Role of antidepressants in fetal synapse formation in Autism Spectrum Disorders

Abstract

Autism is a genetically complex neurodevelopmental disorder in which patients exhibit social deficits in both verbal and non-verbal forms of communication and display restricted and repetitive behaviors. Approximately 1 in 68 children are diagnosed with Autism in the United States². The prevalence of Autism in North Carolina is even greater where 1 in 58 children are diagnosed³. Autism is thought to be influenced by both genetic and environmental factors. Complex interactions between these factors make the creation of therapeutic treatments difficult to achieve. One environmental factor that is being studied in relation to Autism is the anti-depressant Fluoxetine. Fetal exposure to Fluoxetine through maternal ingestion of the drug or consumption of drinking water where the drug is present is thought to interrupt normal fetal brain development. Fluoxetine has previously been show to increase dendritic spine formation, the main location of excitatory synapse development. However, the exact mechanism that causes this dysregulation of the actin cytoskeleton is not fully understood. Post-mortem samples from individuals with Autism also display increased dendritic spine levels. We hypothesize that Fluoxetine acts through the Rac1 pathway to increase dendritic spine density. To examine the impact of Fluoxetine on fetal synapse formation human cortical organoids, or 'mini-brains', were created to recapitulate the second trimester fetal brain. Once the 'mini-brains' reached the appropriate time point in development they were treated either acutely with Fluoxetine, chronically with Fluoxetine, with the Rac1 inhibitor NSC23766 or a combination of Fluoxetine and NSC23766. After 90 days in culture, the 'mini-brains' were harvested, fixed, cryosectioned and stained for pre- and post-synaptic markers. Using ImageJ excitatory synapse density and morphology was analyzed. It was determined that Fluoxetine caused enlargement of synapses that were irregular in shape. The effects of Fluoxetine on synapse formation were reduced when combined with the Rac1 inhibitor NSC23766. In addition to examining excitatory synapse formation, the effects of Fluoxetine and NSC23766 on electrical signal transmission was also observed using micro-electrode technology. Both Fluoxetine and NSC23766 were shown to decrease neuronal activity.