The Effects Of Substrate Stiffness On Ca(2+) Signaling,Synapse Transmission In Cultured Hippocampal Neuronal Network

Purpose:The mechanical property of extracellular matrix and cell-supporting substrates is known to modulate neuronal growth, differentiation, extension and branching. Here we show that substrate stiffness is an important microenvironmental cue, to which mouse hippocampal neurons respond and integrate into synapse transmission in cultured neuronal network. This study aims to investigate the effects of substrate stiffness on Ca2+signaling, synapse transmission in cultured hippocampal neuronal network.Methods:Mouse hippocampal neurons were isolated from one day old pups, then cells were plated and cultured on PDMS substrates fabricated to have similar surface properties but a difference in Young’s modulus. Immunofluorescence staining and Ga2+image were used. Voltage-gated Ca2+channels current and spontaneous postsynaptic currents were examined by patch-clamp recording technique.Results:1)Two types of PDMS substrate preparations had similar surface roughness or topography, but a 10-fold difference in Young’s modulus. 2)Hippocampal neurons cultured on PDMS substrates grew well and a high proportion of them made contacts with each other in the later stage. The confocal image results showed no difference in neuron density, so ma size, and percentage of excitatory and inhibitory neurons on stiff and soft substrates.3)Ca2+oscillations in cultured neuronal network monitored using time-lapse cell imaging increased in both amplitude and frequency among neurons on stiff substrates.4)Voltage-gated Ca2+ channel currents were greater in neurons on stiff substrates than on soft substrates. 5)Spontaneous excitatory postsynaptic activity became greater and more frequent in neurons on stiff substrates, and by contrast no effect on spontaneous inhibitory postsynaptic currents was detected.Conclusion:Stiff substrates up-regulate neuronal Ca2+oscillations, voltage-gated Ca2+channels, and preferentially enhance the excitatory synaptic transmission in cultured hippocampal neuronal network. Substrate stiffness is an important biophysical factor modulating Ca2+signaling and synapse transmission in cultured hippocampal neuronal network.