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 drugs (DREADDs). For DREADDs, we used a mutated human muscarinic receptor type 3 (hM3Dq) which is a Gq-coupled 7-transmembrane receptor that signals calcium mobilization upon activation by its designer ligand clozapine-N-oxide (CNO).20–22 We demonstrate that we can achieve a circuit-level readout of calcium activity in excitatory (glutamate)-inhibitory (GABA) two-way circuits. We also replicate the synaptogenetic property of neuroligin-3 in our device to demonstrate its use in high-throughput morphometric and synapse formation assays on increased synaptogenesis. We optimized these experiments for automated confocal microscopy and reagent dispensing using a commercially available GE IN Cell Analyzer 6000, the industry standard for laser-based confocal imaging. This system enables high-throughput image acquisition for high-content assays and provides a quantitative platform for cell viability and morphological analyses. Incorporation of this plate within the IN Cell system demonstrates the utility of our approach to serve as an HTS platform for neural circuit studies and its potential as a therapeutic development tool.
