To address some of these limitations, we have created a parallelized microfluidic plate-based system designed to be coupled with a liquid handling microscopy platform, the GE IN Cell Analyzer 6000, and demonstrate its ability to assay mini-neurocircuits for functional and morphological properties using human neurons derived from iPS cells. We employed soft lithography techniques to design a 96 well plate bottom which introduces two distinct compartments connected by a series of microchannels into a single well of a 96 well plate. We demonstrate that distinct human neuronal populations can be seeded in each compartment and that these cells can be used for functional and morphological screening assays. As a readout of circuit-level connectivity, we employed lentiviral-mediated expression of the genetically-encoded calcium indicator GCaMP6f to measure neural activity-induced depolarization between connected subpopulations of human neurons.19 To demonstrate HTS capabilities and to confirm synaptic communication between isolated neuronal populations within our circuit models, we used two experimental configurations: 1) glutamate receptor antagonists to silence glutamate signaling from excitatory neurons, as well as 2) excitatory neurons expressing designer receptors exclusively activated by designer
