CRISPR-mediated knockdown of oxytocin receptor in extended amygdala reduces stress-induced social avoidance in female California mice

This is the author accepted manuscrip of Cea Salazar VI, Boender AJ, Seelke AMH, Gaard L, Mederos SL, Rogers S, Gutierrez XZ, Bales KL, Young LJ, Trainor BC. CRISPR-mediated knockdown of oxytocin receptor in extended amygdala reduces stressinduced social avoidance in female California mice. Horm Behav. 2025 Nov;176:105845. doi: 10.1016/j.yhbeh.2025.105845. Epub 2025 Oct 28. PMID: 41151122. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 CRISPR-mediated knockdown of oxytocin receptor in extended amygdala reduces stress-induced social avoidance in female California mice Valentina I. Cea Salazaraa, Arjen J. Boenderb,c Adele M. H. Seelked, Liam Gaardd, Sabrina L. Mederose, Sophia Rogersd, Xiomara Z. Gutierrezd, Karen L. Balesd,f, Larry J. Youngg,h,i, Brian C. Trainora,c,e a Neuroscience Graduate Group, University of California, Davis, USA b Achucarro Basque Center for Neuroscience, Leioa, Spain c Ikerbasque, Basque Foundation for Science, Bilbao, Spain d Department of Psychology, University of California, Davis, USA e Animal Behavior Graduate Group, University of California, Davis, USA f Department of Neurobiology, Physiology, and Behavior, University of California, Davis, USA g Emory National Primate Research Center, Emory University, Atlanta, GA, USA h Center for Translational Social Neuroscience, Emory University, Atlanta, GA, USA I Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, GA, USA Correspondence: [email protected] 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 Abstract Oxytocin receptors (OTRs) within the extended amygdala and nucleus accumbens (NAc) have been implicated in modulating social behaviors, particularly following stress. The effects of OTR could be mediated by modulating the activity of pre-synaptic axon terminals or via receptors in post-synaptic neurons or glia. Using a viral-mediated CRISPR/Cas9 gene editing system in female California mice (Peromyscus californicus), we selectively knocked down OTR in the anteromedial bed nucleus of the stria terminalis (BNST) or NAc to examine their roles modulating social approach and vigilance behaviors. Knockdown of OTR in the BNST attenuated stress-induced decreases of social approach and had less robust effects on vigilance when interacting with a target mouse behind a wire barrier. In this large arena, where mice could control their proximity to a target mouse, BNST OTR knockdown also increased investigation of a non-social stimulus (empty cage). Behavioral effects of BNST OTR knockdown were weaker in the small arena where focal mice physically interacted with target mice. Interestingly, OTR knockdown in the NAc, reduced stress-induced social vigilance without affecting social approach. These effects could mediate altered encoding of socially aversive experiences, as knockdown manipulations were performed before stress exposure. Together, these results highlight effects of local OTR on social behavior that are region-specific. 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 1 Introduction Individuals affected by anxiety disorders experience significant distress in social situations that can lead to self-isolation and avoidance behaviors (Abramowitz & Deacon, 2010). Women are twice as likely to develop anxiety disorders compared to men (Kessler et al., 2012). While there are existing pharmaceutical and behavioral therapeutic interventions, ~40% do not respond to these treatments (Williams & Trainor, 2018). Oxytocin is a neuropeptide that modulates social and emotional behaviors (Gimpl & Fahrenholz, 2001) and has been considered as a potential alternative therapeutic to existing antidepressants (MacDonald & Feifel, 2013 and 2014; Modi & Young., 2012; Gutkowska & Jankowski, 2012; Giovanna et al., 2020; van Zuiden et al., 2017; Schultebraucks et al., 2022; Rashidi et al., 2025). However, the field has struggled to explain findings where intranasal oxytocin sometimes decreases anxiety symptoms but in other cases increases anxiety and other psychiatric symptoms (Tabak et al., 2022; Schultebraucks et al., 2022). A potential explanation for these results is the social salience hypothesis of oxytocin (Bartz et al., 2011; Shamay-Tsoory & Abu-Akel, 2016), which proposes that oxytocin can modulate behaviors by enhancing attention to both positive and negative social contexts. Several lines of evidence indicate that oxytocin promotes salience by acting in discrete neural circuits to drive these dynamic responses (Steinman et al., 2016, 2019a; Nasanbuyan et al., 2018; Osakada et al. 2024; Duque-Wilckens et al., 2018). In appetitive social contexts, activation of oxytocin receptors (OTR) in the mesolimbic dopamine system play a key role in promoting social approach behaviors. The nucleus accumbens (NAc) modulates motivational and learning processes (Salgado & Kaplitt, 2015), and activation of OTR within the NAc facilitates social approach (Williams et al., 2020), social learning (Nardou et al., 2019), pair bonding (Liu & Wang, 2003; Rigney et al., 2025), and social novelty seeking (Smith et al., 2017). Activation of OTR in the ventral tegmental area (VTA) has also been found to modulate social motivation (Borland et al., 2017) and learning (Hung et al., 2017). It was hypothesized that oxytocin acting in the mesolimbic dopamine system facilitates social salience in aversive social contexts (Shamay-Tsoory & Abu-Akel, 2016). However, recent evidence suggests that OTR in the extended amygdala may play an important role during stressful social contexts. Social defeat stress activates a population of oxytocin neurons in the medioventral bed nucleus of the stria terminalis (BNSTmv) in Peromyscus californicus (California mice) (Steinman et al., 2016) and C57Bl/6 J mice (Nasanbuyan et al., 2018). The BNST modulates anxiety-related and social behaviors (Lebow & Chen, 2016) and several subregions have strong molecular (Gegenhuber et al., 2022) and anatomical (Campi et al., 2013; Allen & Gorski, 1990) sex differences. Antisense knockdown of oxytocin in the BNST prevented defeat stress-induced social avoidance and social vigilance in female California mice (Duque-Wilckens et al., 2020). These BNST oxytocin neurons project to the anteromedial BNST (BNSTam) where activation of OTR promotes avoidance and vigilance (Duque-Wilckens et al., 2018; Luo et al., 2022). Oxytocin can also promote aversive responses in non-social contexts, as oxytocin in the dorsolateral BNST (BNSTdl) enhanced fear-potentiated startle (FPS) response in rats (Moaddab & Dabrowska, 2017). Together these data suggested that distinct neural circuits mediate the effects of oxytocin in appetitive and aversive social contexts (Steinman et al., 2019a). 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 Most of the studies reviewed above relied on pharmacological manipulations to target OTR function. Pharmacological approaches target both locally expressed post-synaptic receptors and pre-synaptic receptors. Importantly, at least some behavioral effects of OTR can be mediated by pre-synaptic receptors. Dölen and colleagues showed that the behavioral effects of OTR antagonists infused into the NAc were mediated by OTR expressed on presynaptic terminals originating from the dorsal raphe nucleus (Dölen et al., 2013; Nardou et al., 2023). These studies were performed in C57Bl/6 J mice and utilized transgenic mouse lines to specifically target different populations of OTR. The CRISPR/Cas9 gene-editing systems provides a mechanism to perform similar manipulations in species for which transgenic lines are not available. A viral-mediated CRISPR/Cas9-based tool was developed to target OTR expression across a variety of rodent species used as models to study social behavior (Boender et al., 2023). When a guide RNA targeting the Oxtr gene was expressed with Cas9, OTR binding was reduced across six different rodent species, including California mice. We used this gene editing system to determine whether locally expressed OTR in the BNST or NAc modulate social approach and social vigilance in female California mice. This viral construct was recently used in a male spiny mouse model in the NAc, which reduced huddling behaviors with strangers as well as some feeding behaviors identifying a role for OTRs in non-reproductive contexts (Fricker et al., 2025). California mice are monogamous rodents in which paired males and females defend joint territories. The high levels of aggression in female California mice make this species ideal for studying the effects of social defeat stress (SDS) in both males and females without the use of CD1 aggressive male mice generally used in C57BI/6J models (Steinman & Trainor, 2017; Lake & Trainor, 2024). In this study we assessed the impact of OTR knockdown on behavior before and after stress using two behavioral assays in female California mice. Focal mice were tested in a large arena with an unfamiliar female stress-naïve target mouse confined to a small wire cage, which allows for precise measurements of social approach and social vigilance. Social vigilance is a risk assessment behavior in which an individual avoids but attends to a social context (Walker et al., 2023; Wright et al., 2020). Social approach is assessed by time spent within one body length of the cage. Focal mice were also tested in a smaller arena with freely moving stress-naïve and aggressive SDS target mice. This test is ideal for the observation of affiliative and defensive behaviors. Previous studies showed that social approach and social vigilance are modulated by OTR acting within complementary networks that include the BNST and NAc (Duque-Wilckens et al., 2018 and 2020; Luo et al., 2022; Williams et al., 2020). We used a viral strategy to preform CRISPR-Cas9 editing of the oxytocin receptor target gene. We refer to reductions of OTR binding in CRISPR treated groups as “knockdown” within a specific brain region and examined whether changes in locally expressed OTR corresponded to changes in social behavior before and after social defeat stress. We used two behavioral assays to assess the roles of OTR on social approach as well as ethological measures of social interaction. 2 Materials and methods 2.1 Animals 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 All studies were conducted using California mice at the University of California, Davis (UCD). Protocols were approved by the UCD Institutional Animal Care and Use Committee (IACUC). Adult (90 days old) female California mice were studied. Mice were housed with same-sex cagemates after weaning in groups of 2 to 4 within clear polypropylene cages. Cages were set up with Sani-Chip bedding (Harlan Laboratories, Indianapolis, IN, USA) and 2 nestlets per cage (Ancare, Bellmore, NY, USA). Mice were kept on a 16L:8D light cycle and had ad libitum access to food and water. Viral infusion/surgery were conducted in light cycle, while behavioral testing and stress exposure were performed during the dark cycle. 2.2 Intracranial surgeries Anesthesia was induced via exposure to a 5 % isoflurane mixture with oxygen and then maintained at 2-3.5 %. With a stereotaxic apparatus, mice were bilaterally injected with 300 nL of either the Oxtr guide RNA virus or a scramble sequence virus. Mice were randomly assigned to treatment group (CTRL/KD). Coordinates were calculated using the online California mouse brain atlas at brainmaps.org. For NAc we used, AP: +0.51, ML: ±1.5, DV: -6.0. For BNST we used, AP: + 0.29, ML: ± 1.1, DV: -5.85. Mice assigned to OTR knockdown received a viral cocktail consisting of an AAV9 vector expressing Streptococcus pyogenes Cas9 under the RSV promoter (Fig. 1A, AAV9-RSV-spCas9,stock titer 1.5 X 1010 genomic copies/ul; injected titer 7.5 X 109 gc/ul) and a second AAV9 vector encoding a guide RNA targeting Oxtr (AAV9-U6-gRNA(ΔOXTR.1)-CMVeGFP, 5′-GGTGCTTCATGAAAAAGAAG-3′, stock titer 2.0 X 1010gc/ul stock; injected titer 1.0 X 1010 gc/ul) mixed in a 1:1 (Boender et al., 2023).This guide RNA targets a sequence of the Oxtr gene that is evolutionarily conserved across several rodent species. Control mice received a cocktail consisting of the Cas9 vector and a scrambled gRNA sequence. All mice were given a carprofen injection at the beginning of surgery (1.45 mg/kg dosage) and then 3 daily doses during recovery. Skin staples were used to close the skin and were removed 7 days post-surgery. Surgical controls were not included in the present study. Pre-stress behaviors across all experimental groups were comparable to what we have reported in California mice that did not undergo AAV manipulation (Trainor et al., 2011; Steinman et al., 2016). 2.3 Behavioral timeline Three weeks after surgery, all mice were tested in a Large Arena Social Interaction test (Fig. 1B). In prairie voles 2 weeks of viral expression were sufficient to decrease OTR binding (Boender et al. 2023), so we are confident that mice assigned to knockdown had reduced OTR expression when these behavior tests were conducted. One day later mice were tested in a Small Arena Social Interaction test. In this test target mice are not caged, allowing direct contact with focal mice. Following these behavioral tests all focal mice underwent three consecutive days of social defeat. One week later both the large and small arena social interaction tests were repeated as described above. Mice were never tested with the same Target or aggressive Target mouse (except for post-stress interaction with aggressive Target in the small arena) in the large or small arena tests. 2.4 Large arena test 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 This test consisted of 3 phases: Open Field, Acclimation, and Interaction (Greenberg et al. 2014). Focal mice were placed in the large rectangular arena, made of opaque white acrylic (89 X 63 X 60 cm). Each phase lasted 3 min and was recorded via AnyMaze software. In the Open Field phase, mice were allowed to explore the empty arena. Time spent in the center of the arena and total distance traveled was recorded. This was followed by the Acclimation phase where an empty wire cage (14 X 17 X 14.5 cm) was placed on one end of the arena. Finally, in the last phase an unfamiliar stress-naïve female target mouse was placed inside the wire cage. This test was repeated 1 week after social defeat stress. A different target mouse was used from the prestress test to post-stress testing. 2.5 Small Arena test The small arena test was conducted in an acrylic chamber (51 X 25.4 X 76 cm) connected to a small chamber with a sliding door for introducing target mice. In the Open Field phase, focal mice entered through the attached chamber (13 X 10 X 18 cm) and explored the empty arena for 3 miN. We then assessed interactions with target mice using target mice with (aggressive) or without (naïve) prior experience winning aggressive encounters (Wright et al., 2023; Luo et al., 2024). First a female naïve target mouse was introduced to the arena and interactions with the focal mouse were recorded for 3 min. The target mouse was removed and then an aggressive female target mouse was introduced to the arena for a final 3 min. This test was repeated 1 week after social defeat stress (Fig. 1B). In the post-stress tests, different target mice were used from the prestress tests. However, each focal mouse had interacted with the aggressive target mouse from social defeat encounters. All videos were scored using BORIS by a single trained observer without knowledge of the treatment group status of the focal mouse. Here affiliative (nose-to-nose sniffing, anogenital sniffing, and bodily sniffing), coping (auto-grooming and flipping), defensive (lunging), and monitoring behaviors (social vigilance) were recorded. We define social vigilance as a mouse orienting their head to a conspecific while simultaneously avoiding it. Social approach was not assessed in this test due to the size of the arena and the lack of machine learning to score “passing” behavior versus “approach.” Flipping behavior is a motivated behavior in California mouse that has been identified as a coping behavior (Minie et al., 2021). 2.6 Social defeat stress After testing in the large and small arena tests, all focal mice underwent social defeat stress with aggressive resident females. Each mouse experienced 3 consecutive days of social defeat stress as previously described (Greenberg et al., 2014). On each day, the focal mouse was placed into the home cage of a sexually experienced aggressive resident same-sex mouse. Resident aggressive females were paired with a vasectomized male as this results in more consistent territorial aggression, but were removed during the samesex defeat (Trainor et al. 2013). Each episode of this defeat lasts 7 min or until the intruder/experimental mouse is attacked/defeated a total of 7 times, whichever comes first. After testing, experimental mice are immediately placed back in their homecage. Post-stress testing of focal mice was conducted one week after the last episode of defeat. This 3-day protocol does not induce physical injury to focal or resident mice and 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 results in behavioral changes that can be observed up to 10 weeks after the last episode of defeat (Steinman et al., 2016; Trainor et al., 2011). 2.7 Histology and autoradiography After post-stress behavior testing mice were anesthetized with isoflurane, decapitated, and brains were removed to be flash frozen on dry ice. Brains were stored at -80 °C and then cut with a cryostat at 20 μm and mounted directly on frost plus slides in series. One set of slides was used to determine viral spread by assessing green fluorescent protein (GFP) expression (Fig 1C). These images were used to determine spread of the virus. Adjacent sections were saved on an alternate of slides for OTR autoradiography (as in Hartman et al., 2018). Briefly, slides were removed from -80 °C storage, thawed at room temperature for 1 h, fixed in a light 0.1 % paraformaldehyde (pH 7.4) solution, and washed twice in Tris Buffer, before being incubated in the tracer buffer at room temperature for 60 min. Tracer buffer consisted of 50 mM Tris-HCl buffer (7.4 pH) with 10 mM MgCl2, 0.1 % bovine serum albumin, and 50 pM of radiotracer. For OTR binding, [125I]-ornithine vasotocin analog [125I-OVTA] [vasotocin, d(CH2)5[Tyr(Me)2, Thr4, Orn8, (125I)Tyr9-NH2]; 2200 Ci/mmol] was used (Revvity, Waltham, MA, USA). Previously we observed that the CRISPR tools we used in this study reduced OTR binding but not V1aR binding in California mice (Boender et al., 2023). Following the incubation period, unbound radioligand was removed by 4 washes in 50 mM Tris buffer and 2 % MgCl2, pH 7.4, and then dipped in dH20 and air dried. The slides were dried overnight and exposed to Cytiva Amersha, Hyperfilm (Cytiva Life Sciences, Washington, DC, USA) for 4 days with a set of 125I standard microscales (American Radiolabeled Chemicals, St. Louis, MO, USA). We then used MCID Core Digital Densitometry system (Cambridge, UK) to quantify Oxtr binding in each target brain. For each specimen, optical binding density (OBD) values were calculated for each region of interest (ROI), as well as one background area where no binding was detected. Three separate measurements for each ROI were taken per specimen and averaged. For each specimen, the average background binding value was subtracted from each average ROI measurement to yield normalized OBDs across specimens. There were 2 samples that were damaged in the slide processing and treatment designation for knockdown was determined with GFP photomicrographs of coronal sections of NAc and BNST. Based on oxytocin receptor binding and GFP spread, two mice were reassigned treatment groups (one NAc to BNST, one BNST to NAc) and four mice had OTR binding levels in the BNST and NAc that were below the lowest value observed in control groups. Based on these observations these mice were reassigned to a NAc + BNST knockdown group. We limit our inferences from this group due to the small sample size. 2.8 Statistical analysis Autoradiography data were analyzed using one-way ANOVA in R with planned comparisons to compare treatment groups with controls. For large arena tests behavior was analyzed with one-way ANOVA followed by planned comparisons. We used paired t-test within each treatment group to test if there were differences before versus after stress. For the small arena tests each behavior, we conducted separate analyses by social partner type (naive vs. aggressive target) and timepoint (pre-stress vs. post-stress). For small arena tests each 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 dependent variable was tested across treatment groups using nonparametric Kruskal-Wallis test with pairwise comparisons using the Mann-Whitney U test. 3 RESULTS 3.1 OTR Knockdown in the BNSTam and NAc in female California mice Autoradiography analysis (Fig. 2A) showed that Oxtr CRISPR/Cas9-mediated knockdown altered OTR binding in the BNSTam (Fig. 2B, F3,26 = 13.69, p < 0.001) and NAc (Fig. 2C, F3,26 = 12.18, p < 0.001). Mice assigned to BNST knockdown had reduced OTR binding in BNST (Fig. 2B, planned comparison, p < 0.001, Cohen’s d = 2.36) but not NAc (Fig. 2C) compared to controls. In contrast, mice assigned to NAc knockdown had reduced binding in NAc (Fig. 2C, planned comparison p < 0.001, d = 0.48) but not BNST (Fig. 2B) compared to controls. In some animals binding was reduced in both the BNST and NAc, so these animals were included as a separate group as “BNST + NAc KD.” There were no differences in OTR binding in the LS (Fig. 2D), suggesting that our manipulations were site-specific. 3.2 OTR Knockdown within the BNSTam prevents stress-Induced changes in social approach and vigilance in the large arena Three weeks after bilateral CRISPR/Cas9 injections, adult female California mice were tested in the large arena test to measure baseline behaviors before and after social defeat stress (Fig. 3A). In pre-stress observations there were no differences in social approach (Fig. 3B) or vigilance (Fig. 3C). After stress, OTR knockdown showed a trend to affect social approach behavior (F3,27 = 2.50, p = 0.07). Planned comparisons showed that BNST (p = 0.04, d = 0.43) and BNST+NAc (p = 0.04, d = 0.01) knockdown groups had higher social approach compared to controls (Fig. 3B). For social vigilance there were significant differences between groups after stress (Fig. 3C, F3,27 = 3.50, p = 0.03). Planned comparisons showed lower vigilance in the NAc group (p = 0.01, d = 0.26) and a trend for lower vigilance in the BNST+NAc knockdown (p = 0.052, d = 0.30) compared to controls. Decreased vigilance in the BNST knockdown group compared to control was also at trend level (p = 0.06, d = 0.44). In paired comparisons, controls before and after stress increased their vigilance (p = 0.001, d = -1.19) and decreased their approach (p = 0.008, d = 0.82). Effects of stress were not significant in the other treatment groups. To determine whether the extent of OTR knockdown affected behavior after stress, we examined correlations between receptor density and social approach in the BNST knockdown group and controls (Fig. 4). Receptor density was not significantly correlated with social approach (Fig. 4A, Control: R = -0.16, p = 0.56; BNST knockdown: R = -0.17, p = 0.71) or vigilance (Fig. 4B, Control R = -0.001, p = 0.98; BNST knockdown R = 0.121, p = 0.8). In contrast, a strong negative correlation between social approach and vigilance was observed after stress in controls (R = -0.640, p = 0.007), whereas the same relationship in BNST knockdown mice showed a similar relationship that did not reach significance (R = -0.682, p = 0.09). 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 Osakada, T., Yan, R., Jiang, Y., Wei, D., Tabuchi, R., Dai, B., Wang, X., Zhao, G., Wang, C.X., Liu, J-J., Tsien, R.W., Mar., A.C., Lin, D., 2024. A dedicated hypothalamic oxytocin circuit controls aversive social learning. Nature. 626, 347-356. Paquelet, G.E., Carrion, K., Lacefield, C.O., Zhou, P., Hen, R., & Miller, B.R., 2022. Single-cell activity and network properties of dorsal raphe nucleus serotonin neurons during emotionally salient behaviors. Neuron. 110(16), 2664–2679.e8. https://doi.org/10.1016/j.neuron.2022.05.015. Rashidi, M., Simon, J.J., Bertsch, K., Wegen, G.V., Ditzen, B., Flor, H., Grinevich, V., Wolf, R.C., & Herpertz, S. C., 2025. Effects of intranasal oxytocin on fear extinction learning. Neuropsychopharmacology. 50(3), 548–555. https://doi.org/10.1038/s41386-024-01996-y. Rigney, N., Horie, K., Guo, J.-D., Blumenthal, S.A., Johnson, Z.V., Young, L.J., 2025. Neural connectivity of oxytocin receptor-expressing neurons in the nucleus accumbens and their role in social attachment. Horm. Behav. 171, 105726. https://doi.org/10.1016/j.yhbeh.2025.105726. Salgado, S., & Kaplitt, M. G., 2015. The Nucleus accumbens: A comprehensive review. Stereotact. Funct. Neurosurg. 93(2), 75–93. https://doi.org/10.1159/000368279. Schultebraucks, K., Maslahati, T., Wingenfeld, K., Hellmann-Regen, J., Kraft, J., Kownatzki, M., Behnia, B., Ripke, S., Otte, C., & Roepke, S., 2022. Intranasal oxytocin administration impacts the acquisition and consolidation of trauma-associated memories: a double-blind randomized placebo-controlled experimental study in healthy women. Neuropsychopharmacology. (5), 1046–1054. https://doi.org/10.1038/s41386-021-01247-4. Shamay-Tsoory, S. G., & Abu-Akel, A., 2016. The social salience hypothesis of oxytocin. Biol. Psychiatry. 79(3), 194–202. https://doi.org/10.1016/j.biopsych.2015.07.020. Smith, C.J.W., Mogavero, J.N., Tulimieri, M.T., & Veenema, A. H., 2017. Involvement of the oxytocin system in the nucleus accumbens in the regulation of juvenile social novelty-seeking behavior. Horm. Behav. 93, 94–98. https://doi.org/10.1016/j.yhbeh.2017.05.005. Steinman, M.Q., & Trainor, B.C., 2017. Sex differences in the effects of social defeat on brain and behavior in the California mouse: Insights from a monogamous rodent. Semin. Cell Dev. Biol. 61, 92–98. https://doi.org/10.1016/j.semcdb.2016.06.021. Steinman, M. Q., Duque-Wilckens, N., & Trainor, B. C. (2019). Complementary Neural Circuits for Divergent Effects of Oxytocin: Social Approach Versus Social Anxiety. Biological psychiatry, 85(10), 792–801. https://doi.org/10.1016/j.biopsych.2018.10.008. Steinman, M.Q., Duque-Wilckens, N., Greenberg, G.D., Hao, R., Campi, K.L., Laredo, S.A., Laman-Maharg, A., Manning, C.E., Doig, I.E., Lopez, E.M., Walch, K., Bales, K.L., & Trainor, B.C., 2016. Sex-specific effects of stress on oxytocin neurons correspond with responses to intranasal oxytocin. Biol. Psychiatry. 80(5), 406–414. https://doi.org/10.1016/j.biopsych.2015.10.007. Steinman, M.Q., Duque-Wilckens, N., Trainor, B.C., 2019a. Complementary neural circuits for divergent effects of oxytocin: social approach versus social anxiety. Biol. Psychiatry. 85, 792–801. https://doi.org/10.1016/j.biopsych.2018.10.008. Tabak, B.A., Rosenfield, D., Sunahara, C.S., Alvi, T., Szeto, A., & Mendez, A.J., 2022. Social anxiety is associated with greater peripheral oxytocin reactivity to psychosocial stress. Psychoneuroendocrinology. 140, 105712. https://doi.org/10.1016/j.psyneuen.2022.105712. Taylor, J.H., Walton, J.C., McCann, K.E., Norvelle, A., Liu, Q., Vander Velden, J. W., Borland, J.M., Hart, M., Jin, C., Huhman, K.L., Cox, D. N., & Albers, H.E., 2022. CRISPR-Cas9 editing of the argininevasopressin V1a receptor produces paradoxical changes in social behavior in Syrian hamsters. Proc. Natl. Acad. Sci. U. S. A. 119(19), e2121037119. https://doi.org/10.1073/pnas.2121037119. Terranova, J.I., Song, Z., Larkin, T.E., 2nd, Hardcastle, N., Norvelle, A., Riaz, A., & Albers, H.E., 2016. Serotonin and arginine-vasopressin mediate sex differences in the regulation of dominance and aggression by the social brain. Proc. Natl. Acad. Sci. U. S. A.113(46), 13233–13238. https://doi.org/10.1073/pnas.1610446113. Tidey, J.W., & Miczek, K.A., 1996. Social defeat stress selectively alters mesocorticolimbic dopamine release: an in vivo microdialysis study. Brain Res. 721(1-2), 140–149. https://doi.org/10.1016/00068993(96)00159-x. Trainor, B.C., Pride, M.C., Villalon Landeros, R., Knoblauch, N.W., Takahashi, E.Y., Silva, A.L., & Crean, K.K., 2011. Sex differences in social interaction behavior following social defeat stress in the monogamous California mouse (Peromyscus californicus). PloS one. 6(2), e17405. https://doi.org/10.1371/journal.pone.0017405. 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 Trainor, B.C., Takahashi, E.Y., Campi, K.L., Florez, S.A., Greenberg, G.D., Laman-Maharg, A., Laredo, S.A., Orr, V.N., Silva, A.L., & Steinman, M.Q., 2013. Sex differences in stress-induced social withdrawal: independence from adult gonadal hormones and inhibition of female phenotype by corncob bedding. Horm. Behav. 63(3), 543–550. https://doi.org/10.1016/j.yhbeh.2013.01.011. Walker, S.L., Sud, N., Beyene, R., Palin, N., & Glasper, E.R., 2023. Paternal deprivation induces vigilanceavoidant behavior and accompanies sex-specific alterations in stress reactivity and central proinflammatory cytokine response in California mice (Peromyscus californicus). Psychopharmacology. 240(11), 2317–2334. https://doi.org/10.1007/s00213-023-06354-2. Williams, A.V., & Trainor, B.C., 2018. The impact of sex as a biological variable in the search for novel antidepressants. Front. Neuroendocrinol., 50, 107–117. https://doi.org/10.1016/j.yfrne.2018.05.003. Williams, A.V., Duque-Wilckens, N., Ramos-Maciel, S., Campi, K.L., Bhela, S.K., Xu, C.K., Jackson, K., Chini, B., Pesavento, P.A., & Trainor, B.C., 2020. Social approach and social vigilance are differentially regulated by oxytocin receptors in the nucleus accumbens. Neuropsychopharmacology. 45(9), 1423– 1430. https://doi.org/10.1038/s41386-020-0657-4. Winslow, J.T., Hastings, N., Carter, C.S., Harbaugh, C.R., & Insel, T.R., 1993. A role for central vasopressin in pair bonding in monogamous prairie voles. Nature. 365(6446), 545–548. https://doi.org/10.1038/365545a0. Wright, E.C., Hostinar, C.E., Trainor, B.C., 2020. Anxious to see you: neuroendocrine mechanisms of social vigilance and anxiety during adolescence. Eur. J. Neurosci. 52, 2516–2529. https://doi.org/10.1111/ejn.14628. van Zuiden, M., Frijling, J.L., Nawijn, L., Koch, S.B.J., Goslings, J.C., Luitse, J.S., Biesheuvel, T.H., Honig, A., Veltman, D.J., & Olff, M., 2017. Intranasal oxytocin to prevent posttraumatic stress disorder symptoms: A randomized controlled trial in emergency department patients. Biol. Psychiatry. 81(12), 1030–1040. https://doi.org/10.1016/j.biopsych.2016.11.012. 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 Figure 1: The CRISPR/Cas9 genome-editing system can selectively target the BNST and NAc in female California mice. (A) Schematic of AAV plasmids and CRISPR/Cas9 mechanism of action. (B) Experimental timeline. (C) Representative photomicrographs of viral spread in the BNST and NAc knockdown groups. Scale bar: 100um. LV, lateral ventricle; ACO, anterior commissure; NAc, nucleus accumbens; BNST, bed nucleus of the stria terminalis. 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 Figure 2: Inhibition of Oxtr expression in female Peromyscus californicus in the extended amygdala and nucleus accumbens. (A) Representative images of CRISPR/Cas9 Oxtr manipulation in the BNST and NAc with OTR autoradiography. Control panels show regions quantified for NAc (blue), BNST (green) and LS (purple). (B) Control female mice compared to CRISPR-based knockdown of BNST and other treatment groups. Controls have significantly higher OBD than the BNST KD group (P < 0.001). (C) Control female mice compared to CRISPR-based knockdown of NAc and other treatment groups. Controls are significantly higher from the NAc KD group (P < 0.001). (D) Controls compared to all groups for density comparison in “control” region, the lateral septum. *** P < 0.0001 control vs. knockdown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 Figure 3: Oxytocin receptor knockdown increases social approach and decreases vigilance after stress in the large arena test. (A) Schematic illustration of the Large Arena Social Interaction test. (B) Effects of OTR knockdown on social approach before and after social defeat stress (BNST KD n = 7, NAc KD n = 4, BNST + NAc KD, n = 4, Control n = 16). (C) Effects of OTR knockdown on social vigilance before and after social defeat stress (D) Representative heatmaps for the interaction phase showing reduced social approach determined by time spent in the interaction zone in controls but not other treatment groups.* P < 0.05, # p = 0.06 for BNST KD vs control, # p = 0.052 for BNST +NAc KD vs control, † preversus post-stress paired comparison. Drawings by Dr. Natalia Duque-Wilckens. 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 Figure 4: Correlational analysis of social approach and vigilance for controls and BNST KD. (A) OTR BNST density (OBD) compared to social approach behavior after stress. (B) OTR BNST density (OBD) compared to social vigilance behavior after stress. (C) Social approach compared to social vigilance after stress. 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 Figure 5: Large arena test open field and acclimation phase data. (A) Schematic illustration of the large arena test and the 3 phases: Open Field, Acclimation, and Interaction. (B) Open field locomotion activity for all treatment groups before and after stress (C) Open field center time plotted for all treatment groups before and after stress (D) Acclimation interaction time with an empty cage for all treatment groups before and after stress* P < 0.05 vs control. 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 Figure 6: Small arena test social behaviors before and after stress (A) Schematic illustration for the small arena test. Legend for treatment groups. Nose-to-nose sniffing (C), anogenital sniffing (D), social vigilance (E), lunging (F), and flipping (G) were scored with non-aggressive naïve target mice and then aggressive target mice. Tests were conducted once before social defeat and once after social defeat. † preversus post-stress paired comparison, ** P < 0.01, *** P < 0.001. 826 827 828 829 830