{"paper_id":"25043886-ce9e-41e7-b997-a2de549fa0b4","body_text":"Sex-specific relationships between stress coping and avoidance \nbehavior \n \nKailyn M. Price1 and Abigail M. Polter1* \n \n1Department of Pharmacology and Physiology, George Washington University School \nof Medicine and Health Sciences, Washington, DC 20037 \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n*Corresponding Author \nAbigail M. Polter, Ph.D. \nGeorge Washington University \nDepartment of Pharmacology and Physiology \n2300 Eye St NW, Ste 727 \nWashington, DC, 20037 \nPhone: (202)-994-8172 \nE-mail: ampolter@gwu.edu  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n2 \n \nAbstract 1 \nWhile the experience of stress is ubiquitous, the risk of developing stress-linked 2 \nconditions such as anxiety and depression is related to maladaptive stress responses. 3 \nIn order to probe the relationship between stress coping, sex, and stress-linked 4 \nbehavioral outcomes, we exposed male and female mice to subchronic variable stress 5 \n(SCVS) and measured the correlation between coping during the tail suspension 6 \nstressors (TSS) of SCVS and avoidance behavior in the EPM. We found that females 7 \nengage in more active coping, and there were no sex differences in avoidance or 8 \nlocomotor behavior in the EPM after stress. However, we found that greater active 9 \ncoping predicted greater avoidance in females, but less avoidance in males. The results 10 \ndemonstrate that coping strategies are dynamic across time in males and females, but 11 \nthe relationships between avoidance and coping strategy dynamics are sex-biased. 12 \n 13 \nKeywords: Stress, sex differences, coping, avoidance 14 \n 15 \nPlain English Summary 16 \nThe selection of stress coping strategies is an important component of the stress 17 \nresponse that can impact behavior after stress. Stress coping strategies and behavior 18 \nafter stress can both be sex-biased, but the relationships between them are unclear. 19 \nSCVS is a paradigm that is used to study sex differences in behavior and physiology 20 \nbecause females are specifically vulnerable to SCVS. We recorded behavior during two 21 \nstressors in the SCVS paradigm and found opposite relationships between coping 22 \nbehavior and avoidance behavior after stress in males and females, even though males 23 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n3 \n \nand females exhibit similar dynamics in coping behavior and similar avoidance behavior 24 \nafter stress. These results demonstrate that sex is an important variable in the 25 \nrelationship between coping strategies during stress and behavior after stress. 26 \nIntroduction 27 \nAll living organisms experience stress, broadly defined as challenges to physical 28 \nor emotional homeostasis. Stress often carries a colloquially negative connotation, but it 29 \nis ubiquitous and necessary for learning and adaptation [1, 2]. However, stress can 30 \nprecipitate maladaptive outcomes when it surpasses the adaptive capacities of an 31 \norganism due to the chronicity, intensity, or perception of the stress. In humans, chronic 32 \nstress exposure can increase the risk of developing multiple psychiatric disorders 33 \nincluding depression, anxiety, and substance use-related conditions. Across organisms, 34 \nstress initiates a cascade of physiological mechanisms and behavioral responses. 35 \nAmong these responses are behavioral strategies known as stress coping, which permit 36 \nthe removal, mitigation, and adaptation to a stressor such that the stress response may 37 \nbe primed for more efficient responses in the future [3-5]. Stress coping strategies vary 38 \nbetween conspecifics and across contexts, and some stress coping choices promote 39 \nadaptation while others may be acutely or chronically maladaptive. The appraisal of 40 \nstress and selection of coping strategy is influenced by a range of intrinsic factors 41 \nincluding genetics, early life environment, and circulating hormones, which interact with 42 \nreal time stimuli and shared stress response mechanisms [2, 3]. Consequently, whether 43 \nor not stress coping strategies are adaptive depends on a dynamic interplay of 44 \nnumerous factors including the intensity, frequency, and environmental context of the 45 \nstressor, as well as the animal’s physical abilities and limitations [6-8]. For this reason, 46 \nthe investigation of stress coping strategies can aid the understanding of factors that 47 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n4 \n \ndrive individual differences in vulnerability and resilience for developing stress-linked 48 \nconditions.  49 \nEpidemiological and preclinical data consistently demonstrate that sex and 50 \ngender are key contributing factors in vulnerability for developing stress-linked 51 \nconditions. The global prevalence of depression and anxiety-related conditions in 52 \nwomen is two to three times greater than the prevalence in men, which likely results 53 \nfrom a convergence of genetic, developmental, neurobiological, psychosocial, and 54 \ncultural factors [9-12]. Latent cognitive processes, social support seeking, and anger-55 \nrelated traits are examples of gender-biased coping strategies that can directly 56 \ncontribute to the risk of experiencing new or recurrent depressive episodes [13, 14].  57 \nSex-specific stress coping responses and vulnerability have also been observed 58 \nin model organisms. Female rats exhibit greater corticosterone release and greater 59 \nstruggling behavior over multiple restraint stress sessions [15] and female mice are 60 \nmore susceptible to chronic mild stress as measured by greater immobility in the forced 61 \nswim test and reduced population activity in the ventral tegmental area (VTA) after 62 \nstress [16]. However, in conditioned fear contexts, females adopt the sex-biased 63 \nstrategy of darting, and darting females show reduced freezing during fear extinction 64 \n[17]. These studies suggest that while females can engage in coping behavior during 65 \ninescapable stressors that reflects reduced habituation to stress and impaired 66 \nadaptation in stress pathways, they can also adopt specific behavioral strategies that 67 \npromote adaptation. Thus, the role of behavioral strategies in adaptive responses is 68 \nboth sex and context-dependent.   69 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n5 \n \nThe role of sex in stress-related neurobiological mechanisms and behavioral 70 \noutcomes of stress has received increasing attention in recent years [18-22]. Paradigms 71 \nthat can model convergent and divergent sex-dependent mechanisms across 72 \nbehavioral and physiological endpoints are critical in advancing the understanding of 73 \nlinks between stress, sex, physiology, and maladaptive behavioral outcomes. The 74 \nsubchronic variable stress (SCVS) paradigm models such divergence. This paradigm 75 \nresults in a range of sex-biased behavioral outcomes such as increased avoidance and 76 \nanhedonia as well as physiological changes, including higher corticosterone release 77 \nand changes in neuronal activity and gene expression across reward and limbic circuitry 78 \n[23-30]. SCVS therefore reliably alters post-stress outcomes and physiology in a sex-79 \ndependent fashion, making it a robust platform for investigating whether sex-dependent 80 \ncoping strategies can lead to sexually divergent behavioral outcomes.  81 \nTail suspension stress (TSS) is one of the three hour-long inescapable stressors 82 \nemployed during the SCVS paradigm. The tail suspension test (TST) was initially 83 \ndeveloped as a counterpart to the forced swim test (FST) that increased sensitivity for 84 \ndetecting anti-depressant effects of pharmacological treatments [31]. In its 6-minute 85 \nform, greater immobility in the TST is typically seen as maladaptive- indicative of 86 \nbehavioral despair or overly passive responses. However, over a prolonged stressor, 87 \nimmobility is likely to be dynamic as animals respond to the repeated experience of 88 \nunsuccessful escape attempts and balance the high energy cost of sustained struggling 89 \nagainst the drive to escape. Given the female-specific vulnerability to SCVS, we 90 \nhypothesized that females and males would display distinct patterns of coping during 91 \nthe TSS phases of stress, and that this behavior may predict post-stress behavioral 92 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n6 \n \navoidance. To test this hypothesis, we recorded stress coping behaviors during the TSS 93 \nsessions of SCVS and measured their relationship with exploratory behavior in the EPM 94 \nto examine relationships between sex, stress coping, and avoidance.  95 \nMethods 96 \nAnimals 97 \nAll experiments were conducted in accordance with National Institutes of Health 98 \nGuidelines for the Care and Use of Laboratory Animals and approved by the 99 \nInstitutional Animal Care and Use Committee at The George Washington University. 100 \nMale and female C57BL/6J mice were purchased from the Jackson Laboratory 101 \n(#000664) or bred in-house. Mice were housed in groups of 3-5 in a temperature and 102 \nhumidity-controlled facility with ad libitum access to food and water on a 12:12 light/dark 103 \ncycle for the duration of the experiment.  104 \nSubchronic variable stress 105 \nSCVS was performed as previously described [26, 29]. Briefly, 8 to 11-week-old male 106 \nand female mice were exposed to one hour of foot shock, tail suspension, or restraint 107 \nstress which alternated and repeated once over 6 days. On the first and fourth days, 108 \n100 0.5 mA foot shocks were randomly dispersed over one hour in a sound attenuated 109 \nCoulbourn box. On the second and fifth days, mice were suspended by the tail with tape 110 \napproximately 45 centimeters over the benchtop for 1 hour and behavior was video 111 \nrecorded at 30 fps. A lightweight tube was passed over the tail of the mouse to reduce 112 \ntail climbing. On the third and sixth days, mice were placed in a ventilated 50 mL conical 113 \ntube inside of their home cages for 1 hour. Males and females did not make physical 114 \ncontact with one another during stress or behavior.  115 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n7 \n \nTSS behavioral analysis 116 \nDBScorer, a MATLAB-based behavioral scoring software interface [32], was used to 117 \nanalyze struggling behavior in the TSS sessions. DBScorer reports immobility and 118 \nstruggling behaviors by calculating the change in the area occupied by the animal 119 \nabove a specified threshold between video frames. When the change in area does not 120 \nexceed threshold, the animal is counted as immobile. Videos were analyzed in 10-121 \nminute bins across each tail suspension stressor, excluding the first 60 seconds of the 122 \nfirst 10 minutes of stress. Each video was analyzed with blur image 0.1, 0.8% area 123 \nthreshold, 0s time threshold, and 60s time bin. Immobility behavior is reported in the text 124 \nas the percent of time spent immobile between 0 and 100, where 0 is sustained 125 \nstruggling and 100 is full immobility. We also report immobility bouts, which is the 126 \nnumber of times that the animal stopped struggling. 127 \nElevated Plus Maze 128 \nMice were acclimated to the testing room for 30 minutes before testing. Mice were 129 \nplaced in the center zone of a gray maze with their head facing the open arm opposite 130 \nthe experimenter and allowed to freely explore the maze for 6 minutes. The center zone 131 \nwas illuminated at 116 Lux. Opposing arms were 5.5 cm x 35 cm, raised 48 cm from the 132 \nfloor. Walls of closed arms were 15 cm high. Animals that fell off of the maze (n = 1 133 \nmale, 1 female) were immediately placed back on the maze to complete testing but 134 \nwere excluded from analysis. Behavior was video recorded and analyzed with Any-135 \nmaze software.  136 \nStatistical analysis 137 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n8 \n \nData is reported as mean ± SEM. Statistical analyses were performed with GraphPad 138 \nPrism 10.6.1. Outliers were not removed from the data set, the only excluded animals 139 \nwere those that fell off the EPM. For datasets that did not meet assumptions of normal 140 \ndistribution, non-parametric statistical tests were used. Statistical test and sample size 141 \ndetails are indicated in each figure legend. Statistical significance level was set at p < 142 \n0.05 for all analyses.  143 \nResults 144 \nFemales engage in more active coping during tail suspension stress 145 \nWe first investigated whether males and females exhibited similar patterns of 146 \nstress coping during the tail suspension sessions of SCVS. Average immobility scores 147 \nwere lower in females than males (main effect of sex, F1, 56 = 7.00, p = 0.011; Figure 148 \n1A), and higher during the second tail suspension session (main effect of session, F1, 56 149 \n= 32.78, p < 0.0001), but there was no significant sex x session interaction (F1, 56 = 3.76, 150 \np = 0.057). We also assessed the number of immobility bouts (Figure 1B), and found a 151 \nsignificant main effect of sex (F1, 56 = 6.15, p = 0.016), but no significant effect of 152 \nsession (F1, 56 = 3.01, p = 0.088) or sex x session interaction (F1, 56 = 3.86, p = 0.055). 153 \nThis data suggests that during both TSS sessions, females are making more transitions 154 \nbetween coping states, but are spending less time immobile between struggling bouts. 155 \nThe tail suspension test, a classic test of stress coping strategy and 156 \nantidepressant responses, is traditionally 6-10 minutes in duration [31, 33]. In order to 157 \ntest whether we would have detected sex differences in coping behavior in this duration 158 \nof test, we compared immobility scores and bouts during the first 10 minutes of the first 159 \nTSS session. We found no significant sex difference in immobility score (U = 392.5, p = 160 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n9 \n \n0.67; Figure 1C), but did find that there were more immobility bouts in females (U = 279, 161 \np = 0.028; Figure 1D). These results further suggest that sex differences emerge in 162 \nimmobility behavior over the one-hour tail suspension sessions and would not be 163 \ndetectable by measuring the immobility score in the first 10 minutes of stress alone.  164 \nWhile average immobility scores and total immobility bouts illustrate activity over 165 \nthe entire stressor, they do not demonstrate behavioral changes during prolonged 166 \nstress, an important measure of learning and adaptation. For this reason, we examined 167 \nthe change in immobility scores and immobility bouts within 10-minute bins over the 168 \nduration of both tail suspension sessions (Figure 2A-D). There was a significant main 169 \neffect of time on the immobility score during the first TSS session (F2.94, 164.8 = 46.34, p < 170 \n0.0001; Figure 2A) but no main effect of sex (F1, 56 = 2.46, p = 0.12) or sex x time 171 \ninteraction (F2.94, 164.8 = 0.14, p = 0.94). Immobility scores increased during each 10-172 \nminute bin after the first 10 minutes (Figure 2A). There was also a significant main effect 173 \nof time on immobility bouts during the first TSS session (F3.45, 193.0 = 16.36, p < 0.0001; 174 \nFigure 2B), but no main effect of sex (F1, 56 = 2.33, p = 0.13) or sex x time interaction 175 \n(F3.45, 193.0 = 2.01, p = 0.10). The number of immobility bouts decreased over time, 176 \nsuggesting that animals made fewer transitions between coping states and spent most 177 \nof their time immobile. During the second TSS session, there was a main effect of sex 178 \non immobility score (F1, 56 = 11.13, p = 0.0015; Figure 2C), but not time (F3.85, 215.5 = 179 \n1.56, p = 0.19) or sex x time interaction (F3.85, 215.5 = 0.27, p = 0.89), with females 180 \nspending less time immobile than males for the duration of the stressor. However, there 181 \nwas a significant main effect of both sex (F1, 56 = 7.46, p = 0.0084; Figure 2D) and time 182 \n(F2.69, 150.7 = 4.66, p = 0.0053) on the number of immobility bouts in TSS2, but no 183 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n10 \n \nsignificant sex x time interaction (F2.69, 150.7 = 0.072, p = 0.97). Further analysis 184 \ndemonstrated that the session day significantly contributed to the magnitude of 185 \nimmobility score change (F1, 56 = 74.78, p < 0.0001; Figure 2E) and immobility bout 186 \nchange (F1, 56 = 40.51, p < 0.0001; Figure 2F) within animals. However, changes in 187 \nimmobility score and immobility bouts over time did not differ by sex (immobility score: 188 \nF1, 56 = 0.25, p = 0.62, immobility bouts: F1, 56 = 1.09, p = 0.30) or a sex x session 189 \ninteraction (immobility score: F1, 56 = 0.0038, p = 0.95, immobility bouts: F1, 56 = 1.92, p = 190 \n0.17). These results suggest that changes over time in immobility behavior occur 191 \npredominantly over the first exposure to TSS and are similar in males and females, 192 \ntherefore the sex differences in average immobility scores and bouts in the second TSS 193 \nsession result from sustained coping strategies selected at the beginning of stress, not 194 \nthe rate of adaptation to stress. 195 \nCoping behavior during stress predicts avoidance 196 \nGiven the known roles of sex in risk appraisal, risk taking, and emotional 197 \nreactivity [34, 35], we sought to determine whether the sex-dependent coping behaviors 198 \ndiscovered in the TSS sessions would be associated with post-stress behavior in the 199 \nEPM. We first assessed whether there were sex differences in exploratory behaviors in 200 \nthe EPM. We found no sex differences in total distance traveled (t38 = 0.79, p = 0.43; 201 \nFigure 3A), open arm entries (t38 = 0.50, p = 0.62; Figure 3B), open arm time (t38 = 1.45, 202 \np = 0.15; Figure 3C), or open arm ratio (t38 = 1.63, p = 0.11; Figure 3D).  203 \nOne possible contributor to variability within males and females in avoidance 204 \nbehaviors after stress may be sex differences in the appraisal of and responses to 205 \nstress that directly contribute to the appraisal of threatening contexts after stress. In 206 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n11 \n \norder to test relationships between coping behavior and avoidance, we performed 207 \nsimple linear regressions on average immobility scores and open arm ratios. In males, 208 \nhigher immobility scores during the first TSS session predicted less open arm time, or 209 \ngreater avoidance (Figure 4A; R2 = 0.29, p = 0.014). In the second TSS session, males’ 210 \nimmobility scores and open arm ratios showed a similar relationship but did not reach 211 \nsignificance (Figure 4C; R2 = 0.14, p = 0.10). In females, however, greater immobility 212 \nscores predicted a higher OA ratio, or less avoidance in both the first (Figure 4B; R2 = 213 \n0.27, p = 0.02) and second (Figure 4D; R2 = 0.43, p = 0.0017) TSS sessions. The 214 \nslopes of the immobility score and OA ratio regression were significantly different 215 \nbetween males and females for the first (F1,36 = 12.11, p = 0.0013) and second (F1,36 = 216 \n7.90, p = 0.008) tail suspension sessions. To control for locomotor activity, we 217 \nperformed simple linear regression analyses of distance traveled in the EPM and 218 \nimmobility scores during both tail suspension sessions (Figure 4E-F), and found no 219 \nsignificant relationships in males (TSS1: R2 = 0.062, p = 0.29, TSS2: R2 = 0.13, p = 220 \n0.12) or females (TSS1: R2 = 0.00081, p = 0.91, TSS2: R2 = 0.0047, p = 0.77). 221 \nTogether, these results demonstrate that the relationships between behavior during 222 \nstress and avoidance behavior are both sex and time dependent. 223 \nRelationships between stress coping and avoidance are sex-dependent 224 \nGiven the relationships between avoidance and behavior in the TSS, we tested 225 \nthe collinearity of selected behavioral measures across the tail suspension stressors 226 \nand avoidance behavior (Figure 5). We found that in females, immobility score during 227 \nthe first 10 minutes of TSS1 was positively correlated with the average TSS1 immobility 228 \nscore (r = 0.60, p = 0.005), TSS2 immobility score (r = 0.78, p < 0.0001), and OA ratio (r 229 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n12 \n \n= 0.61, p = 0.005), demonstrating that coping behavior in the first 10 minutes of TSS 230 \ncould predict behavior across both tail suspension sessions in addition to post-stress 231 \navoidance behavior. Immobility scores during the first 10 minutes of the first TSS 232 \nstressor were negatively correlated with the slope of the score over the session (r = -233 \n0.91, p < 0.0001), indicating that lower immobility at the start of the session was 234 \ncorrelated with a greater change in coping over the first stress session. Interestingly, the 235 \nimmobility score slope in females was inversely correlated with OA ratio (r = -0.59, p = 236 \n0.006), which suggests that a higher rate of coping style change in females predicts 237 \nmore avoidance after stress. This is likely explained mostly by the inverse relationship 238 \nbetween immobility in the first 10 minutes of stress and immobility slope, as females 239 \nwho start at a lower immobility score have a higher change in their coping score over 240 \ntime, and the immobility score in the first 10 minutes alone predicts avoidance after 241 \nstress.  242 \nTSS1 and TSS2 immobility scores were positively correlated in males (r = 0.61, p 243 \n= 0.005) and females (r = 0.80, p < 0.0001), which suggests that coping choices within 244 \nanimals are consistent between stress sessions regardless of sex. However, in males, 245 \nwhile immobility in the first 10 minutes of TSS1 was positively correlated with the 246 \naverage immobility score during TSS1 (r = 0.66, p = 0.002) and inversely correlated with 247 \nthe slope of TSS1 immobility score (r = -0.83, p < 0.0001), it did not significantly 248 \ncorrelate with coping behavior in TSS2 (r = 0.24, p = 0.31) or with OA ratio (r = -0.30, p 249 \n= 0.20) unlike the observation in females. Simple linear regression revealed significant 250 \nsex differences between the immobility score in the first 10 minutes of TSS1 and OA 251 \nratio (F1, 36 = 7.68, p = 0.0088), as well as the TSS1 immobility score slope and OA ratio 252 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n13 \n \n(F1, 36 = 4.53, p = 0.040). Taken together, these results suggest that the relationships 253 \nbetween behavioral choices at the beginning of stress and behavior across both stress 254 \nsessions, and the extent to which those behavioral choices predict post-stress 255 \navoidance, are sex-dependent. For females, behavior at the beginning of stress predicts 256 \nthe behavioral profile across both stress sessions and is sufficient to predict avoidance 257 \nafter stress. In males, however, behavior at the beginning of stress is only predictive of 258 \nthe behavioral profile during the first stress session. This suggests that while males and 259 \nfemales engage in similar magnitudes of behavioral flexibility as measured by the 260 \nchange in immobility over TSS, females select a set of behavioral choices at the 261 \nbeginning of stress that are sustained across multiple stress sessions and predict 262 \navoidance after stress, while the initial behavioral strategies in males are not 263 \nnecessarily sustained across both sessions and do not predict avoidance.       264 \n We considered one possible source of behavioral variability contributing to sex 265 \ndifferences in coping behavior- adoption of tail climbing. We assessed whether animals 266 \ntail climbed at any point during TSS sessions and found that 46.67% of females 267 \nengaged in tail climbing at some point during the first tail suspension session and 268 \n36.67% engaged in tail climbing during the second tail suspension session, while only 269 \n7.14% of males engaged in tail climbing during either TSS session (Figure 6). The 270 \nproportion of tail climbing was significantly different between males and females during 271 \nboth the first (p = 0.0010) and second (p = 0.011) TSS sessions (Fisher’s exact test; 272 \nFigure 6A-D). Within females, there was a significant main effect of tail climbing status 273 \non immobility score, with tail climbing females having a significantly lower immobility 274 \nscore than non-tail-climbers (F1, 56 = 64.43, p < 0.0001; Figure 6E). Because DBScorer 275 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n14 \n \ndoes not differentiate between head down and tail climb struggling bouts, it is unclear 276 \nwhat proportion of each total immobility score is attributable to tail climbing vs. head 277 \ndown struggling. However, it is clear that in this configuration tail climbing does not 278 \nresult in a broadly immobile phenotype during tail suspension and that it is a sex-biased 279 \nstrategy that may contribute to adaptation during stress.  280 \n 281 \nDiscussion 282 \nIn this study, we tested whether sex differences in coping strategies emerge 283 \nduring a repeated stressor, and if they are associated with avoidance behaviors after 284 \nstress. Active coping is often seen as a beneficial or adaptive choice that promotes 285 \nresilience after stress, whereas passive coping indicates despair or a “depressive-like\" 286 \nphenotype [36]. However, in an inescapable stressor, the choice to sustain a coping 287 \nstrategy that expends considerable energy may reflect a failure to learn. Sustained 288 \nactive coping may lead to pathophysiological plasticity in neural circuitry that contributes 289 \nto avoidance, reward, motivation, and aversion. This is particularly important in the 290 \nSCVS paradigm where animals are exposed to a series of inescapable stressors and 291 \nmust repeatedly select strategies that promote adaptation across each stressor. We 292 \nfocused on behavior in the TSS sessions of SCVS, but it is likely that behavior is 293 \ninfluenced by previous and ongoing experience across each stressor and would differ 294 \nfrom behavior during isolated tail suspension tests. These prior and ongoing 295 \nexperiences may be important for the observed sex differences in the relationship 296 \nbetween coping and avoidance after stress, specifically given observed sex differences 297 \nin behavioral strategies during other inescapable stressors [15, 17]. 298 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n15 \n \nOne strength of using the SCVS paradigm to ask this question is that each 299 \nstressor is repeated, which allows comparison of behavior during repeated stress 300 \nsessions to ascertain changes in coping strategy. Our data demonstrates that sex 301 \ndifferences in coping strategy are a function of both time and repeated exposure to 302 \nstress. While the choice of coping strategy changed over the duration of the first stress 303 \nsession in both males and females, sex differences were observed in overall immobility 304 \nscore only in the second tail suspension session, where the choice of strategy at the 305 \nbeginning of the stressor was sustained over the duration of the session. This suggests 306 \nthat there may be sex differences in mechanisms that support sustained motivation and 307 \nlearning across repeated inescapable stress sessions.  308 \nOur study demonstrates that relationships between avoidance behavior and 309 \ncoping behavior across stress sessions are a meaningful sex-dependent outcome of 310 \nSCVS and predict behavioral variability within each sex. Despite no sex differences in 311 \nthe overall immobility score during the first TSS session or EPM open arm ratio, higher 312 \nimmobility scores predicted greater avoidance in males but lower avoidance in females. 313 \nOn the second TSS session, when females engaged in more active coping than males, 314 \nhigher immobility scores were only a significant predictor of avoidance in females, but 315 \nnot males. Importantly, some studies have identified sex differences in baseline 316 \nlocomotion of mice and rats during the EPM, which can confound interpretations of 317 \nexploratory behavior [37, 38]. However, our study shows that there is no relationship 318 \nbetween the immobility score and locomotion in the EPM. This suggests that the 319 \nrelationship between coping strategy during tail suspension and avoidance is not 320 \nreducible to sex-biased trends in activity levels. 321 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n16 \n \nAssays like the tail suspension and forced swim tests often report total time spent 322 \nimmobile, or inversely, time spent on escape-oriented behaviors. This data suggests, 323 \nhowever, that the number of transitions between immobility and escape-oriented 324 \nbehavior may reveal important information that total time in each state may not capture. 325 \nDuring the first 10 minutes of the first TSS session, there was no sex difference in the 326 \noverall immobility score, but females made significantly more transitions between 327 \nimmobility and struggling as measured by the number of immobility bouts. The 328 \ndifferences between immobility scores and immobility bouts may reflect a distinction 329 \nbetween action initiation for short escape-oriented bouts in comparison to the 330 \nmotivational vigor required for sustaining longer struggling bouts [39, 40].   331 \nResearchers utilizing the tail suspension test for screening antidepressants and 332 \nmeasuring stress-induced behavioral changes often highlight the challenge of high tail 333 \nclimbing rates in C57BL/6J mice, and many exclude animals who tail climb [33, 41-43]. 334 \nWe did not apply this exclusion criterion in our study for several key reasons. Tail 335 \nclimbing is a coping strategy- it does not permit the animal to escape and requires 336 \nenergy expenditure in a similar way to head-down escape-oriented motion. Importantly 337 \nfor our study, we also found that tail climbing behavior is sex-biased. Nearly half of 338 \nfemales but almost no males exhibit tail climbing at some point during the tail 339 \nsuspension stressor. Removing tail climbing animals would introduce a sex-biased 340 \nexclusion criterion that would preclude a full assessment of how sex-biased coping 341 \nstrategies contribute to behavioral outcomes. It is possible that the vestibular and 342 \nproprioceptive experience in an entirely head-down position is distinct and that repeated 343 \ntail climbing promotes greater motivation to struggle, both of which may be important for 344 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n17 \n \nunderstanding neural mechanisms engaged by struggling behavior. As Shansky and 345 \nMurphy (2021) have emphasized, including females in studies warrants consideration 346 \nthat common behavioral endpoints based on assays that were originally tested 347 \nexclusively in males may not reflect the range of meaningful behavioral strategies in 348 \nfemales [21].    349 \nWhile the EPM is generally understood to test a conserved conflict between an 350 \naversion to open or elevated space and exploration, others argue that it is also testing 351 \nthigmotaxis mediated by the somatosensory system, primarily in the closed arms of the 352 \nmaze [44]. Studies have identified sex differences in thigmotaxis across anxiogenic 353 \nenvironments, which may contribute to sex differences in exploratory behavior in the 354 \nEPM [45, 46]. Neural circuits that assess threat and support coping strategy selection 355 \nduring stress may be altered by repeated unsuccessful escape attempts during stress, 356 \nwhich could directly inform future threat assessments in the EPM. These circuits are 357 \nreliant upon sensory input and interoceptive signals during stress and motivated 358 \nbehavior, and in anxiogenic environments [47-51]. Thus, appraisal of risk and safety 359 \nsignals may be altered by coping choices to promote adaptation after chronic stress. 360 \nFurthermore, each stressor was performed in groups with cage mates, so while animals 361 \ncould not make physical contact in TSS, it is likely that they can see, smell, and hear 362 \ntheir cage mates during stress. Prior work has demonstrated that a physical barrier or 363 \nbeing tested alone does not alter behavior in males during the 6-minute tail suspension 364 \ntest [52], but it is unclear whether this would be true in females, and whether it would 365 \napply in a prolonged stress session. Given the sex-specific roles of social stress on 366 \nphysiological and behavioral outcomes [18], future studies to test the role of social 367 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n18 \n \ncontext and sensory cues during stress on sex-specific coping strategies and avoidance 368 \nbehaviors would be beneficial.  369 \nNeither coping strategies nor avoidance behavior can be interpreted under 370 \nbinaries- the more likely case is that an accumulation of maladaptive choices, 371 \nparticularly when those behaviors are sustained over time, can contribute to reduced 372 \nfitness or pathophysiological states. Why the relationship between coping choices and 373 \navoidance would be sex-biased is likely attributable to differences in the evolutionary 374 \nroles of escape behaviors during inescapable stress and approach behaviors, which 375 \nengage overlapping neural circuitry [53]. Fluctuations in sex hormones across the 376 \nestrous cycle and between animals of different social rank may contribute to baseline 377 \nstress and anxiety levels, and act directly on limbic and striatal circuitry that drive threat 378 \nappraisal and memory [54-57]. The BNST is a known substrate for both stress coping 379 \nand avoidance behaviors [48, 49] with established sex differences in contributions to 380 \nthreat processing [58]. The locus coeruleus and ventral tegmental area, hubs for 381 \nrobustly stress-sensitive noradrenergic and dopaminergic circuitry respectively, exhibit 382 \nstructural and physiological sex differences that may also play direct roles in stress 383 \nprocessing that are particularly relevant in convergent inputs to limbic and cortical 384 \nregions [59]. Future studies exploring specific contributions of sex hormones and sex 385 \nbiased regions to learning and adaptation during stress coping could advance concepts 386 \nof the roles of sex in stress coping and avoidance behavior.  387 \nSummary/ Conclusions 388 \nOur study demonstrates that coping strategies are sex-specific and dynamic across a 389 \nsingle stress session and between repeated stress exposures. Furthermore, coping 390 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n19 \n \nstrategies and the change in coping strategy predicts avoidance behavior in a sex-391 \ndependent fashion. While coping strategies are often not recorded and scored during 392 \nstress, they may provide a key readout for the progression of behavioral changes 393 \nacross chronic stress paradigms, and may contribute to elucidating the engagement of 394 \nmechanisms that promote divergent and convergent sex-specific mechanisms.    395 \nDeclarations 396 \nEthics approval and consent to participate  397 \nNot applicable 398 \nConsent for publication 399 \nNot applicable 400 \nAvailability of data and materials 401 \nThe datasets used and analyzed during the current study are available from the 402 \ncorresponding author on reasonable request. 403 \nCompeting interests 404 \nThe authors declare that they have no competing interests 405 \nFunding 406 \nThis work was funded by NIH grants R01MH122712 and R01MH122712S1. 407 \nAuthor’s contributions 408 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n20 \n \nKP and AMP designed the study; KP collected and analyzed data; KP and AMP 409 \ninterpreted data; KP drafted the paper; AMP edited the paper.   410 \nAcknowledgments 411 \nWe would like to acknowledge Dr. Paul Marvar for use of the Coulbourn boxes. 412 \nAuthor Information 413 \nDepartment of Pharmacology and Physiology, George Washington University School of 414 \nMedicine and Health Sciences, Washington, DC 20037 415 \nReferences 416 \n1. McEwen BS. Mood disorders and allostatic load. Biol Psychiatry. 2003;54 3 :200–7. 417 \n2. McEwen BS, Akil H. Revisiting the stress concept: implications for affective disorders. 418 \nJournal of Neuroscience. 2020;40 1 :12–21. 419 \n3. Douma EH, de Kloet ER. Stress-induced plasticity and functioning of ventral 420 \ntegmental dopamine neurons. 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It is made \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n28 \n \nFigure Legends 589 \n 590 \nFigure 1. Stress coping behavior during the tail suspension phases of SCVS is 591 \nsex-dependent 592 \nA. Experimental schematic for subchronic variable stress (SCVS). Behavior was video 593 \nrecorded during the first and second tail suspension sessions (TSS1 and TSS2). B 594 \nAverage immobility score and C total immobility bouts during TSS1 and TSS2. D 595 \nImmobility score during the first 10 minutes of TSS1. E Total immobility bouts during the 596 \nfirst 10 minutes of TSS1. # indicates significant main effect: # p < 0.05, ## p < 0.01, ### 597 \np < 0.001, #### p < 0.0001. Pairwise comparisons were performed with Mann-Whitney 598 \nU test. N=28 males, 30 females. * p<0.05, ** p<0.01. 599 \n 600 \nFigure 2. Stress coping behavior changes within and between tail suspension 601 \nsessions 602 \nA. Immobility score and B immobility bouts across TSS1, within 10-minute bins. C. 603 \nImmobility score and D Immobility bouts across TSS2, within 10-minute bins. Two-way 604 \nANOVA followed by Dunnett’s multiple comparisons test where significant main effects 605 \nof time were present. E Slopes of immobility score and F slopes of immobility bouts over 606 \ntime for each animal. Two-way ANOVA. # indicates significant main effect: # p < 0.05, 607 \n## p < 0.01, ### p < 0.001, #### p < 0.0001. * indicates significant pairwise difference 608 \nin comparison to first 10 minutes. * p < 0.05, ** p < 0.01, *** p < 0.001, ****p < 0.0001. N 609 \n= 28 males, 30 females. 610 \n 611 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n29 \n \nFigure 3. No sex differences in avoidance behaviors in the elevated plus maze 612 \nAfter SCVS, A. total distance traveled (males: 12.40 ± 0.61 m, females: 11.75 ± 0.55 m) 613 \nin the elevated plus maze. B. open arm entries (males: 11.65 ± 1.64, females: 12.85 ± 614 \n1.77, C. open arm time (males: 39.40 ± 7.39 s, females: 56.78 ± 9.40 s) and D. open 615 \narm ratio (males: 15.05 ± 2.70%, females: 21.92 ± 3.24%) were measured in males and 616 \nfemales. Open arm ratio = [(open arm time/ open arm time + closed arm time) * 100]. N 617 \n= 20 females, 20 males. 618 \n 619 \nFigure 4. Relationships between stress coping and post-stress avoidance 620 \nbehavior are sex-specific 621 \nSimple linear regressions between OA ratio and immobility score during TSS1 in males 622 \nA and females B, and OA ratio and immobility score during TSS2 in males C and 623 \nfemales D. Simple linear regression of distance traveled in the elevated plus maze and 624 \nimmobility score during TSS1 E and TSS2 F. R2 and p-values listed on graph inset. N= 625 \n20 females, 20 males. 626 \n 627 \nFigure 5. Covariance between immobility measures and avoidance behaviors is 628 \nsex-dependent 629 \nPearson r correlation coefficients between the first 10 minutes of TSS1, TSS1 average 630 \nimmobility score, TSS1 immobility slope, TSS2 average immobility score, and open arm 631 \nratio in females A and males B. N= 20 females, 20 males. * p < 0.05, ** p < 0.01, *** p < 632 \n0.001, ****p < 0.0001. 633 \n 634 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n30 \n \nFigure 6. Tail climbing rates are greater in females during tail suspension stress 635 \nProportions of tail climbing at any point during TSS1 in females A and males B and 636 \nduring TSS2 in females C and males D. N = 28 males, 30 females. E. Immobility scores 637 \nare higher in females who do not tail climb at any point during TSS. Two-way ANOVA. 638 \n#### indicates main effect, p < 0.0001. N = 11-19/group. 639 \n 640 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\nA\nB C\nDay 1\nFoot Shock\nDay 2\nTail \nSuspension \n(TSS1)\nDay 3\nRestraint\nDay 4\nFoot Shock\nDay 5\nTail \nSuspension \n(TSS2)\nDay 6\nRestraint\nSubchronic Variable Stress (SCVS)\nT\nS\nS\n \n1\nT\nS\nS\n \n2\n \n0\n2\n0\n0\n4\n0\n0\n6\n0\n0\nF\ne\nm\na\nl\ne\nM\na\nl\ne\n#\nImmobility Bouts\nD\nF\ne\nm\na\nl\ne\n \nM\na\nl\ne\n0\n5\n0\n1\n0\n0\n1\n5\n0\nT\nS\nS\n1\n:\n \nF\ni\nr\ns\nt\n \n1\n0\n \nM\ni\nn\nu\nt\ne\ns\n✱\nImmobility Bouts\nE\nImmobility Score (%)\nF\ne\nm\na\nl\ne\nM\na\nl\ne\n0\n5\n0\n1\n0\n0\nT\nS\nS\n1\n:\n \nF\ni\nr\ns\nt\n \n1\n0\n \nM\ni\nn\nu\nt\ne\ns\nT\nS\nS\n \n1\nT\nS\nS\n \n2\n \n0\n5\n0\n1\n0\n0\nF\ne\nm\na\nl\ne\nM\na\nl\ne\n#\n#\n#\n#\n#Immobility Score (%)\nFig. 1\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\nA\nImmobility Score (%)\nB\nC\nImmobility Bouts\nE\nImmobility Bout Slope\nD\nImmobility Score Slope\nF\n0\n1\n2\n3\n4\n5\n6\n5\n0\n6\n0\n7\n0\n8\n0\n9\n0\n1\n0\n0\nT\nS\nS\n \n1\nF\ne\nm\na\nl\ne\nM\na\nl\ne\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n#\n#\n#\n#\n0\n1\n2\n3\n4\n5\n6\n5\n0\n6\n0\n7\n0\n8\n0\n9\n0\n1\n0\n0\nT\nS\nS\n \n2\nM\na\nl\ne\nF\ne\nm\na\nl\ne\n##\nImmobility Score (%)\n0\n1\n2\n3\n4\n5\n6\n0\n1\n0\n2\n0\n3\n0\n4\n0\n5\n0\n6\n0\nT\nS\nS\n \n2\n1\n0\n-\nM\ni\nn\nu\nt\ne\n \nB\ni\nn\n*\n*\n*\n*\n*\n*\n*\n#\n#\n#\n#\nF\ne\nm\na\nl\ne\nM\na\nl\ne\n##\n0\n1\n2\n3\n4\n5\n6\n0\n1\n0\n2\n0\n3\n0\n4\n0\n5\n0\n6\n0\nT\nS\nS\n \n1\n1\n0\n-\nM\ni\nn\nu\nt\ne\n \nB\ni\nn\n#\n#\n#\n#\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\nF\ne\nm\na\nl\ne\nM\na\nl\ne\nImmobility Bouts\nT\nS\nS\n \n1\nT\nS\nS\n \n2\n \n-\n1\n0\n0\n1\n0\n#\n#\n#\n#\nT\nS\nS\n \n1\nT\nS\nS\n \n2\n \n-\n2\n0\n-\n1\n0\n0\n1\n0\n2\n0\nM\na\nl\ne\n#\n#\n#\n#\nF\ne\nm\na\nl\ne\nM\na\nl\ne\nFig. 2\nF\ne\nm\na\nl\ne\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\nA B\nC D\nF\ne\nm\na\nl\ne\nM\na\nl\ne\n0\n5\n1\n0\n1\n5\n2\n0\nT\no\nt\na\nl\n \nD\ni\ns\nt\na\nn\nc\ne\n \nT\nr\na\nv\ne\nl\ne\nd\nDistance (m)\nF\ne\nm\na\nl\ne\nM\na\nl\ne\n0\n1\n0\n2\n0\n3\n0\n4\n0\nO\np\ne\nn\n \nA\nr\nm\n \nE\nn\nt\nr\ni\ne\ns\nF\ne\nm\na\nl\ne\nM\na\nl\ne\n0\n5\n0\n1\n0\n0\n1\n5\n0\n2\n0\n0\nO\np\ne\nn\n \nA\nr\nm\n \nT\ni\nm\neseconds\nF\ne\nm\na\nl\ne\nM\na\nl\ne\n0\n2\n0\n4\n0\n6\n0\nO\np\ne\nn\n \nA\nr\nm\n \nR\na\nt\ni\no\n \n(\n%\n)\nFig. 3\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\nA B\nOA Ratio (%)\nI\nm\nm\no\nb\ni\nl\ni\nt\ny\n \nS\nc\no\nr\ne\n \n(\n%\n)OA Ratio (%)\n5\n0\n6\n0\n7\n0\n8\n0\n9\n0\n1\n0\n0\n0\n1\n0\n2\n0\n3\n0\n4\n0\n5\n0\n6\n0\n7\n0\nT\nS\nS\n \n2\n:\n \nF\ne\nm\na\nl\ne\ns\nI\nm\nm\no\nb\ni\nl\ni\nt\ny\n \nS\nc\no\nr\ne\n \n(\n%\n)\nR\n2\n=\n0\n.\n4\n3\np\n \n=\n \n0\n.\n0\n0\n1\n7\n5\n0\n6\n0\n7\n0\n8\n0\n9\n0\n1\n0\n0\n0\n1\n0\n2\n0\n3\n0\n4\n0\n5\n0\n6\n0\n7\n0\nT\nS\nS\n1\n:\n \nF\ne\nm\na\nl\ne\ns\nR\n2\n \n=\n \n0\n.\n2\n7\np\n \n=\n \n0\n.\n0\n2\n0\n5\n0\n6\n0\n7\n0\n8\n0\n9\n0\n1\n0\n0\n0\n1\n0\n2\n0\n3\n0\n4\n0\n5\n0\n6\n0\n7\n0\nT\nS\nS\n \n1\n:\n \nM\na\nl\ne\ns\nR\n2\n=\n0\n.\n2\n9\np\n \n=\n \n0\n.\n0\n1\n4\n5\n0\n6\n0\n7\n0\n8\n0\n9\n0\n1\n0\n0\n0\n1\n0\n2\n0\n3\n0\n4\n0\n5\n0\n6\n0\n7\n0\nT\nS\nS\n \n2\n:\n \nM\na\nl\ne\ns\nR\n2\n=\n0\n.\n1\n4\np\n \n=\n \n0\n.\n1\n0\n3\n5\n0\n6\n0\n7\n0\n8\n0\n9\n0\n1\n0\n0\n0\n5\n1\n0\n1\n5\n2\n0\n2\n5\nM\na\nl\ne\nF\ne\nm\na\nl\ne\nR\n2\n \n=\n \n0\n.\n0\n0\n0\n8\n1\np\n \n=\n \n0\n.\n9\n1\nR\n2\n \n=\n \n0\n.\n0\n6\n2\np\n \n=\n \n0\n.\n2\n9EPM Distance (m)\n5\n0\n6\n0\n7\n0\n8\n0\n9\n0\n1\n0\n0\n0\n5\n1\n0\n1\n5\n2\n0\n2\n5\nT\nS\nS\n2\n \nI\nm\nm\no\nb\ni\nl\ni\nt\ny\n \nS\nc\no\nr\ne\n \n(\n%\n)\nM\na\nl\ne\nF\ne\nm\na\nl\ne\nR\n2\n \n=\n \n0\n.\n1\n3\np\n \n=\n \n0\n.\n1\n2\nR\n2\n \n=\n \n0\n.\n0\n0\n4\n7\np\n \n=\n \n0\n.\n7\n7\nTSS1 Immobility Score (%)\nI\nm\nm\no\nb\ni\nl\ni\nt\ny\n \nS\nc\no\nr\ne\n \n(\n%\n)\nI\nm\nm\no\nb\ni\nl\ni\nt\ny\n \nS\nc\no\nr\ne\n \n(\n%\n)\nC D\nEPM Distance (m)\nE F\nOA Ratio (%) OA Ratio (%)\nFig. 4\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\n1\n.\n0\n0\n0\n.\n6\n0\n-\n0\n.\n9\n1\n0\n.\n7\n8\n0\n.\n6\n1\n0\n.\n6\n0\n1\n.\n0\n0\n-\n0\n.\n4\n8\n0\n.\n8\n0\n0\n.\n5\n2\n-\n0\n.\n9\n1\n-\n0\n.\n4\n8\n1\n.\n0\n0\n-\n0\n.\n7\n2\n-\n0\n.\n5\n9\n0\n.\n7\n8\n0\n.\n8\n0\n-\n0\n.\n7\n2\n1\n.\n0\n0\n0\n.\n6\n6\n0\n.\n6\n1\n0\n.\n5\n2\n-\n0\n.\n5\n9\n0\n.\n6\n6\n1\n.\n0\n0\nT\nS\nS\n1\n \nF\ni\nr\ns\nt\n \n1\n0\n \nM\ni\nn\ns\nT\nS\nS\n1\n \nA\nv\ne\nr\na\ng\ne\nT\nS\nS\n1\n \nS\nl\no\np\ne\nT\nS\nS\n2\n \nA\nv\ne\nr\na\ng\ne\nO\nA\n \nR\na\nt\ni\no\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\nI\nm\nm\no\nb\ni\nl\ni\nt\ny\n \na\nn\nd\n \nA\nv\no\ni\nd\na\nn\nc\ne\n:\n \nF\ne\nm\na\nl\ne\ns\n-\n1\n.\n0\n-\n0\n.\n5\n0\n0\n.\n5\n1\n.\n0\nT\nS\nS\n1\n \nF\ni\nr\ns\nt\n \n1\n0\n \nM\ni\nn\ns\nT\nS\nS\n1\n \nA\nv\ne\nr\na\ng\ne\nT\nS\nS\n1\n \nS\nl\no\np\ne\nT\nS\nS\n2\n \nA\nv\ne\nr\na\ng\ne\nO\nA\n \nR\na\nt\ni\no\n1\n.\n0\n0\n0\n.\n6\n6\n-\n0\n.\n8\n3\n0\n.\n2\n4\n-\n0\n.\n3\n0\n0\n.\n6\n6\n1\n.\n0\n0\n-\n0\n.\n3\n1\n0\n.\n6\n1\n-\n0\n.\n5\n4\n-\n0\n.\n8\n3\n-\n0\n.\n3\n1\n1\n.\n0\n0\n0\n.\n0\n8\n0\n.\n1\n0\n0\n.\n2\n4\n0\n.\n6\n1\n0\n.\n0\n8\n1\n.\n0\n0\n-\n0\n.\n3\n7\n-\n0\n.\n3\n0\n-\n0\n.\n5\n4\n0\n.\n1\n0\n-\n0\n.\n3\n7\n1\n.\n0\n0\nT\nS\nS\n1\n \nF\ni\nr\ns\nt\n \n1\n0\n \nM\ni\nn\ns\nT\nS\nS\n1\n \nA\nv\ne\nr\na\ng\ne\nT\nS\nS\n1\n \nS\nl\no\np\ne\nT\nS\nS\n2\n \nA\nv\ne\nr\na\ng\ne\nO\nA\n \nR\na\nt\ni\no\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\nI\nm\nm\no\nb\ni\nl\ni\nt\ny\n \na\nn\nd\n \nA\nv\no\ni\nd\na\nn\nc\ne\n:\n \nM\na\nl\ne\ns\n*\n*\n*\n-\n1\n.\n0\n-\n0\n.\n5\n0\n0\n.\n5\n1\n.\n0\nT\nS\nS\n1\n \nF\ni\nr\ns\nt\n \n1\n0\n \nM\ni\nn\ns\nT\nS\nS\n1\n \nA\nv\ne\nr\na\ng\ne\nT\nS\nS\n1\n \nS\nl\no\np\ne\nT\nS\nS\n2\n \nA\nv\ne\nr\na\ng\ne\nO\nA\n \nR\na\nt\ni\no\nA\nB\nFig. 5\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint \n\nF\ne\nm\na\nl\ne\ns\n \nT\nS\nS\n2\n6\n3\n.\n3\n3\n%\n \n \nN\no\n \nT\na\ni\nl\n \nC\nl\ni\nm\nb\ni\nn\ng\n3\n6\n.\n6\n7\n%\n \n \nT\na\ni\nl\n \nC\nl\ni\nm\nb\ni\nn\ng\nM\na\nl\ne\ns\n \nT\nS\nS\n2\n9\n2\n.\n8\n6\n%\n \n \nN\no\n \nT\na\ni\nl\n \nC\nl\ni\nm\nb\ni\nn\ng\n7\n.\n1\n4\n%\n \n \nT\na\ni\nl\n \nC\nl\ni\nm\nb\ni\nn\ng\nF\ne\nm\na\nl\ne\ns\n \nT\nS\nS\n1\n5\n3\n.\n3\n3\n%\n \n \nN\no\n \nT\na\ni\nl\n \nC\nl\ni\nm\nb\ni\nn\ng\n4\n6\n.\n6\n7\n%\n \n \nT\na\ni\nl\n \nC\nl\ni\nm\nb\ni\nn\ng\nM\na\nl\ne\ns\n \nT\nS\nS\n1\n9\n2\n.\n8\n6\n%\n \n \nN\no\n \nT\na\ni\nl\n \nC\nl\ni\nm\nb\ni\nn\ng\n7\n.\n1\n4\n%\n \n \nT\na\ni\nl\n \nC\nl\ni\nm\nb\ni\nn\ng\nT\na\ni\nl\n \nC\nl\ni\nm\nb\ni\nn\ng\nN\no\nt\n \nT\na\ni\nl\n \nC\nl\ni\nm\nb\ni\nn\ng\n0\n2\n0\n4\n0\n6\n0\n8\n0\n1\n0\n0\nF\ne\nm\na\nl\ne\n \nT\nS\nS\n1\nF\ne\nm\na\nl\ne\n \nT\nS\nS\n2\n#\n#\n#\n#\nImmobility Score (%)\nA B\nC D\nE\nFig. 6\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.31.697182doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}