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Cited by 43Genetics and Neurodevelopmental DisordersNeuroendocrine regulation and behaviorSexual Differentiation and Disorders

Neonatal MeCP2 is important for the organization of sex differences in vasopressin expression

Open access
Forbes-Lorman, Robin M., Rautio, Jared J., Kurian, Joseph R., Auger, Anthony P., Auger, Catherine J.·2012·Epigenetics
DOI 10.4161/epi.7.3.19265PMC PMC3335947
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Abstract

Several neurodevelopmental disorders are marked by atypical Methyl-CpG-binding protein 2 (MeCP2) expression or function; however, the role of MeCP2 is complex and not entirely clear. Interestingly, there are sex differences in some of these disorders, and it appears that MeCP2 has sex-specific roles during development. Specifically, recent data indicate that a transient reduction in MeCP2 within developing amygdala reduces juvenile social play behavior in males to female-typical levels. These data suggest that MeCP2 within the amygdala is involved in programming lasting sex differences in social behavior. In the present study, we infused MeCP2 or control siRNA into the amygdala of male and female rats during the first three days of postnatal life in order to assess the impact of a transient reduction in MeCP2 on arginine vasopressin (AVP), a neural marker that is expressed differentially between males and females and is linked to a number of social behaviors. The expression of AVP, as well as several other genes, was measured in two-week old and adult animals. Two-week old males expressed more AVP and galanin mRNA in the amygdala than females, and a transient reduction in MeCP2 eliminated this sex difference by reducing the expression of both gene products in males. A transient reduction in MeCP2 also decreased androgen receptor (AR) mRNA in two-week old males. In adulthood, control males had more AVP-immunoreactive (AVP-ir) cells than females in the centromedial amygdala (CMA), bed nucleus of the stria terminalis (BST) and in the fibers that project from these cells to the lateral septum (LS). A transient reduction in MeCP2 eliminated this sex difference. Interestingly, there were no lasting differences in galanin or AR levels in adulthood. Reducing MeCP2 levels during development did not alter estrogen receptorα, neurofilament or Foxg1. We conclude that a transient reduction in MeCP2 expression in the developing male amygdala has a transient impact on galanin and AR expression but a lasting impact on AVP expression, highlighting the importance of MeCP2 in organizing sex differences in the amygdala.

AI snapshot

Written from the paper’s full text.

TL;DR

Neonatal infusion of MeCP2 siRNA into the rat amygdala reduced male-typical arginine vasopressin (AVP) expression at postnatal day 14 and eliminated adult sex differences in AVP immunoreactivity in the centromedial amygdala, bed nucleus of the stria terminalis, and lateral septum. The effect was sex-specific, with no lasting changes in galanin or androgen receptor expression in adulthood.

Key findings

  • At PN14, control males had higher relative AVP mRNA in the amygdala than control females (p = 0.011) and MeCP2 siRNA-treated males (p = 0.007); MeCP2 siRNA reduced male AVP mRNA to female-like levels, with no effect in females.
  • MeCP2 siRNA also reduced PN14 galanin mRNA in males: control males > control females (p = 0.005) and > MeCP2 siRNA-treated males (p = 0.008). AR mRNA was lower in MeCP2 siRNA-treated males than control males (p = 0.002), while ERα mRNA was unaffected.
  • In adulthood, control males had more AVP-ir cells than females and MeCP2 siRNA-treated males in the CMA and BST (p < 0.001 for both comparisons in each region) and more AVP-ir fibers in the LS (control males vs females p < 0.001; vs MeCP2 siRNA-treated males p = 0.003). No lasting effects were found for galanin-ir or AR-ir cells, and no treatment effect was seen in the PVT.
  • Serum testosterone did not differ by sex or treatment in gonadectomized, testosterone-implanted adults; average levels were 1.31 ± 0.31 ng/mL in control females, 1.29 ± 0.35 ng/mL in control males, 1.08 ± 0.27 ng/mL in MeCP2 siRNA-treated females, and 1.49 ± 0.25 ng/mL in MeCP2 siRNA-treated males.

Methods

  • Sprague-Dawley rats received bilateral amygdala infusions of 1 μL (100 pmol) MeCP2 or control siRNA on PN0, PN1, and PN2. Experiment 1 used 24 pups from 4 dams (5 male control, 7 male MeCP2 siRNA, 7 female control, 5 female MeCP2 siRNA) and measured mRNA by RT-PCR at PN14 for AVP, galanin, AR, ERα, NF-L, Foxg1, MeCP2, Ywhaz, and HPRT1.
  • Experiment 2 used 40 pups from 5 dams (10 per sex/treatment group), followed the same neonatal infusion paradigm, gonadectomized and implanted testosterone at 6 months, and examined AVP, galanin, and AR immunoreactivity at 7.5 months in CMA, BST, LS, and PVT; testosterone was measured by EIA.
  • Experiment 3 infused SKF 38,393 into the amygdala of PN1 females and measured c-Fos-ir cells in amygdala and BST to assess infusion spread. Statistical comparisons used two-way ANOVA with Fisher LSD post hoc tests, Student’s t-test, and linear regression, with p < 0.05 considered significant.

Limitations

  • The paper states that the mechanism by which a transient reduction in MeCP2 disrupts peptide expression is not clear, and that the functional significance of reduced AVP, including its possible influence on juvenile social play behavior, is not clear.
  • The authors note that it is less clear how amygdala MeCP2 siRNA decreased AVP-ir cell number in the BST; they address possible spread indirectly using SKF 38,393 and hypothalamic AVP mRNA, but state this provides an indirect functional assay.
  • The text notes that adult animals received testosterone-filled implants whereas PN14 animals did not, which could contribute to the lack of an adult galanin sex difference; it also suggests sex differences in galanin may be easier to detect with quantitative PCR. No dedicated limitations section is present in the paper.

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

Integrity signals

Registry

Crossref · 10.4161/epi.7.3.19265

Cites

54 of 54 references verified0 citation errors · 0 not checkable

Statistics

31 tests recomputed · 1 with the reported and recomputed p on opposite sides of .05

Claims the paper makes

9 of 30 cited sources checked · 5 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    MeCP2 siRNA infusion into the amygdala does not reduce MeCP2 levels in the hypothalamus.

    “we have previously confirmed that the MeCP2 siRNA infusion into the amygdala does not reduce MeCP2 levels in the hypothalamus.14”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [14] Kurian, 2008, Mecp2 organizes juvenile social behavior in a sex-specific manner, J Neurosci source
  • Premisecitation

    Systemic SKF 38,393 administration can increase c-Fos-ir labels in both the amygdala and BST.

    “even though systemic SKF 38,393 administration can increase c-Fos-ir labels in both the amygdala and BST.34”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [34] Olesen, 2008, Dopaminergic activation of estrogen receptors induces fos expression within restricted regions of the neonatal female rat brain, PLoS One source
  • Study design and conductcitation

    The amount of MeCP2 siRNA used induced a transient and localized reduction in MeCP2 expression within the amygdala.

    “We have previously shown that this amount of MeCP2 siRNA induced a transient and localized reduction in MeCP2 expression within the amygdala.14”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [14] Kurian, 2008, Mecp2 organizes juvenile social behavior in a sex-specific manner, J Neurosci source
  • Findingown data

    A transient reduction in MeCP2 in the developing male amygdala decreased AR mRNA expression in the amygdala at PN14.

    “Post hoc comparisons indicate that control males expressed higher levels of AR mRNA in the amygdala than MeCP2 siRNAtreated males (p = 0.002).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Control siRNA does not affect gene expression or behavior relative to untreated animals.
    • Rests on an assumption: MeCP2 siRNA does not have off-target effects on other genes that could affect AVP expression.
  • Findingown data

    In adulthood, control males had more AVP-ir fibers in the LS than females.

    “In adulthood, control males had more aVpimmunoreactive (aVp-ir) cells than females in the centromedial amygdala (cMa), bed nucleus of the striaterminalis (BsT) and in the fibers that project from these cells to the lateral septum (Ls).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The number of AVP-immunoreactive cells in adulthood reflects the number of AVP-expressing cells, not differences in detection sensitivity.
  • Classified as probably not a substantial claim. 26
  • May combine two claims. 9
  • The quote only partly states this claim. 1

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

    Findingcitation

    Average testosterone levels in the adult groups fell within the range of typical adult male levels.

    “These levels fall within the range of typical adult male levels of circulating testosterone.33”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [33] Lee, 1975, Variations in serum FSH, LH and testosterone levels in male rats from birth to sexual maturity, J Reprod Fertil source

      The ‘Serum LH and testosterone levels’ section reports circulating testosterone above 230 ng/100 ml in male rats at 70–80 days of age. It provides a reference value for sexually mature rats, but does not define a typical adult male range or report the citing document’s group averages, so the claimed comparison is not established by this paper alone. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    This sex difference in AVP expression is organized during early postnatal life by steroid hormone actions on ER and AR.

    “Moreover, this sex difference is organized during early postnatal life by steroid hormone actions on ER and AR.[24][25][26]”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [24] Han, 2003, Organizational effects of testosterone, estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis, J Neurobiol source
    • cites [25] De Vries, 1994, Sex differences in the effects of testosterone and its metabolites on vasopressin messenger RNA levels in the bed nucleus of the stria terminalis of rats, J Neurosci source

      The Discussion reports that differences in gonadal hormones during the first weeks after birth help determine the sexual differentiation of AVP-producing cells in the rat BST. The paper also discusses estrogen- and androgen-receptor mechanisms affecting AVP expression, but it does not establish that actions on both receptors during early postnatal life organize the sex difference; those receptor mechanisms are discussed in the context of hormone responsiveness and remain partly hypothetical. Page numbers are unavailable in our copy. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [26] Wang, 1993, Sexual differentiation of vasopressin projections of the bed nucleus of the stria terminals and medial amygdaloid nucleus in rats, Endocrinology source

  • May combine two claims.

    Premisecitation

    The BST and CMA are interconnected, and lesions of the BST disrupt AVP expression in the amygdala.

    “More importantly, previous data have shown that the BST and CMA are interconnected, and lesions of the BST disrupt AVP expression in the amygdala.35”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [35] De Vries, 1983, The origin of the vasopressinergic and oxytocinergic innervation of the rat brain with special reference to the lateral septum, Brain Res source

      The lesion results (p. 7) report that unilateral BST lesions reduced vasopressin fiber density in the ipsilateral anterior amygdala. The paper does not establish that the BST and CMA are interconnected or show disrupted AVP expression specifically in the CMA; it measures fiber density in the anterior amygdala. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Many of the consequences of MeCP2 loss can be reversed by postnatal restoration of MeCP2 expression.

    “while many of the consequences of MeCP2 loss can be reversed by postnatal restoration of MeCP2 expression, 11,12”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [11] Guy, 2007, Reversal of neurological defects in a mouse model of Rett syndrome, Science source
    • cites [12] Giacometti, 2007, Partial rescue of MeCP2 deficiency by postnatal activation of MeCP2, Proc Natl Acad Sci U S A source

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

    Premisecitation

    SKF 38,393 is a D1-receptor agonist that induces c-Fos protein in several brain areas, including the amygdala and BST, within an hour following a systemic infusion.

    “SKF 38,393 is a D1-receptor agonist that induces c-Fos protein in several brain areas, 54,55 including the amygdala and BST, 34 within an hour following a systemic infusion”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [54] Meredith, 1997, D1 dopamine receptor agonist (SKF-38393) induction of Fos immunoreactivity in progestin receptor-containing areas of female rat brain, J Neuroendocrinol source
    • cites [55] Shearman, 1997, Widespread expression of functional D1-dopamine receptors in fetal rat brain, Brain Res Dev Brain Res source
    • cites [34] Olesen, 2008, Dopaminergic activation of estrogen receptors induces fos expression within restricted regions of the neonatal female rat brain, PLoS One source

      The Results for Experiment 1 report increased Fos-immunoreactive cells after SKF 38393 treatment in several regions, including the BST and central amygdala (CeA). The Methods identify SKF 38393 as a D1-like receptor agonist, but describe a subcutaneous injection and measurement 2.5 hours later—not induction within one hour following a systemic infusion. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    CBP and phosphorylated CREB are higher in males in several brain areas, and CBP is necessary for the organization of some sex differences in behaviors.

    “For example, CREB-binding protein (CBP) and phosphorylated CREB are both higher in males in several brain areas 44,45 and CBP is necessary for the organization of some sex differences in behaviors.44”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [44] Auger, 2002, Expression of the nuclear receptor coactivator, cAMP response element-binding protein, is sexually dimorphic and modulates sexual differentiation of neonatal rat brain, Endocrinology source
    • cites [45] Auger, 2001, Sex difference in the phosphorylation of cAMP response element binding protein (CREB) in neonatal rat brain, Brain Res source

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationreasoning

    MeCP2 may interact with CREB within the developing amygdala to increase steroid-dependent AVP gene transcription.

    “MeCP2 may interact with CREB within the developing amygdala to increase steroid-dependant AVP gene transcription.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    MeCP2 plays an important role in programming AVP expression into adulthood; however, the influence of MeCP2 on galanin appears transient.

    “These data suggest that MeCP2 plays an important role in programming AVP expression into adulthood; however, the influence of MeCP2 on galanin appears transient.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Control siRNA does not affect gene expression or behavior relative to untreated animals.
    • Rests on an assumption: MeCP2 siRNA does not have off-target effects on other genes that could affect AVP expression.
    • Rests on an assumption: The number of AVP-immunoreactive cells in adulthood reflects the number of AVP-expressing cells, not differences in detection sensitivity.

  • May combine two claims.

    Interpretationcitation

    Higher levels of CREB activity in males within sexually dimorphic brain regions may facilitate MeCP2-induced gene transcription and organize sex differences.

    “These data suggest that higher levels of CREB activity in males within sexually dimorphic brain regions may facilitate MeCP2-induced gene transcription and organize sex differences.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The same sized area was analyzed for each animal.

    “the same sized area was analyzed for each animal.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Juvenile male rats play at higher frequencies than females, and this sex difference is organized by testosterone action within the developing male amygdala.

    “Juvenile male rats play at higher frequencies than females, and this sex difference is organized by testosterone action within the developing male amygdala.15,16”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [15] Meaney, 1981, Neonatal-androgens influence the social play of prepubescent rats, Horm Behav source
    • cites [16] Smith, 1997, The postpubertal change in the playful defense of male rats depends upon neonatal exposure to gonadal hormones, Physiol Behav source

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

    Premisecitation

    AVP is co-expressed in a subset of galanin expressing neurons.

    “Also, as AVP is co-expressed in a subset of galanin expressing neurons, 29 we also examined galanin expression.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [29] Miller, 1993, Extra-hypothalamic vasopressin neurons coexpress galanin messenger RNA as shown by double in situ hybridization histochemistry, J Comp Neurol source

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

    Premisecitation

    CREB binds to the AVP promoter region to activate gene transcription in vitro.

    “Indeed, studies have shown that CREB binds to the AVP promoter region to activate gene transcription in vitro.46”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [46] Iwasaki, 1997, Positive and negative regulation of the rat vasopressin gene promoter, Endocrinology source

  • Classified as probably not a substantial claim.

    Study design and conductown data

    NF-L, Foxg1, and MeCP2 were examined to assess whether changes in gene expression were not simply the impact of overall transcriptional dysregulation.

    “In order assess the possibility that the changes observed in gene expression were not simply the impact of overall transcriptional dysregulation, we examined the effect of neonatal MeCP2 siRNA on neurofilament, light polypeptide (NF-L), MeCP2 and Foxg1 mRNA expression in the amygdala.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    The testosterone EIA has a specificity of 100% for testosterone.

    “The assay speciocity is 100% for testosterone”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Neonatal treatment with dihydrotestosterone, which binds to AR, contributes to male-like AVP mRNA expression in adult rat brain.

    “This is in agreement with data indicating that neonatal treatment with dihydrotestosterone, which binds to AR, contributes to male-like AVP mRNA expression in adult rat brain.24”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [24] Han, 2003, Organizational effects of testosterone, estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis, J Neurobiol source

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

    Study design and conductnone given

    HPRT1 and Ywhaz were used as normalizing genes to control for subtle variations in sample concentrations.

    “HPRT1 and Ywhaz were used as normalizing genes to control for subtle variations in sample concentrations.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    AVP expression within the BST and CMA is involved in a number of social behaviors, including social play behavior.

    “AVP expression within the bed nucleus of the striaterminalis (BST) and the centromedial amygdala (CMA) is known to be involved in a number of social behaviors, [18][19][20] including social play behavior.21”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [18] Winslow, 1993, A role for central vasopressin in pair bonding in monogamous prairie voles, Nature source
    • cites [19] Bielsky, 2005, The V1a vasopressin receptor is necessary and sufficient for normal social recognition: a gene replacement study, Neuron source
    • cites [20] Bielsky, 2004, Oxytocin, vasopressin, and social recognition in mammals, Peptides source

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

    Study design and conductnone given

    Microscope light intensity was kept constant throughout the imaging session for each brain region analyzed to ensure consistent measurements.

    “Microscope light intensity was kept constant throughout the imaging session for each brain region analyzed to ensure consistent measurements.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    MeCP2 binds to the AVP promoter region in the paraventricular nucleus of the hypothalamus to repress gene transcription.

    “Research has shown that MeCP2 binds to the AVP promoter region in the paraventricular nucleus of the hypothalamus to repress gene transcription.22”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22] Murgatroyd, 2009, Dynamic DNA methylation programs persistent adverse effects of early-life stress, Nat Neurosci source

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

    Study design and conductnone given

    The number of AVP or galanin-ir cells in the CMA and BST was counted by an experimenter blind to treatment groups.

    “The number of AVP or galanin-ir cells in the CMA and BST was counted at 10x by an experimenter blind to treatment groups.”
    Quote in the paperNot a substantial claim

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

    Interpretationreasoning

    The number of c-Fos-ir cells in the BST would increase if the SKF 38,393 infusions spread to the BST.

    “suggesting that the number of c-Fos-ir cells in the BST would increase if the SKF 38,393 infusions spread to the BST.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The testosterone EIA has a detection limit of 6 pg/ml.

    “The detection limit of this assay was 6 pg/ml.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    AR is critical in the development of social play behavior.

    “As AR is critical in the development of social play behavior, 15,16,48 a reduction in AR expression during development could also contribute to the lasting decrease in juvenile social play behavior in males.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [15] Meaney, 1981, Neonatal-androgens influence the social play of prepubescent rats, Horm Behav source
    • cites [16] Smith, 1997, The postpubertal change in the playful defense of male rats depends upon neonatal exposure to gonadal hormones, Physiol Behav source
    • cites [48] Meaney, 1983, Sexual differentiation of social play in rat pups is mediated by the neonatal androgen-receptor system, Neuroendocrinology source

  • Classified as probably not a substantial claim. May combine two claims. The quote only partly states this claim.

    Study design and conductown data

    Testosterone EIA was used to ensure that testosterone levels did not differ between sex or treatment groups and were in the physiological range for adult males.

    “We measured testosterone concentrations in adult animals that had been gonadectomized and implanted with testosterone-filled implants in order to ensure that levels did not differ between sex or treatment groups and were in the physiological range for adult males.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Testosterone is metabolized into estradiol and dihydrotestosterone, which bind ER and AR, and these receptors are critical for organizing many sex differences in the brain and behavior.

    “Testosterone is metabolized into two principle ligands: estradiol, which binds to estrogen receptor (ER) and the androgen, dihydrotestosterone, which binds to androgen receptor (AR), 17 and these receptors are critical for organizing many sex differences in the brain and behavior.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [17] Naftolin, 1971, Aromatization of androstenedione by the diencephalon, J Clin Endocrinol Metab source

  • Classified as probably not a substantial claim.

    Study design and conductown data

    AVP mRNA in the hypothalamus was examined to assess whether the infusion of MeCP2 siRNA may have spread to the hypothalamus.

    “In order to assess the possibility that the infusion of MeCP2 siRNA may have spread to the hypothalamus and altered AVP gene expression in that brain region, we examined the effect of neonatal infusion of MeCP2 siRNA into the amygdala on AVP mRNA expression in the hypothalamus.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All of the tissue targeted by each antibody was processed at the same time.

    “All of the tissue targeted by each antibody was processed at the same time.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Sex differences in the levels of AVP are not due to cell death, but rather most likely due to phenotypical differentiation.

    “previous findings from other labs indicate that sex differences in the levels of AVP are not due to cell death, but rather most likely due to phenotypical differentiation.49”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [49] de Vries, 2008, Sexual differentiation of vasopressin innervation of the brain: cell death versus phenotypic differentiation, Endocrinology source

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

    Study design and conductown data

    The testosterone EIA has an intra-assay coefficient of 9.0%.

    “the intra-assay coefficient was 9.0%.”
    Quote in the paperNot a substantial claim
30 of 30

  • The paper relies on this without support.

    Control siRNA does not affect gene expression or behavior relative to untreated animals.

    “Neonatal pups were pooled and assigned to a treatment group (five male control siRNA, seven male MeCP2 siRNA, seven female control siRNA and five female MeCP2 siRNA).”

  • The paper relies on this without support.

    MeCP2 siRNA does not have off-target effects on other genes that could affect AVP expression.

    “We have previously shown that this amount of MeCP2 siRNA induced a transient and localized reduction in MeCP2 expression within the amygdala.14”

  • The paper relies on this without support.

    The number of AVP-immunoreactive cells in adulthood reflects the number of AVP-expressing cells, not differences in detection sensitivity.

    “we examined AVP-ir cell number in adulthood.”

  • The paper relies on this without support.

    Less is known about what genes MeCP2 may regulate within socially-relevant brain regions.

    “less is known about what genes MeCP2 may regulate within socially-relevant brain regions.”

  • The paper relies on this without support.

    MeCP2 disruptions occur in Rett syndrome and several other neurodevelopmental disorders.

    “While numerous studies have confirmed that MeCP2 disruptions occur in Rett syndrome, as well as several other neurodevelopmental disorders”

  • The paper relies on this without support.

    AVP expression is highly regulated by circulating levels of testosterone.

    “as AVP expression is highly regulated by circulating levels of testosterone.”
6 of 6

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?

  • AVP may act via other nuclei not considered in the present study that likely contribute to AVP's actions on play.

    “AVP may act via other nuclei not considered in the present study that likely contribute to AVP’s actions on play, e.g., nucleus circularis, supraoptic nucleus, medial amygdaloid nucleus (Cheng et al., 2008; Kurian et al., 2008; Forbes-Lorman et al., 2012).”

    From Sexually dimorphic role for vasopressin in the development of social play (2014) · cites this paper as Forbes-Lorman et al., 2012

    The paper’s Experiment 1 section says AVP expression in the bed nucleus of the stria terminalis and centromedial amygdala is involved in social play. Its Discussion also notes a correlation between AVP expression and juvenile play, while saying it is unclear whether reduced AVP explains reduced play. Our copy does not link the nucleus circularis or supraoptic nucleus to play, so it supports the general possibility but not the cited examples as a group. (LLM, conf=0.83)

    Experimental — accuracy not yet measured

  • Silencing MeCP2 expression in the MeA reduced social play and AVP mRNA expression in the MeA in males but not females.

    “Likewise, silencing MeCP2 expression in the MeA reduced social play (Kurian et al., 2008) as well as AVP mRNA expression in the MeA (Forbes-Lorman et al., 2012) in males but not in females.”

    From Sex-specific modulation of juvenile social play by vasopressin (2013) · cites this paper as Forbes-Lorman et al., 2012

    Experiment 1 reports that neonatal MeCP2 siRNA reduced AVP mRNA in the amygdala of male rats; the Discussion says it had no effect on female AVP expression. The paper reports the mRNA measurement for the amygdala, not specifically the medial amygdala (MeA). The sentence cites Kurian et al. for the separate social-play finding. (LLM, conf=0.83)

    Experimental — accuracy not yet measured

Statistics that don’t add up

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

  • Inconsistent p-value across alpha = .05: t(7) = 5.17, p = .62

    Major

    The paper reports p = .62 here; recomputing from the test statistic and its degrees of freedom gives p = .001 (two-tailed). The two fall on opposite sides of alpha = .05. The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p = .62 · recomputed p = .001

  • Inconsistent p-value: F(1,31) = 1.23, p = .81

    Minor

    The paper reports p = .81 here; recomputing from the test statistic and its degrees of freedom gives p = .276. The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p = .81 · recomputed p = .276

  • Inconsistent p-value: F(1,31) = 0.06, p = .28

    Minor

    The paper reports p = .28 here; recomputing from the test statistic and its degrees of freedom gives p = .808. The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p = .28 · recomputed p = .808

  • Inconsistent p-value: F(1,29) = 0.002, p = .94

    Minor

    The paper reports p = .94 here; recomputing from the test statistic and its degrees of freedom gives p = .965. The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p = .94 · recomputed p = .965

  • Inconsistent p-value: F(1,32) = 0.08, p = .73

    Minor

    The paper reports p = .73 here; recomputing from the test statistic and its degrees of freedom gives p = .779. The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p = .73 · recomputed p = .779

  • Inconsistent p-value: F(1,32) = 0.13, p = .78

    Minor

    The paper reports p = .78 here; recomputing from the test statistic and its degrees of freedom gives p = .721. The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p = .78 · recomputed p = .721

  • Inconsistent p-value: F(1,29) = 0.006, p = .97

    Minor

    The paper reports p = .97 here; recomputing from the test statistic and its degrees of freedom gives p = .939. The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p = .97 · recomputed p = .939

7 of 7

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 upt(7) = 5.17reported p = .62 · recomputed p = .001The two fall on opposite sides of .05

    “t(7) = 5.17, p = .62”

  • Doesn’t add upF(1, 29) = 0.006reported p = .97 · recomputed p = .939

    “F(1,29) = 0.006, p = .97”

  • Doesn’t add upF(1, 29) = 0.002reported p = .94 · recomputed p = .965

    “F(1,29) = 0.002, p = .94”

  • Doesn’t add upF(1, 31) = 1.23reported p = .81 · recomputed p = .276

    “F(1,31) = 1.23, p = .81”

  • Doesn’t add upF(1, 31) = 0.06reported p = .28 · recomputed p = .808

    “F(1,31) = 0.06, p = .28”

  • Doesn’t add upF(1, 32) = 0.13reported p = .78 · recomputed p = .721

    “F(1,32) = 0.13, p = .78”

  • Doesn’t add upF(1, 32) = 0.08reported p = .73 · recomputed p = .779

    “F(1,32) = 0.08, p = .73”

  • ConsistentF(1, 15) = 6.82reported p = .02 · recomputed p = .020

    “F(1,15) = 6.82, p = .02”

  • ConsistentF(1, 18) = 4.98reported p = .039 · recomputed p = .039

    “F(1,18) = 4.98, p = .039”

  • ConsistentF(1, 18) = 5.41reported p = .032 · recomputed p = .032

    “F(1,18) = 5.41, p = .032”

  • ConsistentF(1, 19) = 8.1reported p = .01 · recomputed p = .010

    “F(1,19) = 8.1, p = .01”

  • ConsistentF(1, 19) = 4.5reported p < .05 · recomputed p = .047

    “F(1,19) = 4.5, p < .05”

  • ConsistentF(1, 19) = 2.17reported p = .16 · recomputed p = .157

    “F(1,19) = 2.17, p = .16”

  • ConsistentF(1, 19) = 0.03reported p = .86 · recomputed p = .864

    “F(1,19) = 0.03, p = .86”

  • ConsistentF(1, 19) = 0.59reported p = .45 · recomputed p = .452

    “F(1,19) = 0.59, p = .45”

  • ConsistentF(1, 18) = 0.07reported p = .79 · recomputed p = .794

    “F(1,18) = 0.07, p = .79”

  • ConsistentF(1, 18) = 0.71reported p = .41 · recomputed p = .411

    “F(1,18) = 0.71, p = .41”

  • ConsistentF(1, 19) = 0.57reported p = .46 · recomputed p = .460

    “F(1,19) = 0.57, p = .46”

  • ConsistentF(1, 18) = 0.09reported p = .77 · recomputed p = .768

    “F(1,18) = 0.09, p = .77”

  • ConsistentF(1, 18) = 0.32reported p = .58 · recomputed p = .579

    “F(1,18) = 0.32, p = .58”

  • ConsistentF(1, 18) = 0.001reported p = .97 · recomputed p = .975

    “F(1,18) = 0.001, p = .97”

  • ConsistentF(1, 18) = 0.33reported p = .57 · recomputed p = .573

    “F(1,18) = 0.33, p = .57”

  • ConsistentF(1, 29) = 8.34reported p = .007 · recomputed p = .007

    “F(1,29) = 8.34, p = .007”

  • ConsistentF(1, 30) = 2.4reported p = .13 · recomputed p = .132

    “F(1,30) = 2.4, p = .13”

  • ConsistentF(1, 30) = 0.15reported p = .7 · recomputed p = .701

    “F(1,30) = 0.15, p = .70”

  • ConsistentF(1, 29) = 0.002reported p = .96 · recomputed p = .965

    “F(1,29) = 0.002, p = .96”

  • ConsistentF(1, 29) = 0.423reported p = .52 · recomputed p = .521

    “F(1,29) = 0.423, p = .52”

  • Consistentt(9) = 2.99reported p = .02 · recomputed p = .015

    “t(9) = 2.99, p = .02”

  • ConsistentF(1, 9) = 6.88reported p = .03 · recomputed p = .028

    “F(1,9) = 6.88, p = .03”

  • ConsistentF(1, 30) = 4.58reported p = .04 · recomputed p = .041

    “F(1,30) = 4.58, p = .04”

  • ConsistentF(1, 31) = 6.55reported p = .016 · recomputed p = .016

    “F(1,31) = 6.55, p = .016”

31 of 31

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.

  • Figure 3B gives different p-values for adult AR-ir cells

    Minor

    The Results report p = 0.13 for sex and p = 0.70 for treatment in adult CMA AR-ir cell counts, but the Figure 3B caption gives p = 0.78 and p = 0.73 for the same comparisons. Both sources describe the effects as nonsignificant, but the reported values conflict.

1 of 1

Cites

Flagged first, then by citations

5 of the 59 references couldn’t be matched to a known paper.

Research works printed but not linked: 2

  1. Han, 2003, Organizational effects of testosterone, estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis, J Neurobiol, 54, 502, 10.1002/neu.10157 · DOI · Paper page
  2. Paxinos G, Watson C.Atlas of Paxinos and Watson(Academic Press,1986).

Cited by

Sorted by citations

Related

Authors
Forbes-Lorman, R. M., Rautio, J. J., Kurian, J. R., Auger, A. P., & Auger, C. J.
Affiliations
University of Wisconsin–Madison (US)
Corresponding author
Auger, Catherine J.
ORCID
Auger, Anthony P.
Venue
Epigenetics
Publisher
Informa UK Limited
Published
2012-03
Volume
7
Issue
3
Pages
230-238
ISSN
  • 1559-2294 (ISSN-L)
  • 1559-2308
DOI
10.4161/epi.7.3.19265
PMC
PMC3335947
OpenAlex
W2085369703
Semantic Scholar
d7e115e7b13732e0e0249b3a72b5dcd87f07e4cb
Cited by
43
References
59
Citations by year
2024: 1 · 2023: 2 · 2022: 2 · 2021: 2 · 2020: 3 · 2018: 1 · 2017: 4 · 2016: 5 · 2015: 6 · 2014: 7 · 2013: 10
FWCI
4.09
Type
journal article
Language
en
Open access
Locations
  • Epigenetics (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion)
  • PubMed Central (submittedVersion)
Funders
  • National Science Foundation (1122074)
  • University of Wisconsin-Madison
  • National Institutes of Health (R01MH072956)
  • National Institute of Mental Health (R01MH072956)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
  • Social Sciences › Psychology › Social Psychology › Neuroendocrine regulation and behavior
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Sexual Differentiation and Disorders
Keywords
MeCP2, vasopressin, sex differences, amygdala, neonatal development, galanin, androgen receptor, centromedial amygdala, bed nucleus of the stria terminalis, lateral septum, social behavior, neurodevelopmental disorders, siRNA knockdown
MeSH terms
Amygdala, Animals, Animals, Newborn, Arginine Vasopressin, Behavior, Animal, Female, Gene Expression Regulation, Male, Receptors, Androgen, Sex Characteristics, Rats, Sprague-Dawley, Galanin, Gene Silencing, RNA, Small Interfering, Rats, Methyl-CpG-Binding Protein 2

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.

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

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