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

This study in mice finds that MDMA (and a selective 5-HT1b receptor agonist) reverse social deficits through increased activity in the nucleus accumbens (NAc). The authors argue that enhancement of 5-HT release or direct 5-HT1b receptor activation may help with treating sociability deficits commonly found in those with autism spectrum disorder (ASD).

Abstract

“Autism spectrum disorder (ASD) is a common set of heterogeneous neurodevelopmental disorders resulting from a variety of genetic and environmental risk factors. A core feature of ASD is impairment in prosocial interactions. Current treatment options for individuals diagnosed with ASD are limited, with no current FDA-approved medications that effectively treat its core symptoms. We recently demonstrated that enhanced serotonin (5-HT) activity in the nucleus accumbens (NAc), via optogenetic activation of 5-HTergic inputs or direct infusion of a specific 5-HT1b receptor agonist, reverses social deficits in a genetic mouse model for ASD based on 16p11.2 copy number variation. Furthermore, the recreational drug MDMA, which is currently being evaluated in clinical trials, promotes sociability in mice due to its 5-HT releasing properties in the NAc. Here, we systematically evaluated the ability of MDMA and a selective 5-HT1b receptor agonist to rescue sociability deficits in multiple different mouse models for ASD. We find that MDMA administration enhances sociability in control mice and reverses sociability deficits in all four ASD mouse models examined, whereas administration of a 5-HT1b receptor agonist selectively rescued the sociability deficits in all six mouse models for ASD. These preclinical findings suggest that pharmacological enhancement of 5-HT release or direct 5-HT1b receptor activation may be therapeutically efficacious in ameliorating some of the core sociability deficits present across etiologically distinct presentations of ASD.”

Authors: Jessica J. Walsh, Pierre Llorach, Daniel F. Cardozo Pinto, Wendy Wenderski, Daniel J. Christoffel, Juliana S. Salgado, Boris D. Heifets, Gerald R. Crabtree & Robert C. Malenka

Summary

INTRODUCTION

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by impairments in social interactions and communication, in addition to stereotyped and restricted behaviors. It is caused by a complex interaction of genetic and environmental factors.

ASD is characterized by deficits in social interaction, and by abnormalities in serotonin (5-HT)-mediated modulation of brain functions. These abnormalities may be related to decreased brain 5-HT production, 5-HT transporter (SERT) binding, and 5-HT receptor binding.

We first became interested in the role of 5-HT in prosocial behaviors because the release of 5-HT in the nucleus accumbens is critical for social reward as assessed by a conditioned place preference assay and juvenile intruder assay. We then used a conditional knockout mouse model of the 16p11.2 deletion syndrome to explore the potential role of 5-HT in ASDs.

These findings suggest that 5-HT signaling may be enhanced by targeting specific 5-HT receptor subtypes in ASD, and that this may be a valuable therapeutic strategy for treating sociability deficits in ASD independent of their underlying etiologies.

MATERIALS AND METHODS Animals

Male and female C57BL/6 mice and the following transgenic lines were used as experimental subjects. All lines were backcrossed extensively to C57BL/6 mice prior to their use in generating mice for this study.

In this study, mice were used with a CD1 background, the Sert-Cre gene, and the 16p11.2flx allele. The Sert-Cre gene was used in mice with the Arid1bflx allele and was used to generate heterozygous mice.

VPA mice were generated as previously described . They were injected subcutaneously with 600 mg/kg of VPA sodium salt on gestational day E12.5 and were housed on a 12-h light/dark cycle with food and water ad libitum.

Design of behavioral tests with drug administration

For all behavioral tests involving MDMA, mice were counterbalanced such that half received drug on the first day and the other half received vehicle. The same cohort of mice was used for all four behavioral tests involving CP-94,253, with one cohort being run over the course of 8 weeks.

Three-chamber sociability test

This assay was performed in an arena with three separate chambers. Mice were habituated to the apparatus for 5 min, and then a conspecific juvenile was placed under a wire mesh cup with square holes that were 0.8 cm x 0.8 cm in one of the outer chambers.

Juvenile interaction test

The juvenile interaction test was performed in the home cage of the test animal, and a novel juvenile mouse was placed into the home cage for 2 min of free interaction. All sessions were video recorded and analyzed manually without knowledge of the experimental manipulation.

CP-94,253 dose-response

Dose-response experiments were conducted using the three-chamber sociability and juvenile interaction assays in two different ways: (1) in a single cohort of mice, and (2) in separate cohorts, with each dose being administered twice per dose (drug or vehicle).

Conditioned place preference (CPP) test

The CPP chamber was an acrylic box with two equal-sized compartments, each with a distinct floor, either clear, textured acrylic or smooth, black acrylic, and distinct walls, vertical lines or circles. Conditioning experiments involving CP-94,253 took place over 4 consecutive days.

We first estimated the center of the distribution of the control cohort administered vehicle (control average) by finding the center of a multivariate t-distribution fit onto the PC1 and PC2 scores for these mice. Then, we calculated the Euclidean distances between each subject and the control average.

Statistical analyses

For all data acquisition and analysis, investigators were blinded to the manipulation that the experimental subject had received and the genotype of the subject. Student’s t-tests, two-way ANOVA, and PCA were used to analyze data, and Sidak’s multiple comparison post hoc test was used when appropriate.

Sociability deficits are conserved across multiple mouse models for ASD

To assess basal sociability behavior, we performed juvenile interaction and three-chamber sociability tests in four different ASD mouse models: 16p11.2flx/flx mice, Fragile X syndrome mice, Cntnap2 knockout mice, and VPA mice. All four lines showed reduced time spent interacting with novel conspecific juvenile mice compared to controls, and all lines exhibited hyperactivity when exploring a novel object.

MDMA reverses sociability deficits in multiple mouse models for ASD

Systemic administration of MDMA reversed the sociability deficits in all four ASD mouse lines while vehicle administration had no effect. MDMA enhanced preference for the social chamber in the three-chamber test, but did not increase the time spent interacting with a juvenile.

MDMA, a drug with a long history of abuse potential, is being tested for treatment of post-traumatic stress disorder and social anxiety in autism spectrum disorder. Drugs that directly target the receptor(s) upon which 5-HT acts in the NAc may recapitulate MDMA’s prosocial effects while mitigating MDMA’s associated health risks. In ASD mouse models, systemic administration of the highly specific 5-HT1b receptor agonist CP-94,253 reversed sociability deficits in both the juvenile interaction and three-chamber tests.

We performed dose-response experiments in wild-type and Cntnap2/ mice to assess if 10 mg/kg of CP-94,253 was an appropriate dose to reverse the observed sociability deficits. The results showed that CP-94,253 dose-dependently reversed the sociability deficits seen in both juvenile interaction and three-chamber tests.

CP-94,252 did not elicit CPP or conditioned place aversion in wild-type or Cntnap2/ mice, but reduced hyperactivity in several mouse lines.

To further examine the surprisingly consistent effects of CP-94,253, we generated mice with heterozygous Arid1b deletion only in 5-HT neurons, which exhibited deficits in sociability in both the juvenile interaction and three-chamber sociability tests. Administration of CP-94,253 reversed these deficits, with no effects on control mice.

Principal component analysis of the behavioral profiles of mouse models for ASD

To assess the overall effect of CP-94,253 administration on the global behavioral profiles of individual mice, we performed dimensionality reduction using PCA. This allowed us to visualize and quantify the impact of CP-94,253 administration for hundreds of behavioral measurements in all six ASD models and their controls.

PC1 and PC2 were most strongly anti-correlated with performance on the sociability assays, and CP-94,253 treatment significantly reduced the distance from the subject’s PCA score under CP-94,253 or vehicle treatment to the control vehicle average.

DISCUSSION

In the present study, we demonstrate that enhancing 5-HT activity with MDMA or a selective 5-HT1b receptor agonist reverses impairments in sociability across multiple mouse models for ASD. The drugs had different effects on sociability and hyperactivity, likely due to their different mechanisms of action.

We intentionally examined multiple ASD mouse models based on different genetic or environmental syndromes, and backcrossed all of them to C57BL/6 mice.

MDMA reversed social deficits in four mouse models for ASD. The mice received systemic administration of MDMA on separate days and showed improved juvenile interaction and three-chamber sociability. Juvenile interaction and three-chamber sociability are significantly reduced in mice with systemic administration of vehicle or MDMA, and are significantly increased in mice with systemic administration of Fmr1 – /y, / MDMA.

Two different drugs, which influence 5-HT signaling, were effective in all models suggesting that 5-HT-mediated modulation of key targets such as the NAc may be one critical convergent target upon which the different causal genetic or environmental factors act.

generated by alterations in brain circuity other than that involving 5-HT neurons.

Previous work has suggested that several ASD mouse models exhibit abnormalities in 5-HT signaling, including decreased SERT mRNA during postnatal development, decreased 5-HT levels, abnormal 5-HT neuron differentiation and innervation patterns, and altered levels of 5-HT receptors.

SSRIs are commonly prescribed to treat symptoms associated with ASD, such as anxiety and depression, but do not appear to ameliorate core ASD symptoms, including social deficits. However, CP-94,253 reverses social deficits in mice with 16p11.2 selectively deleted from 5-HT neurons, suggesting that CP-94,253 is acting centrally, not peripherally.

5-HT1b receptor activation in ASD mouse models may contribute to the prosocial effects of CP-94,253 and MDMA. However, much work remains to be done to fully understand how these drugs mediate their prosocial behavioral effects.

If CP-94,253 elicits long-term effects, they are unlikely to be sensitizing effects, as there were no differences in the dose-response assays between mice given all four doses and mice that received only a single dose.

We hope that our findings will stimulate further work defining the effectiveness of simple, targeted pharmacological interventions in ameliorating some of the core deficits seen in individuals with ASD. We are more enthusiastic about the potential utility of a 5-HT1b receptor agonist in the treatment of ASD.

FUNDING AND DISCLOSURE

This work was supported by the HHMI, NSF, SFARI, and the Stanford University Wu Tsai Neurosciences Institute. RCM is on the scientific advisory boards of MapLight Therapeutics, Cerevance, Cyclerion, AZTherapies, MindMed, and Aelis Farma.

ACKNOWLEDGEMENTS

We thank the Malenka and Heifets labs for helpful comments, Claire Ellis for breeding the mice, and Jason M. Tucciarone for his expertise in timed pregnancies.

AUTHOR CONTRIBUTIONS

JJW performed and analyzed the majority of the experiments with assistance from PL, DFCP designed the PCA code, and DJC performed the PCA analysis. WW and GRC helped select the mouse lines to study.