Sex-specific relationships between stress coping and avoidance behavior

1 While the experience of stress is ubiquitous, the risk of developing stress-linked 2 conditions such as anxiety and depression is related to maladaptive stress responses. 3 In order to probe the relationship between stress coping, sex, and stress-linked 4 behavioral outcomes, we exposed male and female mice to subchronic variable stress 5 (SCVS) and measured the correlation between coping during the tail suspension 6 stressors (TSS) of SCVS and avoidance behavior in the EPM. We found that females 7 engage in more active coping, and there were no sex differences in avoidance or 8 locomotor behavior in the EPM after stress. However, we found that greater active 9 coping predicted greater avoidance in females, but less avoidance in males. The results 10 demonstrate that coping strategies are dynamic across time in males and females, but 11 the relationships between avoidance and coping strategy dynamics are sex-biased. 12 13

Stress, sex differences, coping, avoidance 14 15 Plain English Summary 16 The selection of stress coping strategies is an important component of the stress 17 response that can impact behavior after stress. Stress coping strategies and behavior 18 after stress can both be sex-biased, but the relationships between them are unclear. 19 SCVS is a paradigm that is used to study sex differences in behavior and physiology 20 because females are specifically vulnerable to SCVS. We recorded behavior during two 21 stressors in the SCVS paradigm and found opposite relationships between coping 22 behavior and avoidance behavior after stress in males and females, even though males 23 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 3 and females exhibit similar dynamics in coping behavior and similar avoidance behavior 24 after stress. These results demonstrate that sex is an important variable in the 25 relationship between coping strategies during stress and behavior after stress. 26

27 All living organisms experience stress, broadly defined as challenges to physical 28 or emotional homeostasis. Stress often carries a colloquially negative connotation, but it 29 is ubiquitous and necessary for learning and adaptation [1, 2]. However, stress can 30 precipitate maladaptive outcomes when it surpasses the adaptive capacities of an 31 organism due to the chronicity, intensity, or perception of the stress. In humans, chronic 32 stress exposure can increase the risk of developing multiple psychiatric disorders 33 including depression, anxiety, and substance use-related conditions. Across organisms, 34 stress initiates a cascade of physiological mechanisms and behavioral responses. 35 Among these responses are behavioral strategies known as stress coping, which permit 36 the removal, mitigation, and adaptation to a stressor such that the stress response may 37 be primed for more efficient responses in the future [3-5]. Stress coping strategies vary 38 between conspecifics and across contexts, and some stress coping choices promote 39 adaptation while others may be acutely or chronically maladaptive. The appraisal of 40 stress and selection of coping strategy is influenced by a range of intrinsic factors 41 including genetics, early life environment, and circulating hormones, which interact with 42 real time stimuli and shared stress response mechanisms [2, 3]. Consequently, whether 43 or not stress coping strategies are adaptive depends on a dynamic interplay of 44 numerous factors including the intensity, frequency, and environmental context of the 45 stressor, as well as the animal’s physical abilities and limitations [6-8]. For this reason, 46 the investigation of stress coping strategies can aid the understanding of factors that 47 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 4 drive individual differences in vulnerability and resilience for developing stress-linked 48 conditions. 49 Epidemiological and preclinical data consistently demonstrate that sex and 50 gender are key contributing factors in vulnerability for developing stress-linked 51 conditions. The global prevalence of depression and anxiety-related conditions in 52 women is two to three times greater than the prevalence in men, which likely results 53 from a convergence of genetic, developmental, neurobiological, psychosocial, and 54 cultural factors [9-12]. Latent cognitive processes, social support seeking, and anger-55 related traits are examples of gender-biased coping strategies that can directly 56 contribute to the risk of experiencing new or recurrent depressive episodes [13, 14]. 57 Sex-specific stress coping responses and vulnerability have also been observed 58 in model organisms. Female rats exhibit greater corticosterone release and greater 59 struggling behavior over multiple restraint stress sessions [15] and female mice are 60 more susceptible to chronic mild stress as measured by greater immobility in the forced 61 swim test and reduced population activity in the ventral tegmental area (VTA) after 62 stress [16]. However, in conditioned fear contexts, females adopt the sex-biased 63 strategy of darting, and darting females show reduced freezing during fear extinction 64 [17]. These studies suggest that while females can engage in coping behavior during 65 inescapable stressors that reflects reduced habituation to stress and impaired 66 adaptation in stress pathways, they can also adopt specific behavioral strategies that 67 promote adaptation. Thus, the role of behavioral strategies in adaptive responses is 68 both sex and context-dependent. 69 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 5 The role of sex in stress-related neurobiological mechanisms and behavioral 70 outcomes of stress has received increasing attention in recent years [18-22]. Paradigms 71 that can model convergent and divergent sex-dependent mechanisms across 72 behavioral and physiological endpoints are critical in advancing the understanding of 73 links between stress, sex, physiology, and maladaptive behavioral outcomes. The 74 subchronic variable stress (SCVS) paradigm models such divergence. This paradigm 75

in a range of sex-biased behavioral outcomes such as increased avoidance and 76 anhedonia as well as physiological changes, including higher corticosterone release 77 and changes in neuronal activity and gene expression across reward and limbic circuitry 78 [23-30]. SCVS therefore reliably alters post-stress outcomes and physiology in a sex-79 dependent fashion, making it a robust platform for investigating whether sex-dependent 80 coping strategies can lead to sexually divergent behavioral outcomes. 81 Tail suspension stress (TSS) is one of the three hour-long inescapable stressors 82 employed during the SCVS paradigm. The tail suspension test (TST) was initially 83 developed as a counterpart to the forced swim test (FST) that increased sensitivity for 84 detecting anti-depressant effects of pharmacological treatments [31]. In its 6-minute 85 form, greater immobility in the TST is typically seen as maladaptive- indicative of 86 behavioral despair or overly passive responses. However, over a prolonged stressor, 87 immobility is likely to be dynamic as animals respond to the repeated experience of 88 unsuccessful escape attempts and balance the high energy cost of sustained struggling 89 against the drive to escape. Given the female-specific vulnerability to SCVS, we 90 hypothesized that females and males would display distinct patterns of coping during 91 the TSS phases of stress, and that this behavior may predict post-stress behavioral 92 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 6 avoidance. To test this hypothesis, we recorded stress coping behaviors during the TSS 93 sessions of SCVS and measured their relationship with exploratory behavior in the EPM 94 to examine relationships between sex, stress coping, and avoidance. 95

96 Animals 97 All experiments were conducted in accordance with National Institutes of Health 98 Guidelines for the Care and Use of Laboratory Animals and approved by the 99 Institutional Animal Care and Use Committee at The George Washington University. 100 Male and female C57BL/6J mice were purchased from the Jackson Laboratory 101 (#000664) or bred in-house. Mice were housed in groups of 3-5 in a temperature and 102 humidity-controlled facility with ad libitum access to food and water on a 12:12 light/dark 103 cycle for the duration of the experiment. 104 Subchronic variable stress 105 SCVS was performed as previously described [26, 29]. Briefly, 8 to 11-week-old male 106 and female mice were exposed to one hour of foot shock, tail suspension, or restraint 107 stress which alternated and repeated once over 6 days. On the first and fourth days, 108 100 0.5 mA foot shocks were randomly dispersed over one hour in a sound attenuated 109 Coulbourn box. On the second and fifth days, mice were suspended by the tail with tape 110 approximately 45 centimeters over the benchtop for 1 hour and behavior was video 111 recorded at 30 fps. A lightweight tube was passed over the tail of the mouse to reduce 112 tail climbing. On the third and sixth days, mice were placed in a ventilated 50 mL conical 113 tube inside of their home cages for 1 hour. Males and females did not make physical 114 contact with one another during stress or behavior. 115 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 7 TSS behavioral analysis 116 DBScorer, a MATLAB-based behavioral scoring software interface [32], was used to 117 analyze struggling behavior in the TSS sessions. DBScorer reports immobility and 118 struggling behaviors by calculating the change in the area occupied by the animal 119 above a specified threshold between video frames. When the change in area does not 120 exceed threshold, the animal is counted as immobile. Videos were analyzed in 10-121 minute bins across each tail suspension stressor, excluding the first 60 seconds of the 122 first 10 minutes of stress. Each video was analyzed with blur image 0.1, 0.8% area 123 threshold, 0s time threshold, and 60s time bin. Immobility behavior is reported in the text 124 as the percent of time spent immobile between 0 and 100, where 0 is sustained 125 struggling and 100 is full immobility. We also report immobility bouts, which is the 126 number of times that the animal stopped struggling. 127 Elevated Plus Maze 128 Mice were acclimated to the testing room for 30 minutes before testing. Mice were 129 placed in the center zone of a gray maze with their head facing the open arm opposite 130 the experimenter and allowed to freely explore the maze for 6 minutes. The center zone 131 was illuminated at 116 Lux. Opposing arms were 5.5 cm x 35 cm, raised 48 cm from the 132 floor. Walls of closed arms were 15 cm high. Animals that fell off of the maze (n = 1 133 male, 1 female) were immediately placed back on the maze to complete testing but 134 were excluded from analysis. Behavior was video recorded and analyzed with Any-135 maze software. 136 Statistical analysis 137 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 8 Data is reported as mean ± SEM. Statistical analyses were performed with GraphPad 138 Prism 10.6.1. Outliers were not removed from the data set, the only excluded animals 139 were those that fell off the EPM. For datasets that did not meet assumptions of normal 140 distribution, non-parametric statistical tests were used. Statistical test and sample size 141 details are indicated in each figure legend. Statistical significance level was set at p < 142 0.05 for all analyses. 143

144 Females engage in more active coping during tail suspension stress 145 We first investigated whether males and females exhibited similar patterns of 146 stress coping during the tail suspension sessions of SCVS. Average immobility scores 147 were lower in females than males (main effect of sex, F1, 56 = 7.00, p = 0.011; Figure 148 1A), and higher during the second tail suspension session (main effect of session, F1, 56 149 = 32.78, p < 0.0001), but there was no significant sex x session interaction (F1, 56 = 3.76, 150 p = 0.057). We also assessed the number of immobility bouts (Figure 1B), and found a 151 significant main effect of sex (F1, 56 = 6.15, p = 0.016), but no significant effect of 152 session (F1, 56 = 3.01, p = 0.088) or sex x session interaction (F1, 56 = 3.86, p = 0.055). 153 This data suggests that during both TSS sessions, females are making more transitions 154 between coping states, but are spending less time immobile between struggling bouts. 155 The tail suspension test, a classic test of stress coping strategy and 156 antidepressant responses, is traditionally 6-10 minutes in duration [31, 33]. In order to 157 test whether we would have detected sex differences in coping behavior in this duration 158 of test, we compared immobility scores and bouts during the first 10 minutes of the first 159 TSS session. We found no significant sex difference in immobility score (U = 392.5, p = 160 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 9 0.67; Figure 1C), but did find that there were more immobility bouts in females (U = 279, 161 p = 0.028; Figure 1D). These results further suggest that sex differences emerge in 162 immobility behavior over the one-hour tail suspension sessions and would not be 163 detectable by measuring the immobility score in the first 10 minutes of stress alone. 164 While average immobility scores and total immobility bouts illustrate activity over 165 the entire stressor, they do not demonstrate behavioral changes during prolonged 166 stress, an important measure of learning and adaptation. For this reason, we examined 167 the change in immobility scores and immobility bouts within 10-minute bins over the 168 duration of both tail suspension sessions (Figure 2A-D). There was a significant main 169 effect of time on the immobility score during the first TSS session (F2.94, 164.8 = 46.34, p < 170 0.0001; Figure 2A) but no main effect of sex (F1, 56 = 2.46, p = 0.12) or sex x time 171 interaction (F2.94, 164.8 = 0.14, p = 0.94). Immobility scores increased during each 10-172 minute bin after the first 10 minutes (Figure 2A). There was also a significant main effect 173 of time on immobility bouts during the first TSS session (F3.45, 193.0 = 16.36, p < 0.0001; 174 Figure 2B), but no main effect of sex (F1, 56 = 2.33, p = 0.13) or sex x time interaction 175 (F3.45, 193.0 = 2.01, p = 0.10). The number of immobility bouts decreased over time, 176 suggesting that animals made fewer transitions between coping states and spent most 177 of their time immobile. During the second TSS session, there was a main effect of sex 178 on immobility score (F1, 56 = 11.13, p = 0.0015; Figure 2C), but not time (F3.85, 215.5 = 179 1.56, p = 0.19) or sex x time interaction (F3.85, 215.5 = 0.27, p = 0.89), with females 180 spending less time immobile than males for the duration of the stressor. However, there 181 was a significant main effect of both sex (F1, 56 = 7.46, p = 0.0084; Figure 2D) and time 182 (F2.69, 150.7 = 4.66, p = 0.0053) on the number of immobility bouts in TSS2, but no 183 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 10 significant sex x time interaction (F2.69, 150.7 = 0.072, p = 0.97). Further analysis 184 demonstrated that the session day significantly contributed to the magnitude of 185 immobility score change (F1, 56 = 74.78, p < 0.0001; Figure 2E) and immobility bout 186 change (F1, 56 = 40.51, p < 0.0001; Figure 2F) within animals. However, changes in 187 immobility score and immobility bouts over time did not differ by sex (immobility score: 188 F1, 56 = 0.25, p = 0.62, immobility bouts: F1, 56 = 1.09, p = 0.30) or a sex x session 189 interaction (immobility score: F1, 56 = 0.0038, p = 0.95, immobility bouts: F1, 56 = 1.92, p = 190 0.17). These results suggest that changes over time in immobility behavior occur 191 predominantly over the first exposure to TSS and are similar in males and females, 192 therefore the sex differences in average immobility scores and bouts in the second TSS 193 session result from sustained coping strategies selected at the beginning of stress, not 194 the rate of adaptation to stress. 195 Coping behavior during stress predicts avoidance 196 Given the known roles of sex in risk appraisal, risk taking, and emotional 197 reactivity [34, 35], we sought to determine whether the sex-dependent coping behaviors 198 discovered in the TSS sessions would be associated with post-stress behavior in the 199 EPM. We first assessed whether there were sex differences in exploratory behaviors in 200 the EPM. We found no sex differences in total distance traveled (t38 = 0.79, p = 0.43; 201 Figure 3A), open arm entries (t38 = 0.50, p = 0.62; Figure 3B), open arm time (t38 = 1.45, 202 p = 0.15; Figure 3C), or open arm ratio (t38 = 1.63, p = 0.11; Figure 3D). 203 One possible contributor to variability within males and females in avoidance 204 behaviors after stress may be sex differences in the appraisal of and responses to 205 stress that directly contribute to the appraisal of threatening contexts after stress. In 206 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 11 order to test relationships between coping behavior and avoidance, we performed 207 simple linear regressions on average immobility scores and open arm ratios. In males, 208 higher immobility scores during the first TSS session predicted less open arm time, or 209 greater avoidance (Figure 4A; R2 = 0.29, p = 0.014). In the second TSS session, males’ 210 immobility scores and open arm ratios showed a similar relationship but did not reach 211 significance (Figure 4C; R2 = 0.14, p = 0.10). In females, however, greater immobility 212 scores predicted a higher OA ratio, or less avoidance in both the first (Figure 4B; R2 = 213 0.27, p = 0.02) and second (Figure 4D; R2 = 0.43, p = 0.0017) TSS sessions. The 214 slopes of the immobility score and OA ratio regression were significantly different 215 between males and females for the first (F1,36 = 12.11, p = 0.0013) and second (F1,36 = 216 7.90, p = 0.008) tail suspension sessions. To control for locomotor activity, we 217 performed simple linear regression analyses of distance traveled in the EPM and 218 immobility scores during both tail suspension sessions (Figure 4E-F), and found no 219 significant relationships in males (TSS1: R2 = 0.062, p = 0.29, TSS2: R2 = 0.13, p = 220 0.12) or females (TSS1: R2 = 0.00081, p = 0.91, TSS2: R2 = 0.0047, p = 0.77). 221 Together, these results demonstrate that the relationships between behavior during 222 stress and avoidance behavior are both sex and time dependent. 223 Relationships between stress coping and avoidance are sex-dependent 224 Given the relationships between avoidance and behavior in the TSS, we tested 225 the collinearity of selected behavioral measures across the tail suspension stressors 226 and avoidance behavior (Figure 5). We found that in females, immobility score during 227 the first 10 minutes of TSS1 was positively correlated with the average TSS1 immobility 228 score (r = 0.60, p = 0.005), TSS2 immobility score (r = 0.78, p < 0.0001), and OA ratio (r 229 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 12 = 0.61, p = 0.005), demonstrating that coping behavior in the first 10 minutes of TSS 230 could predict behavior across both tail suspension sessions in addition to post-stress 231 avoidance behavior. Immobility scores during the first 10 minutes of the first TSS 232 stressor were negatively correlated with the slope of the score over the session (r = -233 0.91, p < 0.0001), indicating that lower immobility at the start of the session was 234 correlated with a greater change in coping over the first stress session. Interestingly, the 235 immobility score slope in females was inversely correlated with OA ratio (r = -0.59, p = 236 0.006), which suggests that a higher rate of coping style change in females predicts 237 more avoidance after stress. This is likely explained mostly by the inverse relationship 238 between immobility in the first 10 minutes of stress and immobility slope, as females 239 who start at a lower immobility score have a higher change in their coping score over 240 time, and the immobility score in the first 10 minutes alone predicts avoidance after 241 stress. 242 TSS1 and TSS2 immobility scores were positively correlated in males (r = 0.61, p 243 = 0.005) and females (r = 0.80, p < 0.0001), which suggests that coping choices within 244 animals are consistent between stress sessions regardless of sex. However, in males, 245 while immobility in the first 10 minutes of TSS1 was positively correlated with the 246 average immobility score during TSS1 (r = 0.66, p = 0.002) and inversely correlated with 247 the slope of TSS1 immobility score (r = -0.83, p < 0.0001), it did not significantly 248 correlate with coping behavior in TSS2 (r = 0.24, p = 0.31) or with OA ratio (r = -0.30, p 249 = 0.20) unlike the observation in females. Simple linear regression revealed significant 250 sex differences between the immobility score in the first 10 minutes of TSS1 and OA 251 ratio (F1, 36 = 7.68, p = 0.0088), as well as the TSS1 immobility score slope and OA ratio 252 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 13 (F1, 36 = 4.53, p = 0.040). Taken together, these results suggest that the relationships 253 between behavioral choices at the beginning of stress and behavior across both stress 254 sessions, and the extent to which those behavioral choices predict post-stress 255 avoidance, are sex-dependent. For females, behavior at the beginning of stress predicts 256 the behavioral profile across both stress sessions and is sufficient to predict avoidance 257 after stress. In males, however, behavior at the beginning of stress is only predictive of 258 the behavioral profile during the first stress session. This suggests that while males and 259 females engage in similar magnitudes of behavioral flexibility as measured by the 260 change in immobility over TSS, females select a set of behavioral choices at the 261 beginning of stress that are sustained across multiple stress sessions and predict 262 avoidance after stress, while the initial behavioral strategies in males are not 263 necessarily sustained across both sessions and do not predict avoidance. 264 We considered one possible source of behavioral variability contributing to sex 265 differences in coping behavior- adoption of tail climbing. We assessed whether animals 266 tail climbed at any point during TSS sessions and found that 46.67% of females 267 engaged in tail climbing at some point during the first tail suspension session and 268 36.67% engaged in tail climbing during the second tail suspension session, while only 269 7.14% of males engaged in tail climbing during either TSS session (Figure 6). The 270 proportion of tail climbing was significantly different between males and females during 271 both the first (p = 0.0010) and second (p = 0.011) TSS sessions (Fisher’s exact test; 272 Figure 6A-D). Within females, there was a significant main effect of tail climbing status 273 on immobility score, with tail climbing females having a significantly lower immobility 274 score than non-tail-climbers (F1, 56 = 64.43, p < 0.0001; Figure 6E). Because DBScorer 275 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 14 does not differentiate between head down and tail climb struggling bouts, it is unclear 276 what proportion of each total immobility score is attributable to tail climbing vs. head 277 down struggling. However, it is clear that in this configuration tail climbing does not 278

in a broadly immobile phenotype during tail suspension and that it is a sex-biased 279 strategy that may contribute to adaptation during stress. 280 281

282 In this study, we tested whether sex differences in coping strategies emerge 283 during a repeated stressor, and if they are associated with avoidance behaviors after 284 stress. Active coping is often seen as a beneficial or adaptive choice that promotes 285 resilience after stress, whereas passive coping indicates despair or a “depressive-like" 286 phenotype [36]. However, in an inescapable stressor, the choice to sustain a coping 287 strategy that expends considerable energy may reflect a failure to learn. Sustained 288 active coping may lead to pathophysiological plasticity in neural circuitry that contributes 289 to avoidance, reward, motivation, and aversion. This is particularly important in the 290 SCVS paradigm where animals are exposed to a series of inescapable stressors and 291 must repeatedly select strategies that promote adaptation across each stressor. We 292 focused on behavior in the TSS sessions of SCVS, but it is likely that behavior is 293 influenced by previous and ongoing experience across each stressor and would differ 294 from behavior during isolated tail suspension tests. These prior and ongoing 295 experiences may be important for the observed sex differences in the relationship 296 between coping and avoidance after stress, specifically given observed sex differences 297 in behavioral strategies during other inescapable stressors [15, 17]. 298 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 15 One strength of using the SCVS paradigm to ask this question is that each 299 stressor is repeated, which allows comparison of behavior during repeated stress 300 sessions to ascertain changes in coping strategy. Our data demonstrates that sex 301 differences in coping strategy are a function of both time and repeated exposure to 302 stress. While the choice of coping strategy changed over the duration of the first stress 303 session in both males and females, sex differences were observed in overall immobility 304 score only in the second tail suspension session, where the choice of strategy at the 305 beginning of the stressor was sustained over the duration of the session. This suggests 306 that there may be sex differences in mechanisms that support sustained motivation and 307 learning across repeated inescapable stress sessions. 308 Our study demonstrates that relationships between avoidance behavior and 309 coping behavior across stress sessions are a meaningful sex-dependent outcome of 310 SCVS and predict behavioral variability within each sex. Despite no sex differences in 311 the overall immobility score during the first TSS session or EPM open arm ratio, higher 312 immobility scores predicted greater avoidance in males but lower avoidance in females. 313 On the second TSS session, when females engaged in more active coping than males, 314 higher immobility scores were only a significant predictor of avoidance in females, but 315 not males. Importantly, some studies have identified sex differences in baseline 316 locomotion of mice and rats during the EPM, which can confound interpretations of 317 exploratory behavior [37, 38]. However, our study shows that there is no relationship 318 between the immobility score and locomotion in the EPM. This suggests that the 319 relationship between coping strategy during tail suspension and avoidance is not 320 reducible to sex-biased trends in activity levels. 321 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 16 Assays like the tail suspension and forced swim tests often report total time spent 322 immobile, or inversely, time spent on escape-oriented behaviors. This data suggests, 323 however, that the number of transitions between immobility and escape-oriented 324 behavior may reveal important information that total time in each state may not capture. 325 During the first 10 minutes of the first TSS session, there was no sex difference in the 326 overall immobility score, but females made significantly more transitions between 327 immobility and struggling as measured by the number of immobility bouts. The 328 differences between immobility scores and immobility bouts may reflect a distinction 329 between action initiation for short escape-oriented bouts in comparison to the 330 motivational vigor required for sustaining longer struggling bouts [39, 40]. 331 Researchers utilizing the tail suspension test for screening antidepressants and 332 measuring stress-induced behavioral changes often highlight the challenge of high tail 333 climbing rates in C57BL/6J mice, and many exclude animals who tail climb [33, 41-43]. 334 We did not apply this exclusion criterion in our study for several key reasons. Tail 335 climbing is a coping strategy- it does not permit the animal to escape and requires 336 energy expenditure in a similar way to head-down escape-oriented motion. Importantly 337 for our study, we also found that tail climbing behavior is sex-biased. Nearly half of 338 females but almost no males exhibit tail climbing at some point during the tail 339 suspension stressor. Removing tail climbing animals would introduce a sex-biased 340 exclusion criterion that would preclude a full assessment of how sex-biased coping 341 strategies contribute to behavioral outcomes. It is possible that the vestibular and 342 proprioceptive experience in an entirely head-down position is distinct and that repeated 343 tail climbing promotes greater motivation to struggle, both of which may be important for 344 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 17 understanding neural mechanisms engaged by struggling behavior. As Shansky and 345 Murphy (2021) have emphasized, including females in studies warrants consideration 346 that common behavioral endpoints based on assays that were originally tested 347 exclusively in males may not reflect the range of meaningful behavioral strategies in 348 females [21]. 349 While the EPM is generally understood to test a conserved conflict between an 350 aversion to open or elevated space and exploration, others argue that it is also testing 351 thigmotaxis mediated by the somatosensory system, primarily in the closed arms of the 352 maze [44]. Studies have identified sex differences in thigmotaxis across anxiogenic 353 environments, which may contribute to sex differences in exploratory behavior in the 354 EPM [45, 46]. Neural circuits that assess threat and support coping strategy selection 355 during stress may be altered by repeated unsuccessful escape attempts during stress, 356 which could directly inform future threat assessments in the EPM. These circuits are 357 reliant upon sensory input and interoceptive signals during stress and motivated 358 behavior, and in anxiogenic environments [47-51]. Thus, appraisal of risk and safety 359 signals may be altered by coping choices to promote adaptation after chronic stress. 360 Furthermore, each stressor was performed in groups with cage mates, so while animals 361 could not make physical contact in TSS, it is likely that they can see, smell, and hear 362 their cage mates during stress. Prior work has demonstrated that a physical barrier or 363 being tested alone does not alter behavior in males during the 6-minute tail suspension 364 test [52], but it is unclear whether this would be true in females, and whether it would 365 apply in a prolonged stress session. Given the sex-specific roles of social stress on 366 physiological and behavioral outcomes [18], future studies to test the role of social 367 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 18 context and sensory cues during stress on sex-specific coping strategies and avoidance 368 behaviors would be beneficial. 369 Neither coping strategies nor avoidance behavior can be interpreted under 370 binaries- the more likely case is that an accumulation of maladaptive choices, 371 particularly when those behaviors are sustained over time, can contribute to reduced 372 fitness or pathophysiological states. Why the relationship between coping choices and 373 avoidance would be sex-biased is likely attributable to differences in the evolutionary 374 roles of escape behaviors during inescapable stress and approach behaviors, which 375 engage overlapping neural circuitry [53]. Fluctuations in sex hormones across the 376 estrous cycle and between animals of different social rank may contribute to baseline 377 stress and anxiety levels, and act directly on limbic and striatal circuitry that drive threat 378 appraisal and memory [54-57]. The BNST is a known substrate for both stress coping 379 and avoidance behaviors [48, 49] with established sex differences in contributions to 380 threat processing [58]. The locus coeruleus and ventral tegmental area, hubs for 381 robustly stress-sensitive noradrenergic and dopaminergic circuitry respectively, exhibit 382 structural and physiological sex differences that may also play direct roles in stress 383 processing that are particularly relevant in convergent inputs to limbic and cortical 384 regions [59]. Future studies exploring specific contributions of sex hormones and sex 385 biased regions to learning and adaptation during stress coping could advance concepts 386 of the roles of sex in stress coping and avoidance behavior. 387 Summary/ Conclusions 388 Our study demonstrates that coping strategies are sex-specific and dynamic across a 389 single stress session and between repeated stress exposures. Furthermore, coping 390 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 19 strategies and the change in coping strategy predicts avoidance behavior in a sex-391 dependent fashion. While coping strategies are often not recorded and scored during 392 stress, they may provide a key readout for the progression of behavioral changes 393 across chronic stress paradigms, and may contribute to elucidating the engagement of 394 mechanisms that promote divergent and convergent sex-specific mechanisms. 395 Declarations 396 Ethics approval and consent to participate 397 Not applicable 398 Consent for publication 399 Not applicable 400 Availability of data and materials 401 The datasets used and analyzed during the current study are available from the 402 corresponding author on reasonable request. 403 Competing interests 404 The authors declare that they have no competing interests 405 Funding 406 This work was funded by NIH grants R01MH122712 and R01MH122712S1. 407 Author’s contributions 408 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 20 KP and AMP designed the study; KP collected and analyzed data; KP and AMP 409 interpreted data; KP drafted the paper; AMP edited the paper. 410 Acknowledgments 411 We would like to acknowledge Dr. Paul Marvar for use of the Coulbourn boxes. 412 Author Information 413 Department of Pharmacology and Physiology, George Washington University School of 414 Medicine and Health Sciences, Washington, DC 20037 415

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Physiol 569 Behav. 2017;171:110–9. 570 58. Urien L, Bauer EP. Sex differences in BNST and amygdala activation by contextual, 571 cued, and unpredictable threats. Eneuro. 2022;9 1 . 572 59. Bangasser DA, Wiersielis KR, Khantsis S. Sex differences in the locus coeruleus-573 norepinephrine system and its regulation by stress. Brain Res. 2016;1641:177–88. 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 28 Figure Legends 589 590 Figure 1. Stress coping behavior during the tail suspension phases of SCVS is 591 sex-dependent 592 A. Experimental schematic for subchronic variable stress (SCVS). Behavior was video 593 recorded during the first and second tail suspension sessions (TSS1 and TSS2). B 594 Average immobility score and C total immobility bouts during TSS1 and TSS2. D 595 Immobility score during the first 10 minutes of TSS1. E Total immobility bouts during the 596 first 10 minutes of TSS1. # indicates significant main effect: # p < 0.05, ## p < 0.01, ### 597 p < 0.001, #### p < 0.0001. Pairwise comparisons were performed with Mann-Whitney 598 U test. N=28 males, 30 females. * p<0.05, ** p<0.01. 599 600 Figure 2. Stress coping behavior changes within and between tail suspension 601 sessions 602 A. Immobility score and B immobility bouts across TSS1, within 10-minute bins. C. 603 Immobility score and D Immobility bouts across TSS2, within 10-minute bins. Two-way 604 ANOVA followed by Dunnett’s multiple comparisons test where significant main effects 605 of time were present. E Slopes of immobility score and F slopes of immobility bouts over 606 time for each animal. Two-way ANOVA. # indicates significant main effect: # p < 0.05, 607 ## p < 0.01, ### p < 0.001, #### p < 0.0001. * indicates significant pairwise difference 608 in comparison to first 10 minutes. * p < 0.05, ** p < 0.01, *** p < 0.001, ****p < 0.0001. N 609 = 28 males, 30 females. 610 611 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 29 Figure 3. No sex differences in avoidance behaviors in the elevated plus maze 612 After SCVS, A. total distance traveled (males: 12.40 ± 0.61 m, females: 11.75 ± 0.55 m) 613 in the elevated plus maze. B. open arm entries (males: 11.65 ± 1.64, females: 12.85 ± 614 1.77, C. open arm time (males: 39.40 ± 7.39 s, females: 56.78 ± 9.40 s) and D. open 615 arm ratio (males: 15.05 ± 2.70%, females: 21.92 ± 3.24%) were measured in males and 616 females. Open arm ratio = [(open arm time/ open arm time + closed arm time) * 100]. N 617 = 20 females, 20 males. 618 619 Figure 4. Relationships between stress coping and post-stress avoidance 620 behavior are sex-specific 621 Simple linear regressions between OA ratio and immobility score during TSS1 in males 622 A and females B, and OA ratio and immobility score during TSS2 in males C and 623 females D. Simple linear regression of distance traveled in the elevated plus maze and 624 immobility score during TSS1 E and TSS2 F. R2 and p-values listed on graph inset. N= 625 20 females, 20 males. 626 627 Figure 5. Covariance between immobility measures and avoidance behaviors is 628 sex-dependent 629 Pearson r correlation coefficients between the first 10 minutes of TSS1, TSS1 average 630 immobility score, TSS1 immobility slope, TSS2 average immobility score, and open arm 631 ratio in females A and males B. N= 20 females, 20 males. * p < 0.05, ** p < 0.01, *** p < 632 0.001, ****p < 0.0001. 633 634 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 30 Figure 6. Tail climbing rates are greater in females during tail suspension stress 635 Proportions of tail climbing at any point during TSS1 in females A and males B and 636 during TSS2 in females C and males D. N = 28 males, 30 females. E. Immobility scores 637 are higher in females who do not tail climb at any point during TSS. Two-way ANOVA. 638 #### indicates main effect, p < 0.0001. N = 11-19/group. 639 640 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint A B C Day 1 Foot Shock Day 2 Tail Suspension (TSS1) Day 3 Restraint Day 4 Foot Shock Day 5 Tail Suspension (TSS2) Day 6 Restraint Subchronic Variable Stress (SCVS) T S S 1 T S S 2 0 2 0 0 4 0 0 6 0 0 F e m a l e M a l e # Immobility Bouts D F e m a l e M a l e 0 5 0 1 0 0 1 5 0 T S S 1 : F i r s t 1 0 M i n u t e s ✱ Immobility Bouts E Immobility Score (%) F e m a l e M a l e 0 5 0 1 0 0 T S S 1 : F i r s t 1 0 M i n u t e s T S S 1 T S S 2 0 5 0 1 0 0 F e m a l e M a l e # # # # #Immobility Score (%) Fig. 1 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint A Immobility Score (%) B C Immobility Bouts E Immobility Bout Slope D Immobility Score Slope F 0 1 2 3 4 5 6 5 0 6 0 7 0 8 0 9 0 1 0 0 T S S 1 F e m a l e M a l e * * * * * * * * * * * * * * * * * * * * # # # # 0 1 2 3 4 5 6 5 0 6 0 7 0 8 0 9 0 1 0 0 T S S 2 M a l e F e m a l e ## Immobility Score (%) 0 1 2 3 4 5 6 0 1 0 2 0 3 0 4 0 5 0 6 0 T S S 2 1 0 - M i n u t e B i n * * * * * * * # # # # F e m a l e M a l e ## 0 1 2 3 4 5 6 0 1 0 2 0 3 0 4 0 5 0 6 0 T S S 1 1 0 - M i n u t e B i n # # # # * * * * * * * * * * * * F e m a l e M a l e Immobility Bouts T S S 1 T S S 2 - 1 0 0 1 0 # # # # T S S 1 T S S 2 - 2 0 - 1 0 0 1 0 2 0 M a l e # # # # F e m a l e M a l e Fig. 2 F e m a l e .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint A B C D F e m a l e M a l e 0 5 1 0 1 5 2 0 T o t a l D i s t a n c e T r a v e l e d Distance (m) F e m a l e M a l e 0 1 0 2 0 3 0 4 0 O p e n A r m E n t r i e s F e m a l e M a l e 0 5 0 1 0 0 1 5 0 2 0 0 O p e n A r m T i m eseconds F e m a l e M a l e 0 2 0 4 0 6 0 O p e n A r m R a t i o ( % ) Fig. 3 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint A B OA Ratio (%) I m m o b i l i t y S c o r e ( % )OA Ratio (%) 5 0 6 0 7 0 8 0 9 0 1 0 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 T S S 2 : F e m a l e s I m m o b i l i t y S c o r e ( % ) R 2 = 0 . 4 3 p = 0 . 0 0 1 7 5 0 6 0 7 0 8 0 9 0 1 0 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 T S S 1 : F e m a l e s R 2 = 0 . 2 7 p = 0 . 0 2 0 5 0 6 0 7 0 8 0 9 0 1 0 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 T S S 1 : M a l e s R 2 = 0 . 2 9 p = 0 . 0 1 4 5 0 6 0 7 0 8 0 9 0 1 0 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 T S S 2 : M a l e s R 2 = 0 . 1 4 p = 0 . 1 0 3 5 0 6 0 7 0 8 0 9 0 1 0 0 0 5 1 0 1 5 2 0 2 5 M a l e F e m a l e R 2 = 0 . 0 0 0 8 1 p = 0 . 9 1 R 2 = 0 . 0 6 2 p = 0 . 2 9EPM Distance (m) 5 0 6 0 7 0 8 0 9 0 1 0 0 0 5 1 0 1 5 2 0 2 5 T S S 2 I m m o b i l i t y S c o r e ( % ) M a l e F e m a l e R 2 = 0 . 1 3 p = 0 . 1 2 R 2 = 0 . 0 0 4 7 p = 0 . 7 7 TSS1 Immobility Score (%) I m m o b i l i t y S c o r e ( % ) I m m o b i l i t y S c o r e ( % ) C D EPM Distance (m) E F OA Ratio (%) OA Ratio (%) Fig. 4 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint 1 . 0 0 0 . 6 0 - 0 . 9 1 0 . 7 8 0 . 6 1 0 . 6 0 1 . 0 0 - 0 . 4 8 0 . 8 0 0 . 5 2 - 0 . 9 1 - 0 . 4 8 1 . 0 0 - 0 . 7 2 - 0 . 5 9 0 . 7 8 0 . 8 0 - 0 . 7 2 1 . 0 0 0 . 6 6 0 . 6 1 0 . 5 2 - 0 . 5 9 0 . 6 6 1 . 0 0 T S S 1 F i r s t 1 0 M i n s T S S 1 A v e r a g e T S S 1 S l o p e T S S 2 A v e r a g e O A R a t i o * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * I m m o b i l i t y a n d A v o i d a n c e : F e m a l e s - 1 . 0 - 0 . 5 0 0 . 5 1 . 0 T S S 1 F i r s t 1 0 M i n s T S S 1 A v e r a g e T S S 1 S l o p e T S S 2 A v e r a g e O A R a t i o 1 . 0 0 0 . 6 6 - 0 . 8 3 0 . 2 4 - 0 . 3 0 0 . 6 6 1 . 0 0 - 0 . 3 1 0 . 6 1 - 0 . 5 4 - 0 . 8 3 - 0 . 3 1 1 . 0 0 0 . 0 8 0 . 1 0 0 . 2 4 0 . 6 1 0 . 0 8 1 . 0 0 - 0 . 3 7 - 0 . 3 0 - 0 . 5 4 0 . 1 0 - 0 . 3 7 1 . 0 0 T S S 1 F i r s t 1 0 M i n s T S S 1 A v e r a g e T S S 1 S l o p e T S S 2 A v e r a g e O A R a t i o * * * * * * * * * * * * * I m m o b i l i t y a n d A v o i d a n c e : M a l e s * * * - 1 . 0 - 0 . 5 0 0 . 5 1 . 0 T S S 1 F i r s t 1 0 M i n s T S S 1 A v e r a g e T S S 1 S l o p e T S S 2 A v e r a g e O A R a t i o A B Fig. 5 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint F e m a l e s T S S 2 6 3 . 3 3 % N o T a i l C l i m b i n g 3 6 . 6 7 % T a i l C l i m b i n g M a l e s T S S 2 9 2 . 8 6 % N o T a i l C l i m b i n g 7 . 1 4 % T a i l C l i m b i n g F e m a l e s T S S 1 5 3 . 3 3 % N o T a i l C l i m b i n g 4 6 . 6 7 % T a i l C l i m b i n g M a l e s T S S 1 9 2 . 8 6 % N o T a i l C l i m b i n g 7 . 1 4 % T a i l C l i m b i n g T a i l C l i m b i n g N o t T a i l C l i m b i n g 0 2 0 4 0 6 0 8 0 1 0 0 F e m a l e T S S 1 F e m a l e T S S 2 # # # # Immobility Score (%) A B C D E Fig. 6 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint