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. 2024 Nov 10;60(11):6851–6865. doi: 10.1111/ejn.16595

Female Wistar Kyoto More Immobile rats with genetic stress hyper‐reactivity show enhanced contextual fear memory without deficit in extinction of fear

Aspen M Harter 1, Mariya Nemesh 1, Michelle T Ji 1, Luca Lee 1, Anna Yamazaki 1, Chris Kim 1, Eva E Redei 1,✉
PMCID: PMC11612840  PMID: 39523452

Abstract

The prevalence of post‐traumatic stress disorder (PTSD) is higher in females than males, but pre‐clinical models are established almost exclusively in males. This study is aimed to investigate the stress‐enhanced fear learning model of PTSD in females. The model mirrors PTSD symptomology in males, whereby prior stress leads to extinction resistant exaggerated contextual fear memory. As stress reactivity is highly relevant to the study and risk for PTSD, females of the stress hyper‐reactive Wistar Kyoto More Immobile (WMI) and its nearly isogenic control the Wistar Kyoto Less Immobile (WLI) strains were employed. Prior studies have shown WMI females presenting unchanged or enhanced fear memory in the stress‐enhanced fear learning paradigm compared WLIs. The present study confirmed the enhanced fear memory following contextual fear conditioning in WMIs compared to WLI females, but this increased fear memory was neither exaggerated by prior stress nor showed extinction deficit. The novel stressor of a glucose challenge test resulted in subtle strain‐ and prior stress‐induced differences in plasma glucose responses. However, fasting plasma corticosterone levels were lower, and rose slower in response to glucose challenge in WMI females, suggesting a PTSD‐like dysfunctional stress response. Hippocampal expressions of genes relevant to both learning and memory and the stress response were decreased in stressed WMIs compared to WLI females, further suggesting a marked dysregulation in stress‐related functions like in PTSD. Thus, although WMI females do not show extinction‐resistant enhanced fear memory, they do present other characteristics that are relevant to PTSD in women.

Keywords: glucose challenge, Nr3c1 expression, post‐traumatic stress disorder, restraint stress, stress‐enhanced fear learning

Genetically stress hyperreactive females of the Wistar Kyoto More Immobile (WMI) strain have enhanced contextual fear memory with unimpaired extinction in a paradigm modelling posttraumatic stress disorder. Subsequent fasting plasma corticosterone levels and responses to a novel stressor are flattened in WMIs compared to their nearly isogenic Wistar Kyoto Less Immobile (WLI) controls, together with their unchanged hippocampal expression of learning and memory‐related genes after stress. Thus, WMI females do model aspects of posttraumatic stress disorder, such as enhanced fear memory and flattened stress response.

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Abbreviations

ANOVA

analysis of variance

CFC

contextual fear conditioning

CORT

corticosterone

EDTA

ethylenediaminetetraacetic acid

ELISA

enzyme linked immunosorbent assay

Esr1 and Esr2

oestrogen receptors 1 and 2

Gapdh

glyceraldehyde‐3‐phosphate dehydrogenase

Glut1

glucose transporter isoform 1

GR

glucocorticoid receptor

GTT

intraperitoneal glucose tolerance test

HPA

hypothalamic–pituitary–adrenal

Nr3c1

nuclear receptor subfamily 3 group C member 1

PTSD

posttraumatic stress disorder

RQ

relative quantification

RS

restraint stress

RT

Reverse transcription

SEFL

stress‐enhanced fear learning

TSE

Technical and Scientific Equipment

WLI

Wistar Kyoto Less Immobile

WMI

Wistar Kyoto More Immobile

1. INTRODUCTION

Post‐traumatic stress disorder (PTSD) is a debilitating condition that is characterized by severe emotional distress, intrusive thoughts, nightmares and suicidal ideations and is known to be precipitated by major stressful events (Mann & Marwaha, 2024). Although a large number of both men and women are affected, 7% of the US population, PTSD prevalence rates are doubled in women compared to men, even after correcting for cultural influences or quantity of traumatic events (Kessler et al., 1995; Koenen et al., 2017; Tolin & Foa, 2006). Thus, women may have a greater vulnerability to PTSD than men. In addition to being more prevalent, PTSD in women also tends to be more severe and chronic and has higher comorbidity rates (Christiansen & Berke, 2020). Sex differences relevant to the stress response and PTSD development have been identified at the molecular‐level, receptor levels, and in biomarkers for PTSD (Yehuda et al., 2015). The well‐known physiological stress response, the activation of the hypothalamic–pituitary–adrenal (HPA) axis, is lesser in women than in men (Uhart et al., 2006; Van Cauter et al., 1996) in contrast to rodents, where females have higher levels of HPA responses to stress (Babb et al., 2013; Iwasaki‐Sekino et al., 2009; MacLusky et al., 1996). Susceptibility to stress, such as anxiety, depression or other disorders characterized by HPA axis dysregulation, differ between the sexes and are also risk factors of PTSD, highlighting the importance of exploring differences in PTSD‐associated behaviours between sexes (Thakur et al., 2022). As evaluating sex differences in the behavioural stress responses in humans is difficult, most studies use model systems of stress and then evaluate how accurately the model system mirrors the same behaviour in humans.

Current preclinical models of PTSD emphasize the effects of prior stress on fear memory; however they are focused nearly exclusively on the relevant biological mechanisms in males. One commonly used PTSD model is the stress‐enhanced fear learning (SEFL) paradigm, established almost exclusively with male rodents. The SEFL paradigm produces extinction‐resistant fear memory response by exposing the animal to prior stress before a typical contextual fear conditioning (CFC) test (Perusini & Fanselow, 2015; Rau et al., 2005). Exposure to this prior stressor exaggerates fear memory, which is resistant to extinction. The paradigm's extinction resistance is crucial for the classification of the SEFL paradigm as a model of PTSD as, in humans, PTSD diagnosis includes a difficulty in extinguishing the intense fear memory/trauma response (Maren & Holmes, 2016).

Since the SEFL model depends on the effect of prior stress on CFC, exploring the effect of genetic stress hyper‐reactivity specifically in females would fill gaps in our knowledge of stress and se‐related components of PTSD. Thus, the Wistar Kyoto (WKY) More Immobile (WMI/Eer; WMI) inbred strain was used due to its genetic profile of stress hyper‐reactivity (de Jong et al., 2021). This strain and its nearly isogenic control strain the Wistar Kyoto Less Immobile (WLI/Eer; WLI) were bred bidirectionally from the WKY parental strain, based on immobility measures during a forced swim test (Will et al., 2003). WMIs, both males and females, show increased depression‐like behaviour compared to WLIs (Andrus et al., 2012; Redei et al., 2023). Similar to human depressed patients, WMIs respond to antidepressant treatments (Will et al., 2003) and show dysfunction in resting‐state hippocampal connectivity (Hasler & Northoff, 2011; Williams et al., 2014). Interestingly, the inbred WMIs, but not WLIs, show sex differences in many behaviours, including anxiety (Mehta et al., 2013). Stress‐responses also differ between WLIs and WMIs, and between male and female WMIs in plasma corticosterone (CORT) levels (Przybyl et al., 2021; Schaack et al., 2021), object and social memory (Schaack et al., 2021), and fear memory (Lim, Shi, et al., 2018; Przybyl et al., 2021). In these latter studies, WMI males and WLI females show enhanced fear memory after prior stress, while stressed WMI females show either no effect of stress or significantly attenuated fear memory. This discrepancy in the results of SEFL for WMI females, and the contrasting recent findings showing no sex differences in SEFL paradigms (Conoscenti et al., 2024), occasioned the present study to clarify the effect of SEFL on WMI females. Furthermore, the effect of extinction on the fear memory of stressed WMI and WLI females will be determined as extinction resistant fear memory is the hallmark of this PTSD model.

Prior stressors often alter the responses to subsequent stressors depending on the type and strengths of both (Martí et al., 2001), but the role of genetic stress reactivity differences have not been explored in this regard. Response to a novel stressor following the stress of the SEFL paradigm and extinction would answer whether the fear memory response to the prior stress in the SEFL paradigm could be generalized to another stress response. For this project, an intraperitoneal glucose tolerance test (also known as glucose challenge, GTT) was used as a novel stressor following SEFL, as glucose challenge induces a stress response in rodents (Small et al., 2022). This test is also performed in a novel environment to avoid the context‐based fear, induced in the SEFL model, again examining the generalizability of stress responses in this model. The glucose challenge also served to evaluate potential metabolic dysregulation resulting from the SEFL model of PTSD, as metabolic dysregulation is known to occur in PTSD (Michopoulos et al., 2016; Oroian et al., 2021).

Hippocampal expression of selected fear learning and memory genes will be explored in this study. The glucose transporter isoform 1 (Glut1) is the main mediator of glucose reaching the brain, and its hippocampal expression is known to be associated positively with memory consolidation and learning (Choeiri et al., 2005). Oestrogen receptors, Esr1 and Esr2, are both involved in hippocampus‐related memory as well (Frick et al., 2018; Fugger et al., 1998, 2000). Additionally, increased expression of the glucocorticoid receptor (Nr3c1) is suggested to be a risk factor for PTSD in humans (van Zuiden et al., 2011). Furthermore, Nr3c1 methylation, which inhibits gene expression, is negatively correlated with PTSD risk (Yehuda et al., 2015).

The overall goal of the current study was to test whether the stress hyper‐reactive WMI females will show PTSD phenotypes, such as enhanced fear memory with impaired extinction. Additionally, we hypothesized that WMI females will show abnormal plasma glucose and corticosterone responses to the novel GTT stressor, and that they will show decreased hippocampal expression of Glut1 and Esr1 as well as increased expression of Nr3c1 compared to WLI females.

2. METHODS

2.1. Animals

Animals were housed at Northwestern University Feinberg School of Medicine under the care of the Center for Comparative Medicine. Procedures were approved by the Northwestern Institutional Animal Care and Use Committee. The treatment of the animals, and their conditions, were in accordance with NIH policies. Animals were group housed in temperature and humidity‐controlled environment through a 12‐h light–dark cycle starting at 6:00 AM. Enrichment was not supplied in housing as it alters inherent depression‐like behaviours in WMI animals (Mehta‐Raghavan et al., 2016). Food and water were available ad libitum. Females were 3‐ to 5‐month‐old adult inbred WLI/Eer and WMI/Eer (referred to as WLI and WMI) rats from the 47th to 48th generations. The ages of the animals were balanced across the different experimental groups (Average age of animals in each group were: WLI control, 3.88; WLI stressed, 3.75; WMI control, 3.88; WMI stressed, 3.63 months). All animals were experimentally naive preceding the current experiment.

2.2. Stress‐enhanced fear learning paradigm

The experimental design is shown in Figure 1. Adult 3–5 months old females were either exposed to restraint stress (RS) or left undisturbed (non‐stressed, control group). Forty‐eight hours following the RS test, both groups were exposed to contextual fear conditioning (CFC), followed by an initial fear memory test 24 h later. Subsequently, fear memory was measured every day for a week throughout the extinction period. An intraperitoneal glucose tolerance test (GTT) was performed 24 h after the last extinction trial. Animals were sacrificed by decapitation 120 min after the GTT. Trunk blood and brains were collected.

FIGURE 1.

FIGURE 1

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Overview of experimental procedure. Rats were randomly assigned to a control, non‐stressed group or an experimental group exposed to 2 h of restraint stress (RS). Forty‐eight hours later, both groups were exposed to Day 1 of contextual fear conditioning (CFC). CFC Day 1 consisted of 3 min of chamber habituation followed by one foot shock per minute (.8 mA). CFC Day 2 reintroduced animals to the same context without foot shocks. Starting 24 h after Day 2 of CFC, animals were exposed to an extinction protocol lasting 7 days. Extinction protocol was the same as the second day of CFC. Freezing behaviour and distance traveled measures were collected for CFC and extinction trials as a method of evaluating fear memory. Twenty‐four hours after the last day of extinction, animals were exposed to an intraperitoneal glucose challenge test (GTT), and then 120 min later sacrificed.

2.2.1. Restraint stress

Animals in the experimental group were placed in DecapiCone® (Braintree Scientific, Braintree, MA, USA) plastic containers with an opening to allow free breathing but restrain movement for 2 h. Restraint stress was conducted between 10:00 AM and 4:00 PM. Following restraint, females were returned to their home cages.

2.2.2. Contextual fear conditioning and extinction

Animals were placed into a sound‐attenuated fear conditioning chamber from Technical and Scientific Equipment (TSE, Bad Homburg, Germany). Rats were exposed to 3 min of habituation, followed by three shocks of .8‐mA intensity (1‐s duration once every minute) over the course of 3 min. Twenty‐four hours later, the rats were returned to the same chamber for 3 min without shock. Fear memory was measured by freeze duration and distance traveled on both Day 1 and Day 2 using a computerized infrared beam system (freeze duration detection is every 3 s, making it a maximum of 60. The threshold for distance measured was 1 cm/s). Rats that did not respond to the initial shocks on Day 1 were excluded from the study. The lack of response is defined as freeze duration less than 10 s on Day 1 and Day 2. Beginning 24 h after Day 2 of the CFC, each rat was returned to the same CFC chamber for 3 min without shock, every day for the 7 days of the extinction trial. Fear memory was observed through freezing behaviour and distance traveled as measured by the automated TSE system. Between animals, the chamber was cleaned with 75% ethanol to eliminate behavioural changes caused by odour. The CFC and extinction procedures were conducted between 10:00 AM and 4:00 PM.

Twenty rats across all experimental group showed no responses to fear conditioning on Day 1 of CFC (Control WLI, n = 6; Stressed WLI, n = 5; Control WMI, n = 4, Stressed WMI, n = 5). This was indicated by non‐existent/minimal freezing behaviour during the three 60 second pause periods between foot shocks on CFC Day 1 and confirmed by non‐existent/minimal freezing behaviour during CFC Day 2. These animals were not included in any analysis, and no connection between the animals was identified to explain the failed conditioning beyond the possibility that the animals positioned themselves in a way that shocks were not properly delivered through the machine.

2.3. Glucose tolerance test

Twenty‐four hours after the seventh day of extinction, a GTT was conducted. Prior to the test, the animals were weighed and fasted overnight for 16 h. The next day between 10:00 AM and 12:00 PM, blood was collected from the tail vein to determine fasting glucose levels. Animals were then injected intraperitoneally with 2‐μL/g body weight of 1‐g/mL glucose solution. Tail blood was collected at 30 and 60 min post‐glucose using heparinized capillary tubes. At 120 min, the rats were sacrificed by fast decapitation, and trunk blood and brain samples were collected. Blood samples were collected into EDTA‐coated tubes (.3 μL/.5 mL whole blood, .5 M EDTA), centrifuged at 4°C and 4000 RPM for 10 min, and the plasma was separated for storage at −80°C. Brains were collected in RNAlater™ (Invitrogen, Carlsbad, CA, USA) for dissection later and kept at −80°C.

2.4. Hippocampal quantitative RT‐PCR

Whole hippocampi were dissected using the following coordinates: AP −2.12 to −6.0, ML 0–5.0, DV 5.4–7.6 (Wilcoxon et al., 2005) and placed into RNAlater™. For RNA extraction, tissue was homogenized in TRI Reagent (Sigma‐Aldrich, Saint Louis, MO, USA), and total RNA was isolated using the Direct‐zol RNA Miniprep Plus kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's protocol. RNA quality and concentration were measured using the Nanodrop 1000 Spectrophotometer (ThermoFisher, Waltham, MA, USA). Reverse transcription (RT) was carried out on 1‐μg RNA using Superscript VILO™ Master Mix (Invitrogen, Waltham, MA, USA) as directed by the manufacturer. The 5 ng of cDNA/sample/well was analysed in qPCR using PCR 2X MasterMix Universal for SYBR Green Assay (Lamda Biotech, Saint Louis, MO, USA) in the QuantStudio™ 6 Flex Real‐Time PCR System (Applied Biosystems) in triplicates. Primer pairs were designed by Primer‐BLAST (NCBI, Bethesda, MD, USA). Primer sequences are shown in Table S1. Relative quantification (RQ) of transcripts was determined using Gapdh as the housekeeping gene and a calibrator (non‐stressed WLI cDNA sample) and calculated by the QuantStudio™ Software in which RQ = 2− ΔΔCT.

2.5. Plasma hormone and glucose assays

Blood glucose levels of animals were analysed using the Amplite® Colorimetric Glucose Quantitation Kit (AAT Bioquest, Sunnyvale, USA) from plasma samples diluted to a 1:100 ratio, according to the manufacturer's protocol. The assay was performed in duplicates with a standard curve generated using linear regression from the concentration‐absorbance data using GraphPad Prism version 10.0 (GraphPad Software, La Jolla, CA, USA).

Plasma CORT levels were measured by a commercially available competitive ELISA kit (Corticosterone Competitive ELISA kit, ThermoFisher, USA) according to the manufacturer's protocol. The sensitivity of the assay was 18.6 pg/mL and samples were diluted to a 1:1000 ratio. The ELISA was performed in duplicates with a standard curve generated using linear regression from the log‐transformed concentration–absorbance data (GraphPad Software, La Jolla, CA, USA).

2.6. Statistics

All statistical analyses were performed using GraphPad Prism version 10.3.1 (GraphPad Software, La Jolla, CA, USA) to determine significant differences between the experimental groups. Two‐way ANOVAs (stress and strain) were used to analyse CFC data for Days 1 and 2, for glucose and CORT levels, and gene expression. A three‐way ANOVA with repeated measures (stress, strain, and days) was used to compare fear memory and distance traveled throughout extinction trials. Post hoc analyses were employed after significant ANOVAs, using the two‐stage linear set‐up procedure of Benjamini, Krieger, and Yekutieli (Benjamini et al., 2006). Significance after correction for multiple comparisons was defined as q < .05 and p < .05 for individual p‐values. All data was represented as the mean ± standard error of the mean (SEM). ANOVA results are indicated in the results sections, and post hoc analyses are shown in the figures. Pearson's correlations were performed to determine associations between behaviours, hormones and genes of interest with significance defined as p < .05.

3. RESULTS

3.1. Contextual fear conditioning and extinction

CFC was conducted over 2 days. On the first day, fear conditioning occurred via the pairing of unsignaled foot shocks with the context in which it happened. On Day 1 of the CFC, freeze duration after the foot shocks did not differ significantly between strains and by stress (Figure 2a). However, the inverse of freezing, distance traveled, revealed greater activity of control WMI females than WLIs, which was decreased by stress in WMIs (strain, F[1,26] = 5.06, p < .05; Figure 2b).

FIGURE 2.

FIGURE 2

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Contextual fear conditioning elicits heightened fear memory in Wistar Kyoto More Immobile (WMI) females regardless of stress. (a) Fear learning on Day 1 of contextual fear conditioning (CFC) as measured by freeze duration shows no significant differences between groups. (b) Activity measure of distance traveled on Day 1 is higher in control WMIs compared to Wistar Kyoto Less Immobiles (WLIs) but decreased by stress. (c). Fear memory is greater in WMI females than in WLIs regardless of prior stress. (d) In agreement with fear memory, distance traveled is decreased in WMIs compared to WLIs. However, within the WLI strain, exposure to prior acute stress shows a reduction in distance traveled compared to the control group. Values are shown as mean ± SEM; post hoc group comparisons were carried out by two‐stage linear set‐up procedure of Benjamini, Krieger and Yekutieli following ANOVA, *q < .05, **q < .01. WLI control n = 8; stress n = 8; WMI control n = 8; stress n = 7.

The second day of CFC consists of re‐exposing the animals to the same context without foot shock and evaluating freezing behaviour as a measure of fear memory. Fear memory of WMI females, as measured by freeze duration on Day 2 of CFC, was greater compared to WLI females, regardless of stress (strain, F[1,27] = 21.70, p < .01; Figure 2c). As expected, the decreased distance traveled by WMI females compared to those of WLI females showed an inverse relationship to the increased freezing behaviour, also regardless of stress (strain, F[1,24] = 43.98, p < .01; Figure 2d). Prior stress decreased the distance traveled by the WLI females (stress × strain, F[1,24] = 4.88, p < .05).

During the week of extinction, both WMIs and WLIs showed an attenuation of freeze duration following Day 2 of CFC, which is marked as day 0 of extinction (days, F[7181] = 60.02, p < .01; Figure 3a). Although WMIs had significantly greater freeze duration on extinction Days 0 and 1 compared to WLI females, by Day 3 of extinction, there was no significant difference in freezing behaviour observed between strains. This shows a steeper extinction rate in WMIs compared to WLIs (days × strain, F[7181] = 9.97, p < .01). Moreover, the extinction rate of freeze duration was even steeper in stressed WMI females compared to control WMIs as can be seen through the freezing behaviours of stressed versus control WMIs on Day 2 of extinction (stress, F[1,30] = 3.98, p = .05; days × strain × stress, F[7181] = 2.36; p < .05). By the last day of extinction, there were no significant differences in freeze duration between WMI and WLI females.

FIGURE 3.

FIGURE 3

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Wistar Kyoto More Immobile (WMI) females show no deficit in extinction of fear memories with or without prior stress. (a) Day 0 of extinction is the Day 2 contextual fear conditioning (CFC) data. Freeze duration showed significant differences between strains and/or stress groups through Day 2 of extinction. (b) Distance traveled showed a similar, but inverse pattern of separation between strains through Day 2 of extinction. However, Day 6 and 7 of extinction show a significant decrease in distance traveled in the Wistar Kyoto Less Immobile (WLI) controls when compared to all other groups. Values are shown as mean ± SEM; post hoc were carried out by two‐stage linear set‐up procedure of Benjamini, Krieger and Yekutieli following ANOVA. *q < .05, **q < .01 and ^q < .05, ^^q < .01 control and stressed WLI versus WMI, corrected for multiple comparisons, respectively. + q < .05, ++ q < .01 control versus stressed, within the same strain, corrected for multiple comparisons. Number of animals as in Figure 2.

Distance traveled during extinction showed an inverse relationship to freeze duration, as expected (Figure 3b). Throughout the extinction trials, the distance traveled increased consistently (days, F[7166] = 42.75, p < .01) but remained significantly decreased in WMI compared to WLIs until Day 3 of extinction, confirming a steeper rate of fear memory extinction in WMIs (day × strain, F[7166] = 10.22, p < .01). Additionally, stress increased distance traveled more precipitously in WMIs compared to control WMIs as is evident from the second day of extinction. Meanwhile, stressed WLI females showed increased distance traveled on Days 6 and 7 of extinction (stress, F[1,29] = 3.80, p = .06; day × stress, F[7166] = 3.63, p < .01; day × stress × strain, F[7166] = 2.55, p < .05).

3.2. Plasma glucose and CORT responses to GTT following extinction

It was expected that both plasma glucose and CORT levels would increase after GTT and then return to baseline at 120‐min post‐glucose injection. The strain differences in the pattern of glucose and CORT responses would indicate whether any metabolic disturbances result from the SEFL protocol and the stress hyper‐reactivity of the WMIs.

Analysis of plasma glucose levels following overnight fasting (marked as 0 min in the GTT) showed that prior stress increased fasting glucose levels generally, with no significant main effect of strain or significant interaction between stress × strain (stress, F[1,27] = 6.12, p < .05; Figure 4a).

FIGURE 4.

FIGURE 4

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Glucose challenge test reveals strain and prior stress‐induced differences in plasma glucose and CORT levels of Wistar Kyoto Less Immobile (WLI) and Wistar Kyoto More Immobile (WMI) females. (a) Baseline glucose levels following overnight fasting are heightened in stressed WLI animals. (b) Glucose levels throughout the intraperitoneal glucose tolerance test (GTT) differ by strain and stress after an hour, specifically of WLIs. (c) Plasma CORT levels following overnight fasting are generally lower in WMIs compared to WLIs. (d) CORT responses to the stress of GTT differ between strains: both in the slope of increase after glucose injection, and in the magnitude of the response. Values are shown as mean ± SEM; post hoc comparisons were carried out by two‐stage linear set‐up procedure of Benjamini, Krieger and Yekutieli following ANOVA. *q < .05, **q < .01 and ^q < .05, ^^q < .01 control and stressed WLI versus WMI, respectively, corrected for multiple comparisons. ++ q < .01 time = 0 versus time = 120 min, within strain, corrected for multiple comparisons. Number of animals as in Figure 2.

Glucose levels significantly increased during the GTT between 0 and 60 min, and then returned towards baseline by 120 min (time, F[3,56] = 231.6, p < .01; Figure 4b). While this pattern was maintained between all groups, there were significant differences in the slope of glucose level changes between WMI and WLIs in controls, and in response to prior stress. Prior stress exposure decreased blood glucose levels in WLIs at 60 min compared to control WLIs, while it decreased glucose levels in WMIs only after 120 min compared to controls (time × stress, F[3,71] = 4.98, p < .01; time × stress × strain, F[3,71] = 5.55, p < .01).

Fasting CORT levels differed between WLI and WMI females after prior stress: levels increased in WLI females, but showed no change in WMIs (stress, F[1,24] = 6.98, p < .05; strain × stress, F[1,24] = 5.05, p < .05; Figure 4c).

CORT levels throughout the GTT showed a strain‐dependent pattern. In general, CORT levels increased from 0 to 30 or from 0 to 60 min in WLIs and WMIs, respectively (time, F[3,81] = 50.58, p < .01; time × strain, F[3,81] = 10.60, p < .01; Figure 4d). Specifically, CORT response increased by 30 min in WLIs, with a slight decrease between 30 and 60 min, while CORT responses in WMI females peaked at 60 min, suggesting a somewhat sluggish CORT stress response in WMI females. Elevated plasma CORT levels in response to GTT returned to baseline in WLI control females, but not in control WMIs, where baseline and 120‐min CORT levels differed.

3.3. Hippocampal expression of genes

Expression of relevant genes were measured in the hippocampus, which retrieves and updates contextual fear memories (Maren et al., 2013). Transcript levels of Esr1, Glut1 and Nr3c1 were significantly greater in the hippocampi of control WMIs compared to WLI controls, but stress increased their expression in WLIs while decreasing them in WMIs (Esr1, strain × stress, F[1,24] = 13.55, p < .01; Glut1, strain × stress, F[1,24] = 11.68, p < .01; Nr3c1, strain × stress, F[1,24] = 16.93, p < .01; Figure 5a–c, respectively). Transcript levels of Esr2 increased in WLI hippocampus after prior stress but showed no change in WMIs (strain, F[1,24] = 6.34, p < .05; stress, F[1,24] = 4.58, p < .05; Figure 5d).

FIGURE 5.

FIGURE 5

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Hippocampal gene expression pattern differs between control and stress groups in a strain‐dependent manner. (a and b) Hippocampal expression of Esr1 and Glut1 were significantly lower in control Wistar Kyoto Less Immobile (WLI) females compared to both stressed WLIs and control Wistar Kyoto More Immobiles (WMIs). (c) Nr3c1 transcript levels were higher in control WMIs compared to control WLIs and stressed WMI females. (d) Transcript levels of Esr2 were higher in stressed WLIs compared control WLIs and to stressed WMIs. Values are shown as mean ± SEM. Post hoc group comparisons were carried out by two‐stage linear set‐up procedure of Benjamini, Krieger and Yekutieli following significant ANOVA, *q < .05, **q < .01, corrected for multiple comparisons, #p < .05, individual p value. Number of animals as in Figure 2.

3.4. Correlation of CFC, hormonal measures and hippocampal gene expressions

Pearson correlation analysis of all animals revealed positive correlations between CORT plasma levels at 30 min of the GTT and both fasting CORT (r = .57; p < .01) and fasting glucose levels (r = .41; p = .02) as well as Esr2 transcript levels (r = .37; p = .05; Figure 6a). CORT levels at 30 min were also correlated negatively with freezing behaviour displayed on the second day of contextual fear conditioning (Day 0 of extinction; r = −.41; p = .03). Lastly, Glut1 correlated positively with both Esr2 (r = .70; p < .01) and Nr3c1 (r = .42; p = .03) expressions.

FIGURE 6.

FIGURE 6

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Strain dependent correlations between behaviours, hormone levels and gene expression. (a) Pearson's correlations for all groups regardless of stress status. (b) Correlations for Wistar Kyoto Less Immobile (WLI) females, both controls and stressed. (c) Correlations for Wistar Kyoto More Immobile (WMI) females both controls and stressed. An increasing gradient of colour is used to express increasing strength of correlations; blue represents positive correlations while red represents negative correlations. Values represent Pearson's r values; significance is marked with bolded r values and *p < .05, **p < .01, ˆp < .1. Number of animals as in Figure 2.

Correlations were then further separated and analysed within strains as well. In the WLI females, freezing behaviours on the first and second day of CFC were positively correlated (r = .53; p = .05; Figure 6b). Positive correlations were found between CORT levels at 30 min of the GTT and both fasting CORT (r = .60; p = .02) and fasting glucose (r = .56; p = .02). Fasting glucose was further positively correlated with both fasting CORT (r = .72; p < .01) and the CORT levels measured at 60 min of the GTT (r = .58; p = .02). Lastly, positive correlations were seen between hippocampal transcript levels of Esr1 and Nr3c1 (r = .74; p < .01) and Esr1 and Glut1 (r = .86; p < .01). Interestingly, the positive correlation between Esr1 and Glut1 was the only significant correlation maintained in the WMI correlations from all the overall and WLI analyses (r = .57; p = .03; Figure 6c). No other significant correlations in the WMI strain were observed.

4. DISCUSSION

The results of the study confirm previous findings that WMI females with genetic stress hyper‐reactivity and depression‐like behaviour exhibit exaggerated contextual fear memory compared to their nearly isogenic controls, the WLIs. Prior stress had no effect on these strain differences in fear memory, and no deficit in extinction of fear memory was observed in either strain. The novel stressor of GTT produced subtle strain‐ and prior stress‐induced differences in the plasma glucose responses to the GTT suggesting no metabolic dysregulation in the WMI females. However, fasting plasma CORT levels were lower, and rose slower in response to the metabolic stressor of GTT in WMI females, suggesting a dysfunction of the HPA activation in WMIs like in individuals with PTSD. Hippocampal expressions of the learning‐ and memory‐related genes, Esr1, Nr3c1 and Glut1, were higher in WMIs than in WLIs in control animals, however, this pattern was inverted between the strains in response to prior accumulated stressors. Specifically, hippocampal expression of these genes decreased in stressed WMIs, further suggesting a marked dysregulation in stress‐related functions like in PTSD. Thus, although WMI females do not show extinction‐resistant enhanced fear memory, they do present other characteristics that are relevant to PTSD in women.

4.1. Exaggerated fear memory, but no extinction deficit in WMI females

The current finding of enhanced fear memory on Day 2 of CFC in control female WMIs confirms previous results. Specifically, early adolescent WMI females, and adult WMI females of the same age as in the current study exhibit subtly or significantly enhanced fear memory compared to same age WLIs, respectively (Kim et al., 2021; Przybyl et al., 2021). However, ageing has abolished and even reversed this difference between WLI and WMI females. WMI females of 6–7 months of age show no difference in contextual fear memory, but by 12–13 month of age their fear memory is attenuated compared to same age WLIs (Lim, Shi, et al., 2018, Lim, Wert, et al., 2018). This age dependence in fear memory of naive WMI females suggest that ageing itself, or accumulated minor stressors affect fear memory. However, the lack of stress effect on fear memory via the SEFL paradigm in the present study, particularly in WLIs, conflicts with previous results (Lim, Shi, et al., 2018; Przybyl et al., 2021). These studies have found strain‐dependent effects of stress; exaggerated fear memory in WLI females but unaltered or attenuated fear memory in stressed WMI females (Lim, Shi, et al., 2018; Przybyl et al., 2021). Although there was no increase in fear memory in WLI females after stress in the current study, their activity, as measured by distance traveled, is decreased significantly. This suggest a potential increase in freezing behaviour in WLIs which was not registered by the computerized programme; based on the threshold measures for freeze duration, short freezing periods may not have been recorded. Still, there was no difference in either freeze duration nor distance traveled between WMI females in response to stress, similarly to previous findings (Lim, Shi, et al., 2018).

Throughout extinction, freezing duration and distance traveled decreased and increased, respectively, in both WMI and WLIs. These patterns are both indicative of diminishing fear memory, despite the initial enhanced fear memory response in WMI females. The rapid extinction of fear memory observed in the WMI females indicates that genetic stress hyper‐reactivity does not dampen learning processes. Moreover, the steepness of the WMI female's fear memory extinction suggests improved learning, particularly after stress. These findings would suggest that WMI females are not modelling elevated risk for PTSD. The original SEFL model of PTSD generates a form of ‘extinction resistant’ fear memory (Long & Fanselow, 2012; Rau et al., 2005). While the restraint stress version of the SEFL model is believed to enhance fear learning to the same degree as the original paradigm, extinction was not studied with these modified SEFL paradigms (Cordero et al., 2003; Manzanares et al., 2005; Przybyl et al., 2021). It is possible that prior restraint stress is not sufficient to engrave extinction‐resistant fear, as the magnitude of fear memory might depend on the strength of the initial stressor, as suggested previously (Sandi & Pinelo‐Nava, 2007). The observed attenuation of fear memory in both WLI and WMI strains within only a few days of extinction may also indicate that females require a stronger or different stressor than males to induce lasting extinction‐resistant fear memory. Alternatively, while sex‐specific factors may indeed play a role in learning and memory, genetic stress hyper‐reactivity could affect fear learning and extinction via divergent pathways.

It is important to note that the stress hyper‐reactive WMI females are known to show increased depression‐like behaviours but the same anxiety‐like behaviours as female WLIs (Mehta et al., 2013). These differences are relevant to the current study as PTSD is highly correlated with anxiety symptoms, although it is no longer classified as an anxiety disorder (Breteler et al., 2021; Spinhoven et al., 2014). Thus, the enhanced fear memory of the WMI females compared to WLIs in the SEFL model of PTSD seems to be independent of their anxiety‐like behaviour, but potentially related to their enhanced depression‐like behaviour.

4.2. Blunted and delayed CORT response to glucose challenge in WMI females

Response to a novel stressor following extinction, as measured by plasma glucose response to GTT, suggests a subtle strain‐ and stress‐dependence. While prior stress resulted in a lower glucose peak after GTT in WLI females, no stress effect was seen in WMI females in glucose response. The lack of metabolic dysregulation in WMI females further suggests that they do not model the disrupted glucose metabolism of PTSD patients (Michopoulos et al., 2016; Oroian et al., 2021). In contrast, fasting plasma CORT levels did not rise in previously stressed WMI females in contrast to WLI females, similarly to previous results (Przybyl et al., 2021). Thus, prior stress, whether it is the last day of extinction as in the current study, or the foot shock of fear conditioning as described by Przybyl and colleagues activates the HPA function of WLI, but not WMI females. The pattern of plasma CORT response to GTT showed a slower activation of the HPA axis in WMI females compared to WLIs. Furthermore, CORT levels did not return to baseline by 120 min after the beginning of GTT in the WMI females. This difference in the kinetics of the CORT response between the strains suggest a dysregulation of the stress response in the WMI females. While results of cortisol responsiveness to trauma or to subsequent acute stress differs in PTSD studies, there is a substantial body of evidence suggesting blunted HPA activity being a hallmark of PTSD (Hadad et al., 2020; Meewisse et al., 2007; Morris et al., 2012; Pan et al., 2020; von Majewski et al., 2023; Zaba et al., 2015).

4.3. Hippocampal gene expression

The prolonged CORT response to GTT in WMIs indicates a slow negative feedback regulation of HPA, which agrees with the attenuated hippocampal Nr3c1 expression in stressed WMIs in contrast to those in WLIs. Decreased Nr3c1 expression, as found in the stressed WMI hippocampus, is known to dampen this feedback loop (Palma‐Gudiel et al., 2015). The status of the glucocorticoid receptor (GR = NR3C1) in PTSD is inconsistent with increased, unchanged or decreased levels being found in peripheral mononuclear cells (Hadad et al., 2020). Whether peripheral levels of GR predict those in the hippocampus is not known, although Przybyl et al. (2021) has shown no correlation between the two measured in WLI and WMI rats.

Hippocampal expression of Nr3c1 is positively correlated with transcript levels of Esr1 in WLIs only. Although hippocampal Esr1 expression was generally higher in control WMI females compared to WLIs, it decreased by stress in WMIs but increased in WLIs. Esr1 activity and expression have been linked to synaptogenesis (Foster, 2012) and increased hippocampal activity, resulting in enhanced learning and memory consolidation (Hojo et al., 2011). Esr1 knockout mice display diminished performance in hippocampal‐dependent spatial memory tasks (Fugger et al., 1998, 2000). Inversely, infusion of Esr1 agonist enhances memory consolidation in the hippocampus (Frick et al., 2018). Thus, the increased hippocampal expression of Nr3c1 and Esr1 in the stressed WLI females, which also show augmented CORT response to fasting and classical CORT response kinetics, suggests that these measures are characteristics of a regulated HPA response to stress.

Highly significant correlation has been seen between Esr1 and Glut1 expression in the hippocampus of females of both strains, similarly to those seen in other tissues (Laudański et al., 2004). Glucose transport across the blood brain barrier participates in the facilitation of memory‐related mechanisms (Cruz et al., 2022; Sajadi et al., 2023). The glucose transporter isoform 1 provides a critical component of neuron health and activity (Mergenthaler et al., 2013; Takata et al., 1997; Uldry & Thorens, 2004). Increased hippocampal transcript levels of Glut1 have also been associated with improved memory consolidation and learning (Choeiri et al., 2005). Inversely, inhibition of glucose transport in the hippocampus decreases fear learning and memory, further supporting the role of glucose transport in memory facilitation (Kong et al., 2017). Despite increased expressions of Nr3c1, Esr1 and Glut1 in the stressed WLI hippocampus, but decreased transcript levels of these genes in the stressed WMI, expression of none of these genes correlated with freezing behaviour on either day of CFC. One possible explanation for this may be that hippocampal levels of these transcripts are not involved in fear‐based learning and memory, but rather characteristics of the WMI strain.

It is worth noting the limitations of the current study. All hormone and gene analyses were performed 8 days after the completion of Day 2 CFC and following repeated exposure to the fear‐associated context. As such, the significance of these findings is related to the strain differences in response to the accumulated stress effects, such as restraint, CFC, extinction trials and GTT. Further questions, regarding differences in immediate stress responses following the SEFL procedure, can be answered in the Przybyl et al. (2021), in which some animals were also sacrificed immediately after day 1 CFC. Furthermore, while the control animals may be used as a comparison against the stress‐exposed females, they were not handled and not exposed to CFC, extinction and GTT. Thus, they cannot be considered complete controls for interpreting the hormonal and gene expression results. The study subjects were female animals, but estrous cycle differences were not taken into consideration. Since memory deficits induced by prolonged stress have shown to be dependent on the estrous cycle of female rats (do Nascimento et al., 2019), the current study does not provide a definitive answer without considering differences in the effect of estrous cycle on fear memory in these strains.

5. CONCLUSIONS

Despite the limitations, the current findings provide clear indication that genetic stress hyper‐reactivity leads to exaggerated fear memory in females that is not resistant to extinction. Although prior stress did not alter fear memory in either strain, WMI females presented phenotypes suggesting similarity to PTSD. Specifically, the blunted and slower stress responses and the decreased hippocampal expressions of genes intimately involved in the stress response suggest that WMI females might model some aspects of PTSD. As no animal model can mimic the complexity of a disorder such as PTSD, this study suggests that dissecting complex phenotypes to ‘sets’ (Berkemeier & Page, 2023) formed from any particular defining behavioural or molecular components, could aid in identifying causative relationships.

AUTHOR CONTRIBUTIONS

Conceptualization, A. H. and E. E. R.; methodology and validation, A. H., M. N., M. J., L. L., A. Y. and C. K.; analysis, A. H. and E. E. R.; writing–original draft preparation, E. E. R. and A. H.; writing–review and editing, A. H., M. N., M. J., L. L., A. Y., C. K. and E. E. R.; funding acquisition, E. E. R and A. H. All authors have read and agreed to the published version of the manuscript.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

PEER REVIEW

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer-review/10.1111/ejn.16595.

Supporting information

Table S1: Primer Sequences for Quantitative PCR.

EJN-60-6851-s001.docx (12.5KB, docx)

ACKNOWLEDGEMENTS

This work was supported by the Davee Foundation to E. E. R. and by grants from the Office of Undergraduate Research, Weinberg College of Arts and Sciences to A. H.

Harter, A. M. , Nemesh, M. , Ji, M. T. , Lee, L. , Yamazaki, A. , Kim, C. , & Redei, E. E. (2024). Female Wistar Kyoto More Immobile rats with genetic stress hyper‐reactivity show enhanced contextual fear memory without deficit in extinction of fear. European Journal of Neuroscience, 60(11), 6851–6865. 10.1111/ejn.16595

Edited by: Miriam Melis

DATA AVAILABILITY STATEMENT

Raw data supporting the findings described in this work will be made available upon request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1: Primer Sequences for Quantitative PCR.

EJN-60-6851-s001.docx (12.5KB, docx)

Data Availability Statement

Raw data supporting the findings described in this work will be made available upon request.

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