Novel technology & techniques to study long-term neural dynamics of living neural networks cultured in-vitro
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The fundamental mechanisms of information processing and storage, and network plasticity for in-vitro LNNs are studied by monitoring extracellular electrochemical neuronal activity via a Multi-Electrode Array (MEA). MEAs monitor action potentials, and enable observing morphological activities of in-vitro dissociated LNNs at the network level. Cortical neurons (extracted from 18-day old rat embryos) are dissociated and plated onto a grid of 59 electrodes on the center of an MEA culture dish. Once plated, the neurons reform synaptic connections with one another to become a single functional network, displaying highly correlated activity among constituent neurons. MEAs can be used as two-way interface between the outside world and LNNs. In-vitro LNNs serve as excellent models for long-term experiments studying the development of network circuitry. This specific investigation focused on designing novel technologies aimed at studying a specific form of neural network plasticity: synaptic scaling in homeostatic plasticity. This endeavor is broken into 3 thrusts: Thrust I: Cell Incubation System: The Hotbox is a custom-built environmental control device that modulates temperature, CO₂, and humidity levels inside a custom built environmental enclosure. Temperature is monitored with thermocouple, and a PID controller is used to drive heating elements. CO₂ monitored with CO₂ sensors; bang-bang controller is used to turn CO₂ air supply on/off. Thrust II: Electrophysiology—Alleviating Bursting in LNN: Attempts to scale short-term Burst-Inhibiting phenomena to longer time scales and induce more pronounced and permanent network modifications were successful. However, the integrity of LNN health was compromised at 17 DIV; thus this experiment can only serve as a preliminary experiment for future experiments attempting to demonstrate Burst-Inhibiting and Synaptic Scaling phenomena. Thrust III: Enabling Two-Photon Microscopy Imaging—Transfection Protocols Electroporation: based transfection techniques were assessed for their effectiveness for fluorescent microscopy imaging cases. Although some electroporation parameters yielded successful transfections, all treatments yielded low transfection efficiency and low cell viability.