Effects of Sex,Estradiol,and Acute Stress on Spontaneous Recovery of a Conditioned Touchscreen Response in Rats

Abstract

The present study examined sex differences and the effects of estradiol on acquisition and extinction using an appetitive reinforcement schedule in operant touchscreen chambers. Additionally, it aimed to clarify how acute stress influences response inhibition, as assessed by the spontaneous recovery of operant responses following extinction, in males, females, and ovariectomized females with and without estradiol treatment. In Experiment 1, male and female rats exhibited equal rates of acquisition. While male and female rats extinguished their response also at similar rates, female rats showed transient but significantly greater extinction compared to male rats during the first day of extinction. Acute stress administered immediately prior to the extinction retention test, conducted 21 days following extinction phase, enhanced spontaneous recovery of the learned response in both male and female rats, but also led to a more rapid decline in response rates across the re-extinction session. In Experiment 2, ovariectomized female rats receiving chronic estradiol or cholesterol (control) treatment showed no significant differences in acquisition or extinction learning. Unlike Experiment 1, both estradiol-treated and non-treated ovariectomized stressed rats exhibited lower spontaneous recovery throughout the extinction retention test, indicating that naturally cycling female hormones may modulate stress effects on response recovery. Additionally, non-stressed rats treated with estradiol demonstrated reduced responding in the early stages of the extinction retention test compared to controls. These findings suggest that acute stress enhances response recovery in intact, cycling female and male rats. However, in ovariectomized female rats, acute stress attenuates the spontaneous recovery of an extinguished response in a manner partially dependent on estradiol treatment. These results provide insight into how acute stress influences response inhibition in an appetitive learning paradigm and highlight the role of sex and gonadal hormone status in learning, memory, and response inhibition, with implications for stress-related disorders and addiction behavior.

Keywords

sex differences acute stress estradiol touchscreen spontaneous recovery

Introduction

Stress plays a critical role in the acquisition and expression of many learned behaviors.¹ Other factors, such as sex² and estrogens,^3,4 have also shown to modulate learned behaviors, and possibly interacts with stress effects.^5
-7 The purpose of this study was to investigate how acute stress affects male and female rats’ ability to inhibit a learned response and to separately examine whether estradiol modulates the effect of stress on response inhibition in female rats.

Response inhibition, also referred to as response suppression, inhibitory control, or behavioral inhibition, is the ability to withhold a response, which has significant adaptive value. For example, suppressing responses to distracting stimuli is essential for maintaining focus on a task, whereas excessive response suppression can hinder goal attainment. In addition, response inhibition plays a crucial role in suppressing addictive behaviors,⁸ which are known to be modulated by both stress^9,10 and sex differences.¹¹ In this study, we used appetitively motivated operant conditioning, extinction, and extinction recall of a touchscreen response to examine response inhibition in rats.

Traditionally, response inhibition has been assessed using Go/No-Go and Stop-Signal tasks, in which the organism learns to execute a response to 1 type of cue and inhibit responding to a different type of cue. Human studies have shown mixed results regarding the effects of acute stress on such response inhibition. Some research suggests that stress impairs inhibitory control, as measured by tasks like the Go/No-Go and Stop-Signal tasks,^12,13 while others indicate enhanced inhibitory performance on the same tasks under stress.^14,15 Evidence from rodent studies is more limited but suggests that acute physiological stressors impair response inhibition on tasks like the Stop-Signal task.¹⁶ In addition to the traditional Stop-Signal type tasks rodent research frequently uses extinction paradigms to measure inhibitory learning.

Extinction in instrumental learning involves forming a new association in which a conditioned stimulus (CS) no longer predicts the unconditioned stimulus (US), competing with the original CS-US association. Relapse phenomena, such as renewal (context change), reinstatement (CS-US reintroduction), and spontaneous recovery (response reemergence over time), influence which association is expressed behaviorally.^17,18 Response inhibition has been suggested as a fundamental component of extinction learning, enabling the suppression of previously learned fear responses.¹⁹ Importantly, the response inhibition required during extinction training most closely parallels the behavioral suppression targeted in the treatment of addiction. The present study examined response inhibition by analyzing the spontaneous recovery of an extinguished appetitively conditioned touchscreen response (CR).

Research on stress and extinction suggests that acute stress can impair extinction recall in both rats and humans.²⁰ In humans, for example, acute cold pressor stress²¹ and exogenous administration of the stress hormone cortisol²² impaired retrieval of extinguished associations in a predictive learning task. Similarly, acute cold pressor stress reduced extinction retrieval of conditioned fear responses relative to controls.²³ Rodent studies likewise demonstrate that acute stress can disrupt extinction recall; however, the majority of this work has been conducted using fear-based (aversive) paradigms. For instance, acute immobilization stress in male mice decreased recall of extinguished fear-conditioned responses,²⁴ and acute elevated platform stress in male rats increased renewal of conditioned fear responses, also reflecting impaired extinction recall.²⁵ Importantly, fear-based (aversive) and non–fear-based (appetitive) learning paradigms differ fundamentally in their motivational properties and underlying neural circuitry.^26,27 Aversive tasks rely on avoidance of a negative outcome (eg, footshock), whereas appetitive tasks are driven by the pursuit of a positive outcome (eg, food reward). Because fear-based paradigms are themselves inherently stressful, the impact of an acute stressor on extinction processes may differ substantially from its effects in appetitive paradigms that lack an intrinsic aversive component. Consequently, caution is warranted when generalizing findings from fear-conditioning studies to appetitive learning. Despite this distinction, the effects of acute stress on extinction recall in non–fear-based learning paradigms, particularly in rodents, remain poorly understood. The present study aimed to address this gap by examining how acute stress influences extinction memory of appetitively conditioned responses in male and female rats.

There are clear sex differences in response inhibition and its neural underpinnings.²⁸ However, the direction of these behavioral differences has been inconsistent across human and animal studies, often depending on the type of task used to assess inhibitory control.²⁹ Studies examining sex differences in extinction learning and memory as measures of inhibitory control generally suggest that males outperform females in extinction recall. For example, female rats show resistance to extinction of conditioned fear response compared to male rats,³⁰ and exhibit stronger renewal, reinstatement, and spontaneous recovery of conditioned fear following extinction.³¹ Despite these findings, it remains unclear whether similar sex differences exist in the extinction learning and retention of non-fear-based conditioned responses. One study showed that female rats take longer than males to learn to inhibit responses to an appetitively conditioned excitor when paired with an inhibitor, indicating impaired conditioned inhibition.³² To clarify sex differences in extinction learning, this study examines sex differences in the extinction and retention of an appetitively conditioned touchscreen response.

Although females tend to show reduced extinction of conditioned fear, naturally cycling estrogens, as well as estradiol-treatment appear to enhance the extinction of fear memories.^33,34 However, estradiol-treatment impairs conditioned inhibition of fear in females³⁵ and response inhibition on an appetitive differential reinforcement of low rates of responding (DRL) task.³⁶ Similarly, to the sex differences, it is not known how estrogens, specifically estradiol, would affect extinction of an appetitively conditioned response. Therefore, in addition to examining sex difference during extinction of an appetitive touchscreen response, the present study tested effects of estradiol replacement in ovariectomized (OVX) female rats, to test the influence of estrogens on extinction learning and retrieval of the extinguished response after a retention interval.

Acute stress has been shown to interact with sex and estrogens to influence learning and memory in a variety of tasks. For instance, acute stress enhances acquisition of trace eyeblink conditioning in male rats but impairs it in females.⁵ Stress also increases hippocampal spine density in males while decreasing it in females.³⁷ Similar sex-specific effects are observed in humans, with stress impairing spatial memory retrieval in women but not men.³⁸ Importantly, these effects are mediated by gonadal hormones, as stress-induced impairments in prefrontal cortex-dependent tasks occur only in females with naturally high estrogens levels.^6,7 In our lab we also found that acute stress impairs spatial learning in female rats specifically during proestrus (characterized by high estrogens levels) but not during phases when estrogens levels are low.³⁹ In contrast, cortisol impaired extinction of memory retrieval in a predictive learning task specifically in men, with slightly reversed effects observed in women.²²

Together, these findings indicate that the effects of acute stress on learning and memory are modulated by sex and estradiol, with the direction of these effects varying by task. Based on the literature reviewed above, we hypothesized that acute stress would impair recovery of an extinguished instrumental response in all rats, with a greater impairment in female rats relative to males, and in estradiol-treated OVX females relative to cholesterol-treated OVX females.

In summary, the goal of the present study was to delineate how acute stress influences response inhibition. Response inhibition was assessed by measuring the recovery of a previously learned associative response after extinction in a rodent model, a paradigm that has been primarily examined in fear-based tasks, with very limited work in rodents addressing the effects of acute stress on recovery of an appetitive response. Because the effects of acute stress on cognitive performance are highly sensitive to biological factors, including sex and circulating estrogen levels, Experiment 1 examined whether the effects of acute stress differed between male and female rats, whereas Experiment 2 provided a mechanistic extension by testing whether acute stress interacted with estradiol treatment in ovariectomized females. In this study, we trained male and female rats in operant touchscreen chambers to associate a visual cue with sucrose reinforcement, followed by extinction and retention of the extinguished response test (ie, spontaneous recovery test) after a 3-week retention period. Ovariectomized female rats with and without subcutaneous estradiol (most common and potent naturally circulating estrogen during reproductive years) implants were included to examine estrogen’s role in these processes. To assess how acute stress differentially affects male and female rats and interacts with estradiol, rats were exposed to restraint stress immediately prior to the spontaneous recovery test. This study aims to examine the interaction between sex, stress, and estradiol in modulating extinction and response inhibition in appetitively conditioned learning.

Methods

Experiment 1

Subjects

Sixteen male and 16 intact female Sprague-Dawley rats, approximately 60 days old, were obtained from Charles River Laboratories. Subjects were pair-housed in flat-bottom cages with standard rodent bedding. The rats were maintained on a 12-hour light-dark cycle, with all behavioral testing conducted during the light cycle. Housing conditions were controlled at ambient temperatures between 71°F and 76°F, with humidity levels around 40%. Throughout all experimental stages, except during the retention interval, the rats were maintained on a food-restricted diet, which included 1 hour of free-feeding every 24 hours. A food-restricted diet was initiated 5 days prior to the start of behavioral training. Daily monitoring ensured that the rats’ weight did not drop below 85% of their baseline weight.

Apparatus

The study utilized the Bussey-Saksida Touch Screen 4-Chamber System (Model 80004) for rats, obtained from Lafayette Instruments (Lafayette, IN). Operant control was managed using the Animal Behavior Environment Test (ABET II) software. This computer-automated behavioral apparatus allowed precise conditioning and measurement of associative learning. Each chamber had internal dimensions of 12’’L × 10.25”D × 7.9”H. Four rats were tested simultaneously during experimental sessions.

Procedure

The training and extinction procedures followed protocols outlined by Bussey et al⁴⁰ and Mar et al,⁴¹ ensuring precise stimulus control and the ability to test multiple rats concurrently. Each animal was assigned to a specific chamber and trained/testing in that same chamber throughout the experiment. All chambers were cleaned after every session throughout all phases of training and testing, including acquisition, extinction, and spontaneous recovery, to ensure consistent conditions across sessions. All behavioral procedures were conducted between approximately 9:00 a.m. and 4:00 p.m., with rats tested in groups of 4, under ambient temperatures ranging from 71°F to 76°F and humidity levels of approximately 40%. Sample sizes were determined a priori guided by a power analysis and established practice in the touchscreen literature, where rat cohorts include approximately 6 to 10 animals per condition. Accordingly, groups in the present study contained an average of 8 rats per condition, consistent with prior studies using similar touchscreen-based paradigms.^42,43

Preliminary Training in the Touchscreen Chambers

The pre-training phase consisted of 2 habituation schedules:

Habituation Day 1: The magazine light illuminated and a 45 mg sucrose pellet was released into the magazine area. Subsequent illumination and pellet release would occur 30 seconds after the animal had exited the magazine area. The rat could earn a maximum of 10 pellets.

Habituation Day 2: A visual training image (a white square) appeared at a random location on the touchscreen. If a response to the target was made (ie, the image was touched) within 30 seconds, the image disappeared, the magazine light turned on, and the animal received three 45 mg sucrose pellets. If a response was not made within 30 seconds, the image still disappeared, the magazine light turned on, but only 1 sucrose pellet was dispensed. A 10-second inter-trial interval (ITI) followed reward collection. The session ended when 100 trials were completed or 60 minutes had elapsed.

Acquisition of an Instrumental Touchscreen Response

During acquisition training, conducted on a fixed-ratio 1 (FR1) schedule, a conditioned stimulus (CS; a white square) was presented for 30 seconds on either the right or left side of the touchscreen. Side presentation was randomized by the computer such that each side was presented an equal number of times within each 10-trial block and no more than 3 consecutive trials occurred on the same side. If a response to the CS was made, the image disappeared, the magazine light turned on, and the animal received 1 sucrose pellet. A 10-second ITI followed reward collection. If a response to the CS was not made, the image disappeared, the house light illuminated for 10 seconds, and the ITI followed. Each session ended after 100 trials or 60 minutes, whichever came first. Trials with target and non-target responses were recorded. Trials with non-target responses included both omission trials (ie, failure to make a touch within 30 seconds) and blank touches (ie, touching a non-CS area of the screen). The acquisition criterion was set at 80 responses to the target trials for 2 consecutive days. Once reached, the animal progressed to extinction training the following day.

Extinction of an Instrumental Touchscreen Response

During extinction training, the CS appeared for 30 seconds and disappeared regardless of the animal’s response. If a response to the target CS was not made, a 10-second ITI followed, classifying the trial as a “no-touch trial.” If a response to the target CS was made, the image disappeared without food reinforcement. The house light remained off throughout extinction training. Each session consisted of 60 trials, with a maximum duration of 40 minutes. Extinction training continued until the animal achieved at least 46 no-touch trials out of 60. Upon meeting this criterion, animals underwent a 21-day retention interval without testing.

Spontaneous Recovery Test

After the 21-day retention interval, all rats were returned to the 1-hour-per-day free-feeding restriction regimen for 5 consecutive days prior to the spontaneous recovery test to ensure that body weight and motivational state were re-stabilized to levels comparable to those during acquisition and extinction. The animals were then re-tested on the extinction schedule to assess spontaneous recovery. Half of the male and female rats (counterbalanced) were exposed to acute restraint stress in the testing room before testing.

Acute Restraint Stress Manipulation

Half of the subjects in each sex group were exposed to a 30-minute physical restraint stressor in a restraining tube (Stoelting Co., Wood Dale, IL). Rats were pseudo randomly assigned to restraint stress conditions, counterbalanced for sex, running squad, and operant chamber, prior to the start of any behavioral training to avoid potential confounds. Cage mates were assigned to the same stress condition. The restraint occurred in the testing room, adjacent to the training chambers. During restraint stress administration, only rats assigned to the stress condition were present in the testing room. Non-stressed rats remained in the housing facility until all animals were ready to be tested. No behavioral testing occurred in the touchscreen chambers during restraint stress administration. Immediately following restraint, animals were tested for spontaneous recovery. This stress procedure has been previously shown to induce a physiological corticosterone response.⁴⁴

Experiment 2

Subjects

Experiment 2 followed the same procedure as Experiment 1 but exclusively used 32 ovariectomized (OVX) female Sprague-Dawley rats, approximately 60 days old upon arrival. Housing conditions were identical to those in Experiment 1.

Apparatus

The same Bussey-Saksida touchscreen chamber system and software were used as in Experiment 1.

Procedure

Acute restraint stress manipulation and touchscreen behavioral testing were conducted following the same protocols as described in Experiment 1, with 1 exception. Because in Experiment 1 the rats showed high response performance (~65%) on the first day of acquisition following Habituation Day 2, the number of Habituation Day 2 trials was reduced. In Experiment 2, the session ended when the animal completed 40 (instead of 100) trials or 60 minutes had elapsed, whichever occurred first. Rats were pseudo randomly assigned to restraint stress and estradiol treatment conditions, counterbalanced across all manipulated variables, running squads, and operant chambers, prior to the start of behavioral training to avoid potential confounds.

Estradiol Manipulations

Ovariectomies (OVX) were performed by Charles River Laboratories prior to the animals’ arrival at the CNU animal facilities. Hormone capsule implantation occurred approximately 10 days following OVX. Estradiol was administered via a 10 mm silastic capsule (0.058’’ ID, 0.077” OD, Fisher Scientific) containing 10% 17β-estradiol crystals and 90% cholesterol (Sigma, Inc., St. Louis, MO) for estradiol-replaced rats, while the control group received 100% cholesterol implants. Half of the animals received a 17β-estradiol implant, while the other half received a cholesterol implant as a control. Five days prior to the start of the food-restricted diet, in preparation for operant touchscreen training, all rats received a subcutaneous implant under the scruff of the neck. The implant procedure was performed using clean field techniques. For this procedure, rats were anesthetized using Isoflurane 1% to 5% gas delivered in O2 at a rate of 0.7 l/minute. After shaving and disinfecting a 2 × 2 cm area in the back of the rat’s neck a small (~1 cm) incision was made with a scalpel blade. A silastic capsule was then inserted into the incision. The incision was closed using sterile suture wound clips, and the rats received a sub-q injection of 5 mg/kg of Carprofen/Rimadyl (SQ) for post-surgery pain management. Rats were monitored until they have recovered from anesthesia and then subsequently daily for 3 days after surgery. This method has been successfully used in prior research^45,46 for chronic estradiol administration, and demonstrated that these implants produce continuous circulating 17β-estradiol levels of approximately 40 pg/ml, comparable to normal proestrus levels.⁴⁵ This method provides long-term, stable estradiol release compared to oral administration, daily injections, or commercially available slow-release pellets.^47
-49 The chronic estradiol treatment used in this study is analogous to birth control in premenopausal women and hormone replacement therapy in postmenopausal women.⁵⁰ After implantation, rats were allowed a 5-day recovery period before initiation of a food-restricted diet. Behavioral testing began 5 days after the onset of food restriction, resulting in approximately 20 days between OVX and the start of behavioral testing.

Corticosterone Quantification

After all the behavioral testing was completed, trunk blood was collected from all rats in Experiment 2, during euthanasia (ie, decapitation conducted under isoflurane anesthesia).

Thirty-minutes (approximately of when the middle of the behavioral test would be administered) prior to sacrificing the rats, previously non-stressed were exposed to the identical stress the stressed rats received prior to behavioral test. This procedure was meant to mimic the hormonal response to the acute stress we used during the spontaneous recovery test. The reason we exposed rats to the stress that did not receive it before is because this study focused on acute stress effects and previous experience with a stressor has the potential of changing the physiological response to that stress when experienced again. The blood was allowed to coagulate at room temperature for 30 minutes, and then centrifuged at 2000g and 4°C for 10 minutes. The resulting supernatant (blood serum) was removed and immediately stored at −80°C until further analysis. An ELISA (ENZO life sciences, Inc.; Farmingdale, NY) kit was used to measure corticosterone levels in rats’ blood serum with detection sensitivity of 27.0 pg/ml (range 32-20 000 pg/ml). Levels of corticosterone in stressed rats were compared to non-stressed rats. One of the 32 samples from No Stress Cholesterol-treated group was lost during the extraction process and thus was not included in the analysis. The purpose of measuring corticosterone levels was to determine the level of the physiological response during behavioral testing following restraint stress conducted in this experiment during the spontaneous recovery test.

Data Analysis

The rats’ touch (ie, either a nose-poke or a paw touch) on the touchscreen were detected by an infrared touchscreen assembly surrounding the screen. Responses were recorded using the ABET II software throughout the training and testing sessions. The primary dependent measure was the percentage of touchscreen responses (CR) to the visual stimulus (CS) within each 10-trial block during acquisition, extinction, and the spontaneous recovery test. Statistical analyses were conducted using SPSS software to assess significant differences between groups. For both acquisition and extinction, the number of daily sessions required to reach criterion was analyzed using independent-samples t-tests comparing males and females. To further examine group differences in learning rate, a General Linear Model (GLM) repeated-measures ANOVA was used comparing percent conditioned responses (%CR) across 10-trial blocks across the first 2 sessions of acquisition and for each of the first 3 sessions of extinction (Experiment 1: Block × Sex; Experiment 2: Block × Treatment). Learning curves were also presented visually to facilitate comparison of acquisition and extinction rates between groups. Only the first 2 acquisition sessions and the first 3 extinction sessions were included in the statistical analyses and visualizations because rats reached acquisition and extinction criteria at different times. While data were available for all rats on these days, extending the curves beyond these sessions would have resulted in missing data for some animals and potentially misleading comparisons. Notably, in Experiment 1, 11 out of 32 rats reached acquisition criterion within 2 sessions, and 8 out of 32 rats reached extinction criterion within 3 sessions, while in experiment 2, 10 out of 32 rats reached acquisition criterion within 2 sessions, 9 out of 32 rats reached extinction criterion within 3 sessions and 2 rats reached extinction criterion within 2 sessions. Repeated-measures ANOVA was also used to analyze the effects of sex and stress across six 10-trial blocks during the single session of the spontaneous recovery test. Significant interactions involving block, stress, or sex were followed up with Bonferroni-corrected pairwise comparisons.

Results

Experiment 1

On average, male rats required 3.31 sessions to reach the acquisition criterion, whereas female rats required 3.06 sessions. An independent-samples t-test revealed no significant sex difference in the number of sessions required to reach the acquisition criterion, t(30) = 0.46, P = .65. A repeated measures ANOVA (Block: 1-20 × Sex: Male vs Female) revealed that the percent touchscreen responses significantly increased in both male and female rats across the 10-trial blocks of the first 2 sessions of acquisition, as indicated by a main within-subjects effect of blocks, F(19, 342) = 2.12, P < .01, demonstrating that all rats improved over the first 2 days of learning. No significant main effect of sex or session by sex interaction was observed, suggesting that male and female rats acquired conditioned touchscreen responses at the same rate (Figure 1a).

Figure 1.

Experiment 1 group averages for male versus female percent touchscreen stimulus responses per 10-trial block. (a) response rates across the first two daily acquisition sessions of an instrumental response, (b) response rates across the first three daily extinction training sessions, and (c) Response rates during the final block of the third extinction session, followed by a 21-day retention interval and response rates across six blocks of the spontaneous recovery test conducted either with or without the stressor.

On average male rats needed 4.43 sessions to reach extinction criterion, and female rats 4.81 number of sessions. An independent-sample t-test revealed no significant sex differences in the number of sessions male and female rats needed to reach extinction criterion, t(30) = −0.84, P = .41. A repeated measures ANOVA (Block: 1-6 × Sex: Male vs Female) indicated that both male and female rats decreased responding across the six 10-trial blocks on the first, second and third sessions of extinction, F(5, 150) = 26.26, P < .01; F(5, 150) = 36.51, P < .01; F(5, 150) = 29.07, P < .01, respectively (Figure 1b). This analysis also revealed a significant block by sex interaction across the first session of extinction, F(5, 150) = 2.3, P < .05, and a significant between-subject effect of sex, F(1, 30) = 6.34, P = .017. Bonferonni-corrected pairwise comparison indicated that male rats responded significantly more than female rats on blocks 4 and 5 (both PBonf < .05), whereas no sex differences were observed during blocks 1, 2, 3 or 6 (Figure 1b).

A repeated measures ANOVA (Block: extinction block 18 vs spontaneous recovery block 1 × Sex: Male vs Female × Stress: Stress vs No Stress) comparing the last block of the third day of extinction and the first block of the spontaneous recovery test after a 21-day retention period showed that touchscreen responding increased in all rats, F(1, 28) = 201.46, P < .01, suggested by the main effect of block. Additionally, a significant block by stress interaction was observed, F(1, 28) = 4.50, P = .04, showing that acute stress increased response recovery in both male and female rats (Figure 1c).

A repeated measures ANOVA (Block: 1-6 × Sex: Male vs Female × Stress: Stress vs No Stress) revealed a significant decrease in the touchscreen responding across the 6 blocks of the spontaneous recovery test, indicated by a main within-subjects effect of block, F(5, 140) = 36.98, P < .01. Furthermore, there was a significant block by stress interaction, F(5, 140) = 5.95, P < .01, revealing that the decreased response rate was greater in stressed rats compared to non-stressed rats (Figure 1c), suggesting a greater persistence of the spontaneous recovery in stressed rats. Follow-up Bonferroni-corrected pairwise comparisons revealed significant effects of Stress during early and late blocks. During Blocks 1 and 2, stressed animals exhibited significantly higher responding than non-stressed animals (both PBonf < .05). No group differences were observed during Blocks 3 to 5 (all PBonf > .05). In contrast, during Block 6, non-stressed animals exhibited significantly higher responding than stressed animals (PBonf = .040; Figure 1c).

Experiment 2

On average, cholesterol treated OVX female rats required 3.18 sessions to reach the acquisition criterion, whereas estradiol treated rats required 3.56 sessions. An independent-samples t test revealed no significant sex difference in the number of sessions required to reach the acquisition criterion, t(30) = −0.82, P = .42. A repeated measures ANOVA (Block: 1-20 × Treatment: Estradiol vs Cholesterol) indicated that touchscreen response rates significantly increased in both treatment groups (ie, cholesterol and estradiol) across the 10-trial blocks of the first 2 sessions of acquisition, as supported by a significant within-subjects effect of blocks, F(19, 475) = 19.29, P < .01. However, no main effect of estradiol treatment was observed, indicating that the treatment group did not influence the acquisition rate (Figure 2a).

Figure 2.

Experiment 2 group averages for estradiol- versus cholesterol-treated OVX female rats’ percent touchscreen stimulus responses per 10-trial block. (a) Response rates across the first two daily acquisition sessions of an instrumental response, (b) Response rates across the first three daily extinction training sessions, and (c) Response rates during the final block of the third extinction session, followed by a 21-day retention interval and response rates across six blocks of the spontaneous recovery test conducted either with or without the stressor.

On average cholesterol treated OVX female rats needed 4.44 number of sessions to reach extinction criterion, and estradiol treated rats required 4.25 number of sessions. An independent-sample t-test revealed no significant sex differences in the number of sessions male and female rats needed to reach extinction criterion, t(30) = 0.43, P = .38. A repeated measures ANOVA (Block: 1-6 × Treatment: Estradiol vs Cholesterol) indicated that during extinction training, response rates of both treatment groups significantly decreased across the six 10-trial blocks on the first, second and third sessions of extinction, F(5, 150) = 26.38, P < .01, F(5,150) = 27.00, P < .01, F(5,140) = 15.27, P < .01, respectively, with no significant block by treatment interaction effects (Figure 2b).

A repeated measures ANOVA (Block: last block of the third day of extinction vs first block of spontaneous recovery × Treatment: Estradiol vs Cholesterol × Stress: Stress vs No Stress) revealed a significant main effect of Block, F(1, 26) = 19.81, P < .01, showing strong spontaneous recovery of responding (Figure 1c). The Block × Treatment × Stress interaction approached significance, F(1, 26) = 4.18, P = .051, suggesting a trend toward differential spontaneous recovery depending on the combination of treatment and stress. Analysis of between-subjects effects revealed significant main effects of Treatment, F(1, 26) = 5.66, P = .025, and Stress, F(1, 26) = 6.43, P = .018, indicating overall differences in responding between treatment and stress groups when averaged across blocks. The Treatment × Stress interaction was not significant, F(1, 26) = 2.87, P = .10.

During spontaneous recovery, repeated-measures ANOVA (Block: 1-6 × Treatment: Estradiol vs Cholesterol × Stress: Stress vs No Stress) revealed a significant main effect of Block, F(5,140) = 23.42, P < .01, reflecting a decrease in conditioned responses across the session (Figure 2c). There was also a significant main effect of Stress, F(1,28) = 13.24, P < .01, with stressed rats exhibiting lower responding than non-stressed rats in both cholesterol and estradiol-treated OVX rats. No main effect of Estradiol Treatment, or Estradiol Treatment × Stress interaction, F(1,28) = 0.02, P = .901, was observed. The Block × Estradiol Treatment × Stress interaction approached significance, F(5,140) = 2.20, P = .058, suggesting the possibility of differential block-dependent stress and estradiol effects between treatment groups. Although the 3-way interaction did not reach statistical significance (P = .058), the pattern of means suggested a potential early block-dependent estradiol effect under no-stress conditions. Estradiol-treated rats appeared to exhibit lower responding than cholesterol-treated controls during the first block, with group differences diminishing across subsequent blocks and not evident under stress conditions (Figure 2c).

Levels of Corticosterone Change in Response to Restrained Stress

The mean serum (±SEM) corticosterone levels obtained 30 minutes following restrained stress was 32.17 ng/ml (±7.16) for cholesterol-treated rats in the control group, 56.39 ng/ml (±23.02) for Estradiol-treated rats in the control group, 195.29 ng/ml (±28.45) for cholesterol-treated rats in the stress group, and 179.75 ng/ml (±19.5) for estradiol-treated rats in the stress group.

A 2 (stress) × 2 (hormone treatment) ANOVA revealed a significant main effect of stress, showing an increase in corticosterone levels in stressed compared to control rats, F(1, 27) = 48.73, P < .01. There was no significant main effect of hormone treatment, F(1,27) = 0.045, P = .83, indication no difference in the level of corticosterone between cholesterol and estradiol treated rats, nor a significant interaction effect, F(1,27) = 0.94, P = .34, indicated that stress did not induce a different physiological response in cholesterol-treated compared to estradiol-treated rats.

Discussion

Summary of Main Results

This study examined the effects of stress, sex, and estradiol on appetitively conditioned touchscreen responding in rats. In Experiment 1, no sex differences were observed in response rates during acquisition, indicating that both male and female rats learned the task at similar rates. However, during the first day of extinction learning female rats show transiently increased rate of extinction compared to male rats. Notably, acute stress administered before the spontaneous recovery test initially increased spontaneous recovery in both sexes compared to controls. However, stressed rats also exhibited a more rapid decline in responding toward the end of the test, suggesting that acute stress may temporarily enhance responding before facilitating extinction.

In Experiment 2, no response differences were observed between the estradiol and cholesterol treatment female rats during acquisition. Estradiol did not show an effect on extinction in females, indicating that estradiol did not affect associative appetitively driven touchscreen response acquisition nor extinction. However, unlike in Experiment 1, stressed rats exhibited lower spontaneous recovery across all trial blocks, regardless of treatment group. Additionally, estradiol-treated non-stressed rats displayed lower response rates during the initial trials of the spontaneous recovery test compared to cholesterol-treated non-stressed rats, suggesting that estradiol may have an initial inhibitory effect on responding or better memory of extinction.

Effects of Acute Stress on Response Recovery

Multiple factors can influence changes in response rates in these tasks, including stress, sex differences, and contextual changes. Prior research indicates that stress enhances freezing behavior in fear-based extinction recall tests²⁴ but impairs response inhibition and other cognitive functions such as working memory.^16,51 Additionally, sex differences in stress effects vary by task. For instance, when estrogens’ effects are blocked or mitigated, sex differences in fear conditioning are.^33,52 However, estrogens appear to modulate these differences, as female rats in proestrus (characterized by high circulating estrogens) exhibit lower levels of freezing in fear conditioning tasks.^33,53

Our findings show that acute restraint stress increased spontaneous recovery in both male and female rats, consistent with previous research indicating that acute stress impairs extinction recall in both rats and humans.²⁰ Thus, our results support the idea that the effects of acute stress on extinction recall are similar in appetitively conditioned paradigms to those in fear-based paradigms.^24,25 Moreover, research on extinction recall in female rats or sex comparisons in extinction recall, is very limited,⁵⁴ and to our knowledge, sex comparison studies specifically examining the effects of acute stress on extinction recall are especially scarce. Our findings demonstrate that acute stress increases spontaneous recovery (ie, reduces extinction recall) in intact male and female rats.

Changes in Context and Response Recovery

Context plays a crucial role in modulating learned associations. Bouton’s research team emphasizes that learned responses are heavily influenced by the original training context, which includes both external factors (eg, objects, smells) and internal states (eg, hormone levels, mood)⁵⁵ The renewal effect demonstrates that learned behaviors are context-dependent, with extinction being particularly sensitive to contextual changes.¹⁸ While alterations in the learning context do not significantly impact performance, extinction learning is highly context-specific.⁵⁵ It is possible that the physiological state of the animal induced by the acute stressor changed the internal context for the animal and thus led to increase in spontaneous recovery in intact male and female rats.

Estradiol and Stress Effects on Spontaneous Recovery

Experiment 1 results suggest that acute stress impairs memory for the extinction of an operant response in intact male and female rats, while Experiment 2 demonstrates that acute stress generally enhances extinction memory in ovariectomized female rats. Although previous research indicates that females show reduced extinction of conditioned fear compared to males, naturally cycling estrogens and exogenously replaced estradiol have been shown to enhance fear extinction recall.^33,34,56 In our study, non-stressed rats treated with estradiol exhibited lower spontaneous recovery during the initial 10-trial block compared to cholesterol-treated controls, also suggesting stronger recall of extinction.

The more surprising finding emerged regarding the effects of acute stress. The acute stress-induced enhancement of spontaneous recovery observed in intact female and male rats in Experiment 1 appeared to be reversed in ovariectomized female rats in Experiment 2. Specifically, acute stress reduced spontaneous recovery in these ovariectomized females. This represents a novel finding, given the limited literature on the direct comparison and interaction between estradiol and acute stress in the extinction recall of an appetitively motivated response. It demonstrates that the presence of circulating gonadal hormones can significantly influence how acute stressors impact extinction recall. A follow-up study comparing the effects of acute stress on spontaneous recovery in intact versus gonadectomized females and males within the same experimental design could offer further insight into this interaction.

Physiological Influences of Acute versus Chronic Stress

It is important to note that this experiment specifically examined the effects of acute stress on the retrieval of an extinguished response, as acute and chronic stressors differ in their physiological mechanisms and in their effects on learning and memory. Chronic stress often impairs cognitive functions, whereas acute stress can enhance them.⁵⁷ For instance, stressed rats exhibited reduced food consumption, with the effect being more pronounced in females, even after stress removal.⁵⁸ Similarly, chronic stressors negatively affect other behavior, leading to increased immobilization in the forced-swim test and higher rates of drug reinstatement.^59,60 Acute stressors, on the other hand, activate the immune system and heighten arousal, while chronic stress suppresses immune function, which may account for differences in their effects.⁶¹ Acute stressors also promote neuronal growth, whereas chronic stress inhibits neurogenesis.⁶¹ Glucocorticoids, a class of corticosteroids, play a key role in the stress response and contribute to the cognitive benefits of acute stress.⁵⁷ Other hormones, such as epinephrine, may also mediate these effects, though further research is.⁶²

Study Limitations

This study has several limitations that should be considered when interpreting the findings. One limitation is the absence of direct measurements of circulating estradiol levels and estrous cycle tracking in intact females. Although established estradiol treatment protocols were used, the lack of hormonal verification limits more precise mechanistic conclusions regarding estrogenic modulation of stress effects on spontaneous recovery. In addition, intact and ovariectomized females were examined in separate experiments rather than within a single factorial design, in part due to practical constraints on the number of animals that can be run concurrently in our laboratory. A fully crossed design including both intact and ovariectomized females within the same cohort will be important in future studies to more directly test hormone × stress interactions.

A further limitation is that potential group differences in appetite or motivational state were not directly assessed. Acute stress exposure, hormonal manipulations, and sex-related differences in body weight may influence food motivation, which could in turn affect performance in food-reinforced touchscreen tasks. All rats were maintained on the same food-restriction regimen, consisting of 1 hour of free feeding every 24 hours, which allows animals to regulate their individual intake and helps accommodate hormone-related changes in metabolic demand. Moreover, all groups successfully acquired the task and completed the maximum number of trials during extinction and spontaneous recovery. Nevertheless, because no independent measures of food consumption or incentive motivation (eg, progressive-ratio testing) were included, we cannot fully exclude the possibility that some of the observed differences in responding were influenced in part by motivational factors. However, the transient and condition-specific nature of the effects suggests that general appetite differences alone are unlikely to fully account for the observed behavioral pattern. Future studies incorporating direct measures of food motivation will be important for disentangling these influences.

Despite these limitations, the present results provide important theoretical insights into how stress and ovarian hormones interact to influence the recovery of inhibited reward-seeking behavior.

Implications and Theoretical Considerations

Response inhibition is frequently discussed in the context of drug addiction because of the substantial societal and personal costs associated with substance use disorders. In the United States alone, substance use disorder is estimated to cost tens of billions of dollars annually in crime, lost productivity, foster care, and other social consequences.⁶³ Neuroimaging studies of chronically addicted individuals indicate that compulsive drug use and elevated relapse risk are linked to dysfunction in neural systems that support inhibitory control.⁸ Impaired response inhibition is not unique to substance use disorders but is also implicated in other maladaptive behaviors, including food addiction⁶⁴ and compulsive buying or shopping disorder.⁶⁵ The present findings suggest that stress influences response inhibition and may increase the return of previously learned responses, implicating stress as a potential trigger for relapse-like addictive behaviors.

Stress is a well-established trigger for relapse,^9,10 including behaviors such as food addition,^10,66 and because stressors are highly prevalent in daily life, individuals are more likely to engage in unhealthy or impulsive behaviors during stressful periods than under non-stressful conditions. Basic animal research using classical and operant conditioning paradigms has long contributed to the development and refinement of behavioral therapies for addiction and related disorders.⁶⁷ The present findings extend this literature by showing that acute stress enhances the spontaneous recovery of an extinguished operant response in intact male and female rats. In contrast, acute stress reduces spontaneous recovery in ovariectomized females, suggesting that naturally circulating ovarian hormones may lower stress-induced relapse-like behavior. However, a factorial follow-up study directly testing hormone × stress interactions will be necessary to confirm this interpretation. Importantly, these effects were observed in an appetitively motivated operant task, which is particularly relevant to reward-seeking and habit-like behaviors, and complements the extensive literature based on fear-conditioned paradigms. Understanding how stress and hormonal status interact to influence the recovery of inhibited responses is a critical step toward improving strategies for preventing relapse in a broad range of maladaptive behaviors.

Several brain regions likely contribute to the learned behaviors examined in the present experiments. Most relevant, extinction learning and extinction recall are regulated by distinct subregions of the medial prefrontal cortex (mPFC). The prelimbic cortex is primarily involved in the expression of learned responses, whereas the infralimbic cortex (IL-mPFC) is crucial for learning to suppress these responses.⁶⁸ Moreover, extinction learning and extinction recall may themselves involve different brain subregions, and noradrenergic activation of the basolateral amygdala (BLA) appears to specifically influence extinction recall under acute stress conditions.¹ The current findings suggest that, similar to stress hormones, gonadal hormones also mediate the recall of extinguished responses and interact with acute stress. The effect of gonadal hormones on extinction recall is possibly mediated by the infralimbic region of the medial prefrontal cortex (IL-mPFC) and other subregions involved in the recall of response suppression. Further investigation is needed to identify the specific neural mechanisms underlying this regulation.

Much of the current research focuses on the influence of sex, stress, and gonadal hormones on extinction and extinction recall (ie, inhibitory processes) within the context of fear learning. However, understanding the neural mechanisms underlying reward processing is equally critical, as exogenous substances such as drugs can activate these pathways and contribute to the development of addiction. By investigating the factors that modulate reward circuitry, we can gain deeper insight into the complex nature of addiction and identify potential therapeutic targets.

Conclusion

In conclusion, the present findings demonstrate that the recovery of an extinguished, reward-seeking operant response is modulated by the acute stress and gonadal hormone status. While acute stress increased the initial spontaneous recovery in intact male and female rats, this effect was reversed in ovariectomized females, indicating that circulating ovarian hormones play a critical role in determining how stress influences extinction recall. These results extend the current literature on stress and extinction in fear-based paradigms to appetitively motivated behaviors that more closely resemble maladaptive habits and substance-seeking. Moreover, the block-dependent effects of estradiol suggest that during initial trials of response recovery, where estradiol has the potential to decrease spontaneous recovery and thus enhance extinction recall. Together, these findings highlight the importance of internal physiological context in shaping relapse-like behavior and underscore the need to consider sex and hormonal status when developing and evaluating interventions aimed at strengthening response inhibition and reducing relapse across a range of addictive and compulsive disorders.

Footnotes

Acknowledgements

This research was conducted with funds and equipment provided by Christopher Newport University.

ORCID iDs

Olga Lipatova

Matthew M. Campolattaro

Emery W. Harlan

Brisa B. Rasmussen

Ethical Considerations

No human subjects were involved in the present study. This research utilized rodent animal models. The project was reviewed and approved by the Christopher Newport University Institutional Animal Care and Use Committee (IACUC), which is composed of 3 scientists, 1 non-scientist, 1 community member, and a veterinarian (Protocol #2019-9). All research procedures were conducted in accordance with the ethical standards for the use of animals in research as outlined in the USDA Guide. Christopher Newport University maintains a Public Health Service (PHS) Assurance and adheres to all associated guidelines and requirements.

Author Contributions

Olga Lipatova and Matthew Campolattaro contributed to the conception and design of the study. Emery Harlan, Srinidhi Gopal, and Brisa Rasmussen contributed to data collection. Olga Lipatova and Emery Harlan performed the data analysis and prepared the initial draft of the manuscript. Olga Lipatova and Matthew Campolattaro critically revised the manuscript for important intellectual content. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work, ensuring its accuracy and integrity.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: All experimental research described in this manuscript was supported by internal grants from Christopher Newport University for Faculty Professional Development.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

Data can be made available upon request.

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