Qualitative and quantitative estimation of comprehensive synaptic connectivity in short- and long-term cultured rat hippocampal neurons with new analytical methods inspired by Scatchard and Hill plots

To investigate comprehensive synaptic connectivity, we examined Ca2+ responses with quantitative electric current stimulation by indium-tin-oxide (ITO) glass electrode with transparent and high electro-conductivity. The number of neurons with Ca2+ responses was low during the application of stepwise increase of electric current in short-term cultured neurons (less than 17 days in-vitro (DIV)). The neurons cultured over 17 DIV showed two-type responses: S-shaped (sigmoid) and monotonous saturated responses, and Scatchard plots well illustrated the difference of these two responses. Furthermore, sigmoid like neural network responses over 17 DIV were altered to the monotonous saturated ones by the application of the mixture of AP5 and CNQX, specific blockers of NMDA and AMPA receptors, respectively. This alternation was also characterized by the change of Hill coefficients. These findings indicate that the neural network with sigmoid-like responses has strong synergetic or cooperative synaptic connectivity via excitatory glutamate synapses.