Mechanism of Stiff Substrates up-Regulate Cultured Neuronal Network Activity

Our previous work provided compelling evidence showing that substrate stiffness is crucial for regulating synaptic connectivity and excitatory synaptic transmission among neurons in the neuronal network. However, the underlying mechanisms remain elusive. In our study, polydimethylsiloxane (PDMS) substrates with different stiffness have been fabricated to investigate the mechanisms by which the substrate stiffness upregulates the formation and activity of the cultured neuronal network. Here we report that stiff substrate increased both the number of synapses and the efficacy of excitatory synaptic transmission. More colocalization of synaptotagmin and PSD-95 was observed in the neuronal network on stiff substrate, which indicated the synapse number has increased. We also found that the increased synapse number was mediated by Hevin and SPARC that are secreted from astrocyte. The increased efficacy of excitatory synaptic transmission induced by stiff substrate was explored in three aspects. First, stiff substrate enhanced the presynaptic activity through increasing the vesicular release probability (Pr) of neurotransmitters as well as the calcium influx. Second, stiff substrate reduced voltage-dependent Mg²⁺ blockade to N-methyl-d-aspartate receptor (NMDAR) channels, which led to higher postsynaptic activity. Third, our work suggested that the increased excitatory synaptic transmission in the neural network on stiff substrate involved the upregulated synaptic glutamate concentration. Taken together, these findings may provide a molecular mechanism underlying substrate stiffness regulation of excitatory synaptic transmission in the cultured neural network.