | 31.08 (5.73) | 31.49 (5.09) |
Freezing during the CS presentations in the conditioning session was analyzed with a 2 (sex: male vs. female) × 2 (retention interval: 1 vs. 28 days) × 3 (CS presentation) repeated-measures ANOVA. This revealed a significant effect of CS presentation [
Burst activity during the pre-CS period and during the shocks can be seen in
Overall mean freezing in Contexts A and B during the context tests is presented in
To further probe contextual fear retrieval, we also examined freezing across 4, 1-min bins during the context tests with a 4 (bin: 1–4) × 2 (context: A vs. B) × sex × retention interval repeated-measures ANOVA. There was a significant main effect of bin [
Mean percent freezing across the four sessions of Context B re-exposure is shown in
Mean percent freezing across sessions of tone retrieval/extinction can be seen in
Mean percent freezing (± SEM) during the tone retrieval/extinction tests in the Remote [upper panel
For session 1, ANOVA revealed a significant effect of block [
For session 2, the effects of block and sex were significant [
The sex difference in the Remote condition that was observed in session 2 continued in session 3. There was a significant main effect of block [
In session 4, there was a significant main effect of block [
Similarly, no significant differences were observed between groups in either session 5 or session 6. The only significant effect in each session was that of block [Session 5:
In order to examine the progression of extinction across sessions between groups, we also examined mean percent freezing during tone presentations for each session, averaged over all 30 CS presentations [see
Overall mean percent freezing (±SEM) during sessions of tone retrieval/extinction in the Remote [
There was a significant main effect of session [
There is some evidence that, in addition to freezing, rats exhibit a darting response to conditioned fear cues, and that female rats do so at higher rates than males. In order to assess darting, we manually scored the presence of rapid, escape-like movements across the conditioning chamber during the 10 s tone for all rats across all tone presentations during extinction/retrieval sessions. We observed a single instance of darting, exhibited by one Female Recent rat during session 1; no other instances were observed. Thus, there was no discernable difference between males and females in this measure in the current study.
Understanding sex differences in fear conditioning is critically important for appropriate treatment of many psychopathologies that involve dysregulated fear learning, such as PTSD. The purpose of this study was to compare retrieval of auditory fear conditioning between male and female rats that were tested at either a recent (1 day) or remote (28 days) time-point. Our results are relevant to both initial tone retrieval and subsequent extinction. We observed no group differences in retrieval of auditory fear during the early portion of session 1 [see also
During initial conditioning we observed group differences that were not anticipated: there was a small but significant difference in baseline freezing between Recent and Remote groups (Remote groups showed lower freezing at baseline), while Recent groups exhibited significantly lower freezing during the second tone than Remote groups. It should be noted that, while all testing for context and tone retrieval/extinction occurred when rats were the same age, conditioning occurred when Recent groups were a few weeks older than Remote groups. The effects of age on fear conditioning are most frequently attributed to conditioning that occurs in adolescence vs. adulthood (e.g.,
Our analysis of the first session of tone retrieval/extinction showed that initial retrieval did not differ based on sex or retention interval (see
The primary finding from our study was that, despite initial retrieval being relatively equivalent between sexes, a dissociation emerged between recent and remote memories that was sensitive to sex: remotely conditioned females showed higher levels of freezing in sessions 2 and 3 relative to their male counterparts, while there was no difference between recently conditioned males and females throughout the entirety of tone extinction/retrieval. One possibility is that the excitatory tone-shock association was more strongly encoded/consolidated in females in the Remote group, resulting in stronger resistance to extinction in those rats. Indeed, there are several lines of research indicating sex differences in the neurobiological processes involved in the consolidation of fear memory (e.g.,
One of the limitations of our study was that we did not see differential retrieval in the fear-conditioning context (A) vs. the neutral context (B). This was an unexpected result of this study: our laboratory has previously observed differential responding across contexts with other behavioral paradigms using very similar arrangements of contextual cues (e.g.,
Previous studies have indicated that female rats have a higher propensity than males to exhibit darting, an escape-like response to fear cues (
While our study was not adequately powered to investigate the role of gonadal hormones in the sex difference we observed [see also
Relevant to our study were findings from a study by
In summary, the present experiment extends prior work investigating sex differences in recently vs. remotely acquired memory (
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
This animal study was reviewed and approved by University of Vermont Institutional Animal Care and Use Committee.
All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.
This work was supported by the National Institute of Mental Health of the National Institutes of Health under award numbers K01MH116158 and R01MH118734 (TT).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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