We then assessed neuronal maturation and synaptic formation in organoids at TD31. Quantification of microtubule-associated protein 2 (MAP2) showed a significant increase in its density in ASD-derived neurons (Figure 3E, F). Moreover, synapse number quantification revealed a significant increase in Synapsin I-immunoreactive (SynI)+ puncta in ASD-derived neurons, suggesting increased neuronal maturation and synaptic overgrowth (Figure 3E, G). This is in agreement with the upregulated expression of the blue and brown modules (Figure 2A), whose signatures suggested accelerated or increased neuronal differentiation and synaptic connections in probands. Furthermore, quantification of the inhibitory VGAT (vesicular GABA transporter) and excitatory VGLUT1 (vesicular glutamate transpoter-1) immunoreactivity revealed a significant increase in VGAT-immunoreactive puncta (Figure 3H, I) and no significant changes in VGLUT1 puncta (Figure 3J), suggesting an increase in the number of inhibitory synapses in ASD-derived neurons.
