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

Abstract

Mouse models of neuropsychiatric diseases provide a platform for mechanistic understanding and development of new therapies. We previously demonstrated that knockout of the mouse homolog of CNTNAP2 (contactin-associated protein-like 2), in which mutations cause cortical dysplasia and focal epilepsy (CDFE) syndrome, displays many features that parallel those of the human disorder. Because CDFE has high penetrance for autism spectrum disorder (ASD), we performed an in vivo screen for drugs that ameliorate abnormal social behavior in Cntnap2 mutant mice and found that acute administration of the neuropeptide oxytocin improved social deficits. We found a decrease in the number of oxytocin immunoreactive neurons in the paraventricular nucleus (PVN) of the hypothalamus in mutant mice and an overall decrease in brain oxytocin levels. Administration of a selective melanocortin receptor 4 agonist, which causes endogenous oxytocin release, also acutely rescued the social deficits, an effect blocked by an oxytocin antagonist. We confirmed that oxytocin neurons mediated the behavioral improvement by activating endogenous oxytocin neurons in the paraventricular hypothalamus with Designer Receptors Exclusively Activated by Designer Drugs (DREADD). Last, we showed that chronic early postnatal treatment with oxytocin led to more lasting behavioral recovery and restored oxytocin immunoreactivity in the PVN. These data demonstrate dysregulation of the oxytocin system in Cntnap2 knockout mice and suggest that there may be critical developmental windows for optimal treatment to rectify this deficit.

Figure 1. Oxytocin administration rescues social behavior in the Cntnap2 mouse model

(A) Reciprocal social interaction test for WT (left) and KO (right) drug or vehicle treated mice by i.p. injection. The time spent engaged in social interaction for each pair of mice is shown (n=6 pairs, 3M-3F, per genotype/condition). Two-way ANOVA F_11,60=2.89, p=0.004, treatment effect F_5,60=4.13, p=0.0027, treatment/genotype interaction F_5,60=2.23, p=0.0627, followed by Dunnett pairwise comparisons to controls WT_vehicle-KO_vehicle p=0.03, KO_vehicle-KO_OXT p=0.03, KO_vehicle-KO_AVP p=0.04. (B) Social approach (3 chamber) test. Time spent interacting with an empty cup or a cup with a stranger mouse inside is shown for each genotype when treated i.p. with vehicle, OXT or AVP (n=8–10, 4/5M-4/5F, mice per genotype/condition). Paired Student t test comparing 'mouse' to 'empty' as a measure of sociability within each group. WT_vehicle p=0.01, WT_OXT p=0.02, KO_OXT=0.03 (C) Reciprocal social interaction test when OXT and AVP are administered intranasally (n=8, 4M-4F, pairs per genotype/condition). Two-way ANOVA F_5,42=15.88, p<0.0001, treatment effect F_2,42=21.08, p<0.0001, genotype/treatment interaction F_2,42=17.01, p≤0.0001 followed by Dunnett pairwise comparisons to controls WT_vehicle-KO_vehicle p<0.0001, KO_vehicle-KO_OXT p<0.0001, KO_vehicle-KO_AVP p<0.0001. (D) Reciprocal social interaction test (n=8 pairs of mice, 4M-4F, per genotype/condition). One-way ANOVA F_5,41=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/condition). One-way ANOVA F_4,35=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 as a plus (+) sign.

Figure 2. Pharmacological stimulation of oxytocin 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/condition). Two-way ANOVA genotype/treatment interaction F_1,20=4.87, p=0.04, followed by Dunnett pairwise comparisons to controls, WT_vehicle-KO_vehicle p=0.04, KO_vehicle-KO_MC4Rag. p=0.02. (B) Reciprocal social interaction test in Cntnap2 KO (n=7 pairs of mice, 4M-3F, per condition). One way ANOVA F_2,18=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 as a plus (+) sign.

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

(A) Representative images of OXT immunoreactivity in different anterio-posterior 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 t-test, p=0.03. (C) Quantification of OXT levels in whole brain extracts by radioimmunoassay at P30 (n=10 mice, 5M-5F, per genotype). Student 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 as a plus (+) sign.

Figure 4. Oxytocin immunoreactivity in Cntnap2 mutants at P7 is normal

(A) Representative images of OXT immunoreactivity at different anterio-posterior 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 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 as a plus (+) sign.

Figure 5. Early postnatal oxytocin treatment restores peptide levels and improves social behavior at P30

(A) Social approach (3 chamber) test shown for WT and KO mice at P30 treated early with vehicle or OXT (n=10–14 mice/group). Paired Student t test comparing 'mouse' to 'empty' within each group as a measure of sociability, WT_saline p=0.006 (n=10, 4M-6F), WT_OXT p=0.006 (n=14, 8M-6F), KO_saline p=0.3 (n=12, 6M-6F), KO_OXT 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 t test, p<0.001 (C) Quantification, as detected by radioimmunoassay, of OXT levels in whole brain extracts for KO mice treated with saline or with OXT at P30 (n=6, 3M-3F; n=8, 4M-4F, respectively). Student 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 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: 100µm.). 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 as a plus (+) sign.

Figure 6. Evoked oxytocin release improves social behavior in the Cntnap2 mouse

(A) Schematic representation of the construct used to express designer receptors in OXT cells (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 oxytocin neuron. CNO (5 µM) was perfused in the bath for 250 seconds, which resulted in consistent depolarization of membrane potential and an increase in action potential firing (D) Social approach (3 chamber) test in saline or CNO (5mg/kg) treated KO and WT mice (n=6 male mice per genotype/condition). Paired Student t test comparing 'mouse' to 'empty' within each group as a measure of sociability, WT_saline p<0.001, WT_CNO p=0.03, KO_saline p=0.13, KO_CNO 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 as a plus (+) sign.