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Cited by 34Stress Responses and CortisolNeuroendocrine regulation and behaviorSexual Differentiation and Disorders

Sexual differentiation of neural mechanisms of stress sensitivity during puberty

Open access
Wright, Emily C., Luo, Pei X., Zakharenkov, Hannah C., Serna Godoy, Alexandra, Lake, Alyssa A., Prince, Zhana D., Sekar, Shwetha, Culkin, Hannah I. +7 more·2023·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.2306475120PMC PMC10614610

Published version of a preprint

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Abstract

Anxiety disorders are a major public health concern and current treatments are inadequate for many individuals. Anxiety is more common in women than men and this difference arises during puberty. Sex differences in physiological stress responses may contribute to this variability. During puberty, gonadal hormones shape brain structure and function, but the extent to which these changes affect stress sensitivity is unknown. We examined how pubertal androgens shape behavioral and neural responses to social stress in California mice ( Peromyscus californicus ), a model species for studying sex differences in stress responses. In adults, social defeat reduces social approach and increases social vigilance in females but not males. We show this sex difference is absent in juveniles, and that prepubertal castration sensitizes adult males to social defeat. Adult gonadectomy does not alter behavioral responses to defeat, indicating that gonadal hormones act during puberty to program behavioral responses to stress in adulthood. Calcium imaging in the medioventral bed nucleus of the stria terminalis (BNST) showed that social threats increased neural activity and that prepubertal castration generalized these responses to less threatening social contexts. These results support recent hypotheses that the BNST responds to immediate threats. Prepubertal treatment with the nonaromatizable androgen dihydrotestosterone acts in males and females to reduce the effects of defeat on social approach and vigilance in adults. These data indicate that activation of androgen receptors during puberty is critical for programming behavioral responses to stress in adulthood.

AI snapshot

Written from the paper’s full text.

TL;DR

In California mice, pubertal androgens organize adult behavioral and neural sensitivity to social defeat: prepubertal castration increased defeat-induced social avoidance/vigilance and generalized BNST threat responses, while pubertal testosterone or DHT replacement reduced these effects. BNST calcium activity tracked immediate social threats/aggression rather than freezing.

Key findings

  • Juvenile social defeat at PN50 reduced social approach and increased social vigilance in both males and females (approach: F1,27 = 47.09, P < 0.001; vigilance: Kruskal–Wallis = 16.33, P < 0.001), contrasting with the adult sex-specific pattern.
  • Prepubertal castration made adult males susceptible to defeat: castrated males exposed to defeat had reduced social approach vs. controls (P < 0.0001, d = 2.6) and increased social vigilance (Mann–Whitney U = 23.5, P < 0.01, d = 1.6), whereas sham or no-surgery males showed no effect of defeat.
  • Ventral BNST GCaMP activity increased when focal mice were within one body length of target mice and the target was in central or peripheral vision, but not when facing away; no changes in ΔF/F occurred during freezing. Prepubertally castrated males showed larger delayed BNST ΔF/F after nose-to-nose sniffing with naive mice (β = 13.2, z = 2.23, P = 0.03) and after anogenital sniffing (β = 6.12, z = 2.06, P = 0.04).
  • Pubertal androgen replacement reversed castration effects in stressed males: testosterone and DHT increased social approach and lowered social vigilance; in stressed females, DHT increased social approach vs. empty implants (P = 0.02, d = 0.1). The oxytocin receptor antagonist L-368,899 increased social approach and decreased social vigilance in juvenile males and females.

Methods

  • California mice of both sexes were used. Juvenile social defeat occurred PN34–36 with social interaction testing at PN50. Adult males underwent prepubertal castration, sham, or no surgery between PN35–40, then social defeat at PN90–92 and testing at PN106; separate groups received testosterone, DHT, or empty silastic implants.
  • Fiber photometry of ventral BNST GCaMP was performed during free interaction with naive or aggressor target mice; DeepLabCut tracked distance and orientation. Immunohistochemistry measured oxytocin/c-fos colocalization in anterior and posterior PVN and c-fos in anteromedial BNST. Oxytocin receptor antagonist injections were given before social interaction testing.
  • Behavioral and cell-count data were analyzed with ANOVA and planned comparisons; social vigilance and oxytocin/c-fos colocalization used nonparametric tests. Photometry analysis methods are described in the SI Appendix.

Limitations

  • The paper states that silastic implants released hormones during both puberty and adulthood, and it did not test whether pubertal androgen effects are maintained in the absence of androgens in adults. It also states that social defeat still has strong effects on male California mice, so androgen blunting of defeat effects is limited.
  • Evident from the text: the BNST calcium imaging castration comparison was conducted only in males, and female pubertal hormone manipulation used intact females given DHT or empty implants, so female pubertal androgen effects are less directly isolated. The paper also notes that estrogen receptors may play a complementary role but were not directly tested.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1073/pnas.2306475120

Cites

119 of 120 references verified1 citation errors · 0 not checkable

Statistics

9 tests recomputed · 0 with the reported and recomputed p on opposite sides of .05

Claims the paper makes

76 of 103 cited sources checked · 44 supported · 2 flagged · accuracy not yet measured

  • Premisecitation

    In adults, sex differences in behavioral responses to defeat are independent of gonadal hormones.

    “In adults, sex differences in behavioral responses to defeat are independent of gonadal hormones (42, 43), suggesting that sex differences are organized during development.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [42] B. C. Trainor, Sex differences in social interaction behavior following social defeat stress in the monogamous California mouse (peromyscus californicus). PLoS One 6, e17405 (2011). 21364768 PMC3045459 source

      The Discussion section, “Effects of Defeat on Corticosterone Levels” (page not available), says that ovariectomy ameliorated defeat-related changes in social interaction behavior. It concludes that ovarian hormones may mediate sex differences in responses to defeat, contrary to the claim that those differences are independent of gonadal hormones. (LLM, conf=0.89)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [43] B. C. Trainor, Sex differences in stress-induced social withdrawal: Independence from adult gonadal hormones and inhibition of female phenotype by corncob bedding. Horm. Behav. 63, 543– 550 (2013). 23384773 PMC3637973 source

      The Results (Experiment 1) show that defeat reduced social interaction in both intact and ovariectomized female California mice, but not males; gonadectomy had no effect on that social-withdrawal pattern. The Discussion concludes that adult gonadal hormones do not appear to mediate this sex difference. The citing sentence states the conclusion more broadly, without limiting it to California mice and the social-withdrawal behavior studied. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Premisecitation

    Adult gonadal hormones have little impact on stress-induced social avoidance.

    “our previous work clearly demonstrates that adult gonadal hormones have little impact on stress-induced social avoidance (43, 50).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [43] B. C. Trainor, Sex differences in stress-induced social withdrawal: Independence from adult gonadal hormones and inhibition of female phenotype by corncob bedding. Horm. Behav. 63, 543– 550 (2013). 23384773 PMC3637973 source
    • cites [50] B. C. Trainor, Sex differences in social interaction behavior following social defeat stress in the monogamous California mouse (Peromyscus californicus). PLoS One 6, e17405 (2011). 21364768 PMC3045459 source

      The Discussion (Effects of Defeat on Corticosterone Levels; page unavailable) says that ovariectomy ameliorated defeat-induced changes in social interaction in follow-up experiments. Rather than demonstrating little impact, the paper suggests ovarian hormones are involved in the response to defeat; it identifies those follow-up findings as unpublished. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
  • Premisecitation

    The extended amygdala, including the BNST, is especially sensitive to steroid hormones.

    “The extended amygdala, which includes the bed nucleus of the stria terminalis (BNST), is especially sensitive to steroid hormones (28).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [28] B. Gegenhuber, M. V. Wu, R. Bronstein, J. Tollkuhn, Gene regulation by gonadal hormone receptors underlies brain sex differences. Nature 606, 153– 159 (2022). 35508660 PMC9159952 source

      The 'Genomic targets of ERα in the brain' section reports that oestradiol regulates genes in the BNSTp and identifies the posterior medial amygdala as another region where ERα regulates behaviour. This supports hormone responsiveness in the regions studied, but the paper does not characterize the extended amygdala as a whole or establish that it is especially sensitive relative to other regions. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Premisecitation

    During puberty, stressors have stronger behavioral effects in females than males.

    “Preclinical studies show that during puberty, stressors have stronger behavioral effects on behavior in females than males (20–22).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [20] P. Asgari, G. McKinney, T. E. Hodges, C. M. McCormick, Social instability stress in adolescence and social interaction in female rats. Neuroscience 477, 1– 13 (2021). 34619317 source
    • cites [21] A. Graf, Acute and long-term sex-dependent effects of social instability stress on anxiety-like and social behaviours in Wistar rats. Behav. Brain Res. 438, 114180 (2023). 36349601 source
    • cites [22] C. H. Bourke, G. N. Neigh, Behavioral effects of chronic adolescent stress are sustained and sexually dimorphic. Horm. Behav. 60, 112– 120 (2011). 21466807 PMC3112263 source
  • Premisecitation

    Gonadal hormones shape brain structure, function, and gene expression networks that could influence stress responses.

    “Although the cause of these differences is unknown, other studies show that gonadal hormones shape brain structure (23–26), function (27), and gene expression networks (28) that could influence stress responses.”
    Quote in the paperSubstantial claim5 of 6 sources checked
    • cites [23] E. I. Ahmed, Pubertal hormones modulate the addition of new cells to sexually dimorphic brain regions. Nat. Neurosci. 11, 995– 997 (2008). 19160494 PMC2772186 source
    • cites [24] K. Delevich, D. Piekarski, L. Wilbrecht, Neuroscience: Sex hormones at work in the neocortex. Curr. Biol. 29, R122– R125 (2019). 30779899 source
    • cites [25] C. M. Drzewiecki, J. Willing, J. M. Juraska, Synaptic number changes in the medial prefrontal cortex across adolescence in male and female rats: A role for pubertal onset. Synapse 70, 361– 368 (2016). 27103097 PMC4945496 source
    • cites [26] K. M. Schulz, C. L. Sisk, The organizing actions of adolescent gonadal steroid hormones on brain and behavioral development. Neurosci. Biobehav. Rev. 70, 148– 158 (2016). 27497718 PMC5074860 source
    • cites [27] K. Delevich, A. W. Thomas, L. Wilbrecht, Adolescence and “late blooming” synapses of the prefrontal cortex. Cold Spring Harb. Symp. Quant. Biol. 83, 37– 43 (2019), 10.1101/sqb.2018.83.037507. 30674651 source
    • cites [28] B. Gegenhuber, M. V. Wu, R. Bronstein, J. Tollkuhn, Gene regulation by gonadal hormone receptors underlies brain sex differences. Nature 606, 153– 159 (2022). 35508660 PMC9159952 source
  • Classified as probably not a substantial claim. 19
  • May combine two claims. 28
  • The quote only partly states this claim. 3

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Functional MRI studies in humans show BNST activity is correlated with trait anxiety.

    “Functional MRI studies in humans show that activity within the BNST is correlated with trait anxiety (32) and is increased in response to unpredictable threats (33).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [32] L. H. Somerville, Interactions between transient and sustained neural signals support the generation and regulation of anxious emotion. Cereb. Cortex 23, 49– 60 (2013). 22250290 PMC3513951 source

  • Classified as probably not a substantial claim.

    Premisecitation

    Puberty is characterized by cortical and subcortical reorganization.

    “Puberty is a key developmental stage characterized by changes in physiological stress responses (16) as well as cortical and subcortical reorganization (17–19).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [17] A.-L. Goddings, A. Beltz, J. S. Peper, E. A. Crone, B. R. Braams, Understanding the role of puberty in structural and functional development of the adolescent brain. J. Res. Adolesc. 29, 32– 53 (2019). 30869842 source
    • cites [18] M. Chini, I. L. Hanganu-Opatz, Prefrontal cortex development in health and disease: Lessons from rodents and humans. Trends Neurosci. 44, 227– 240 (2021). 33246578 source
    • cites [19] A. Caballero, A. Orozco, K. Y. Tseng, Developmental regulation of excitatory-inhibitory synaptic balance in the prefrontal cortex during adolescence. Semin. Cell Dev. Biol. 118, 60– 63 (2021). 33714681 PMC8429511 source

      The Introduction and “The E-I balance in humans” section describe prefrontal cortical maturation and synaptic pruning during adolescence. They do not establish that puberty specifically is characterized by both cortical and subcortical reorganization; the paper’s discussion of subcortical inputs concerns their influence on the prefrontal cortex, not a general reorganization of subcortical structures. No page numbers are available in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Fiber photometry allows for data collection in freely moving animals, expanding the behavioral repertoire that can be studied.

    “Fiber photometry allows for data collection in freely moving animals (36), expanding the behavioral repertoire that can be studied.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [36] C. A. Siciliano, K. M. Tye, Leveraging calcium imaging to illuminate circuit dysfunction in addiction. Alcohol 74, 47– 63 (2019). 30470589 PMC7575247 source

  • Classified as probably not a substantial claim.

    Premisecitation

    Previous immediate early gene analyses show that social stress increases neural activity within the BNST.

    “Previous immediate early gene analyses shows that social stress increases neural activity within the BNST (53, 54).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [53] C. Numa, Social defeat stress-specific increase in c-Fos expression in the extended amygdala in mice: Involvement of dopamine D1 receptor in the medial prefrontal cortex. Sci. Rep. 9, 16670 (2019). 31723165 PMC6854085 source
    • cites [54] D. Kollack-Walker, A. Watson, Differential expression of c- fos mRNA within neurocircuits of male hamsters exposed to acute or chronic defeat. J. Neuroendocrinol. 11, 547– 559 (1999). 10444312 source

  • May combine two claims.

    Premisecitation

    These results contrast with adults, where social defeat decreases social approach and increases social vigilance in females but not males.

    “These results contrast with observed sex differences in adults, which show that social defeat decreases social approach (38, 42, 43, 51) and increases social vigilance (40) in females but not males.”
    Quote in the paperSubstantial claim5 of 5 sources checked
    • cites [38] G. D. Greenberg, Sex differences in stress-induced social withdrawal: Role of brain derived neurotrophic factor in the bed nucleus of the stria terminalis. Front. Behav. Neurosci. 7, 223 (2014). 24409132 PMC3885825 source

      The Introduction reports that three episodes of social defeat induce social aversion in female California mice but not males, and the Methods specify that the mice were adults (>3 months old). That supports the decreased-social-approach portion of the claim. The passages found do not report increased social vigilance; in the citing sentence, that point has a separate citation (40). (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [42] B. C. Trainor, Sex differences in social interaction behavior following social defeat stress in the monogamous California mouse (peromyscus californicus). PLoS One 6, e17405 (2011). 21364768 PMC3045459 source
    • cites [43] B. C. Trainor, Sex differences in stress-induced social withdrawal: Independence from adult gonadal hormones and inhibition of female phenotype by corncob bedding. Horm. Behav. 63, 543– 550 (2013). 23384773 PMC3637973 source
    • cites [51] G. D. Greenberg, M. Q. Steinman, I. E. Doig, R. Hao, B. C. Trainor, Effects of social defeat on dopamine neurons in the ventral tegmental area in male and female California mice. Eur. J. Neurosci. 42, 3081– 94 (2015). 26469289 PMC4715680 source
    • cites [40] N. Duque-Wilckens, Oxytocin receptors in the anteromedial bed nucleus of the stria terminalis promote stress-induced social avoidance in female California mice. Biol. Psychiatry 83, 203– 213 (2018). 29066224 PMC5743604 source

  • Classified as probably not a substantial claim.

    Premisecitation

    Previous work in California mice shows changes in BNST drive stress-induced social avoidance and social vigilance.

    “Previous work in California mice (Peromyscus californicus) shows that social defeat stress has stronger effects on BNST structure and function in adult females than males (37, 38) and that these changes drive stress-induced social avoidance and social vigilance (39–41).”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [39] N. Duque-Wilckens, Extra-hypothalamic oxytocin neurons drive stress-induced social vigilance and avoidance. Proc Natl. Acad. Sci. U.S.A. 117, 26406– 26413 (2020). 33020267 PMC7585015 source
    • cites [40] N. Duque-Wilckens, Oxytocin receptors in the anteromedial bed nucleus of the stria terminalis promote stress-induced social avoidance in female California mice. Biol. Psychiatry 83, 203– 213 (2018). 29066224 PMC5743604 source
    • cites [41] P. X. Luo, Oxytocin receptor behavioral effects and cell types in the bed nucleus of the stria terminalis. Horm. Behav. 143, 105203 (2022). 35636023 PMC9827713 source

      The Discussion reports that activating oxytocin receptors in the BNST reduced social approach and increased social vigilance in California mice, while BNST receptor blockade had the opposite effects in stressed females. This supports a role for BNST function in those behaviors, but the paper does not show that stress-induced changes in BNST structure drive them; its carbetocin experiments used stress-naïve mice. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Functional MRI studies have limited temporal resolution (~3 s) and require participants to remain immobile.

    “These studies have limited temporal resolution (~3 s) and participants must remain immobile during observations (34).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [34] G. H. Glover, Overview of functional magnetic resonance imaging. Neurosurg. Clin. N. Am. 22, 133– 139 (2011). 21435566 PMC3073717 source

      The ‘Temporal resolution’ section says fMRI has low temporal resolution and gives a typical BOLD-response width of about 3 seconds, but it also says some temporal inferences can reach 100 ms; 3 seconds is not an absolute resolution limit. ‘Basis for fMRI’ says head motion can introduce noise, supporting a need to minimize movement, but the paper does not say participants must remain entirely immobile. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Early work suggested that the BNST encodes diffuse or remote threats.

    “Early work suggested that the BNST encodes diffuse or remote threats (72) while more recent work suggests that the BNST is highly responsive to immediate threats (65).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [72] M. Davis, D. L. Walker, L. Miles, C. Grillon, Phasic vs. sustained fear in rats and humans: Role of the extended amygdala in fear vs. anxiety. Neuropsychopharmacology 35, 105– 135 (2010). 19693004 PMC2795099 source

  • May combine two claims.

    Interpretationcitation

    These data inform current debates on BNST function and are consistent with reports of increased reactivity of the BNST in humans diagnosed with anxiety disorders.

    “These data inform current debates on BNST function (65) and are consistent with reports of increased reactivity of the BNST in humans diagnosed with anxiety disorders (66).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [65] A. J. Shackman, A. S. Fox, Contributions of the central extended amygdala to fear and anxiety. J. Neurosci. 36, 8050– 8063 (2016). 27488625 PMC4971357 source
    • cites [66] J. A. Clauss, S. N. Avery, M. M. Benningfield, J. U. Blackford, Social anxiety is associated with BNST response to unpredictability. Depress Anxiety 36, 666– 675 (2019). 30953446 PMC6679811 source

      The Results (BNST Connectivity) associate higher social anxiety with altered BNST connectivity and a greater BNST-than-amygdala response to certain unpredictable images. But the Methods (Participants) describe a sample spanning social-anxiety levels, only nine of whom met criteria for an anxiety disorder; the Results (BNST Activation) say social anxiety did not moderate BNST activation. Thus the paper does not establish generally increased BNST reactivity in people diagnosed with anxiety disorders. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Sex differences in stress sensitivity emerge at puberty in humans and other animals, coinciding with an increased incidence of anxiety in women.

    “Sex differences in stress sensitivity emerge at puberty in both humans (63, 64) and other animals (20–22), coinciding with an increased incidence of anxiety in women (10, 12, 14, 15).”
    Quote in the paperSubstantial claim3 of 9 sources checked
    • cites [63] C. E. Hostinar, A. E. Johnson, M. R. Gunnar, Parent support is less effective in buffering cortisol stress reactivity for adolescents compared to children. Dev. Sci. 18, 281– 297 (2015). 24942038 PMC4270957 source

      The Puberty and Social-Developmental Changes During Adolescence section reports studies finding sex differences in puberty-related HPA activity in humans, which supports the human portion of the cited sentence. It also says several studies found no such differences; the Discussion reports no sex difference in cortisol reactivity among this study’s adolescents. The source therefore does not establish that sex differences in stress sensitivity generally emerge at puberty. The animal and anxiety clauses have separate citations in the citing sentence. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [64] E. C. Wright, C. E. Hostinar, B. C. Trainor, Anxious to see you: Neuroendocrine mechanisms of social vigilance and anxiety during adolescence. Eur. J. Neurosci. 52, 2516– 2529 (2020). 31782841 PMC7255921 source

      The Introduction (page not supplied) says that sex differences in social-anxiety prevalence emerge during human adolescence and discusses pubertal changes that may affect susceptibility. It does not establish that sex differences in stress sensitivity emerge specifically at puberty in both humans and other animals; the Conclusions (page not supplied) describe adolescent sex differences in animal models as an understudied area. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [20] P. Asgari, G. McKinney, T. E. Hodges, C. M. McCormick, Social instability stress in adolescence and social interaction in female rats. Neuroscience 477, 1– 13 (2021). 34619317 source
    • cites [21] A. Graf, Acute and long-term sex-dependent effects of social instability stress on anxiety-like and social behaviours in Wistar rats. Behav. Brain Res. 438, 114180 (2023). 36349601 source
    • cites [22] C. H. Bourke, G. N. Neigh, Behavioral effects of chronic adolescent stress are sustained and sexually dimorphic. Horm. Behav. 60, 112– 120 (2011). 21466807 PMC3112263 source
    • cites [10] P. Munk-Jørgensen, Prevalence of generalized anxiety disorder in general practice in Denmark, Finland, Norway, and Sweden. PS 57, 1738– 1744 (2006). 17158488 source
    • cites [12] K. Beesdo, Incidence of social anxiety disorder and the consistent risk for secondary depression in the first three decades of life. Arch. Gen. Psychiatry 64, 903– 912 (2007). 17679635 source
    • cites [14] C. G. Davey, M. Yücel, N. B. Allen, The emergence of depression in adolescence: Development of the prefrontal cortex and the representation of reward. Neurosci. Biobehav. Rev. 32, 1– 19 (2008). 17570526 source
    • cites [15] R. Wesselhoeft, C. B. Pedersen, P. B. Mortensen, O. Mors, N. Bilenberg, Gender–age interaction in incidence rates of childhood emotional disorders. Psychol. Med. 45, 829– 839 (2015). 25110838 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Testosterone can exert acute anxiolytic effects in a variety of behavioral assays.

    “Similarly, testosterone can exert acute anxiolytic effects in a variety of behavioral assays (94–96).”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [94] J. L. Aikey, J. G. Nyby, D. M. Anmuth, P. J. James, Testosterone rapidly reduces anxiety in male house mice (Mus musculus). Horm. Behav. 42, 448– 460 (2002). 12488111 source
    • cites [95] C. A. Frye, A. M. Seliga, Testosterone increases analgesia, anxiolysis, and cognitive performance of male rats. Cogn. Affective Behav. Neurosci. 1, 371– 381 (2001). 12467088 source

      The Elevated Plus-Maze results report less anxiety-like behavior in testosterone-replaced male rats than in gonadectomized rats. But the Surgery section describes implants providing sustained testosterone throughout testing, not an acute dose. The Discussion also says there was no statistically significant evidence of reduced anxiety in all assays; the other significant anxiety-related differences involved intact rats, not a benefit of testosterone replacement. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [96] N. Carrier, M. Kabbaj, Extracellular signal-regulated kinase 2 signaling in the hippocampal dentate gyrus mediates the antidepressant effects of testosterone. Biol. Psychiatry 71, 642– 651 (2012). 22265242 PMC3307821 source

      The Experimental Design and Results sections describe testosterone replacement over weeks and effects on depressive-like behavior in sucrose-preference and forced-swim tests. A search for acute anxiolytic effects, anxiety-like behavior, and anxiety assays found no passage in our copy addressing the attributed claim. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    More recent work suggests that the BNST is highly responsive to immediate threats.

    “Early work suggested that the BNST encodes diffuse or remote threats (72) while more recent work suggests that the BNST is highly responsive to immediate threats (65).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [65] A. J. Shackman, A. S. Fox, Contributions of the central extended amygdala to fear and anxiety. J. Neurosci. 36, 8050– 8063 (2016). 27488625 PMC4971357 source

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Nonsurgery controls were included to control for possible effects of early life exposure to the anesthesia isoflurane.

    “Nonsurgery controls were included to control for possible effects of early life exposure to the anesthesia isoflurane (107).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [107] K. L. Murphy, M. G. Baxter, Long-term effects of neonatal single or multiple isoflurane exposures on spatial memory in rats. Front. Neurol. 4, 87 (2013). 23847588 PMC3703565 source

  • May combine two claims.

    Premisecitation

    In adult California mice, androgens do not affect social approach regardless of stress exposure even though they blunt acute corticosterone responses to defeat.

    “Interestingly, in adult California mice, androgens do not affect social approach regardless of stress exposure even though they blunt acute corticosterone responses to defeat (43).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [43] B. C. Trainor, Sex differences in stress-induced social withdrawal: Independence from adult gonadal hormones and inhibition of female phenotype by corncob bedding. Horm. Behav. 63, 543– 550 (2013). 23384773 PMC3637973 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Estrogens can enhance anxiety-related behaviors by activating Esr1 or exerting anxiolytic effects via Esr2.

    “Estrogens can enhance anxiety-related behaviors by activating Esr1 or exerting anxiolytic effects via Esr2 (100).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [100] A. P. Borrow, R. J. Handa, Estrogen receptors modulation of anxiety-like behavior. Vitam. Horm. 103, 27– 52 (2017). 28061972 PMC5815294 source

  • May combine two claims.

    Findingown data

    Neural activity in the BNST is increased by social threats but not defensive responses like freezing.

    “Using calcium imaging we show that neural activity in the bed nucleus of the stria terminalis (BNST) is increased by social threats but not defensive responses like freezing.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Stress-induced acute increases in ΔF/F after attacks by naive target mice, but attacks by experienced aggressors induced both acute and delayed increases in ΔF/F.

    “In this study, stress-induced acute increases in ΔF/F after attacks by naive target mice, but attacks by experienced aggressors induced both acute and delayed increased in ΔF/F.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After stress, attacks by naive target mice induced an acute increase in ΔF/F with no change in activity during the delayed (3 to 6 s) period.

    “After stress, attacks by naive target mice induced an acute increase in ΔF/F (Fig. 4C, β = 2.81, P = 0.045) with no change in activity during the delayed (3 to 6 s) period.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Castrated male California mice exposed to defeat had reduced social approach vs controls, whereas there was no effect of defeat in sham or no-surgery males.

    “Castrated males exposed to defeat had reduced social approach vs. controls (P < 0.0001, d = 2.6), whereas there was no effect of defeat in sham or no-surgery males.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    Oxytocin/c-fos colocalizations in posterior PVN were negatively correlated with social approach but not social vigilance.

    “and oxytocin/c-fos colocalizations were negatively correlated with social approach (Fig. 5D”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There were no differences in locomotor behavior in the open-field phase, time spent in the center, or vigilance behavior during acclimation.

    “There were no differences in locomotor behavior in the open-field phase (Fig. 1G), time spent in the center (SI Appendix, Fig. S2A), or vigilance behavior during the acclimation phase (SI Appendix, Fig. S2B).”
    Quote in the paperNot a substantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    Oxytocin/c-fos colocalizations in anterior PVN were negatively correlated with social approach and positively correlated with social vigilance.

    “Oxytocin/c-fos colocalizations were negatively correlated with social approach (Fig. 5C”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    During the acclimation phase, female California mice spent more time investigating an empty cage than males, with no effects of stress.

    “During the acclimation phase, females spent more time investigating an empty cage than males (Fig. 1F, F1,27 = 6.64, P = 0.016) with no effects of stress.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Increased GCaMP6f fluorescence response (ΔF/F) in BNST was observed when focal mice were within one body length (8 cm) of target mice and within the central or peripheral visual fields but not if the focal mouse was facing away from target mice.

    “Increased GCaMP6f fluorescence response (ΔF/F) in BNST was observed when focal mice were within one body length (8 cm) of target mice and within the central (Fig. 3D, 0° ≥ 40°, β = −0.438, z = −9.783, P > 0.001) or peripheral visual fields (40° ≥ 100°, β = −0.486, z = −10.356, P > 0.001) but not if the focal mouse was facing away from target mice (100° ≥ 180°).”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    The number of c-fos cells in anteromedial BNST was negatively correlated with social approach but not social vigilance.

    “The number of c-fos cells was negatively correlated with social approach (Fig. 5E”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Effects of hormone treatment on social vigilance in female California mice were weaker, with a post hoc test indicating lower levels of social vigilance in DHT-treated mice vs. empty implant.

    “Effects on social vigilance were weaker (Kruskal–Wallis = 5.04, P = 0.08), with a post hoc test indicating lower levels of social vigilance in DHT-treated mice vs. empty implant (Mann-Whitney = 11, P = 0.028, d = 1.2).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In female California mice, before stress exposure, there were no differences in social approach, social vigilance, acclimation, or open-field behavior with hormone treatment.

    “In social interaction tests performed before stress exposure, there were no differences in social approach (Fig. 6E), social vigilance (Fig. 6F), acclimation (SI Appendix, Fig. S7D), or open-field behavior (SI Appendix, Fig. S7 E and F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Prepubertally castrated males exposed to defeat exhibited more freezing and heightened BNST calcium transients in the presence of naive target mice.

    “Prepubertally castrated males exposed to defeat exhibited more freezing and heightened BNST calcium transients in the presence of naive target mice.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In both males and females exposed to social defeat, an i.p. injection of 5 mg/kg of the oxytocin receptor antagonist L368,899 increased social approach and decreased social vigilance.

    “In both males and females, treatment with 5 mg/kg i.p. (intraperitoneal) of the oxytocin receptor antagonist L-368,899 30 min before testing increased social approach (Fig. 5G, main effect of dose F2,42 = 8.42, P < 0.001) and decreased social vigilance (Fig. 5H, Kruskal–Wallis = 15.36, P < 0.009).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Although pubertal DHT and testosterone treatment had similar effects on social approach in stressed males, the effect of DHT on social vigilance was weaker.

    “Although pubertal DHT and testosterone treatment had similar effects on social approach in stressed males, the effect of DHT on social vigilance was weaker.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Puberty is a key developmental stage characterized by changes in physiological stress responses.

    “Puberty is a key developmental stage characterized by changes in physiological stress responses (16) as well as cortical and subcortical reorganization (17–19).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [16] C. E. DePasquale, M. P. Herzberg, M. R. Gunnar, The pubertal stress recalibration hypothesis: Potential neural and behavioral consequences. Child Dev. Perspect. 15, 249– 256 (2021). 34925549 PMC8680280 source

  • May combine two claims.

    Interpretationown data

    BNST ΔF/F is enhanced by social threats and this response is more generalized in prepubertally castrated males.

    “These results suggest that BNST ΔF/F is enhanced by social threats and that this response is more generalized in prepubertally castrated males.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Oxytocin neurons in the PVN project to the BNST, suggesting that these outcomes could be functionally linked.

    “Oxytocin neurons in the PVN project to the BNST (82), suggesting that these outcomes could be functionally linked.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [82] H. S. Knobloch, Evoked axonal oxytocin release in the central amygdala attenuates fear response. Neuron 73, 553– 566 (2012). 22325206 source

  • May combine two claims.

    Interpretationcitation

    Androgens may impede overgeneralization of threat, which is a key characteristic of anxiety disorders.

    “Thus, androgens may impede overgeneralization of threat, which is a key characteristic of anxiety disorders (76).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [76] S. Lissek, Classical fear conditioning in the anxiety disorders: A meta-analysis. Behav. Res. Therapy 43, 1391– 1424 (2005). 15885654 source

  • May combine two claims.

    Interpretationown data

    These findings are in line with our observations that prepubertal castration led to increased c-fos/oxytocin colocalizations in the PVN and c-fos expression in anteromedial BNST.

    “These findings are in line with our observations that prepubertal castration led to increased c-fos/oxytocin colocalizations in the PVN and c-fos expression in anteromedial BNST.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Silastic implants released hormones both during puberty and adulthood.

    “Silastic implants released hormones both during puberty and adulthood.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The study did not test whether effects of pubertal androgen exposure were maintained in the absence of androgens in adults.

    “We did not test whether effects of pubertal androgen exposure were maintained in the absence of androgens in adults”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Androgens act during puberty to influence how social behavior is impacted by stress, suggesting that even in species where androgens do not have an overt role in regulating stress sensitivity in adults, androgens can program behavioral responses during adolescence.

    “Here, we show that androgens act during puberty to influence how social behavior is impacted by stress, suggesting that even in species where androgens do not have an overt role in regulating stress sensitivity in adults, androgens can program behavioral responses during adolescence.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Normality was assessed via QQPlot.

    “Normality was assessed via QQPlot.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Intact females were randomly assigned to receive a DHT or empty implant at PN35-PN40.

    “Intact females were randomly assigned to receive a DHT or empty implant at PN35-PN40 as described for males.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Nose-to-nose sniffing, anogenital sniffing, freezing, aggressive behavior directed towards focal mice, and aggressive behavior directed towards target mice were scored by an observer without knowledge of the treatment group.

    “Nose-to-nose sniffing, anogenital sniffing, freezing, aggressive behavior directed towards focal mice, and aggressive behavior directed towards target mice were scored by an observer without knowledge of the treatment group using BORIS.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Social vigilance and oxytocin/c-fos colocalization data had heterogeneous variability, so nonparametric analyses were used.

    “Social vigilance and oxytocin/c-fos colocalization data had heterogeneous variability, so nonparametric analyses were used.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    A Flinger-Killen test was used to assess the homogeneity of variance.

    “A Flinger–Killen test was used to assess the homogeneity of variance.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Male juveniles were randomly assigned to castration, sham surgery, or no-surgery control between PN35 and PN40.

    “Male juveniles were randomly assigned to castration, sham surgery, or no-surgery control between PN35 and PN40 (61).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [61] B. C. Trainor, C. A. Marler, Testosterone promotes paternal behaviour in a monogamous mammal via conversion to oestrogen. Proc. R Soc. Lond. Ser. B Biol. Sci. 269, 823– 829 (2002). PMC1690962 11958714 source

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    In the first study, male juveniles were randomly assigned to castration or sham surgery after weaning.

    “In the first study, male juveniles were randomly assigned to castration or sham surgery after weaning.”
    Quote in the paperSubstantial claim
45 of 45

Statistics that don’t add up

Each reported p-value recomputed from its test statistic and degrees of freedom.

  • Inconsistent p-value: z = −10.356, p > .001

    Minor

    The paper reports p > .001 here; recomputing from the test statistic and its degrees of freedom gives p < .001 (two-tailed). The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p > .001 · recomputed p < .001

  • Inconsistent p-value: z = −9.783, p > .001

    Minor

    The paper reports p > .001 here; recomputing from the test statistic and its degrees of freedom gives p < .001 (two-tailed). The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p > .001 · recomputed p < .001

2 of 2

Statistics recomputed

Every reported test result, recomputed from its test statistic and degrees of freedom. A result shows as not adding up only once an independent check has confirmed it.

  • Doesn’t add upz = -9.783reported p > .001 · recomputed p < .001

    “z = −9.783, p > .001”

  • Doesn’t add upz = -10.356reported p > .001 · recomputed p < .001

    “z = −10.356, p > .001”

  • Consistentz = 2.05reported p = .04 · recomputed p = .040

    “z = 2.05, p = .04”

  • Consistentz = 2.23reported p = .03 · recomputed p = .026

    “z = 2.23, p = .03”

  • Consistentz = 2.06reported p = .04 · recomputed p = .039

    “z = 2.06, p = .04”

  • Consistentz = 2.56reported p = .01 · recomputed p = .010

    “z = 2.56, p = .01”

  • Consistentz = 2.67reported p = .008 · recomputed p = .008

    “z = 2.67, p = .008”

  • Consistentz = 5.33reported p < .001 · recomputed p < .001

    “z = 5.33, p < .001”

  • Consistentz = 3.89reported p < .001 · recomputed p < .001

    “z = 3.89, p < .001”

9 of 9

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Male hormone-replacement sample sizes do not match ANOVA degrees of freedom

    Minor

    The male hormone-replacement analysis has three treatment groups and reports F2,22, implying 25 observations. The Fig. 6 caption says there were 7 to 8 males per group, allowing at most 24 across those three groups.

  • Different calcium indicators are named for the same imaging study

    Minor

    The results describe the BNST calcium imaging as using GCaMP6f, but the methods say the mice received a virus encoding GCaMP6s. These are different indicators.

  • Fig. 3 proximity P-value threshold conflicts with its caption

    Minor

    The results give P > 0.001 for both visual-field effects in Fig. 3D, whereas the figure caption marks the proximity comparison as P < 0.001. The reported z values, both near −10, also support the smaller P-value.

3 of 3

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. 10.1371/journal.pone.0017405 · DOI · Paper page
  2. S. Leary AVMA Guidelines for the Euthanasia of Animals: 2020 Edition (AVMA 2020 ) pp. 1–121.

Cited by

Sorted by citations

Related

Authors
Wright, E. C., Luo, P. X., Zakharenkov, H. C., Serna Godoy, A., Lake, A. A., Prince, Z. D., Sekar, S., Culkin, H. I., Ramirez, A. V., Dwyer, T., Kapoor, A., Corbett, C., Tian, L., Fox, A. S., & Trainor, B. C.
Affiliations
  • University of California, Davis (US)
  • Department of Psychology, University of California, Davis, CA 95616
  • University of Wisconsin–Madison (US)
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2023-10-17
Volume
120
Issue
43
Pages
e2306475120-e2306475120
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.2306475120
PMC
PMC10614610
OpenAlex
W4387700788
Cited by
34
References
120
Citations by year
2026: 6 · 2025: 15 · 2024: 10 · 2023: 1 · 2021: 2
FWCI
4.41
Type
journal article
Language
en
Open access
Licence
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by-nc-nd)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
Funders
  • National Science Foundation (IOS 1937335)
  • HHS | NIH | National Institute of Mental Health (R01MH121829, F32MH125597)
  • HHS | NIH | NIH Office of the Director (P51OD011107)
  • HHS | NIH | National Institute of Neurological Disorders and Stroke (U01NS120820)
Topics
  • Life Sciences › Neuroscience › Behavioral Neuroscience › Stress Responses and Cortisol
  • Social Sciences › Psychology › Social Psychology › Neuroendocrine regulation and behavior
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Sexual Differentiation and Disorders
Keywords
sex differences, puberty, social stress, social defeat, anxiety, gonadal hormones, androgens, dihydrotestosterone, androgen receptor, social approach, social vigilance, California mouse, Peromyscus californicus, stress sensitivity
MeSH terms
Adult, Androgens, Female, Humans, Male, Puberty, Septal Nuclei, Sex Differentiation, Gonadal Hormones

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-12.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-12.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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