Research ArticleAutism

Exogenous and evoked oxytocin restores social behavior in the Cntnap2 mouse model of autism

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Science Translational Medicine  21 Jan 2015:
Vol. 7, Issue 271, pp. 271ra8
DOI: 10.1126/scitranslmed.3010257
  • Fig. 1. Oxytocin administration rescues social behavior in the Cntnap2 mouse model.

    (A) Reciprocal social interaction test for wild-type (WT) (left) and KO (right) drug- or vehicle-treated mice by intraperitoneal injection. The time spent engaged in social interaction for each pair of mice is shown (n = 6 pairs, 3M-3F, per genotype per condition). Two-way ANOVA F11,60 = 2.89, P = 0.004; treatment effect F5,60 = 4.13, P = 0.0027; treatment/genotype interaction F5,60 = 2.23, P = 0.0627, followed by Dunnett’s pairwise comparisons to controls WTvehicle-KOvehicle P = 0.03, KOvehicle-KOOXT P = 0.03, KOvehicle-KOAVP P = 0.04. (B) Social approach (three-chamber) test. Time spent interacting with an empty cup or a cup with a stranger mouse inside is shown for each genotype when treated intraperitoneally with vehicle, OXT, or AVP (n = 8 to 10, 4/5M-4/5F, mice per genotype per condition). Paired Student’s t test comparing “mouse” to “empty” as a measure of sociability within each group. WTvehicle P = 0.01, WTOXT P = 0.02, KOOXT P = 0.03. (C) Reciprocal social interaction test when OXT and AVP are administered intranasally (n = 8, 4M-4F, pairs per genotype per condition). Two-way ANOVA F5,42 = 15.88, P < 0.0001; treatment effect F2,42 = 21.08, P < 0.0001; genotype/treatment interaction F2,42 = 17.01, P ≤ 0.0001, followed by Dunnett’s pairwise comparisons to controls WTvehicle-KOvehicle P < 0.0001, KOvehicle-KOOXT P < 0.0001, KOvehicle-KOAVP P < 0.0001. (D) Reciprocal social interaction test (n = 8 pairs of mice, 4M-4F, per genotype per condition). One-way ANOVA F5,41 = 22.02, P < 0.0001, followed by Bonferroni post hoc test, modified significance level P = 0.003, all significant comparisons P < 0.0001. (E) Reciprocal social interaction test showing time course of a single acute dose of intranasal OXT administration (n = 8 pairs of mice, 4M-4F, per genotype per condition). One-way ANOVA F4,35 = 23.16, P < 0.0001, followed by Bonferroni post hoc test, modified significance level P = 0.005, all significant comparisons P < 0.0001. M, male; F, female. Box-plots represent the median plus the 25th and 75th percentiles. Whiskers represent the minimum and maximum values. The mean is represented by a plus sign. Asterisks represent a statistically significant difference between indicated groups.

  • Fig. 2. Pharmacological stimulation of OXT release improves social behavior in the Cntnap2 model.

    (A) Reciprocal social interaction test in vehicle- or drug-treated animals (n = 6 pairs of mice, 3M-3F, per genotype per condition). Two-way ANOVA genotype/treatment interaction F1,20 = 4.87, P = 0.04, followed by Dunnett’s pairwise comparisons to controls, WTvehicle-KOvehicle P = 0.04, KOvehicle-KOMC4Rag P = 0.02. (B) Reciprocal social interaction test in Cntnap2 KO (n = 7 pairs of mice, 4M-3F, per condition). One-way ANOVA F2,18 = 14.6, treatment effect P ≤ 0.0001, followed by Bonferroni post hoc test, modified significance level P = 0.017, vehicle-agonist P = 0.002, agonist-antagonist P < 0.0001. M, male; F, female. Box-plots represent the median plus the 25th and 75th percentiles. Whiskers represent the minimum and maximum values. The mean is represented by a plus sign. Asterisks represent a statistically significant difference between indicated groups.

  • Fig. 3. Cntnap2 mutant mice show reduced central OXT levels at P30.

    (A) Representative images of OXT immunoreactivity in different anteroposterior levels of the PVN in Cntnap2 KO and WT controls at P30. Scale bar, 100 μm. (B) Stereological counts of OXT-positive cells in the PVN region of both genotypes at P30 (n = 4 mice, 2M-2F, per genotype). Student’s t test, P = 0.03. (C) Quantification of OXT levels in whole-brain extracts by RIA at P30 (n = 10 mice, 5M-5F, per genotype). Student’s t test, P = 0.01. M, male; F, female. Box-plots represent the median plus the 25th and 75th percentiles. Whiskers represent the minimum and maximum values. The mean is represented by a plus sign. Asterisks represent a statistically significant difference between indicated groups.

  • Fig. 4. Oxytocin immunoreactivity in Cntnap2 mutants at P7 is normal.

    (A) Representative images of OXT immunoreactivity at different anteroposterior levels of the PVN in Cntnap2 KO and WT controls at P7. Scale bar, 100 μm. (B) Stereological counts of OXT-positive cells in the PVN region of both genotypes at P7 (n = 4 mice, 2M-2F, per genotype). No differences were found with Student’s t test (P = 0.53). M, male; F, female. Box-plots represent the median plus the 25th and 75th percentiles. Whiskers represent the minimum and maximum values. The mean is represented by a plus sign.

  • Fig. 5. Early postnatal OXT treatment restores peptide levels and improves social behavior at P30.

    (A) Social approach (three-chamber) test shown for WT and KO mice at P30 treated early with vehicle or OXT (n = 10 to 14 mice per group). Paired Student’s t test comparing “mouse” to “empty” within each group as a measure of sociability, WTsaline P = 0.006 (n = 10, 4M-6F), WTOXT P = 0.006 (n = 14, 8M-6F), KOsaline P = 0.3 (n = 12, 6M-6F), KOOXT P = 0.004 (n = 13, 6M-7F). (B) Reciprocal social interaction test shown in pairs of KO mice at P30 treated early with saline or with OXT (n = 7 and 8 pairs of mice, 4M-3/4F, respectively). Student’s t test, P < 0.001. (C) Quantification, as detected by RIA, of OXT levels in whole-brain extracts for KO mice treated with saline or OXT at P30 (n = 6, 3M-3F; n = 8, 4M-4F, respectively). Student’s t test, P = 0.02. (D) Stereological quantification of the number of OXT immunoreactive cells in Cntnap2 KO mice treated with either saline or OXT at P30 (n = 4 mice, 2M-2F, per condition). Student’s t test, P = 0.005. (E) Representative images of OXT immunoreactivity in the PVN of saline- or OXT-treated KO animals at P30. Scale bar, 100 μm. M, male; F, female. Box-plots represent the median plus the 25th and 75th percentiles. Whiskers represent the minimum and maximum values. The mean is represented by a plus sign. Asterisks represent a statistically significant difference between indicated groups.

  • Fig. 6. Evoked OXT release improves social behavior in the Cntnap2 mouse.

    (A) Schematic representation of the construct used to express designer receptors in OXT cells. WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; ITR, inverted terminal repeat. (B) mCherry fluorescence in the PVN of AAV2-injected mice colocalizes with OXT immunoreactivity. (C) Representative trace of whole-cell, current-clamp recordings from an mCherry-labeled OXT neuron. CNO (5 μM) was perfused in the bath for 250 s, which resulted in consistent depolarization of membrane potential and an increase in action potential firing. (D) Social approach (three-chamber) test in saline- or CNO-treated (5 mg/kg) KO and WT mice (n = 6 male mice per genotype per condition). Paired Student’s t test comparing “mouse” to “empty” within each group as a measure of sociability, WTsaline P < 0.001, WTCNO P = 0.03, KOsaline P = 0.13, KOCNO P = 0.001. Box-plots represent the median plus the 25th and 75th percentiles. Whiskers represent the minimum and maximum values. The mean is represented by a plus sign. Asterisks represent a statistically significant difference between indicated groups.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/271/271ra8/DC1

    Fig. S1. No OXT effect on non-core–associated behavioral deficits in the Cntnap2 mouse model.

    Fig. S2. Cntnap2/OXT colocalization in the PVN.

    Fig. S3. Total number of neurons in the PVN area.

    Fig. S4. Vasopressin immunoreactivity in the PVN.

    Fig. S5. Map and sequence of the hM3Dq-mCherry construct.

    Table S1. Drugs used for pharmacological testing.

    Table S2. Raw data of Fig. 1A.

    Table S3. Raw data of Fig. 1B.

    Table S4. Raw data of Fig. 1C.

    Table S5. Raw data of Fig. 1D.

    Table S6. Raw data of Fig. 1E.

    Table S7. Raw data of Fig. 2A.

    Table S8. Raw data of Fig. 2B.

    Table S9. Raw data of Fig. 3.

    Table S10. Raw data of Fig. 4.

    Table S11. Raw data of Fig. 5A.

    Table S12. Raw data of Fig. 5B.

    Table S13. Raw data of Fig. 5C.

    Table S14. Raw data of Fig. 5D.

    Table S15. Raw data of Fig. 6.

    Table S16. Raw data of fig. S1A.

    Table S17. Raw data of fig. S1B.

    Table S18. Raw data of fig. S1C.

    Table S19. Raw data of fig. S1D.

    Table S20. Raw data of fig. S3.

    Table S21. Raw data of fig. S4.

  • Supplementary Material for:

    Exogenous and evoked oxytocin restores social behavior in the Cntnap2 mouse model of autism

    Olga Peñagarikano, María T. Lázaro, Xiao-Hong Lu, Aaron Gordon, Hongmei Dong, Hoa A. Lam, Elior Peles, Nigel T. Maidment, Niall P. Murphy, X. William Yang, Peyman Golshani, Daniel H. Geschwind*

    *Corresponding author. E-mail: dhg{at}ucla.edu

    Published 21 January 2015, Sci. Transl. Med. 7, 271ra8 (2015)
    DOI: 10.1126/scitranslmed.3010257

    This PDF file includes:

    • Fig. S1. No OXT effect on non-core–associated behavioral deficits in the Cntnap2 mouse model.
    • Fig. S2. Cntnap2/OXT colocalization in the PVN.
    • Fig. S3. Total number of neurons in the PVN area.
    • Fig. S4. Vasopressin immunoreactivity in the PVN.
    • Fig. S5. Map and sequence of the hM3Dq-mCherry construct.
    • Table S1. Drugs used for pharmacological testing.
    • Table S2. Raw data of Fig. 1A.
    • Table S3. Raw data of Fig. 1B.
    • Table S4. Raw data of Fig. 1C.
    • Table S5. Raw data of Fig. 1D.
    • Table S6. Raw data of Fig. 1E.
    • Table S7. Raw data of Fig. 2A.
    • Table S8. Raw data of Fig. 2B.
    • Table S9. Raw data of Fig. 3.
    • Table S10. Raw data of Fig. 4.
    • Table S11. Raw data of Fig. 5A.
    • Table S12. Raw data of Fig. 5B.
    • Table S13. Raw data of Fig. 5C.
    • Table S14. Raw data of Fig. 5D.
    • Table S15. Raw data of Fig. 6.
    • Table S16. Raw data of fig. S1A.
    • Table S17. Raw data of fig. S1B.
    • Table S18. Raw data of fig. S1C.
    • Table S19. Raw data of fig. S1D.
    • Table S20. Raw data of fig. S3.
    • Table S21. Raw data of fig. S4.

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