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Updated: Sep 26, 2025
Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model
Published on: October 18, 2015
Evaluating spatial and network properties of NMDA-dependent neuronal connectivity in mixed cortical cultures
Catherine P Rojvirat1, Joshua R Berlin2, Tuan D Nguyen3
1Department of Pharmacology, Physiology and Neuroscience, New Jersey Medical School, Rutgers University, 185 South Orange Avenue, Newark, NJ 07101-1709, United States; School of Graduate Studies, Rutgers Biomedical and Health Sciences Campus-Newark, Rutgers University, 185 South Orange Avenue, Newark, NJ 07101-1709, United States.
Abstract:
A technique combining fluorescence imaging with Ca2+ indicators and single-cell laser scanning photostimulation of caged glutamate (LSPS) allowed identification of functional connections between individual neurons in mixed cultures of rat neocortical cells as well as observation of synchronous spontaneous activity among neurons. LSPS performed on large numbers of neurons yielded maps of functional connections between neurons and allowed calculation of neuronal network parameters. LSPS also provided an indirect measure of excitability of neurons targeted for photostimulation. By repeating LSPS sessions with the same neurons, stability of connections and change in the number and strength of connections were also determined. Experiments were conducted in the presence of bicuculline to study in detail the properties of excitatory neurotransmission. The AMPA receptor inhibitor, 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), abolished synchronous neuronal activity but had no effect on connections mapped by LSPS. In contrast, the NMDA receptor inhibitor, 2-Amino-5-phosphono-pentanoic acid (APV), dramatically decreased the number of functional connections between neurons while also affecting synchronous spontaneous activity. Functional connections were also decreased by increasing extracellular Mg2+ concentration. These data demonstrated that LSPS mapping interrogates NMDA receptor-dependent connectivity between neurons in the network. In addition, a GluN2A-specific inhibitor, NVP-AAM077, decreased the number and strength of connections between neurons as well as neuron excitability. Conversely, the GluN2A-specific positive modulator, GNE-0723, increased these same properties. These data showed that LSPS can be used to directly study perturbations in the properties of NMDA receptor-dependent connectivity in neuronal networks. This approach should be applicable in a wide variety of in vitro and in vivo experimental preparations.
