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. Author manuscript; available in PMC: 2025 Feb 1.
Published in final edited form as: Alcohol Clin Exp Res (Hoboken). 2024 Jun 20;48(8):1586–1599. doi: 10.1111/acer.15397

Effects of early social isolation and adolescent single prolonged stress on anxiety-like behaviors and voluntary ethanol consumption in female Long Evans rats

Stacy R Pitcairn 1, Olivia A Ortelli 1, Jeffrey L Weiner 1
PMCID: PMC11568547  NIHMSID: NIHMS2028962  PMID: 39031683

Abstract

Background:

Exposure to stress during childhood and adolescence is a risk factor for alcohol use disorder (AUD) and comorbid conditions, including posttraumatic stress disorder (PTSD). We previously established an adolescent social isolation (SI) model that leads to the emergence of a wide range of behavioral risk factors for AUD, including increased anxiety-like behavior, locomotor activity, and ethanol consumption in male and female rats. Here, we sought to test the hypothesis that SI may increase vulnerability to single prolonged stress (SPS), a rodent model of PTSD.

Methods:

Female Long Evans rats (n = 8/group) were either single-housed or group-housed (GH) (4/cage) on postnatal day 21. One week later, rats underwent testing in the open field test (OFT), elevated plus-maze (EPM), and successive alleys test (SAT). Following initial behavioral testing, a subset of SI/GH rats were exposed to SPS. All rats were then tested on the novelty-suppressed feeding test (NSFT) followed by fear conditioning and home cage two-bottle choice to assess ethanol consumption.

Results:

SI significantly increased activity in the OFT and anxiety-like behavior on the SAT, but not the EPM. While SI and SPS alone had no effect on the NSFT, exposure to both stressors significantly increased approach latency. Complex effects of stress history were observed across a 3-day fear conditioning paradigm and no group differences were observed with home cage ethanol consumption, regardless of prior ethanol exposure.

Conclusions:

The results from this study provide novel evidence that SI interacts with SPS in female rats to influence behavior in assays of unconditioned anxiety-like behavior (NSFT) and conditioned fear. Surprisingly, stress exposure had no effect on home cage ethanol consumption. Ultimately, these models provide useful avenues to examine the interaction between stressful experiences, alcohol exposure, biological sex, and the neurobiological adaptations underlying potential risk factors for psychiatric conditions.

Keywords: alcohol use disorder, early life stress, posttraumatic stress disorder, single prolonged stress, social isolation

INTRODUCTION

Alcohol use disorder (AUD) and posttraumatic stress disorder (PTSD) are chronic psychiatric conditions that often present together in the clinical population (Carlson & Weiner, 2021; Debell et al., 2014; Smith & Cottler, 2018). The frequent comorbidity of AUD and PTSD can exacerbate symptoms of both disorders, thus complicating treatment and often resulting in poorer patient outcomes than either disorder in isolation (Flanagan et al., 2018; Gilpin & Weiner, 2017). Although it has been hypothesized that shared neural circuit adaptations contribute to the etiology of these disorders (see Gilpin & Weiner, 2017 for review), there remain critical gaps in the understanding of the neurobiology of comorbid PTSD and AUD.

For example, striking sex differences are observed in both AUD and PTSD, perhaps suggesting that women may be particularly vulnerable to this comorbidity. Epidemiological research repeatedly shows that women are about twice as likely as men to meet diagnostic criteria for PTSD, even after controlling for trauma type (Blanco et al., 2018; Breslau, 1997; Christiansen, 2015; Hiscox et al., 2023; Kilpatrick et al., 2013; McLean et al., 2011; Olff, 2017; Tolin & Foa, 2006). Additionally, women with PTSD are at a greater risk for developing AUD compared to women without PTSD. Early data from the National Comorbidity Survey showed that the prevalence of alcohol misuse/dependence in women with PTSD was 27.9% compared to 13.5% in women without PTSD (Kessler et al., 1995). More recent studies have reported the same, such that women with PTSD had a significantly higher risk of developing AUD compared to women without PTSD, and this relationship was not fully explained by other comorbid psychiatric conditions or trauma exposure that did not result in PTSD (Sartor et al., 2010). Sex differences have also been observed in the bidirectional relationship between AUD and PTSD, such that PTSD is more likely to precede alcohol dependence for women, whereas for men the opposite was observed (Sonne et al., 2003). Recent work has shown that bidirectional associations between PTSD and alcohol dependence were stronger for women compared with men (Berenz et al., 2017). Additionally, this is supported by sex-specific analyses of data from Wave III of the National Survey on Alcohol and Related Conditions that suggest that the bidirectional relationship between PTSD and substance use disorders is often greater in women (Peltier et al., 2022). Taken together, these studies emphasize the critical importance of examining biological sex as a risk factor for AUD/PTSD.

Exposure to stress during childhood and adolescence is another major risk factor for AUD alone (Dube et al., 2002), PTSD alone (Breslau, 1997; Copeland et al., 2007), and the AUD/PTSD dual diagnosis (Müller et al., 2015; Lee et al., 2018; see Carlson & Weiner, 2021 for review). In the human population, early life stress can involve both acute and chronic stressors, such as experiencing violence (physical and/or sexual), witnessing violence and/or death, war, serious accidents, natural disasters, among others (Copeland et al., 2007; Lee et al., 2018; Smith & Pollak, 2020). During critical developmental periods of the nervous system, these experiences can lead to enduring alterations in stress responding, which may play a role in stress- and trauma-related disorders, such as PTSD (Lee et al., 2018).

From a prognostic standpoint, these events are additive, such that exposure to multiple traumatic events seems to be strongly associated with the likelihood of developing PTSD and substance use disorders (Copeland et al., 2007; Khoury et al., 2010). One longitudinal study by Copeland and colleagues followed a representative sample of children ages 9–13, conducted regular interviews until age 16, and found that exposure to potentially traumatic events is fairly common in children and rarely results in DSM-5 childhood PTSD symptoms. However, after multiple traumatic experiences or a history of anxiety, exposure to potentially traumatic events was more likely to result in PTSD symptoms (Copeland et al., 2007; Khoury et al., 2010). Additionally, children who have experienced stress during early life show lower amygdala and hippocampal volumes that are associated with the magnitude of cumulative stress (Hanson et al., 2015). Given that development is a period marked by constant neurobiological changes, many researchers and clinicians have proposed that the developing brain may be more sensitive to stress-induced disruptions (Schneider, 2013; see Lee et al., 2018 for review). Taken together, these observations emphasize the critical need to understand the effects of early life stress on neural development and vulnerability to PTSD.

Our lab has leveraged these compelling epidemiological findings to establish an adolescent social isolation model of vulnerability to both AUD and PTSD in male rats. During this manipulation, rats arrive on postnatal day (PND) 21 and are separated into socially isolated (SI) or group-housed (GH) conditions from PND28–70. In male rats, SI leads to the emergence of a wide range of behavioral risk factors for AUD and PTSD, including increased anxiety-like behavior (McCool & Chappell, 2009; Skelly et al., 2015; Yorgason et al., 2013), deficits in fear extinction in a fear-potentiated startle paradigm (Skelly et al., 2015), increased locomotor activity (Chappell et al., 2013; Skelly et al., 2015), and increased home cage and operant ethanol consumption (Chappell et al., 2013; McCool & Chappell, 2009; Skelly et al., 2015). Interestingly, none of these behaviors developed in female rats following this SI paradigm (Butler et al., 2014). While this 6 week single housing paradigm failed to elicit behavioral phenotypes, such as increases in anxiety-like behavior and escalations in ethanol drinking in females, we recently discovered that the vulnerability window to adolescent single housing stress may be sexually dimorphic, as an earlier and shorter isolation window did lead to the expression of these behaviors in females (Ortelli et al., 2023). Given the dearth of preclinical studies on AUD/PTSD-related behaviors in females, the present study sought to test the hypothesis that this novel early social isolation paradigm may interact with single prolonged stress, a rodent model of PTSD, to modulate behavioral phenotypes associated with AUD/PTSD vulnerability in female Long Evans rats.

MATERIALS AND METHODS

Subjects

All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Wake Forest University Institutional Animal Care and Use Committee. A total of 32 female Long Evans rats (Inotiv, Indianapolis, IN, USA) were used in two separate studies with 16 rats in each experiment. The design of the experiments was identical except for the home cage drinking procedures (as described below). All rats arrived on PND 21 and were randomly separated into group-housed (GH; 4 rats/cage, n = 16) or socially isolated (SI) conditions (n = 16) on the day of arrival. GH rats were housed in large cages (33.0 × 58.7 cm; Nalgene, Rochester, NY, USA) while SI rats were housed in standard cages (20.3 × 26.7 cm; Allentown Inc., Allentown, NJ, USA) and all rats were given ad libitum access to standard rodent chow (Prolab RMH 3000 Lab Diet 5P00 obtained from Lab Supply, Durham, NC, USA) and water. Rats in SI and GH groups lived in identical environmental conditions and were housed in the same room. Rats were maintained on a 12-h reverse light/dark cycle with lights off at 9:00 am. Rats were handled once weekly for weighing and marking. When drinking procedures began, rats were weighed daily to ensure accurate measures of ethanol consumption (g/kg/day).

Behavioral assays

All behavioral procedures began 1 h into the dark cycle. Rats were brought into the testing room 20 min prior to each behavioral assay to allow for acclimation. Housing condition groups were counterbalanced in each assay, such that the same group did not run first in each assay.

Open field test

Subjects were run in the open field test (OFT) on PND 30. Rats were placed in a standard locomotor activity chamber that was illuminated with one 7.5 watt bulb mounted 48 cm over the center of the arena (42 cm × 42 cm × 30 cm) for a duration of 30 min to assess locomotor activity (Digiscan animal activity monitors; Omnitech, Columbus, OH, USA). General locomotor activity was measured by the total distance traveled.

Elevated plus-maze

On PND 32, rats were placed in a standard elevated plus-maze (EPM; Med Associates, St. Albans, VT, USA) consisting of two closed arms and two open arms (each arm 10.2 cm wide by 50.8 cm long). Lighting setup was identical to previous experiments (approximately 0 lux for closed arms, 90–120 lux for open arms). Rats were placed in the junction facing the open arm and recorded in the assay for 5 min. In this assay, the percent of total time spent in the open arms and number of entries into the open arms were used as measures of anxiety-like behavior. Number of entries into the closed arms was used as a measure of general locomotor activity.

Successive alleys test

On PND 36, rats underwent the successive alleys test (SAT) as an additional assay of anxiety-like behavior. The SAT consists of four segments, each of which are 45 cm in length. The first segment is enclosed and dark gray in color (9.0 cm width, walls 29.0 cm height), the second segment is dark gray with lower walls (9.0 cm in width, walls 2.5 cm height), the third segment is light gray (6.7 cm width, walls 0.5 cm height), and the final segment is white (3.5 cm width, contains walls 0.3 cm in height). The SAT is illuminated by one 60 watt bulb positioned 120 cm above the arena. In this assay, locomotor activity was measured by recording the total distance traveled. Anxiety-like behavior was assessed using, number of entries into the lighter zones (dark gray, light gray, and white), percent of total time spent in the lighter zones, and latency to enter the lighter zones. Rats were recorded in this assay for 5 min and were tracked using Ethovision software (Noldus, Leesburg, VA, USA).

Single prolonged stress (SPS)

Following initial behavioral testing, SI and GH rats were randomly subdivided to undergo single prolonged stress (SPS) or the control condition (n = 8/group). On PND 46, SPS was conducted in a novel testing room with overhead lights off, a red lit lamp illuminated, and a repeating auditory tone (1 kHz, 70 dB). Control rats were placed in a smaller room behind the testing room throughout the duration of SPS with identical environmental conditions. Rats in the SPS group were first placed in hard plastic cylindrical restraints for a duration of 2 h. Following restraint, rats (n = 4 at a time) were placed into a 15-min forced group swim in a plastic tub (21 in width × 21in length × 17 in height) filled 2/3 full with 24°C water. Following the forced swim, rats underwent a 10-min rest period on dry cloth towels. Ether exposure occurred under a fume hood to four rats at a time in a standard cage with a ventilated lid. Cotton balls with diethyl ether (Sigma-Aldrich, St. Louis, MO, USA) were placed in the cage at a rate of 3 mL/min in a plastic barrier that prevented physical contact with the rats but allowed for vapor dissipation. GH rats underwent the swim and ether exposure with their cage mates, while SI rats underwent the swim and ether with a unique group of four SI rats. Rats were removed upon loss of consciousness and lack of righting response, placed in a clean cage, and monitored for recovery. Rats remained in their initial housing conditions following ether exposure and were returned to their housing room for a 1-week undisturbed period.

Novelty suppressed feeding test (NSFT)

All rats underwent the NSFT on PND 57. Rats were placed in the corner of a clear acrylic arena (26.6 in × 34 in). Bright light sources were placed at each of the four corners of the arena and a shortbread cookie (Lorna Doone; Nabisco, East Hanover, NJ, USA) was placed in the center. The setup of these light sources was identical to previous experiments in our lab (approximately 120–165 lux in the center, 40–50 lux in the margin). Rats were recorded in the assay for 10 min and videos were recorded and analyzed using Ethovision software (Noldus). Total distance traveled was used as a measure of general locomotor activity. Latency to enter the food zone was used as a measure of anxiety-like behavior. The food zone was delineated in Ethovision as the area of the arena covered only by the food dish and an entry was recorded when the nose-point crossed into this zone. On a separate day at the identical time, a shortbread cookie was placed in the home cage and latency to approach food was measured. Latency to enter food zone in the home cage was used to control for differences in general motivation to eat (i.e., hunger) in a non-anxiogenic environment.

Fear conditioning

On PND 59, all rats underwent a 3-day fear conditioning (FC) paradigm. The first day was an acquisition day. Rats were placed in fear conditioning chambers (42 cm × 42 cm × 30 cm) with an electric grid floor and tone speakers (Omnitech, Columbus, OH, USA). Following a 5-min habituation period in the chamber, the tone (8 kHz) was presented for 29.5 s followed by a shock (0.25 mA) for 0.5 s. The acquisition day consisted of five presentations of the tone + shock pairing. The second and third days were extinction days, where the tone was presented alone with no shock. Rats were presented with 14 presentations of the tone alone on each extinction day to assess extinction of conditioned fear. Percent of the 30 s stimulus presentation spent freezing was used as the measure of fear.

Home cage ethanol drinking: Experiment 1

In the first experiment, drinking began on PND 64. All rats were single housed throughout drinking procedures in order to accurately assess individual ethanol consumption. GH rats were separated 3 days prior to the start of drinking procedures to allow acclimation to single housing. Ethanol consumption was assessed in all rats in both experiments using a two-bottle choice paradigm. For the first 8 days, rats had continuous access to ethanol and water in their home cage that was available for 24 h. Water and ethanol bottles were alternated daily to account for side preference and weighed daily in order to obtain measures of ethanol consumption (g/kg/day). Ethanol concentration was increased over the 8-day period with 2 days at each concentration (3% (v/v), 5%, 7%, and 10%). This gradual increase of ethanol concentration was used because a prior experiment with female Long Evans rats showed an escalation of ethanol consumption with this regimen (Ortelli et al., 2023).

Home cage ethanol drinking: Experiment 2

Following the first experiment, we hypothesized that exposure to ethanol prior to the stressor may be important to the development of drinking phenotypes following stress (Anderson et al., 2016; Siegmund et al., 2005; for review see Becker, 2017). Therefore, in the second experiment, rats underwent an additional 8-day continuous access two-bottle choice procedure during the period following the SAT and prior to SPS, from PND 37-PND 45, in addition to the 8 days of continuous access on PND 64 (similar to Experiment 1). Because rats must be single-housed for drinking procedures, rats remained in single housed conditions from PND 36 through the completion of the experiment. NSFT, fear conditioning, and later drinking procedures occurred as described for Experiment 1.

Statistical analyses

All statistical analyses were performed in GraphPad Prism version 10.1.2 for Windows (GraphPad Software, Boston, MA, USA) or R (Version 1.4.1106, http://www.r-project.org/). For the OFT, repeated measures two-way ANOVA was used to analyze total distance traveled by housing condition (levels = SI, GH) and Time (5-min time bins from 0 to 30 min). Sidak’s multiple comparisons test was used to determine the specific time bins where SI and GH rats were significantly different. For the total distance traveled on the OFT averaged across time bins, Welch’s t-tests were used to examine differences between SI and GH groups. For the EPM, Welch’s t-tests were used to evaluate effects of SI or GH housing condition on time spent in the open arm and number of entries into the closed arm. For number of entries into the open arm, Shapiro–Wilk normality test failed (p < 0.05) and Mann–Whitney Rank Sum Test was used. OFT and EPM data were included in a larger analyzed data set that has been previously published (Ortelli et al., 2023). For the SAT, a repeated measures two-way ANOVA was used to analyze number of entries to each zone (levels = enclosed, dark gray, light gray, and white) time spent in each zone, and latency to each zone by housing condition (levels = SI, GH). Behavioral data from both cohorts was combined for OFT, EPM, and SAT.

To examine if earlier access to ethanol in Experiment 2 affected subsequent behavioral tests (NSFT, FC, and later drinking), likelihood ratio tests (LRTs) were conducted to compare full models versus partial models omitting ethanol exposure (two levels: ethanol exposure, no ethanol exposure). For NSFT latency, the full model included housing (two levels: SI, GH), SPS exposure (two levels: SPS, no SPS), ethanol access (two levels: ethanol, no ethanol) and all interactions. The LRT comparing the two models revealed no significant difference between the full and partial models (F(4,20) = 1.36, p = 0.283); therefore, we decided to combine both cohorts for NSFT analyses. Specifically, two-way ANOVAs with housing condition as the first factor (levels = SI, GH) and SPS exposure (levels = SPS, no SPS) as the second factor to examine effect of each stressor on latency to approach food in the NSFT.

A similar approach using LRTs to compare full models vs. partial models omitting ethanol history was used to assess if ethanol history impacted FC and later drinking. However, because FC and later drinking analyses include repeated measures, rat ID was included as a random factor and the Kenward–Roger method for calculating degrees of freedom was used. For FC, the full model also included fixed effects of housing (two levels: SI, GH), SPS exposure (two levels: SPS, no SPS), and ethanol access (two levels: ethanol, no ethanol). LRTs comparing both models revealed no significant difference between full and partial models on all 3 days of fear conditioning (Day 1: F(1,28) = 0.47, p = 0.498; Day 2: F(1,28) = 0.005, p = 0.942; Day 3: F(1,28) = 1.39, p = 0.249), therefore, behavioral data from both cohorts were combined for FC. Specifically, repeated measures three-way ANOVAs were used to determine group differences in time spent freezing on each day of fear conditioning, using SPS exposure and housing condition as between-subjects factors and stimulus presentation as a within-subjects factor. The Greenhouse–Geisser correction was applied when the assumption of sphericity was violated. When a significant Housing × SPS interaction was observed, simple main effect and interaction analyses were performed.

Finally, the same approach used to assess whether ethanol history impacted FC was taken to compare later drinking, however, an additional fixed effect of ethanol concentration (four levels: 3, 5, 7, 10 (%)) was included in both the full and partial models. The LRT revealed a significant difference between these models (F(1,28) = 4.20, p = 0.0498), therefore drinking data for experiments 1 and 2 were graphed and analyzed separately (n = 16/experiment). For the 8 days of continuous access drinking following fear conditioning, SPS exposure (levels = SPS, no SPS) and housing (levels = SI, GH) served as between-subjects factors while concentration (levels = 3, 5, 7, 10 (%)) served as a within-subject factor. For Experiment 2 where rats were given 8 days of access prior to SPS, a repeated measures two-way ANOVA was used with housing as the between-subjects factor and concentration as the within-subject factor. Outliers were assessed using the ROUT method (Q = 1%). All data are expressed as mean ± SEM. The minimal level of significance was p < 0.05 for all analyses. For fear conditioning and later drinking data, the lme4 (Bates et al., 2015) and pbkrtest (Halekoh & Højsgaard, 2014) packages in R were used to conduct mixed effects models and LRTs, respectively.

RESULTS

Social isolation led to distinct behavioral phenotypes compared to group-housed counterparts

A repeated measures two-way ANOVA revealed a strong main effect of housing condition on distance traveled in the OFT, with SI rats showing increased locomotion compared to their GH counterparts (Figure 1B; F(1,30) = 13.02, p = 0.001). There were also significant differences in Time Bin (Figure 1B; F(5,150) = 90.30, p < 0.0001) and a Time Bin × Housing interaction (F(5,150) = 2.472, p = 0.035). When distance traveled per rat was averaged across time bins, Welch’s t-test revealed that SI rats traveled significantly more than GH rats (Figure 1C; t(29.07) = 4.43, p = 0.0001), such that SI rats (527.5 ± 51.36 cm) traveled over double that of GH rats (231.1 ± 42.85 cm).

FIGURE 1.

FIGURE 1

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Behavior on open field test and elevated plus-maze. (A) Experimental timeline. (B) Distance traveled on open field test (OFT) by 5-min time bins between socially isolated (SI) and group-housed (GH) rats. (C) Total distance traveled throughout the 30-min OFT session. (D) Percent of total time spent in the open arm of the elevated plus-maze (EPM). (E) Entries into the open arm of the EPM. (F) Entries into the closed arm of the EPM. Experimental timeline created with Biorender. Data are presented as mean ± SEM. ***p < 0.001, **p < 0.01, #p < 0.1.

In the EPM, SI rats were trending toward decreased time in the open arms (Figure 1D; t(23.26) = 1.83, p = 0.080), with GH rats spending nearly double the time in the open arms than SI rats (36.3 ± 7.96 s vs. 19.7 ± 4.36 s). No significant difference was observed between SI and GH animals in the number of entries into the open arms (Figure 1E; Mann–Whitney U = 88.000, p = 0.129). There was also no significant difference between groups in the number of entries into the closed arms, which is used as a measure of general locomotor activity (Figure 1F; t(28.64) = 0.4616, p = 0.648).

For the SAT, a repeated measures two-way ANOVA was implemented to examine the effect of housing condition on latency to enter each zone of the SAT, number of entries to each zone, and percent of total time spent in each zone as measures of anxiety-like behavior. For latency to each zone, there was a significant effect of Housing (Figure 2B; F(1,30) = 13.33, p = 0.001), Zone (F(1.475,44.26) = 46.96, p < 0.0001), and Zone × Housing interaction (F(2,60) = 5.398, p = 0.007). Sidak’s multiple comparisons test revealed that SI rats showed longer latency to approach the light gray zone (padj = 0.008) and the white zone (padj = 0.0007), the two most anxiogenic regions of the assay. On average, SI rats had a latency of 225.9 ± 23.40 s to enter the light gray zone, while GH rats had an average latency of 100.9 ± 30.06 s to the light gray zone. Additionally, SI rats took twice as long to approach the white zone compared to GH rats (270.2 ± 14.61 s vs. 132.6 ± 27.86 s). Regarding number of entries to each zone, the analysis revealed a significant effect of Zone (Figure 2C; F(1.932,57.97) = 139.6, p < 0.0001) and a significant Zone × Housing interaction (Figure 2C; F(3,90) = 5.962, p = 0.001). No effect of Housing alone was observed (F(1,30) = 0.4709, p = 0.498). Sidak’s multiple comparisons test revealed that SI rats made more entries into the enclosed zone of the SAT compared to group-housed rats (padj = 0.049), which is considered the least anxiogenic zone of the assay. SI rats made on average 38.4 ± 2.17 entries into the enclosed zone, while GH rats made on average 30.5 ± 2.00 entries into the enclosed zone. Percent time spent in each zone was calculated as (time spent in zone/total assay time) × 100. Analysis revealed that there was a significant effect of Zone (Figure 2D; F(1.270,38.11) = 74.36, p < 0.0001) and a significant Zone × Housing interaction (F(3,90) = 3.917, p = 0.011). There was no significant main effect of Housing alone (F(1,30) = 1.000, p = 0.325). Sidak’s multiple comparisons test did not reveal significant effect of housing within zone, but GH rats trended to increased percent of time spent in light gray (padj = 0.066) and white (padj = 0.098) compared to SI rats. Differences between SI and GH rats on these measures was not driven by differences in general locomotion, as there were no significant differences between the housing groups on total distance traveled in the session (Figure 2E; t(30) = 1.59, p = 0.121).

FIGURE 2.

FIGURE 2

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Behavior on the successive alleys test. (A) Schematic of the successive alleys test (SAT). (B) Latency to enter each zone between SI and GH rats. Maximum assay duration is 300 s. (C) Number of entries to each zone of the SAT. (D) Percent of total time spend in each zone of the SAT. (E) Total distance traveled throughout the test. Schematic created with Biorender. Data are presented as mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05, #p < 0.1.

SI and SPS showed synergistic effects on anxiety-like behavior in the NSFT

To examine the effect of multiple stress exposures on latency to approach the food in the NSFT, a two-way ANOVA was conducted with housing condition (2 levels = SI, GH) and SPS exposure (2 levels = SPS, no SPS). One outlier was excluded from the GH alone, one outlier was excluded from GH + SPS, and two outliers were excluded from the SI alone group. There was a significant effect of Housing condition (F(1,24) = 9.203, p = 0.006) and a significant effect of SPS (F(1,24) = 12.84, p = 0.002). There was also a significant Housing × SPS interaction (F(1,24) = 11.44, p = 0.003). Sidak’s multiple comparisons test revealed a significant difference between GH alone and the SI + SPS group (Figure 3A; padj = 0.0004), a difference between GH + SPS and SI + SPS groups (Figure 3A; padj = 0.0005), and a difference between SI alone and SI + SPS group (Figure 3A; padj = 0.0003). GH alone showed an average latency of 50.4 ± 10.24 s, GH + SPS showed an average latency of 61.0 ± 10.92 s, and SI alone showed an average latency of 32.1 ± 11.43 s. The dual stressor group, SI + SPS, showed an average latency of 400.0 ± 89.73 s. Latency to approach food in the home cage was analyzed as a control. One value from the GH + SPS group was determined to be an outlier and was excluded. No effect of Housing condition (Figure 3B; F(1,27) = 2.403, p = 0.133), SPS exposure (Figure 3B; F(1,27) = 0.2914, p = 0.594), or interaction effect was observed (F(1,27) = 1.323, p = 0.260) on latency to approach food in the home cage.

FIGURE 3.

FIGURE 3

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Behavior on the novelty-suppressed feeding test. (A) Latency to approach food in the novelty-suppressed feeding test (NSFT). Gray symbols indicate values that were detected as outliers using the ROUT method (1% IQR) and excluded. Total assay time was 600 seconds. (B) Latency to approach food in the home cage. Data are presented as mean ± SEM. ***p < 0.001.

Effects of SI and SPS and acquisition and extinction of conditioned fear

A repeated measures three-way ANOVA revealed significant main effects of SPS (Figure 4A; F(1,28) = 6.12, p = 0.02) and stimulus presentation (Figure 4A; F(4,112) = 31.11, p < 0.0001), but not housing (Figure 4A; F(1,28) = 1.49, p = 0.23) on percent freezing during fear acquisition. There were no significant interactions (ps > 0.05). These results suggest that regardless of housing condition, subjects who had experienced SPS spent significantly less time freezing during fear conditioning acquisition compared to non-SPS rats (33.1 ± 3.83% vs. 47.9 ± 3.45%).

FIGURE 4.

FIGURE 4

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Fear conditioning behavior. (A) Percent of 30-s period spent freezing during fear acquisition session (tone + shock) across stimulus presentations. (B, C) Percent of 30-s tone duration spent freezing during fear extinction sessions (tone alone). Data are presented as mean ± SEM. ****p < 0.001, *p < 0.05.

Next, a repeated measures three-way ANOVA was used to determine if there were differences depending on SPS and housing in percent time spent freezing across the 14 stimulus presentations on the first extinction day, in which the tone was presented without a shock. Similar to the findings from day 1, we report a significant main effect of Stimulus Presentation (Figure 4B; F(6.97,195.09) = 7.46, p < 0.0001). Unlike day 1, we report a significant main effect of housing (Figure 4B; F(1,28) = 5.78, p = 0.02) but not SPS (F(1,28) = 0.005, p = 0.94), as well as a significant Housing × SPS interaction (F(1,28) = 10.18, p = 0.003). Simple main effect analyses, grouped by SPS history, revealed a significant main effect of housing among non-SPS controls (F(1,14) = 24.7, p < 0.001), but not among SPS rats (F(1,14) = 0.23, p = 0.64). Indeed, descriptive statistics revealed that across all stimulus presentations, non-SPS + GH subjects froze nearly 2× that of non-SPS + SI rats (47.4 ± 2.94% vs. 26.1 ± 2.10%) while SPS + GH and SPS + SI rats had comparable freezing behavior (35.5 ± 2.25% vs. 38.5 ± 2.68%). Stimulus presentation remained a significant main effect for both non-SPS (F(5.49,76.8) = 6.87, p < 0.0001) and SPS (F(13,182) = 2.38, p = 0.006) subjects, with all groups decreasing time spent freezing as the tone alone was presented.

Finally, a repeated measures three-way ANOVA analyzed group differences in percent time freezing on the final extinction day. Like the previous day, housing served as a significant main effect (Figure 4C; F(1,28) = 9.97, p = 0.004), suggesting that regardless of SPS history, GH subjects (37.0 ± 1.75%) froze more than SI rats (27.1 ± 1.36%) when collapsed across all stimulus presentations. SPS exposure was not a significant main effect (Figure 4C; F(1,28) = 0.01, p = 0.91). When collapsed across housing condition, SPS (31.9 ± 1.63%) and non-SPS rats (32.2 ± 1.57%) had indistinguishable freezing behavior. For the first time, Stimulus Presentation was not a statistically significant main effect (Figure 4C; F(13,364) = 1.58, p = 0.09), suggesting that freezing behavior did not change across the session. There were no significant interactions (ps > 0.10).

SI and SPS have no effect on ethanol consumption, regardless of prior ethanol exposure

Rats in Experiment 1 had no prior exposure to ethanol and began home cage drinking following fear conditioning (Figure 5A). A repeated measures three-way ANOVA revealed no significant effect of either SPS exposure (Figure 5B; F(1,12) = 3.361, p = 0.090) or housing (Figure 5B; F(1,12) = 0.101, p = 0.756). A significant main effect of concentration was observed (Figure 5B; F(1.69,20.29) = 37.634, p < 0.0001). There were no significant interactions (ps > 0.05).

FIGURE 5.

FIGURE 5

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Ethanol consumption in experiments 1 and 2. (A) Timeline of ethanol access in Experiment 1. (B) Experiment 1 home cage ethanol consumption (g/kg) at increasing ethanol concentrations. (C) Timeline of ethanol access in Experiment 2. (D) Experiment 2 home cage ethanol consumption (g/kg) at increasing ethanol concentrations prior to SPS (PND 37–45). (E) Experiment 2 home cage ethanol consumption (g/kg) following SPS, NSFT, and fear conditioning. Data are presented as mean ± SEM. Timelines created with Biorender. EPM, elevated plus-maze; FC, fear conditioning; GH, group housed; NSFT, novelty-suppressed feeding test; OFT, open field test; PND, postnatal day; SAT, successive alleys test; SI, socially isolated; SPS, single prolonged stress. ****p < 0.001, *p < 0.05.

In Experiment 2, animals received continuous access to ethanol for 8 days prior to SPS (Figure 5C). In contrast to recent findings from our lab in which early SI significantly increased ethanol drinking in females (Ortelli et al., 2023), no significant effect of housing was observed on ethanol consumption prior to SPS (Figure 5D; F(1,14) = 0.969, p = 0.342). A significant effect of concentration was once again observed (Figure 5D; F(1.89,26.45) = 5.061, p = 0.015). Following SPS and the completion of behavioral testing, rats underwent the same 8-day continuous access paradigm (Figure 5C). As in Experiment 1, analysis revealed no effect of SPS (Figure 5E; F(1,12) = 0.053, p = 0.823), housing (Figure 5E; F(1,12) = 2.243, p = 0.160) or any interactions (ps > 0.05). Again, only a significant effect of concentration was observed (Figure 5E; F(3,36) = 11.787, p < 0.0001).

Finally, we assessed whether there were group differences in the percent change of ethanol consumption (pre vs. post SPS) across the concentrations (data not shown). A three-way ANOVA did not reveal a significant main effect of SPS, suggesting that SPS-exposed females did not demonstrate an increase in drinking compared to non-SPS controls, regardless of housing history (F(1,12) = 1.67, p = 0.22, data not shown). Surprisingly, we did observe a significant main effect of housing, such that females with a history of social isolation (regardless of SPS) showed decreases in ethanol consumption when comparing the two drinking periods, however GH females did not (F(1,12) = 5.18, p = 0.04). There was no significant main effect of concentration nor any significant interactions (ps > 0.05).

DISCUSSION

The present studies demonstrate that early social isolation interacts with SPS in adolescence to influence behavior in assays of unconditioned anxiety-like (e.g., novelty-suppressed feeding) and conditioned fear behavior (e.g., fear conditioning) without affecting home cage ethanol consumption. Prior to SPS, we observed that SI rats showed increased locomotor behavior in the OFT, increased anxiety-like behavior in the SAT, and a trend toward increased anxiety-like behavior in the EPM, compared to GH rats. Following SPS, we observed a synergistic effects of SI and SPS exposure on approach latency to food in the NSFT arena. In a fear conditioning paradigm, we observed complex effects of stress history on freezing behavior during both acquisition and extinction. Despite these findings, we did not observe any group differences in ethanol consumption, regardless of prior ethanol experience.

The OFT and EPM data reported here replicate a larger analysis previously reported by our lab (Ortelli et al., 2023). We have previously speculated on possible interpretations of the observed behavioral phenotypes, particularly the lack of a significant difference in open arm time on the EPM. To further characterize unconditioned anxiety-like behavior, the present study also assessed behavior on the SAT, an assay that expands upon the premise of the EPM and similarly exploits rats’ natural tendency to avoid brightly lit, open areas of a novel environment (Deacon, 2013; Treit & Fundytus, 1988). The SAT creates a gradient of anxiogenic conditions by including a decline in wall height, decline in alley width, and increasing brightness, in contrast to the EPM where changes in wall height, alley width, and brightness are binary (Deacon, 2013). We observed that SI rats show anxiogenic phenotypes on the SAT, such that they made more entries into the enclosed zone (i.e., the least anxiogenic zone) and showed longer latency to enter the light gray and white zones (i.e., the more anxiogenic zones) compared to GH rats. For time spent in each zone, SI rats displayed a trend of increased anxiety-like behavior, such that they trended toward lower percent of their total time in the light gray and white (i.e., more anxiogenic) zones compared to GH rats. The anxiety-like behavior observed on the SAT aligns with previous work from our lab, where alcohol-exposed female rats showed anxiety-like behavior on the SAT, but not the EPM (Bach et al., 2021). Although more studies are needed to parse out the circuitry underlying behavior in this specific task, the observed significant effects may support its use to assess anxiety-like behavior following stress, particularly in females who may not exhibit the same stress-induced anxiety-like behavioral phenotypes as male rats (Ortelli et al., 2023; Weiss et al., 2004).

Novelty-suppressed feeding was used as an additional behavioral assay, because unlike the EPM and SAT, this assay is used to measure both anxiety- and depressive-like behavior (Samuels & Hen, 2011). We observed a synergistic effects of SI and SPS, such that the rats with a history of both SI and SPS showed longer latency to approach than either of the single stress conditions or a control cohort not exposed to either stressor. Other preclinical models of early life stress, such as maternal separation (Qin et al., 2021) and chronic unpredictable stress (Chaby et al., 2015), have been shown to increase latency to food on the NSFT. However, studies specifically examining early social isolation report mixed results, although different species and social isolation windows were used across these studies. One study isolated male and female mice from PND21–70 and showed that single-housing increased the latency to eat in male mice yet decreased this measure in females (Oliver et al., 2020). In contrast, a study in rats reported that females isolated from PND19–73 showed increased latency to food in the NSFT (Hermes et al., 2011). Given that this was the only assay in the present study where synergistic effect of both stressors was observed, it is possible that the double-hit primarily exacerbates depressive/anhedonia-like measures, rather than fear or anxiety-like behavior per se. Indeed, Francois et al. (2022) reported that among mice socially isolated at 5 weeks of age (mid-adolescence, not postweaning), both SI males and SI females had a longer latency to eat in the novel environment compared to the home cage, but when they examined the amount of food consumed, they observed that adolescent social isolation leads to consistent and significant hypophagic (i.e., reduced feeding) responses in females. In males, caloric intake was not significantly decreased in the novel cage, which lends support to the idea that perhaps females are more anhedonic. However, future studies are warranted to examine additional measures of anhedonia or depressive-like behaviors following multiple hits of stress throughout development.

Fear conditioning is extensively used in combination with stress paradigms due to the fact that stress experience can affect the neural circuitry underlying fear and extinction learning (Akirav & Maroun, 2007; Rodrigues et al., 2009). SPS is used as a model of PTSD for its ability to recapitulate behavior relevant to PTSD pathology, specifically deficits in the extinction of conditioned fear (Souza et al., 2017). Therefore, we wanted to examine how stress history affected fear acquisition and extinction learning in a fear conditioning task. We hypothesized that SPS rats would spend more time freezing during the tone presentation and show impaired extinction relative to the non-SPS rats. Similar to SPS, early life social stress has been shown to affect fear acquisition and extinction across various fear conditioning paradigms. For example, males isolated from PND21–42 have shown transient increases in freezing to a conditioned stimulus relative to GH males (Lukkes et al., 2009). Females exposed to adolescent social instability stress froze more than control females during cue extinction trials, but they did not differ in cue extinction when tested 2 days later (McCormick et al., 2013). Additionally, other nonsocial chronic stress paradigms (e.g., chronic restraint stress) have been shown to impair fear extinction in both males and females (Baran et al., 2009; Hoffman et al., 2014). We also speculated that perhaps the SI and SPS stress condition would show a synergistic effect of both stressors, such that rats in the dual stressor condition would exhibit greater fear behavior (i.e., more time spent freezing) and stronger deficits in extinction than either stress condition alone.

Despite these prior findings, we observed that rats who experienced SPS spent less time freezing on the acquisition day, regardless of their housing condition. On the second day (extinction), the tone alone was presented with no shock and we observed that non-SPS GH subjects froze more than the non-SPS SI rats. On the third and final extinction day, we report that regardless of SPS history, GH subjects froze more than SI rats. Given that freezing is typically interpreted as a measure of fear, these results did not align with our initial hypothesis. However, these unexpected findings may be explained by the behavioral coping strategy used by the rats during the paradigm. Recent work has suggested that, while male rats tend to freeze in response to the conditioned stimulus, a subset of rats exhibit darting behavior, which is characterized by at least one rapid movement at a velocity exceeding 20 cm/s during one or more tone presentations (Gruene et al., 2015). Importantly, this darting phenotype is more common in females than males (Gruene et al., 2015). In the fear conditioning chambers used in the present study, rat movement (or lack of movement) was measured by beam breaks rather than video recording. Therefore, our unexpected result that rats with a history of stress spent less time freezing may actually be a reflection of them darting more, a behavioral strategy we were unable to accurately capture. Future work examining the effects of stress history on both male and female fear behavior should take into account differences in behavioral coping strategies in response to the conditioned stimulus.

The final aim of this study was to assess the independent and synergistic effects that social isolation and SPS paradigms can have on home cage ethanol drinking. In Experiment 1, we employed an 8 day continuous access paradigm beginning at PND 64. To our surprise, we did not observe a significant main effect of housing, suggesting that regardless of SPS exposure, SI and GH females had comparable ethanol consumption. These findings contrast with extensive prior work from our lab in males, and recent findings from us and others in females, showing that social isolation results in long-lasting increases in ethanol drinking in various self-administration paradigms (McElroy et al., 2023; Ortelli et al., 2023). Interestingly, the SI females in the current study had similar consumption when compared to the aforementioned study, yet GH females in the current experiment had almost double the ethanol consumption than the GH females did in Ortelli et al. (2023) (≥4 g/kg vs. ≤2 g/kg at ethanol concentrations of 5%–10%). It’s possible that this discordance may be due to the fact that subjects in Experiment 1 started drinking at PND 64, almost 1 month later than drinking began in our earlier study. To account for this methodological difference, subjects in Experiment 2 began the continuous access drinking paradigm at PND 37 comparable to our original study (PND 39). However, we once again observed no difference in drinking between SI and GH females, with GH females again having consumption 2× that observed in Ortelli et al. (2023) (≥4 g/kg vs. ≤2 g/kg at ethanol concentrations of 5%–10%). There is a limited, yet growing, amount of alcohol research that includes both sexes and even less research that solely characterizes female drinking. Therefore, it is difficult to speculate as to why the GH females in the current study had greater home cage ethanol consumption compared to our previous studies. For example, it is possible that gonadal hormones modulate the relationship between stress and ethanol consumption and that these relationships are sexually dimorphic across different phases of development (see Erol et al., 2019 for review). Future studies investigating the effects of sex on ethanol drinking behaviors in both sexes, but particularly in females, is imperative for developing more effective treatments for both stress- and alcohol-related disorders.

We were also surprised that SPS exposure did not result in heightened ethanol drinking compared to non-SPS controls, regardless of housing condition, in either experiment. Interestingly, within-subject analyses did not reveal any significant increases in drinking among SPS-exposed females compared to the 8 day continuous access baseline (Experiment 2). Our hypothesis that SPS-exposed subjects would have increased drinking was grounded in the self-medication hypothesis (Hawn et al., 2020; Khantzian, 1997), as we expected that subjects would drink more to alleviate increases in negative emotion-like behavior, as observed in the NSFT. It is important to highlight that while the critical, pathological switch from positive (i.e., drinking for euphoric effects) to negative reinforcement (i.e., drinking to alleviate negative emotions), and the associated neurobiological changes tend to occur sooner in women compared to men, this transition still takes years to occur (Towers et al., 2023). This temporal incongruence may explain why many preclinical stress models, including the one employed in the current study, do not reliably produce robust increases in ethanol drinking (for detailed review see Becker et al., 2011). Future studies would benefit from investigating how longer ethanol exposure durations may interact with one or dual stressors, as it’s possible that preclinical stress models would result in more robust increases in drinking if subjects had a longer drinking history, as is more common with the human experience.

Finally, few studies have yet examined how SPS exposure interacts specifically with alcohol, let alone whether biological sex further moderates this relationship. While Yu et al. (2016) reported that males with a history of SPS developed greater preference for ethanol when assessed in the conditioned place preference assay compared to non-SPS controls, no studies have sought to replicate these findings in female subjects. Therefore, it is possible that biological sex mediates the behavioral phenotypes that emerge as a consequence of the SPS paradigm, similar to what our lab has observed when investigating the social isolation paradigm. Indeed, studies have reported that estradiol may mitigate the behavioral effects of SPS (Lisieski et al., 2018), further highlighting the need to investigate how sex hormones may adapt as a result of SPS and whether these adaptations can have sex-specific interactions with alcohol.

Although the present experiments did not assess neurobiological changes, cortical and limbic regions likely play a role in the synergistic effect of these stressors. The neural circuitry underlying reward and stress is highly interconnected and includes regions, such as the prefrontal cortex, amygdala, hippocampus, and nucleus accumbens, among others (for review, see Carlson & Weiner, 2021). Prior research has demonstrated that both social isolation (Hermes et al., 2011; Zhang et al., 2012) and SPS (Schoenfeld et al., 2019) can alter latency behavior in the NSFT and lead to neurobiological changes. For example, female rats socially isolated from PND19–73 have shown longer latency to food in the NSFT and decreased levels of glutamate receptor subtypes (e.g., NR1 and GluR1) and synaptic-associated proteins (e.g., synapsin I and PSD95) in the prefrontal cortex compared to GH rats (Hermes et al., 2011). Thus, future studies should continue to examine subcortical and cortical regions as contributors to the synergistic effects of multiple stressful experiences throughout development.

CONCLUSIONS

The results from this study provide new evidence that early social isolation combined with a later stressor, such as SPS, can result in a synergistic effect of both stressors on negative emotion-like behavior in the novelty-suppressed feeding test. Interestingly, SI alone or SPS alone did not show an effect, but the dual-stressor condition resulted in longer approach latency on this assay. Additionally, we observed that social isolation in female rats led to increased anxiety-like behavior on the SAT. These findings support the notion that early life stress can increase sensitivity to phenotypes that emerge following exposure to additional stressors later in life. Ultimately, these models provide useful avenues to probe the neurobiological adaptations that lead to these behaviors and allow for better understanding of the interaction between stressful experiences, alcohol exposure, biological sex, and other potential risk factors for psychiatric conditions.

FUNDING INFORMATION

This work was supported by National Institutes of Health Grants [P50 AA026117, R37 AA17531, R01 AA26551 (JLW), T32 AA7565 (SRP), T32 NS115704 (OAO)].

Footnotes

CONFLIC T OF INTEREST STATEMENT

All authors have no conflicts of interest to report.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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