Systemic enhancement of serotonin signaling reverses social deficits in multiple mouse models for ASD

A range of transgenic and environmental mouse models were used, all aged 8–16 weeks and of both sexes. Lines included conditional deletion of the 16p11.2 syntenic region from 5-HT neurons (Sert-Cre +/-:16p11.2 flx/flx), constitutive Fmr1 knockouts, Cntnap2 knockouts, prenatal valproic acid (VPA) exposure, Actl6b -/- mice, and mice with heterozygous Arid1b deletion restricted to 5-HT neurons (Sert-Cre +/-:Arid1b flx/+). Most lines were backcrossed >8 generations to C57BL/6; one line had a mixed C57BL/6–CD1 background. Controls were littermates without the targeted deletion or, for Fmr1 females, age-matched in-house C57BL/6. Novel juvenile conspecifics for social assays were 3–5 weeks old and drug-naïve. Animals were housed on a 12-h light/dark cycle, all procedures were approved by institutional panels, and experiments were performed blinded and randomised by cage. Sample sizes were not determined by formal power calculation but chosen from prior experience with the assays. Drugs were administered intraperitoneally at 0.01 ml/g in saline. MDMA was given at 7.5 mg/kg and CP-94,253 at 10 mg/kg for the principal experiments; dose‑response testing for CP-94,253 used 1, 3, 10 and 30 mg/kg. Behavioural testing used within-subject, counterbalanced designs in which each mouse received drug on one test day and vehicle on another, 20 minutes prior to testing. Different cohorts were used for the MDMA experiments; the same cohort underwent multiple CP-94,253 experiments across up to 8 weeks, with counterbalancing to avoid order effects. Behavioural endpoints included the three-chamber sociability assay, juvenile interaction in the subject's home cage, novel object interaction and open-field locomotion. Three-chamber sociability was quantified as time spent near a juvenile under an enclosure versus an empty enclosure; juvenile interaction scored active social behaviours (pursuit, grooming, sniffing) during 2 min free interaction. Locomotion was tracked in a 40 cm × 40 cm arena for 18 min. Conditioned place preference (CPP) protocols assessed reinforcing properties of CP-94,253 over a 4-day paradigm. For data reduction across many measures, principal component analysis (PCA) was applied to scaled, centred behavioural data; the authors quantified rescue by computing Euclidean distances from each subject's PCA score (vehicle or drug) to the control vehicle average. Statistical analyses were blinded; t-tests, two-way ANOVA with Sidak post hoc tests, and three-way ANOVA (drug × genotype × sex) were employed as appropriate. Sexes were pooled in reported analyses because no sex differences were detected. Data were tested for normality and equal variances; Prism and R were used for analyses.

Baseline characterisation showed conserved sociability deficits across the four initial ASD models. In juvenile interaction and three-chamber assays, Sert-Cre +/-:16p11.2 flx/flx, Cntnap2 -/-, Fmr1 -/y, -/- and VPA‑exposed mice all spent less time interacting with or near a novel conspecific than controls. These deficits were statistically robust (figure legend reports t-values with P < 0.001 for multiple comparisons). Novel object exploration was unchanged across lines, while locomotor activity was increased in all lines except the Sert-Cre +/-:16p11.2 flx/flx mice. Systemic MDMA (7.5 mg/kg i.p.) reversed sociability deficits in all four ASD models in both juvenile interaction and three-chamber assays when administered 20 min before testing; vehicle had no effect. In control mice MDMA increased preference for the social chamber in the three-chamber test but did not alter time spent in juvenile interaction. MDMA did not affect novel object interaction. Systemic administration of the selective 5-HT1b agonist CP-94,253 (10 mg/kg i.p.) also reversed sociability deficits across the genetic ASD models in both assays. In VPA mice CP-94,253 reversed juvenile interaction deficits and mitigated social aversion in the three-chamber test but did not further enhance sociability in that assay. Dose‑response testing in Cntnap2 -/- mice showed a dose-dependent rescue across 1–30 mg/kg when doses were administered progressively in the same cohort; doses up to 10 mg/kg produced little effect in wild-type mice while 30 mg/kg elicited a modest sociability increase. Separate single‑dose cohort experiments produced similar results, arguing against carry-over effects. To assess reinforcing potential, CPP assays showed that CP-94,253 did not induce conditioned place preference or aversion in wild-type or Cntnap2 -/- mice. CP-94,253 reduced hyperactivity in several ASD lines (Cntnap2 -/-, Fmr1 -/y, -/-, and VPA) but did not alter locomotion in control mice or in the Sert-Cre +/-:16p11.2 flx/flx line; novel object exploration remained unaffected. Extending the approach, CP-94,253 reversed sociability deficits and reduced hyperactivity in Actl6b -/- mice, and reversed sociability deficits in mice with Arid1b heterozygous deletion restricted to 5-HT neurons (Sert-Cre +/-:Arid1b flx/+), without altering novel object interaction or locomotion in that Arid1b line. PCA of the full behavioural profiles indicated that vehicle-treated ASD models shared a similar multivariate profile and were distinct from controls (PC1 and PC2 explained ~66–72% of variance; PC1 correlated inversely with sociability measures). After CP-94,253 treatment the ASD models' profiles overlapped with controls, and Euclidean distance from each subject's score to the control vehicle average was significantly reduced for ASD cohorts but unchanged in controls, indicating a global normalisation of measured behaviours by CP-94,253.

Walsh and colleagues interpret the data to mean that enhancing 5-HT signalling—either by promoting 5-HT release with MDMA or by selectively activating 5-HT1b receptors with CP-94,253—can acutely reverse sociability deficits across multiple, etiologically diverse mouse models of ASD. They highlight two consistent differences between the drugs: MDMA enhanced sociability in control mice and can increase locomotion and has reinforcing properties at higher doses, whereas CP-94,253 did not enhance sociability in controls, reduced hyperactivity in several ASD lines, and lacked evidence of acute reinforcement in CPP assays. The investigators attribute these distinctions to different mechanisms of action: MDMA causes broad, supraphysiological 5-HT release via SERT and also increases dopamine, whereas CP-94,253 is a selective 5-HT1b receptor agonist that should have minimal off-target effects on other 5-HT receptor subtypes. The authors situate their findings within prior literature suggesting altered 5-HT signalling in several ASD models and humans with ASD, and they argue that the NAc and 5-HT1b receptors constitute a plausible convergent node through which diverse genetic and environmental perturbations impair social behaviour. They note evidence that deleting 16p11.2 or Arid1b selectively from 5-HT neurons produces sociability deficits, supporting a central, 5-HT‑linked mechanism rather than a purely peripheral effect. Mechanistically, 5-HT1b receptor activation inhibits excitatory synaptic transmission in the NAc, and modulation of excitatory inputs to NAc has been implicated in reversal of social deficits in other ASD models, suggesting a candidate circuit-level explanation. Key limitations acknowledged by the authors include the possibility that residual background genetic differences could contribute to observed behaviours despite extensive backcrossing, and the fact that only acute drug effects were tested. They emphasise caution regarding developmental effects of 5-HTergic agents if translated to children. Nevertheless, they propose that a selective 5-HT1b agonist may represent a more feasible therapeutic strategy than MDMA for ameliorating sociability deficits in ASD because it produced broad behavioural normalisation across models without acute reinforcing effects. The authors call for further work to define mechanisms, longer-term effects, and translational potential while noting the need to weigh developmental safety concerns.