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[Preprint]. 2025 Jun 12:2024.08.12.607663. Originally published 2024 Aug 15. [Version 3] doi: 10.1101/2024.08.12.607663

Male and female mice may respectively form stronger social aversive memories with same and different sex conspecifics

Jasmin N Beaver a,b,c, Marissa M Nicodemus a,b, Isabella R Spalding a, Lauren R Scrimshaw a,b, Sohini Dutta b,d, Aaron M Jasnow e, Lee M Gilman a,b,c
PMCID: PMC11343151  PMID: 39185229

Abstract

Mice offer a wealth of opportunities for investigating brain circuits regulating multiple behaviors, largely due to their genetic tractability. Social behaviors are translationally relevant, considering both mice and humans are highly social mammals, and human social behavior disruptions are key symptoms of myriad neuropsychiatric disorders. Stresses related to social experiences are particularly influential in the severity and maintenance of neuropsychiatric disorders like anxiety disorders, and trauma and stressor-related disorders. Yet, induction and study of social stress in mice has disproportionately focused on males, influenced heavily by their inherent territorial nature. Conspecific-instigated stress (i.e., defeat), while ethologically relevant, is quite variable and predominantly specific to males, making rigorous and sex-inclusive studies challenging. In pursuit of a controllable, consistent, high throughput, and sex-inclusive method for social stress elicitation, we modified a paradigm to train male and female F1 129S1/SvlmJ × C57BL/6J mice to associate (via classical conditioning) same or different sex C57BL/6J conspecifics with a mild, aversive stimulus. While further paradigm optimization is required, social interaction testing 24 h after conditioning indicates males socially conditioned better to male conspecifics by exhibiting reduced social interaction, whereas females socially conditioned better to male conspecifics. Serum corticosterone levels inversely corresponded to social avoidance after different sex, but not same sex, conditioning, suggesting corticosterone-mediated arousal influences cross sex interactions. These current outcomes reveal why past pursuits to develop same sex female social stress paradigms may have met with limited success. Future research should expand investigation of utilizing male mouse conspecifics to instigate social stress across sexes.

Significance Statement

Validated paradigms to study social stress in female mice, and across sexes, are needed. We modified a published male mouse protocol by using classical conditioning to pair an aversive stressor with a conspecific. Our goal was to create a uniform, cross-sex, high-throughput social stress technique to advance future research. Though our modified paradigm requires future improvements, we did acquire evidence that both males and females socially conditioned in this way exhibit stronger associations when a male conspecific is used. Future research seeking to induce social stress in female mice may meet with more success using male, rather than female, conspecifics. This work, while not achieving our goal, provides useful information to advance future sex-inclusive social stress investigations.

Introduction

Social interaction behavior is a cross-mammalian phenomenon seen in humans, non-human primates, rats, hamsters, and mice, among other animals (Bloomberg et al., 1994; Kamps et al., 2001; Leung, 2011; Monfils and Agee, 2019; Lee et al., 2020) [author citations omitted for double-blind review]. Shifts in social interaction behavior can be informative of the physical and/or emotional state of the mammal (Ago et al., 2014; Wilson and Koenig, 2014; Venniro et al., 2018). In humans, disruptions in typical social interaction behavior are characteristic of numerous neuropsychiatric conditions including trauma and stress-related disorders, mood and anxiety disorders, and autism spectrum disorder. (American Psychiatric Association, 2022).

Rodent studies examining social behavior and socially associated stressors within various contexts have advanced identification of regulating neural pathways, and assisted development of new therapeutic approaches (Coccurello et al., 2009; Smith and Wang, 2012; Caldwell et al., 2017; Bergamini et al., 2018; Donovan et al., 2020; Sahani et al., 2022; Venniro et al., 2022) [author citations omitted for double-blind review]. However, social behavior research in the genetically tractable mouse species (Mus musculus) has been relatively limited in studying behavioral and neurophysiological consequences of aversive social interactions in female mice (Kondrakiewicz et al., 2019; Kuske and Trainor, 2021). This is largely due to capitalization of male-specific territorial aggression in most rodent social conditioning paradigms (Blanchard et al., 2001; Peña et al., 2019; Lopez and Bagot, 2021; Furman et al., 2022; Lyons et al., 2023; Pantoja-Urbán et al., 2024) [author citations omitted for double-blind review]. A breadth of social stress techniques has been employed in hamsters, mice, and rats (Miyashita et al., 2006; McCormick et al., 2007; Bernberg et al., 2008; Kercmar et al., 2011; McCormick et al., 2013; Iñiguez et al., 2018; Sterley et al., 2018; Lee et al., 2020; Furman et al., 2022; Pan et al., 2023). Of these, the most prevalent paradigm in mice is that of social defeat (both acute and chronic) (Golden et al., 2011; Bonnefil et al., 2019; Wang et al., 2021) [author citations omitted for double-blind review]. Aside from the male-centric nature of social defeat, additional concerns of reproducibility arise from the inherently variable range of aversive social experiences each ‘defeated’ experimental mouse encounters. Such variability, mostly beyond experimenter control, plus the single sex bias of social defeat left us wanting to explore an improved paradigm. For this, we paired controllable, uniform, aversive unconditioned stimuli with the presence of a social stimulus (conspecific; conditioned stimulus) to study aversive social conditioning across sexes in mice using a higher throughput approach (requiring 1 conditioning day, rather than multiple days-weeks).

To accomplish this, we modified a paradigm previously utilized in male mice, involving manual administration of an aversive stimulus (mild foot shock) selectively when a male mouse actively investigates a conspecific, with the goal of attenuating subsequent social engagement (Toth et al., 2012; Zoicas et al., 2014; de la Zerda et al., 2022; Grossmann et al., 2024). Rather than continuing this operant-style approach, where a mouse’s behavior dictates an outcome, we shifted to a classical conditioning approach in which shock delivery is standardized across individual mice. We anticipated experimental mice would associate the presence of the conspecific with the aversive unconditioned stimulus. Additionally, all mice would receive the same number of shocks, instead of variable numbers based upon behavior.

Here, we evaluated how this paradigm affected social engagement and fear behaviors across sexes after mice were exposed to a novel conspecific. Specifically, mice were socially conditioned (SC) with an aversive stimulus (mild foot shock) when in the presence of their assigned conspecific, independent of investigative behavior. Then, mice were tested for social engagement and fear behavior in a separate environment with their assigned conspecific present, followed by testing of freezing in the conditioning environment in the absence of any conspecific. The former test enabled evaluation of both social and fear behaviors, while the latter test assessed contextual memory sans social stimuli. These behaviors were tested under circumstances when the experimental and conspecific mice were the same sex, and when they were different sexes. We hypothesized SC mice would exhibit reduced social engagement (indexed as time spent directly adjacent to the conspecific enclosure) when tested with their assigned conspecific in a novel environment, regardless of if the SC and conspecific mice were the same or different sexes. Our broad goal was to develop a paradigm that would facilitate rigorous future investigations of social behavior across mouse sexes, which are currently underrepresented in literature (Kuske and Trainor, 2021).

Methods

Mice

Hybrid F1 offspring (i.e., 129SB6F1/J) of both sexes resulting from pairing female 129S1/SvlmJ (RRID:IMSR_JAX:002448) and male C57BL/6J (RRID:IMSR_JAX:000664) mice (hereafter experimental mice), and male and female C57BL/6J mice (hereafter conspecifics), all ≥9 weeks old or older, were group-housed (2–5 per cage) within sex. Experimental mice were hybrid F1 offspring of 129S1/SvlmJ and C57BL/6J mice because these two strains are the most prevalent among transgenic mice, and because hybrids provide broader generalizability (Kumar et al., 2021; Keady et al., 2022). Such hybrids are used for a variety of physiology and neuroscience research purposes (e.g., (Curley et al., 2010; Kelley et al., 2011; Agrimson et al., 2017; Niibori et al., 2020; Correia et al., 2024)). Conspecifics were C57BL/6J mice housed in a separate room from experimental mice. C57BL/6J mice were used as conspecifics to ensure they were entirely novel to experimental mice through separate housing; because C57BL/6Js are often used as the primary or background strain for testing social behaviors in mice (see reviews (Laman-Maharg and Trainor, 2017; Varholick et al., 2021; Takahashi, 2025)); and because our F1 breeding was limited to generation of experimental mice. All mice had ad libitum access to food and water in rooms maintained on a 12:12 light/dark cycle with lights on at 07:00 local standard time (i.e., Zeitgeber time 0), and temperature maintained at 22 ± 2°C. All mice were fed LabDiet 5001 rodent laboratory chow (LabDiet, Brentwood, MO) and were kept on 7090 Teklad Sani-chip bedding (Envigo, East Millstone, NJ) in cages containing Nestlets (Ancare, Bellmore, NY) and huts (Bio-Serv, Flemington, NJ) for enrichment. Experiments were approved by [authors’ university] Institutional Animal Care and Use Committee, and adhered to the National Research Council’s Guide for the Care and Use of Laboratory Animals, 8th Ed. (National Research Council, 2011).

Social Conditioning Paradigm

The entire paradigm spans four consecutive days. Procedures for each individual day are outlined below in order. On Days 0 and 1, experimental mice underwent different experimental manipulations according to their specific treatment group. On Days 2 and 3, all experimental mice experienced identical testing conditions regardless of their treatment group (Fig. 1).

Figure 1. Study timeline and treatment groups.

Figure 1.

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Five groups were used in this study for each experiment (same sex experiment, and different sex experiment): one socially conditioned (SC) group and four Control groups. Day 0 was for pre-exposure; Day 1 was when social conditioning occurred; Day 2 tested for social engagement; Day 3 tested for context-specific freezing behavior. On Day 0, SC mice (above horizontal lines) encountered their assigned conspecific in the social conditioning context, where olfactory, visual, and auditory cues could be exchanged while tactile interactions were minimized. No aversive stimulus was applied on Day 0, but on Day 1, experimental mice and their assigned conspecific were returned to this context. SC (but not conspecific) mice then experienced five mild foot shocks to associate their conspecific with that aversive experience. SC mice were then tested for social engagement with that same conspecific on Day 2, followed by testing for context fear behavior on Day 3 in the absence of their assigned conspecific. Each Control group (below horizontal lines) was composed of different mice. Shock Control (A) mice underwent the same exact procedure as detailed for the SC group, except no foot shock was administered on Day 1. Social Control (B) mice similarly experienced the same procedure as mice in the SC group, save that Social Control mice did not encounter any conspecific until Day 2 for testing of social engagement. Pre-Contingency Control mice encountered their conspecific 4 h before pre-exposure and social conditioning, meaning no conspecific was present for Pre-Contingency Control mice when they were in the social conditioning chamber for pre-exposure or social conditioning. To encounter their assigned conspecific, Pre-Contingency Control mice were placed into a clean cage with a clear acrylic divider separating them from their conspecific. This divider allowed visual, olfactory, and auditory exchanges with the conspecific but minimized tactile interactions, the same as for SC mice and Shock Controls when in the social conditioning context. On Day 0, Pre-Contingency Control mice encountered their conspecific for 5 min; on Day 1, for 9 min; these were timed to match the length of conspecific exposure that mice in the SC group experienced. Post-Contingency Control mice were treated the same as Pre-Contingency Control mice, except the former’s encounters with their assigned conspecifics occurred 4 h after, rather than before, pre-exposure and social conditioning.

Pre-exposure (Day 0)

Pre-exposure was used to help reduce the novelty of the transparent enclosure and/or the conspecific to the experimental mouse, plus minimize any potential sex differences in acquisition (Siegfried et al., 1987; Wiltgen et al., 2001; Guzmán et al., 2009; Brown et al., 2011). On Day 0, experimental mice were placed in Coulbourn Instruments chambers (17.8 cm D × 17.8 cm W × 30.5 cm H; Allentown, PA) with (Shock Control and SC mice) or without (Social, Pre-Contingency, and Post-Contingency Controls) a conspecific (age-matched; either same sex or different sex relative to experimental mouse, depending on if assigned to same sex or different sex experiment) for pre-exposure (Fig. 1). Chambers comprised two opposing aluminum walls each adjoining two clear acrylic walls. Conspecifics were put inside a transparent enclosure placed within a corner of the social conditioning chamber. The transparent enclosure included 23 holes (each 0.32 cm diameter) on each of the 2 sides facing the SC mouse to enable olfactory cue exchanges. Previous work demonstrates that olfaction is the most important modality when recognizing and interacting with novel conspecifics (see review (Sterley and Bains, 2021)); tactile (whiskers) and auditory modalities appear to only be recruited when recognizing cage mates (de la Zerda et al., 2022) in male mice (females were not studied). Chambers and enclosures were cleaned with 70% ethanol before and after each session; chambers had visible illumination and one clear acrylic wall was marked with a blue dotted pattern. These components contributed olfactory and visual cues to the context, in addition to the tactile cue of the stainless steel grid floor for the experimental mouse. During pre-exposure, experimental mice were allowed to explore the chamber and conspecific enclosure for 5 min, then both mice were returned to their respective home cages. No aversive stimulus was presented.

Social Conditioning (Day 1)

On Day 1, experimental mice were placed in the same social conditioning chamber as Day 0 and received five, 1 s mild foot shocks (1.0 mA) in the presence of the same conspecific that they encountered on Day 0 (Fig. 1). This was to have experimental mice form associations between aversive foot shocks and their respective conspecific (Fig. 1). Conditioning lasted for 9 minutes; after a 2 minute baseline, shocks were then administered at 120, 210, 300, 390, and 480 s. Conspecifics did not receive foot shocks. Cameras mounted above the chambers were used to record movements, and freezing behavior (≥0.25 s) during conditioning was quantified using FreezeFrame software (v. 5.201, Actimetrics, Wilmette, IL). Freezing – ceasing all movement save breathing – is an innate behavior expressed by mice in response to a real or perceived threat, enabling threat assessment while minimizing detection (see review (Blanchard and Blanchard, 1988)). All videos were manually reviewed to ensure the software satisfactorily distinguished between freezing behavior and mere absence of locomotion.

Social Engagement Testing (Day 2)

On Day 2, experimental mice were placed in a novel room containing open arenas (41.9 cm W × 41.9 cm D × 39.6 cm H). Social engagement testing intentionally occurred in a novel environment with a novel conspecific enclosure to ensure sensitivity of our social interaction measure while minimizing potential contextual confounds; in other words, to isolate effects on social engagement from those of context fear learning (the latter assessed on Day 3). Mice acclimated to the room in their home cage for 30 min prior to behavior testing commencing. Each open arena had one empty PVC tube (8.9 cm outer diameter) in a single corner of the square chamber with wire mesh (0.25 × 0.25 cm openings) covering a rectangular portion (11.4 cm W × 4.1 cm H) cut out of the tube bottom. Experimental mice were placed in the corner of the arena opposite the PVC tube and could investigate freely for 2.5 minutes (pre-test). Immediately after these 2.5 min, the conspecific that each experimental mouse had previously been conditioned with on Day 1 was then placed in the PVC tube in the corner. Experimental mice were allowed to continue investigating the arena for an additional 5 min (post-test; Fig. 1). Because of the mesh at the bottom of the PVC tubes containing conspecifics, exchanges of visual, auditory, and olfactory cues between experimental mice and conspecifics were possible, but tactile interactions were minimized. Social interaction behavior was quantified as post-test duration when at least 80% of an experimental mouse’s body was in the interaction zone, a 7 cm radius zone (~223 cm2 – 240 cm2, i.e., 14–15% of the open arena) around the base of the PVC tube. Freezing behavior, regardless of arena location, lasting at least 250 ms during the post-test was also measured, then converted into a percentage of the post-test time. Social and freezing behaviors were detected using ANY-maze software (v. 7.09 Stoelting Co., Wood Dale, IL). Proprietary incompatibilities and video file encoding precluded analyses of freezing behavior during social interaction testing from being evaluated with FreezeFrame software; this is why ANY-maze software was used instead here. Post-test social interaction time was log-transformed (Y=log(Y+0.001) to account for 3 mice with 0 s post-test social interaction) so that data would be normally distributed for statistical analyses. Females were intentionally not assessed for estrus cycle stage for five reasons: 1) to minimize mouse usage (Russell and Burch, 1959), we did not power our studies for assessment of estrus; 2) with the goal of developing a social stress paradigm, we are seeking effects robust enough to not depend upon estrus in intact, randomly cycling females; 3) we intentionally focused here on fear and social behaviors, and did not measure sexual behaviors (e.g., lordosis); 4) evidence that overall mouse behaviors are not affected by estrus stage (Prendergast et al., 2014; Levy et al., 2023), (but see (Chari et al., 2020)); 5) cross-species evidence indicates vaginal lavage to determine estrus cycle is stressful (Becegato et al., 2021; Bahadır-Varol et al., 2022), and we sought to minimize stress confounds here.

Context Testing (Day 3)

On the last day of the social conditioning paradigm, experimental mice were placed in the social conditioning chamber for behavior testing in the absence of any conspecific (Fig. 1). The conspecific enclosure was still present to keep the context consistent with conditioning. All other tactile, visual, and olfactory cues from Day 1 were present. Testing lasted 10 min and did not involve any foot shocks. Freezing behavior was again quantified using FreezeFrame software.

Treatment Groups

The SC group involved mice that underwent social conditioning in the presence of a non-shocked conspecific (either same sex or different sex relative to SC mouse, depending upon experiment), with the hypothesis that SC mice would associate the conspecific with this aversive experience. We planned four Control groups for this one SC group; each experiment (same sex or different sex) had its own respective set of four Control groups for its respective SC group. The goals of these were to control for: 1) foot shock exposure, i.e., Shock Controls; 2) presence of conspecific during pre-exposure & foot shock, i.e., Social Controls; 3) exposure to conspecific temporally distal to foot shock exposure, such that social encounters still occurred in a manner not contingent with foot shock nor the conditioning context, i.e., Pre- and Post-Contingency Controls (Fig. 1). Shock Controls experienced procedures identical to those of the SC group, except a foot shock was never administered on Day 1 (Fig. 1). Social Controls were treated the same as the SC group, except experimental mice never encountered their assigned conspecific until testing on Day 2 (Fig. 1). Pre- and Post-Contingency Controls involved experimental mice encountering conspecifics either 4 h before (Pre-Contingency Controls) or 4 h after (Post-Contingency Controls) pre-exposure (Day 0) and social conditioning (Day 1); in other words, conspecifics were not present in the conditioning chamber during these two periods. Instead, experimental mice were exposed to their conspecific in a separate room for 5 minutes or 9 minutes (the same amount of time as pre-exposure or social conditioning, respectively) 4 h before (Pre-Contingency Controls) or 4 h after (Post-Contingency Controls) the experimental mice were exposed to the conditioning chamber. This exposure involved experimental mice and their respective conspecific being placed in a clean mouse cage separated with an acrylic divider to allow for visual, auditory, and olfactory exchange, but minimizing tactile interaction, mirroring the experiences of SC mice and Shock Controls within the social conditioning context on Days 0–1. Mice in Pre- or Post-Contingency Control conditions still experienced foot shocks on Day 1, and encountered their assigned conspecific for testing on Day 2 (Fig. 1). This temporal separation of 4 h was to minimize consolidation of social encounters from interfering/intermingling with consolidation of the aversive foot shock encounter in the social conditioning chamber, while still making execution of these experiments feasible within a 12 h lights-on period (Wanisch et al., 2008; Cai et al., 2016) [author citations omitted for double-blind review] (see reviews (Abel and Lattal, 2001; Sheppard et al., 2018)).

Serum Corticosterone

Thirty minutes after all experimental mice underwent contextual fear testing on Day 3, they were briefly anesthetized with isoflurane then rapidly decapitated for trunk blood collection. Blood clotted at room temperature for 10 minutes, then was spun at 3500 rpm for 1 h at 4°C. Serum was collected and stored at −80°C until corticosterone analyses could be performed. Serum corticosterone was measured using Enzo Life Sciences corticosterone enzyme-linked immunosorbent assay (ELISA) kits (Farmingdale, NY). Assays were run according to the manufacturer's instructions using their small volume protocol. Plates were read at 405 nm with correction at 580 nm. The sensitivity of the assay was 26.99 pg/mL. After serum corticosterone levels were interpolated from each plate’s standard curve, they were log-transformed to account for the typical skewness of serum corticosterone (Teilmann et al., 2014; Uarquin et al., 2016) [author citations omitted for double-blind review]. Log-transformed serum corticosterone is hereafter referred to as “cort”.

Statistical Analyses

Data were graphed with GraphPad Prism 10.3.0 (442), Beta (GraphPad Software, San Diego, CA), and analyzed using IBM SPSS Statistics 28.0.0.0 (IBM, Armonk, NY), with the significance threshold set a priori at p<0.05. Non-significant trends (p<0.10) were mentioned only when the associated partial η2 (ηp2) was ≥0.060. Data were graphed as the mean ± standard error of the mean (SEM). Details of identified outliers (greater than the mean ± 4 standard deviations) are provided in the Supplemental Dataset. The same sex experiment and different sex experiment each included their own SC group plus accompanying four Control groups. Experiments were analyed separately. Social conditioning acquisition was analyzed using a 3-way repeated measures (RM) general linear model (GLM; time × treatment group × sex of experimental mouse) and pairwise comparisons with Bonferroni correction. Greenhouse Geisser corrections were utilized for within-subjects analyses. Measurements of contextual fear expression average, log-transformed post-test social interaction time, percent time freezing during social interaction testing, and cort were analyzed with a 2-way GLM (treatment group × sex of experimental mouse) and pairwise comparisons with Bonferroni correction.

Results

Social Conditioning Acquisition

Social conditioning acquisition was examined in experimental mice in either the presence or absence of a conspecific. One experiment used same-sex conspecifics, and the other experiment used different sex conspecifics; each of these experiments included its own SC group plus four corresponding Control groups (Fig. 1). Shock Control mice never received any foot shocks during social ‘conditioning’, to distinguish the effects of receiving mild foot shocks on subsequent social and fear behaviors. The influence of encountering any novel conspecific prior to Day 2 and 3 testing was assessed with Social Control mice, which did not have a conspecific present during either pre-exposure or ‘social’ conditioning. To assess how social interaction with a novel conspecific prior to Day 2 and 3 testing, but temporally separate from pre-exposure and ‘social conditioning’, Pre-Contingency Control mice encountered their assigned conspecific 4 h prior to pre-exposure and ‘social conditioning’. Post-Contingency Control mice evaluated the same thing as Pre-Contingency Control mice, except Post-Contingency Control mice encountered their assigned conspecific 4 h after pre-exposure and ‘social conditioning’. Social conditioning acquisition is illustrated in Fig. 2A, 2B for mice in the same sex conspecific experiment, and in Fig. 2C, 2D for mice in the different sex conspecific experiment. All mice receiving a foot shock expressed >40% freezing during at least one post-shock period during social conditioning.

Figure 2. Day 1 acquisition during social conditioning procedure.

Figure 2.

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Percent time freezing during social conditioning acquisition for mice in same sex (Panels A, B) and different sex (Panels C, D) experiments for male (A, C) and female (B, D) mice. Numbers of mice graphed within Panels A-D in order: Socially conditioned (SC; n=8, 9, 9, 8); Shock Control (8, 8, 7, 7); Social Control (n=8 for all); Pre-Contingency Control (n=7, 8, 8, 9); Post-Contingency Control (n=7, 8, 8, 8). Average freezing for the first two minutes, prior to commencement of acquisition, is plotted on the x-axis as baseline. The average percent freezing for each 30 second period following each of the five mild foot shocks are thereafter plotted along x-axis (Post-shock Periods 1–5). Alphabetically, within experiment, sex, and time point: aindicates (Shock Control vs. SC) p<0.001, p=0.007, p<0.001, p=0.025, p<0.001, p<0.001. bindicates (Social Control vs. SC) p=0.026, p=0.005, p=0.009, p=0.038. cindicates (Pre-Contingency Control vs. SC) p=0.032. eindicates (Shock Control vs. Social Control) p<0.001, p<0.001, p=0.003, p<0.001. findicates (Shock Control vs. Pre-Contingency Control) p=0.004, p<0.001, p=0.024, p<0.001, p<0.001. gindicates (Shock Control vs. Post-Contingency Control) p=0.002, p=0.002, p=0.047, p<0.001, p=0.002. hindicates (Social Control vs. Pre-Contingency Control) p=0.043. jindicates (Pre-Contingency Control vs. Post-Contingency Control) p=0.028. *indicates p<0.001 Social, Pre-Contingency, and Post-Contingency Controls and SC mice vs. Shock Controls. Alphabetically, within experiment, group, and time point: Bindicates (Social Controls male vs. female) p=0.005. Cindicates (Pre-Contingency Controls male vs. female) p=0.019, p=0.006, p=0.012. Dindicates (Post-Contingency Controls male vs. female) p=0.012. Sindicates (SC male vs. female) p=0.007, p=0.031, p=0.006, p=0.012, p=0.012. Data graphed as mean ± SEM.

For acquisition in the same sex experiment, no three-way interaction of time × sex × group (of which there are five: SC mice plus four Control treatment groups) occurred (Table 1). We observed a significant two-way interaction between time × group, and a non-significant trend for group × sex was noted (p=0.070; Table 1). A significant main effect of sex was also found for mice in the same sex experiment (Table 1). Pairwise comparisons indicated that same sex conspecific experiment SC males exhibited less freezing at post-shock periods 1–4 compared to SC females in the same experiment (Fig. 2A, 2B). In contrast, sex differences within the four same sex Control groups were not observed at any timepoint in the same sex experiment, with the sole exception being Post-Contingency Control females freezing more than Post-Contingency Control males at post-shock period 3 (Fig. 2A, 2B). Within males, SC plus Social, Pre-Contingency, and Post-Contingency Controls in the same sex experiment froze significantly more than Shock Control males at post-shock periods 4 and 5. At post-shock period 3, Social, Pre-Contingency, and Post-Contingency Controls were freezing more than Shock Control males, but SC males weren’t (Fig. 2A). SC males exposed to same sex conspecifics during acquisition temporarily exhibited less freezing at post-shock periods 3 and 4 specifically compared to Social Control males, which had no conspecifics present during acquisition (Fig. 2A). Similar to males, female SC mice and Social, Pre-Contingency, and Post-Contingency Controls in the same sex experiment all exhibited significantly greater freezing compared to Shock Control mice at post-shock periods 3–5, as expected (Fig. 2B). At post-shock period 2, SC mice and Social and Post-Contingency Controls displayed higher freezing compared to Shock Control mice, whereas Pre-Contingency Control mice did not (p=0.268; Fig. 2B).

Table 1.

Two-way RM GLM on learning of social conditioning for mice of both sexes with same sex conspecifics.

Same Sex Experiment – Social Conditioning Acquisition
F Statistic p value ηp2
Time F(4.18,288)=135.9 <0.001 0.663
Group F(4,69)=45.09 <0.001 0.723
Sex F(1,69)=13.54 <0.001 0.164
Time × Group F(16.7,288)=9.518 <0.001 0.356
Time × Sex F(4.18,288)=1.714 0.144 0.024
Group × Sex F(4,69)=2.275 0.070 0.117
Time × Group × Sex F(16.7,288)=0.787 0.706 0.044
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Social conditioning acquisition for mice in the different sex experiment is shown in Fig. 2C, 2D. A significant three-way interaction of time × sex × group was observed (Table 2). Only the second post-shock period was different across sexes in SC mice in the different sex experiment (Fig. 2C, 2D), with females exhibiting greater freezing than males. Social Control female mice in the different sex experiment exhibited higher freezing than Social Control males solely at the third post-shock period. For Pre-Contingency Control mice, females displayed greater freezing for the first three post-shock periods than males in the different sex experiment (Fig. 2C, 2D). SC males exhibited an initial higher level of freezing at post-shock period one compared to Shock, Social, and Pre-Contingency Controls, but not Post-Contingency Controls (Fig. 2C), but this was not sustained as a pattern at later post-shock periods. Females at post-shock period one in the SC and Pre-Contingency Control groups froze more than Shock Control females, partially mirroring the males’ initial acquisition response (Fig. 2D). At post-shock period two, male Post-Contingency Control mice were the only group that froze more than male Shock Controls (Fig. 2C). The same time point in females in the different sex experiment revealed a more divergent pattern continuing what was observed in that sex at post-shock period one.

Table 2.

Two-way RM GLM on learning of social conditioning for mice of both sexes with different sex conspecifics.

Different Sex Experiment – Learning Social Conditioning
F Statistic p value ηp2
Time F(4.35,304)=184.2 <0.001 0.725
Group F(4,70)=38.88 <0.001 0.690
Sex F(1,70)=6.694 0.012 0.087
Time × Group F(17.4,304)=12.67 <0.001 0.420
Time × Sex F(4.35,304)=3.317 0.009 0.045
Group × Sex F(4,70)=0.892 0.474 0.048
Time × Group × Sex F(17.4,304)=1.658 0.048 0.087
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Specifically, different sex experiment SC and Pre-Contingency Control females continued to freeze more than female Shock Controls, plus the two former exhibited significantly greater freezing than female Social Controls (Fig. 2D). However, this clustering pattern of Pre-Contingency Control and SC females, and Shock and Social Control females, disappeared from post-shock period three onwards. Post-Contingency Control mice briefly exhibited less freezing than Pre-Contingency Control mice at post-shock period three, but this was the only such occurrence in females (Fig. 2D). Social, Pre-Contingency, and Post-Contingency Controls and SC mice of both sexes in the different sex experiment froze significantly more than same sex Shock Control mice at post-shock periods 3–5 (Fig. 2C, 2D). Combined with same sex experiment acquisition, these findings indicate that social exposure – whether concurrent with or temporally distal from an aversive unconditioned stimulus – can have transient impacts on the freezing behavior exhibited by experimental mice across sexes. All conditioning procedures here, though, culminated in similar final freezing levels across sexes of both experimental mice and their conspecifics. This facilitates subsequent comparisons of the different conditioning manipulations on subsequent social and freezing behaviors by minimizing potential acquisition confounds.

Social Interaction Testing

Mice were tested for social engagement and freezing behavior using the social interaction test 24 h after social conditioning concluded. Three mice did not interact at all with their conspecific, and thus were excluded from social interaction analyses, but were retained for freezing behavior analyses (1 different sex Shock Control female; 1 same sex Social Control female; 1 same sex SC male; see Supplemental Dataset).

Social conditioning in the same sex experiment did not result in a sex × group interaction on social engagement, but a main effect of group was found (Table 3). Pairwise comparisons indicated that SC males exhibited less social interaction than SC females, Shock Control males, and Pre-Contingency Control males in the same sex experiment (Fig. 3A).

Table 3.

Two-way GLM on social interaction for mice of both sexes with same sex conspecifics.

Same Sex Experiment – Log-transformed Social Interaction
F Statistic p value ηp2
Group F(4,65)=4.357 0.003 0.211
Sex F(1,65)=0.535 0.467 0.008
Group × Sex F(4,65)=1.746 0.151 0.097
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Figure 3. Day 2 social interaction and fear behaviors following social conditioning.

Figure 3.

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Log-transformed social interaction (Panels A, B) and percent time freezing during post-test social interaction (Panels C, D) data are shown for mice in the same (A, C) and different (B, D) sex experiments. Numbers of mice graphed within each panel, left to right: A) n=7, 7, 8, 7, 7, 9, 8, 6, 8, 8; B) n=9, 6, 8, 8, 8, 8, 6, 7, 9, 7; C) n=8, 7, 8, 7, 7, 9, 8, 7, 8, 8; D) n=9, 6, 8, 8, 8, 8, 7, 7, 9, 7. Y axes for C, D were split to facilitate clearer visualization of the low freezing levels exhibited during social engagement testing. Left to right, top to bottom: ap=0.016, cp=0.037, Sp=0.021, Sp=0.019, bp=0.013, Bp=0.019, ap=0.005, ep<0.001, hp=0.002, iindicates (Social Control vs. Pre-Contingency Control) p=0.008. Data graphed as mean ± SEM.

Fear behavior, indexed as percent time freezing during the social interaction post-test, was also evaluated. While the goal of our modified paradigm was to ultimately decrease social engagement following conditioning, we also assessed the level of freezing expressed in the novel social testing arena. In the same sex experiment, no sex × group interaction occurred, but main effects of both group and sex were observed (Table 4). Pairwise comparisons indicated that freezing was higher in Social Control females compared to males (Fig. 3C). SC and Social Control females froze more than Shock Control females. Similarly, Pre and Post--Contingency Control females exhibited less fear behavior than Social Control females (Fig. 3C). Overall, though, freezing levels during social interaction testing were minimal as compared to those occurring during acquisition (Fig. 2) and context testing (Fig. 4A–B).

Table 4.

Two-way GLM on percent time freezing during social interaction for mice of both sexes with same sex conspecifics.

Same Sex Experiment – % Freezing During Social Interaction
F Statistic p value ηp2
Group F(4,67)=10.55 <0.001 0.387
Sex F(1,67)=5.244 0.025 0.073
Group × Sex F(4,67)=0.730 0.575 0.042
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Figure 4. Day 3 social conditioning context fear expression averages and subsequent cort levels.

Figure 4.

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Average context fear behavior (Panels A, B) and cort (Panels C, D) data are shown for mice in all groups in the same sex (A, C) and different sex (B, D) experiments. Numbers of mice graphed within Panels A-D in order, left to right: A) n=8, 8, 8, 7, 7, 9, 8, 8, 8, 8; B) n=8, 7, 8, 7, 7, 8, 7, 8, 9, 8; C) n=7, 8, 8, 7, 7, 9, 8, 8, 8, 8; D) n=9, 7, 8, 8, 8, 8, 7, 8, 9, 8. Left to right, top to bottom: Sp=0.049, dindicates (Pre-Contingency Control vs. SC) p=0.004, ap=0.004, fp=0.045, gp=0.012, ap=0.033, ep=0.044, Sp=0.007. *p<0.001 Social, Pre-Contingency, and Post-Contingency Controls and SC mice vs. Shock Control. Data graphed as mean ± SEM.

As with the same sex experiment, there was no significant interaction nor a main effect of sex in the different sex experiment, but there was a significant group effect on social engagement (Table 5). Pairwise comparisons demonstrated that, opposite to the same sex experiment, SC females in the different sex experiment exhibited significantly reduced social interaction, both compared to female Social Control mice and to SC male mice (Fig. 3B). Combined, these findings indicate that the social engagement of female mice is more affected by temporally concurrent aversive associations with males, whereas males are more socially affected by temporally concurrent aversive associations with their own sex.

Table 5.

Two-way ANOVA on social interaction for mice of both sexes with different sex conspecifics.

Different Sex Experiment – Log-transformed Social Interaction
F Statistic p value ηp2
Group F(4,66)=2.903 0.028 0.150
Sex F(1,66)=0.740 0.393 0.011
Group × Sex F(4,66)=1.474 0.220 0.082
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Unlike the same sex experiment, neither an interaction nor main effects of sex and group were observed with freezing behavior during social interaction testing for the different sex experiment (Table 6). Moreover, no significant pairwise comparisons were observed (Fig. 3D). The predominant consistency across experiments for freezing was that expression of fear was minimal during the post-test of social interaction, as compared to freezing quantified in the conditioning context (Figs. 2, 4A–B).

Table 6.

Two-way ANOVA on percent time freezing during social interaction for mice of both sexes with different sex conspecifics.

Different Sex Experiment – % Freezing During Social Interaction
F Statistic p value ηp2
Group F(4,67)=0.698 0.596 0.040
Sex F(1,67)=3.414 0.069 0.048
Group × Sex F(4,67)=0.491 0.743 0.028
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Social Conditioning Context Fear Testing

After Day 2’s social interaction evaluation of mice’s association between their assigned conspecific and the aversive stimulus they experienced on Day 1, we next assessed their fear response to the social conditioning context on Day 3. This was accomplished by quantifying percent of time spent freezing in the conditioning context, in the absence of any conspecific. Because of the natural extinction process that can occur during testing, we specifically examined behavioral expression of social conditioning context fear during minutes two through six of testing, to best capture fear expression with minimal confounds from extinction processes [author citations omitted for double-blind review]. This testing further allowed us to determine if social engagement testing on Day 2 might have any carry-over effects on contextual fear expression on Day 3 (Tables 7–8, Fig. 4A–B).

Table 7.

Two-way GLM on both sexes’ context fear testing average with same sex conspecifics.

Same Sex Experiment – Fear Expression Average
F Statistic p value ηp2
Group F(4,69)=108.5 <0.001 0.863
Sex F(1,69)=2.859 0.095 0.040
Group × Sex F(4,69)=0.676 0.611 0.038
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Table 8.

Two-way GLM on both sexes’ context fear testing average with different sex conspecifics.

Different Sex Experiment – Fear Expression Average
F Statistic p value ηp2
Group F(4,67)=155.9 <0.001 0.903
Sex F(1,67)=0.008 0.929 0.000
Group × Sex F(4,67)=1.220 0.311 0.068
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For the same sex experiment, no two-way interaction between group × sex was found, but a significant main effect of group emerged (Table 7). Mostly, this was due to Shock Control mice, as expected, freezing significantly less than all mice in the same sex experiment that experienced foot shocks during conditioning (Fig. 4A). Male SC mice exhibited significantly less average freezing in the conditioning context compared to female SC mice (Fig. 4A).

In the different sex experiment, no significant group × sex interaction was found, nor was a significant effect of sex (Table 8). A significant group effect was similarly observed for the different sex experiment, again attributable to the low freezing levels by Shock Control mice across sexes (Fig. 4B). Male SC mice also froze significantly less than Post-Contingency Control males (Fig. 4B). Integrated with the results from social interaction testing, these findings indicate that conditioned shifts in social engagement are distinct from conditioned fear to the social conditioning context. In parallel, freezing behavior was relatively minimal during social interaction testing compared to testing in the conditioning context, suggesting both that freezing behavior did not confound social interaction outcomes and that mice were able to sufficiently discriminate between contexts. Curiously, interaction of male SC mice with familiar conspecifics in a novel environment on Day 2 might lead to mild reductions in contextual fear expression on Day 3.

Serum Corticosterone

Thirty minutes after Day 3 testing concluded, blood was collected for subsequent analyses of serum corticosterone levels. Cort levels of mice in the same sex experiment exhibited no significant interaction of group × sex, but a main effect of group was observed (Table 9, Fig. 4C). Pairwise comparisons showed that, across sex, SC mice in the same sex experiment exhibited elevated cort levels compared to their respective same sex Shock Control mice (Fig. 4C). While this was not surprising, male Pre- and Post-Contingency Control mice also exhibited higher cort levels than Shock Control mice, whereas Social Control mice did not (Fig. 4C). Conversely, female Social Control mice had higher cort levels than Shock Control mice, but female Pre- and Post-Contingency Control mice did not (Fig. 4C). Given no conspecific was present during Day 3 testing, these findings suggest enduring and sex-specific physiological differences following social conditioning, despite minimal behavioral shifts. Specifically, any social interaction with their assigned conspecific on Day 1, whether concurrent or temporally separate with foot shock exposure, elevated male cort levels but attenuated female cort levels after context testing on Day 3 in the absence of any conspecifics. Only SC females did not align with this pattern, exhibiting heightened cort levels on Day 3 similar to those of Social Control females (Fig. 4C).

Table 9.

Two-way GLM on cort for mice of both sexes with same sex conspecifics.

Same Sex Experiment – Log-transformed Serum Corticosterone
F Statistic p value ηp2
Group F(4,68)=7.098 <0.001 0.295
Sex F(1,68)=0.813 0.370 0.012
Group × Sex F(4,68)=1.207 0.316 0.066
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Cort levels of mice in the different sex experiment, while not exhibiting a significant sex × group interaction, did display significant main effects of both group and sex (Table 10). In contrast to the same sex experiment, pairwise comparisons indicated that within each sex, levels of cort did not differ across groups, including relative to Shock Control mice (Fig. 4D). The only difference that reached significance was male SC mice having lower cort levels compared to female SC mice (Fig. 4D). Combined with cort data from the same sex experiment (Fig. 4C), these findings illustrate how same sex conspecific interactions on the same day as an aversive learning experience can elicit sex-specific and enduring hormone changes. Moreover, these lasting physiological shifts are not mirrored after different sex conspecific interactions. This indicates the combination of conspecific and experimental sex, when temporally concurrent or adjacent to a fear learning event, influences endocrine responses to context days later.

Table 10.

Two-way GLM on cort for mice of both sexes with different sex conspecifics.

Different Sex Experiment – Log-transformed Serum Corticosterone
F Statistic p value ηp2
Group F(4,70)=3.841 0.007 0.180
Sex F(1,70)=8.708 0.004 0.111
Group × Sex F(4,70)=0.709 0.589 0.039
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Discussion

Our goal in executing these experiments was to begin establishing a paradigm for inducing consistent, reproducible socially paired stress in mice across sexes. While our results indicate this specific paradigm does not yet accomplish our goal, we want to share our approach and findings so that others can utilize this information to efficiently prioritize future efforts in this domain. Moreover, despite a mostly negative outcome towards our overarching goal, we nevertheless uncovered sex-specific patterns of behavioral and physiological stress responsivity that are useful to fields incorporating sex as a biological variable, and/or seeking to employ different sex social stressors in mice.

All mice that underwent social conditioning with mild foot shocks (Social, Pre-Contingency, and Post-Contingency Controls plus SC mice) ultimately ended their acquisition stage at similar freezing levels. Subsequent outcomes in behavioral testing for social engagement and contextual fear expression, as well as for serum corticosterone, could, therefore, be interpreted with minimal concern for acquisition confounds. Conversely, this consistency might have contributed to our negative findings. Most of the statistically significant differences between Social, Pre-Contingency, and Post-Contingency Controls and SC mice occurred during the first three post-shock periods, both in the same sex and different sex experiments. These well might have been flukes. Alternatively, they might indicate that our social conditioning protocol resulted in a ceiling effect. Additional studies would need to parse this out.

Our social interaction findings were not as robust as anticipated. For our same sex experiment, we found that male SC mice engaged less during social testing compared to Shock Control mice, and compared to Social Control mice that never encountered a conspecific during training. Additionally, male SC mice exhibited less social engagement than female SC mice in same sex experiment, whereas the converse was true for the different sex experiment. This reveals that male mice likely form stronger associations when aversive experiences occur in conjunction with the presence of another male mouse, whereas female mice appear to associate male mice more strongly when their presence is concurrent with aversive stimuli. Such a sex difference in response to conspecific sex could help explain some of the challenges in developing social stress paradigms using female laboratory mice (Mus musculus). Relatedly, this could indicate that, in the presence of a potential sexual partner, threat assessment is suppressed. The inverse has been demonstrated in female rats; that is, fear attenuates sexual behaviors, at least in part through amygdala and hypothalamus estrogen receptor signaling (Moëne et al., 2019). Indirect evidence in male rats housed with females post-context fear conditioning suggests that encounters with different sexes may suppress threat assessment. These male rats subsequently exhibited attenuated fear expression, involving dopamine receptor signaling in the hippocampus (Bai et al., 2009). However, a different group studying mice reported that ejaculation by males was required for retention of extinguished social fear conditioning (Grossmann et al., 2024). Continued evaluations are needed to determine whether our observations – under conditions where copulation is impossible – that male exposure to a female can diminish threat assessment behaviors are reproducible. Evidence for social transmission of stress in rodents suggests an alternative interpretation (Brandl et al., 2022). Instead, it could be that because the conspecific for each SC mouse did not experience the same stress (mild foot shock), this in turn affects the SC mouse’s perception of their own aversive experience (Guzmán et al., 2009), likely in a sex-specific manner.

These sex-specific behavior patterns correspond to evidence that accumbal dopamine signaling dynamics in mice are sex-dependent, both upon the sex of the studied mouse and the sex of their assigned conspecific (Dai et al., 2022). Our findings align with the success of using social defeat to stress male mice. Additionally, they indicate that future studies seeking to suppress female mice’s social interaction behavior through a socially associated stimulus will meet with more success if using male, rather than female, conspecifics. Indeed, others have encountered challenges when trying to elicit behavioral shifts in female mice, even after 4 weeks of stress that involved only female conspecifics (Díez-Solinska et al., 2023). One group reported that female mice consistently prefer a socially paired food reinforcer, even if the pairing was with a same sex conspecific that had just previously undergone acute stress (mild foot shock) (Kietzman et al., 2022). Because males’ food preference was not studied by the researchers, and neither did their females encounter stressed different sex conspecifics, it remains unclear if these effects are sex-specific, and if they would generalize to different sex interactions. Future studies will also need to determine how socially conditioning female mice to male conspecifics subsequently affects interactions with female conspecifics, and vice versa.

One day after social engagement testing, mice were tested for fear expression in the social conditioning context in the absence of any conspecifics. As expected, Shock Control mice across experiments exhibited little freezing, given they never experienced a foot shock in this environment. Indeed, Shock Controls drove most of the group differences across sexes and experiments. This indicates social conditioning did not have a consistent effect upon fear expression, measured during minutes two through six to minimize extinction confounds [author citations omitted for double-blind review]. The only semi-consistent group difference in fear expression was between male SC and Post-Contingency Control mice in the different sex experiment. This suggests that when male mice are in the vicinity of a possible sexual partner, they could experience impaired encoding of context cues. However, the absence of significant differences of male SC mice relative to Social and Pre-Contingency Controls argues against this interpretation.

A sex difference in context fear expression was observed between SC mice in the same sex experiment, but not in the different sex experiment. This is the only directionally comparable outcome with social engagement, in that male SC mice in the same sex experiment both exhibited less context fear and less social engagement versus female SC mice. This could be interpretated in at least two ways. Male SC mice might have found their male conspecific more salient than the context, thereby leading them to form stronger associations between the mild foot shocks and their conspecific as compared to female SC mice. Alternatively, the presence of the male conspecific may have led to social buffering of the mild foot shocks for male SC mice, whereas this process did not occur for female SC mice in the same sex experiment. Because our goal was for mice to associate the aversive mild foot shock with their assigned conspecific, any social buffering of social conditioning context fear could counteract our goal. This is why our use of conspecifics of a different strain, that were never housed in the same room as experimental mice, ensured they had never previously been encountered (even via olfaction) by the mice tested here. Given the critical contribution of attachment to social buffering (see review (Kiyokawa and Hennessy, 2018)), we can be reasonably confident that there were no social buffering effects upon social conditioning context fear in same sex experiment mice. Thus, we are inclined toward the first interpretation.

Serum was collected thirty minutes after context fear testing, 24 h after experimental mice’s last encounter with their assigned conspecific. We were interested to discover that sex-specific group differences in cort levels in the same sex experiment appeared to be best explained by Day 1 group conditions, rather than behavior on Days 1–3. For example, male Shock Control mice’s cort levels were lower than Pre- and Post-Contingency Controls and SC groups – the three conditions where both conspecific exposure and foot shocks occurred within an 8 h window on Day 1. Social Control male mice only experienced foot shocks, and did not encounter a conspecific, on Day 1. Shock Control males similarly only encountered conspecifics, and did not experience foot shocks on Day 1; and male Social Control cort levels did not differ from Shock Control males. Like males in the same sex experiment, female SC mice had elevated cort relative to Shock Control females. In contrast, however, with this experiment’s females, only Social Control females had higher cort levels than Shock Control females. Pre- and Post-Contingency Control females’ cort was not different from Shock Controls, and the former groups encountered conspecifics 4 h before or after, respectively, experiencing foot shocks. Collectively, this suggests social encounters temporally distal from the foot shock, but still occurring within the same day, might augment males’ and mitigate females’ cort responses to a stressful experience two days later. In contrast, coinciding foot shocks and conspecific exposure in SC mice obscured this sex difference in endocrine response.

Unlike the same sex experiment, the different sex experiment mice’s cort levels did not map onto groups in either sex, despite a significant group effect being detected. Also distinct from the same sex experiment is that cort levels of SC mice in the different sex experiment had a sex difference. Specifically, female SC mice had greater cort levels than male SC mice. This inversely maps onto their social engagement on Day 2, in that female SC mice engaged less with their male conspecifics than male SC mice did with their female conspecifics. However, average context fear expression was not different between these two groups on Day 3. This cort level difference could, therefore, be an enduring endocrine effect of encountering their assigned conspecifics on Day 2. That said, the absence of any inverted patterns between social engagement and cort levels across all Controls in the different sex experiment makes this possibility seem less plausible than what we observed with cort levels in the same sex experiment.

To ensure we rigorously evaluated this modified paradigm’s hypothesized utility as a higher throughput approach for more consistent induction of social stress across sexes, we included four separate controls. Had we only included one control group, e.g., Shock Controls, we might have incorrectly concluded that our modified paradigm successfully reduced social engagement in females, if not across sexes. Through the inclusion of four control groups, however, we have concluded that our findings do not support our hypothesis, and that we cannot endorse this paradigm in its current state. Instead, our data indicate that this protocol requires additional optimization to achieve its overarching goal. Nevertheless, we are sharing our approach and findings so that others with similar procedure goals can learn from our observations, and improve upon our study’s limitations. If we had been able to continue our investigations, we would have next reduced the mild foot shock amplitude, the number of foot shocks, or some combination thereof. Given the group differences in freezing that emerged early during our social conditioning, milder and/or less aversive stimuli could uncover enduring effects of group conditions. To begin though, it was important here that all our groups attained similar starting points to minimize confounds for testing interpretations. We would also advise other researchers to consider optimizing their apparatus and software, if possible, to facilitate consistent analyses of interaction and freezing, if their conditioning/stress component generates freezing. We used one software to administer foot shocks while simultaneously recording and quantifying freezing, and another software to track animal location and identify time spent within specific arena locations, each in separate apparatus. Both software programs excel at their purposes, but each have unique – and unfortunately incompatible – video formats plus requirements regarding lighting, contrast, and background for optimal behavior/location detection.

Another limitation is that we could not evaluate cort levels over time without introducing additional confounding stressors to repeatedly sample blood. Daily blood sampling would reveal if our cort observations were attributable to specific days, how cort levels mapped on to each day’s behavior measure, and/or if cort differences are transient or persistent. Our experiments were all performed in sexually naïve mice. We recognize that sexually experienced mice (for example (Newman et al., 2019)) of both sexes might respond, behaviorally and physiologically, in a manner distinct from what we report here. Earlier practices of dividing mice into ‘susceptible’ and ‘resilient’ groups following social stress have more recently fallen out of favor, given categorization of those responses are context-specific, require substantial rodent numbers for optimal data confidence, and neither phenotype can be universally deemed ‘better’ or ‘worse’ (Koolhaas et al., 2017; Lyons et al., 2023). We therefore evaluated all SC mice as a single group. Still, bimodal distribution of social engagement in SC mice looks possible in all but the different sex SC males. Increasing numbers to establish definitive thresholds with sufficient power for phenotypic grouping could prove informative, particularly for the same sex experiment. The same limitations as listed above would still apply, however. Finally, our studies used a brief protocol spanning only four days, intended for high throughput and maximal comparison to other social stress literature. We therefore cannot speak to whether these outcomes persist for weeks or months, timeframes that are ethologically relevant for social learning and memory. Once a short-term protocol has been optimized, duration of effects would be a critical next step in determining validity of the paradigm.

Here, we have reported a novel foray into sex-inclusive efforts to develop a controllable, less labor-intensive, and higher throughput social stress paradigm in mice. These experiments have provided utilizable insights regarding how mice of both sexes associate a same- or different-sex conspecific with an aversive experience. We report that these associations seem strongest in males after same sex encounters, and in females after different sex encounters. Such distinctive responses are probably evolutionarily favorable. While our investigation has generated new information, our modified paradigm did not accomplish what we anticipated. We share our negative findings here to encourage and assist other labs to achieve their own positive outcomes, and to spare them (and mice) the time and effort of duplicating the present work. Development of a sex-inclusive social stress paradigm for mice remains a worthwhile pursuit, as such would be a boon across multiple disciplines. Perhaps the most salient contribution our study provides to the broader literature is that future studies focused on socially stressing females might utilize male conspecifics for optimal outcomes.

Supplementary Material

Supplement 1
media-1.xlsx (61.2KB, xlsx)

Acknowledgements:

We gratefully acknowledge the mice used in this study, the unrivaled veterinary care by Stan Dannemiller, DVM, MS, DACLAM, and the dedicated work of our vivarium caretakers. Figure 1 was created with BioRender.com (Toronto, ON).

Funding sources:

This work was supported by R15 MH118705 to AMJ and LMG, and by Kent State University.

Footnotes

Conflict of Interest: The authors declare no competing financial interests.

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