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Cited by 104Neuroendocrine regulation and behaviorStress Responses and CortisolNeuropeptides and Animal Physiology

Ucn3 and CRF-R2 in the medial amygdala regulate complex social dynamics

Open access
Shemesh, Yair, Forkosh, Oren, Mahn, Mathias, Anpilov, Sergey, Sztainberg, Yehezkel, Manashirov, Sharon, Shlapobersky, Tamar, Elliott, Evan +13 more·2016·Nature Neuroscience
DOI 10.1038/nn.4346
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Abstract

Social encounters are associated with varying degrees of emotional arousal and stress. The mechanisms underlying adequate socioemotional balance are unknown. The medial amygdala (MeA) is a brain region associated with social behavior in mice. Corticotropin-releasing factor receptor type-2 (CRF-R2) and its specific ligand urocortin-3 (Ucn3), known components of the behavioral stress response system, are highly expressed in the MeA. Here we show that mice deficient in CRF-R2 or Ucn3 exhibit abnormally low preference for novel conspecifics. MeA-specific knockdown of Crfr2 (Crhr2) in adulthood recapitulated this phenotype. In contrast, pharmacological activation of MeA CRF-R2 or optogenetic activation of MeA Ucn3 neurons increased preference for novel mice. Furthermore, chemogenetic inhibition of MeA Ucn3 neurons elicited pro-social behavior in freely behaving groups of mice without affecting their hierarchal structure. These findings collectively suggest that the MeA Ucn3-CRF-R2 system modulates the ability of mice to cope with social challenges.

AI snapshot

Written from the paper’s full text.

TL;DR

The medial amygdala (MeA) Ucn3–CRF-R2 system bidirectionally regulates social coping in mice: loss of function reduces preference for novel conspecifics, while activation increases it. Chemogenetic inhibition of MeA Ucn3 neurons in groups promotes pro-social behavior without disrupting dominance hierarchy, and MeA Crfr2 mRNA is downregulated in mouse models of social avoidance.

Key findings

  • Germline Crfr2 KO mice (n = 7) spent more time with familiar mice (P = 0.015) and less with novel mice (P = 0.0005) than WT controls; Ucn3 KO mice (n = 6) also avoided novel conspecifics (P = 0.0076). Nonsocial exploration and novel-object preference were unchanged.
  • MeA-specific Crfr2 knockdown recapitulated the KO phenotype: more time with familiar mice (P = 0.033) and less with novel mice (P = 0.0441), whereas lateral septum Crfr2 knockdown had no effect.
  • MeA mUcn3 delivery increased novel-mouse investigation and decreased familiar-mouse investigation; this was blocked by the CRF-R2 antagonist Astressin 2B. Optogenetic activation of MeA Ucn3 neurons similarly increased approach to novel mice and was also blocked by Astressin 2B.
  • In groups of four male mice, chemogenetic inhibition of MeA Ucn3 neurons increased time outside the nest (P = 0.001), increased approaches (P = 0.001), and specifically increased approaches toward equal-rank mice (P = 0.0001), while social rank remained stable. MeA Crfr2 mRNA was downregulated in chronic social defeat (P = 0.0221) and Shank3 KO (P = 0.028) models, with Ucn3 levels unchanged.

Methods

  • Male mice were used across experiments, including Crfr2 KO, Ucn3 KO, C57BL/6J, Ucn3::Cre, and Crfr2::Cre lines. Behavioral testing used a three-chamber dyadic social approach test (30 min) with familiar and novel conspecifics, plus open field, dark-light transfer, locomotor, and novel-object preference tests.
  • Circuit manipulations included germline KO, lentiviral MeA Crfr2 knockdown, intra-MeA mUcn3 and Astressin 2B infusion, optogenetic activation of MeA Ucn3 neurons, and DREADD-mediated chemogenetic inhibition in a semi-natural group arena with automated tracking.
  • Additional methods included patch-clamp electrophysiology, viral tracing of inputs/outputs, immunohistochemistry, cFos quantification, double in situ hybridization, and real-time PCR. Statistical analyses used repeated-measures ANOVA, t-tests, Mann-Whitney tests, Wilcoxon tests, and permutation tests for group behavior.

Limitations

  • Stated in the paper: experimenters were not blinded to treatment in any experiment, and no statistical methods were used to predetermine sample sizes, though sample sizes were said to be similar to those generally used in the field.
  • Evident from the text: all experiments used male mice, limiting generalizability to females; some optogenetic and tracing experiments had very small n values (e.g., n = 3 vs. 4 mice for optogenetic behavior; n = 2–3 mice for tracing).
  • Evident from the text: the Crfr2 mRNA reductions in social avoidance models are correlational, and the study does not establish causality between those expression changes and social avoidance behavior.

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

Integrity signals

Registry

Crossref · 10.1038/nn.4346

Cites

45 of 45 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

0 of 26 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Crfr2 KO mice display stronger preference for familiar mice relative to wild-type controls.

    “We found that Crfr2 KO mice displayed stronger preference for familiar mice relative to wild-type (WT) controls (Fig. 1a)”
    • Also stated in: § DISCUSSION · plain
      “with the latter also increasing interest in familiars.”
    Quote in the paperSubstantial claim
  • Findingown data

    MeA-specific knockdown of Crfr2 in adulthood recapitulates the Crfr2 KO phenotype of increased interest in familiar mice and reduced interest in novel mice.

    “MeA-specific knockdown of Crfr2 (Crhr2) in adulthood recapitulated this phenotype.”
    • Also stated in: § MeA Crfr2 knockdown mice avoid novel conspecifics · plain
      “Like Crfr2 KO mice, MeA Crfr2 KD mice showed more interest in familiar mice and reduced interest in novel mice (Fig. 3b).”
    • Also stated in: § DISCUSSION · plain
      “Knockdown of Crfr2 specifically in the MeA was sufficient to recapitulate the Crfr2 KO phenotype.”
    Quote in the paperSubstantial claim
  • Findingown data

    Mice deficient in CRF-R2 or Ucn3 exhibit abnormally low preference for novel conspecifics.

    “Here we show that mice deficient in CRF-R2 or Ucn3 exhibit abnormally low preference for novel conspecifics.”
    • Also stated in: § Germline Ucn3 KO and Crfr2 KO mice avoid novel conspecifics · plain
      “both Crfr2 KO and Ucn3 KO mice avoided novel conspecifics relative to the WT controls (Fig. 1a,b).”
    • Also stated in: § DISCUSSION · plain
      “germline KOs of Ucn3 and its receptor Crfr2 resulted in reduced interest in novel conspecifics”
    Quote in the paperSubstantial claim
  • Findingown data

    Acute CNO administration significantly reduces cFos expression in the MeA of Ucn3::Cre+ mice expressing inhibitory DREADD.

    “We found that cFos expression in the MeA of Ucn3::Cre + mice was significantly reduced (Supplementary Fig. 10b,c).”
    Quote in the paperSubstantial claim
  • Findingown data

    Inhibition of MeA Ucn3 neurons significantly increases the number of approaches in group-housed mice.

    “In addition, we found a significant increase in the number of approaches (Fig. 7e).”
    Quote in the paperSubstantial claim
  • May combine two claims. 24
  • Classified as probably not a substantial claim. 21

  • May combine two claims.

    Findingown data

    MeA Ucn3 and MeA CRF-R2 neurons both receive inputs from and project to limbic regions that are part of the mouse social brain network.

    “These findings demonstrate that, as expected, Ucn3 and CRF-R2 cells in the MeA both receive inputs from and project to limbic regions that are part of the mouse social brain network (Fig. 2e,f).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Crfr2 KO and Ucn3 KO mice are similar to WT mice in nonsocial exploratory behavior and novelty-seeking behavior.

    “By contrast, Crfr2 KO and Ucn3 KO mice were similar to WT mice in their nonsocial exploratory behavior (Supplementary Fig. 1c,d) and novelty-seeking behavior (Fig. 1c,d and Supplementary Fig. 1b)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mice treated with mUcn3 into the MeA spend more time near novel mice and less time near familiar mice compared to controls.

    “Mice treated with mUcn3 spent more time near novel mice and less time near familiar mice, as compared to controls (Fig. 4g).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MeA Crfr2 KD and control mice do not differ significantly in nonsocial anxiety-like behavior, dark-light transfer, or home-cage locomotor activity.

    “We found no significant differences between MeA Crfr2 KD and control mice in nonsocial anxiety-like behavior in the open field test, in the darklight transfer test or in home-cage locomotor activity (Fig. 3d,e and Supplementary Fig. 5a-d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    mUcn3 application increases neuronal input resistance and action potential firing in response to positive current injections in MeA CRF-R2 neurons.

    “This effect was accompanied by an increase in the neuronal input resistance (Fig. 4c,e).”
    • Also stated in: § MeA Crfr2 knockdown mice avoid novel conspecifics · plain
      “we further found that the neurons fired more action potentials (APs) in response to positive current injections following exposure to mUcn3 (Fig. 4c,f).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    During the third day, in hM4D-expressing mice, dominant mice approached subordinates more than vice versa, and group members of similar social rank approached each other the least.

    “During the third day, in the hM4D-expressing mice, dominant mice approached subordinates more than vice versa, and group members of similar social rank approached each other the least (Fig. 7f).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In both chronic social defeat and Shank3 knockout mouse models, Crfr2 mRNA levels are significantly downregulated in the MeA, while Ucn3 levels remain unchanged.

    “In both models, Crfr2 mRNA levels were significantly downregulated in the MeA (Fig. 6a,b), while Ucn3 levels remained unchanged (data not shown).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    During the first day of CNO administration (fourth day overall), approaches toward individuals of similar social rank became most prominent, with no significant change in approaches toward subordinates or higher rank mice.

    “By contrast, in these groups, during the first day of CNO administration (the fourth day overall), approaches made toward individuals of similar social rank became most prominent, but there was no significant change in approaches toward subordinates or higher rank mice (Fig. 7f).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Treatment groups showed a significant increase in time spent outside the nest during CNO administration, while control groups spent more time inside the nest.

    “The treatment groups showed a significant increase in the time outside (two-tailed permutation test, P = 0.001, n = 20), while the control groups spent more time inside the nest (two-tailed permutation test, P = 0.015, n = 12).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MeA mUcn3-administered mice spent significantly less time investigating a familiar mouse and more time investigating a novel mouse compared to saline-injected controls.

    “The MeA mUcn3 mice spent significantly less time investigating a familiar mouse (one-tailed t-test, P = 0.0024, t = 3.42, d.f.= 22) and more time investigating a novel mouse (one-tailed t-test, P = 0.0258, t = 2.059, d.f.= 22) compared to controls.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingown data

    2 of 6 Ucn3::Cre+ mice injected with helpers and rabies viruses showed clear EYFP expression in the injection site.

    “We injected 6 Ucn3::Cre + mice with both the helpers and rabies viruses; 2 of the 6 showed clear EYFP expression in the injection site, and their brains were further analyzed for sites that project to the MeA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cre+ mice made more approaches following CNO administration, whereas Cre- mice did not change their approach rate significantly.

    “Cre + mice made more approaches following CNO administration (two-tailed permutation test, P = 0.001, n = 20), whereas Cre -mice did not change their approach rate significantly (two-tailed permutation test, P = 0.077, n = 20 and 12 respectively).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MeA Crfr2 KD mice spent significantly more time investigating familiar mice and less time investigating novel mice compared to controls.

    “The MeA Crfr2 KD mice spent significantly more time investigating the familiar mice (one-tailed t-test, P = 0.033, t = 1.957, d.f.= 18) and less time investigating differed from controls in the amount of time spent in each chamber the novel mice (onetailed t-test, P = 0.0441, t = 1.803, d.f.= 18) compared to controls.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The number of approaches toward equals increased significantly during CNO administration, while approaches to mice of different rank remained similar.

    “The number of approaches toward equals increased significantly (two-tailed permutation test, P = 0.0001, n = 22 and 26), while approaches to mice of different rank remained similar (permutation test, P = 0.072 and P = 0.53 n = 20 and 12 for approaches toward dominant and subordinate accordingly, n = 19 and 17 respectively).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The MeA Ucn3-CRF-R2 circuit is important in socioemotional balance and may be dysregulated in mouse models of social avoidance.

    “The results we presented strongly suggest that the MeA Ucn3-CRF-R2 circuit is important in socioemotional balance and may be dysregulated in mouse models of social avoidance.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The mechanisms underlying adequate socioemotional balance are unknown.
    • Rests on an assumption: Maintaining a proper balance between the processing of social signals and emotional responses to social challenges is essential for normal social performance.

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

    Premisecitation

    Oxytocin not only motivates in-group favoritism, but also has a role in the emergence of intergroup conflict and violence.

    “It has been reported that oxytocin not only motivates in-group favoritism, but also has a role in the emergence of intergroup conflict and violence 40 .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] De Dreu, C.K., Greer, L.L., Van Kleef, G.A., Shalvi, S. & Handgraaf, M.J. Oxytocin promotes human ethnocentrism. Proc. Natl. Acad. Sci. USA 108, 1262–1266 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Activation of MeA CRF-R2 or Ucn3 neurons promotes social investigation of novel conspecifics, while loss-of-function promotes social investigation of familiar conspecifics.

    “Thus, the results of the dyadic social test suggest that activation of MeA CRF-R2 or Ucn3 neurons promotes social investigation of novel conspecifics while loss-of-function promotes social investigation of familiar conspecifics.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Reduction in MeA Crfr2 mRNA expression in both environmental and genetic models of social disorders points to possible involvement of stress system components in the amygdala in core symptoms of autism in humans, as well as social deficits in other psychopathologies such as social anxiety disorder and schizophrenia.

    “Finally, the reduction in expression levels of MeA Crfr2 mRNA in both an environmental model and a genetic model of social disorders in mice points to a possible involvement of stress system components in the amygdala in core symptoms of autism in humans, as well as in social deficits in other psychopathologies, such as social anxiety disorder 41 and schizophrenia.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [41] Miskovic, V. & Schmidt, L.A. Social fearfulness in the human brain. Neurosci. Biobehav. Rev. 36, 459–478 (2012). source

      Not checked in the free run.

    • Rests on an assumption: The mechanisms underlying adequate socioemotional balance are unknown.

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

    Premisecitation

    The PVN produces and secretes arginine vasopressin and oxytocin.

    “For example, the PVN, which we found received projections from both the MeA Ucn3 and CRF-R2 cells, is a region producing and secreting arginine vasopressin and oxytocin 26 .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] Stoop, R. Neuromodulation by oxytocin and vasopressin. Neuron 76, 142–159 (2012). source

      Not checked in the free run.

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

    Premisecitation

    Dysregulation of the CRF system has been linked to stress-related emotional disorders such as anxiety and depression.

    “Dysregulation of the CRF system has been linked to stressrelated emotional disorders such as anxiety and depression 6 .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [6] Sztainberg, Y. & Chen, A. Neuropeptide regulation of stress-induced behavior: insights from the crf/urocortin family. in Handbook of Neuroendocrinology (eds Fink, G., Pfaff, D.W. & Levine, J.) Ch. 15 (Academic Press, 2011).

      Not checked in the free run.

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

    Premisecitation

    In rich and complex environments, mice usually live in groups consisting of a few subordinate males and breeding and nonbreeding females, all dominated by a single male.

    “In rich and complex environments, mice usually live in groups consisting of a few subordinate males and breeding and nonbreeding females, all dominated by a single male 36 .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] Bronson, F.H. The reproductive ecology of the house mouse. Q. Rev. Biol. 54, 265–299 (1979). source

      Not checked in the free run.

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

    Study design and conductown data

    Stability of optogenetic Ucn3 + neuron activation was determined by a repeated measures ANOVA on the average number of evoked spikes per stimulation, calculated in 10-s bins (50 stimulations).

    “Stability of optogenetic Ucn3 + neuron activation was determined by a repeated measures ANOVA on the average number of evoked spikes per stimulation, calculated in 10-s bins (50 stimulations).”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    CRF plays a well-established role in the regulation of the hypothalamic-pituitary-adrenal axis under basal and stress conditions and is proposed to integrate autonomic, metabolic, and behavioral responses to stressors.

    “CRF plays a well-established role in the regulation of the hypothalamic-pituitary-adrenal axis under basal and stress conditions and is proposed to integrate the autonomic, metabolic, and behavioral responses to stressors 6 .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [6] Sztainberg, Y. & Chen, A. Neuropeptide regulation of stress-induced behavior: insights from the crf/urocortin family. in Handbook of Neuroendocrinology (eds Fink, G., Pfaff, D.W. & Levine, J.) Ch. 15 (Academic Press, 2011).

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Mice that did not show GFP at the aimed injection location were excluded from the data.

    “Mice that did not show GFP at the aimed injection location were excluded from the data.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    The tolerance of the dominant male to intruders or to in-group subordinates varies widely and depends on the structure of the habitat.

    “The tolerance of the dominant male to intruders or to in-group subordinates varies widely and depends on the structure of the habitat 36 .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] Bronson, F.H. The reproductive ecology of the house mouse. Q. Rev. Biol. 54, 265–299 (1979). source

      Not checked in the free run.

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

    Premisecitation

    Individuals having poor social competence and high social anxiety are at greater risk of developing psychopathology than normally developing individuals, both in childhood and in adult life.

    “Individuals having poor social competence and high social anxiety are at greater risk of developing psychopathology than normally developing individuals, both in childhood and in adult life 37,38 .”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [37] Selten, J.-P., van der Ven, E., Rutten, B.P. & Cantor-Graae, E. The social defeat hypothesis of schizophrenia: an update. Schizophr. Bull. 39, 1180–1186 (2013). source

      Not checked in the free run.

    • cites [38] van Os, J., Kenis, G. & Rutten, B.P. The environment and schizophrenia. Nature 468, 203–212 (2010). source

      Not checked in the free run.

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

    Premisecitation

    The central CRF system helps mediate the neuroendocrine and behavioral responses to stressful challenges.

    “The central corticotropin-releasing factor (CRF) system helps mediate the neuroendocrine and behavioral responses to stressful challenges 6 .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [6] Sztainberg, Y. & Chen, A. Neuropeptide regulation of stress-induced behavior: insights from the crf/urocortin family. in Handbook of Neuroendocrinology (eds Fink, G., Pfaff, D.W. & Levine, J.) Ch. 15 (Academic Press, 2011).

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Only animals with the tip of the cannula located at the MeA were included in the analysis.

    “Only animals with the tip of the cannula located at the MeA were included in the analysis.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    To exclude mice that failed to express hM4D-DREAD in the MeA, we stained MeA sections from each brain for mCherry.

    “To exclude mice that failed to express hM4D-DREAD in the MeA, we stained MeA sections from each brain for mCherry”
    Quote in the paperSubstantial claim
    • Rests on an assumption: DREADDs can be selectively activated by the otherwise inactive clozapine analog clozapine-Noxide (CNO).

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    Following behavioral tests, confirmation of the accuracy of the injection site for the lenti-shCrfr2 was done by immunostaining using biotinylated anti-GFP antibody and for the AAV5-EF1α-DIO-ChR2-EYFP by EYFP expression.

    “Following behavioral tests, confirmation of the accuracy of the injection site for the lenti-shCrfr2 was done by immunostaining using biotinylated anti-GFP antibody and for the AAV5-EF1α-DIO-ChR2-EYFP by EYFP expression.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    In nature, social dominance is frequently determined by fighting among members of the group, which in turn determines access to mating opportunities, food, and territory.

    “In nature social dominance is frequently determined by fighting among members of the group, which in turn determines access to mating opportunities, food, and territory 36 .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] Bronson, F.H. The reproductive ecology of the house mouse. Q. Rev. Biol. 54, 265–299 (1979). source

      Not checked in the free run.

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

    Study design and conductnone given

    When parametric tests were used, data were tested for normality.

    “When parametric tests were used, data were tested for normality.”
    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 conductcitation

    For all experiments, no statistical methods were used to predetermine sample sizes, but sample sizes are similar to those generally employed in the field.

    “For all experiments, no statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those generally employed in the field 8 .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [8] Deussing, J.M. et al. Urocortin 3 modulates social discrimination abilities via corticotropin-releasing hormone receptor type 2. J. Neurosci. 30, 9103–9116 (2010). source

      Not checked in the free run.

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

    Study design and conductnone given

    In none of the experiments in this work were the experimenters blinded to the treatment.

    “In none of the experiments in this work were the experimenters blinded to the treatment.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Individual mice were excluded from all behavioral analysis if data differed more than 2 s.d. from mean.

    “Individual mice were excluded from all behavioral analysis if data differed more than 2 s.d.from mean.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    In the Crfr2 KD experiment, mice were randomly assigned to Crfr2 KD or control groups.

    “In the Crfr2 KD experiment, mice were randomly assigned to Crfr2 KD or control groups.”
    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 conductnone given

    In the pharmacological Ucn3 administration experiment, mice were cannulated and randomly assigned to saline and mUcn3 groups.

    “In the pharmacological Ucn3 administration experiment, mice were cannulated and randomly assigned to saline and mUcn3 groups.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Equal variance was verified between control and treated groups in all experiments.

    “Equal variance was verified between control and treated groups in all experiments.”
    Quote in the paperNot a substantial claim
38 of 38

  • The paper relies on this without support.

    The mechanisms underlying adequate socioemotional balance are unknown.

    “The mechanisms underlying adequate socioemotional balance are unknown.”

  • The paper relies on this without support.

    Understanding of the molecular mechanisms and neurobiological circuits that regulate the socioemotional balance is limited, and our ability to intervene and treat maladaptive social stress is poor.

    “Understanding of the molecular mechanisms and neurobiological circuits that regulate the socioemotional balance is limited, and our ability to intervene and treat maladaptive social stress is poor.”

  • The paper relies on this without support.

    Social encounters are associated with varying degrees of emotional arousal and stress.

    “Social encounters are associated with varying degrees of emotional arousal and stress.”

  • The paper relies on this without support.

    Moderate levels of social apprehension are essential for ensuring safe and successful social engagement.

    “However, from an evolutionary point of view, moderate levels of social apprehension are essential for ensuring safe and successful social engagement.”

  • The paper relies on this without support.

    Maintaining a proper balance between the processing of social signals and emotional responses to social challenges is essential for normal social performance.

    “Thus, maintaining a proper balance between the processing of social signals and emotional responses to social challenges is essential for normal social performance.”

  • The paper relies on this without support.

    DREADDs can be selectively activated by the otherwise inactive clozapine analog clozapine-Noxide (CNO).

    “DREADDs can be selectively activated by the otherwise inactive clozapine analog clozapine-Noxide (CNO).”

  • The paper relies on this without support.

    The fur dyes used for mouse identification do not affect behavior.

    “The dyes used-namely, Electric Banana (HCR 11012), Pillarbox Red (HCR 11020), and Raven (HCR 11007), from Tish & Snooky's NYC Inc., New York-were composed of natural ingredients.”

  • The paper relies on this without support.

    The automated tracking software correctly identifies individual mice based on fur colors.

    “Mice were identified and tracked automatically according to their fur colors, which were learned from labeled data.”

  • The authors state this as an assumption.

    The dominance hierarchy among mice is linear.

    “David's score assumes a linear hierarchy, where each pair from the group contains a dominant and a subordinate individual.”

  • The paper relies on this without support.

    The sample sizes used are adequate to detect the effects studied.

    “For all experiments, no statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those generally employed in the field 8 .”
10 of 10

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • The authors previously developed an experimental system that allows animals to behave freely in a large enriched environment while their location is tracked automatically over days and dozens of behavioral readouts are extracted.

    “we previously developed an experimental system that allows animals to behave freely in a large environment, enriched with hiding places, elevated platforms, bedding, feeders, and water sources, while their location is tracked automatically over days, and dozens of behavioral readouts are extracted”

    From Wireless Optogenetic Stimulation of Oxytocin Neurons in a Semi-natural Setup Dynamically Elevates Both Pro-social and Agonistic Behaviors (2020) · cites this paper as Shemesh et al., 2016

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.

  • ConsistentF(119, 714) = 0.7reported p = .99 · recomputed p = .992

    “F(119,714) = 0.7, p = .99”

1 of 1

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.

  • Behavioral optical stimulation frequency differs

    Major

    The optical-stimulation Methods specify 20 Hz pulse trains, while the Figure 5g caption specifies 5 Hz for the behavioral light-on condition.

  • Figure 3 caption reverses the knockdown result

    Minor

    The figure title says MeA Crfr2 knockdown increases preference for novel conspecifics, whereas the Results say it reduces interest in them.

  • Knockdown testing time differs between Results and Methods

    Minor

    The Results place behavioral testing 10 days after the lentiviral injection, while the viral-injection Methods say mice recovered for 2 weeks before behavioral testing.

  • Optogenetics mouse count does not match its breakdown

    Minor

    The stated groups contain 3 and 4 mice, totaling 7 rather than the reported 17.

    Reported 17 · recomputed 7

4 of 4

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. Duda, R.O., Hart, P.E. & Stork, D.G. Pattern Classification and Scene Analysis 2nd ed. (Wiley Interscience, 1995).
  2. Breiman, L., Friedman, J., Stone, C.J. & Olshen, R.A. Classification and Regression Trees (CRC Press, 1984).

Cited by

Sorted by citations

Related

Authors
Shemesh, Y., Forkosh, O., Mahn, M., Anpilov, S., Sztainberg, Y., Manashirov, S., Shlapobersky, T., Elliott, E., Tabouy, L., Ezra, G., Adler, E. S., Ben-Efraim, Y. J., Gil, S., Kuperman, Y., Haramati, S., Dine, J., Eder, M., Deussing, J. M., Schneidman, E., Yizhar, O., & Chen, A.
Affiliations
  • Max Planck Institute of Psychiatry (DE)
  • Weizmann Institute of Science (IL)
  • Bar-Ilan University (IL)
Corresponding author
Chen, Alon
ORCID
Venue
Nature Neuroscience
Publisher
Springer Science and Business Media LLC
Published
2016-07-18
Volume
19
Issue
11
Pages
1489-1496
ISSN
  • 1097-6256 (ISSN-L)
  • 1546-1726
DOI
10.1038/nn.4346
OpenAlex
W2498537245
Semantic Scholar
d86c787b213c813181bc85cf813f542bccc3860b
Cited by
99
References
45
Citations by year
2026: 3 · 2025: 10 · 2024: 14 · 2023: 7 · 2022: 13 · 2021: 14 · 2020: 8 · 2019: 7 · 2018: 13 · 2017: 9 · 2016: 1
FWCI
15.70
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Neuroscience (publishedVersion)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, other-oa)
  • Max Planck Digital Library (submittedVersion)
  • MPG.PuRe (Max Planck Society) (submittedVersion)
Funders
  • Perlman Family Foundation
  • European Commission (321919)
  • Weizmann Institute of Science
  • Israel Science Foundation (1565/15, 1629/12, 1916/12, 1351/12)
  • Israeli Centers for Research Excellence (1916/12)
  • Planning and Budgeting Committee of the Council for Higher Education of Israel
  • FP7 Ideas: European Research Council (260463, 311238, 337637)
Topics
  • Social Sciences › Psychology › Social Psychology › Neuroendocrine regulation and behavior
  • Life Sciences › Neuroscience › Behavioral Neuroscience › Stress Responses and Cortisol
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuropeptides and Animal Physiology
Keywords
urocortin 3, corticotropin-releasing factor, medial amygdala, social behavior, stress response, prosocial behavior, social hierarchy, optogenetic stimulation, chemogenetic inhibition, mice
MeSH terms
Amygdala, Animals, Behavior, Animal, Corticotropin-Releasing Hormone, Inhibition, Psychological, Neurons, Social Behavior, Receptors, Corticotropin-Releasing Hormone, Mice, Knockout, Mice, Urocortins

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 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

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.

What counts as a claim ›How we measure confidence ›How we check this ›

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