Discussion
A critical balance between segregation and integration was observed when the mask was removed at DIV 10, allowing for the formation of a strong connectivity among the still-separated compartments, thus demonstrating the existence of a time window in network development in which it is possible to achieve a balance between segregation and integration.
Methods
We designed a cross-shaped polymeric mask to initially establish four independent sub-populations of cortical neurons and analyzed how the timing of its removal affected network development. We evaluated the morphological and functional features of the networks from 11 to 18 days in vitro (DIVs) with immunofluorescence techniques and micro-electrode arrays (MEAs).
Results
The removal of the mask at different developmental stages of the network lead to strong variations in the degree of intercommunication among the four assemblies (altering the segregation/integration balance), impacting firing and bursting parameters. Early removal (after 5 DIVs) resulted in networks with a level of integration similar to homogeneous controls (without physical constraints). In contrast, late removal (after 15 DIVs) hindered the formation of strong inter-compartment connectivity, leading to more clustered and segregated assemblies.