c-Jun N-terminal kinase and phosphatidylinositide 3-kinases signaling pathways mediate synapse formation and spontaneous network activities in primary neurons

The cellular signals controlling synapse formation and neuronal network function remain poorly understood. This study investigated the canonical signaling pathways in neurons and their effects on network development using pharmacological assays to inhibit multiple signaling pathways in cultured primary neurons. Immunofluorescence was applied to detect synapse-related proteins in the neurons. Microelectrode arrays (MEAs) have been used to investigate the developmental characteristics of neuronal networks. Ribonucleic acid (RNA) sequencing was conducted to analyze gene expression profiles related to multiple signaling pathways. We found that the c-Jun N-terminal kinase (JNK) pathway was essential for pre- and postsynaptic formation, while the phosphatidylinositide 3-kinases (PI3K) pathway was crucial for postsynaptic formation and influenced vesicular glutamate transporter 1 (VGlut1) puncta size. Inhibition of the JNK pathway significantly reduced the mean firing rate (MFR), network burst frequency (NBF), and firing regularity after four weeks, but did not influence network synchrony. During network development, the PI3K pathway modulates longer burst durations and decreases network synchrony. The inhibition of these pathways disrupts the signaling related to neurodevelopment and synapses. Additionally, inhibition of the PI3K pathway alters voltage-gated ion channel activity, synaptic transmission, and synaptic plasticity. Overall, this study revealed that the JNK and PI3K pathways play distinct roles in synapse formation and significantly influence neuronal network development. These findings enhance current understanding of the intracellular signaling pathways involved in synapse formation and neuronal network development.