Prolonging life in chick forebrain-neuron culture and acquiring spontaneous spiking activity on a microelectrode array

Abstract

Various types of animal neurons were cultured on a microelectrode array (MEA) platform to form biosensors to detect potential environmental neurotoxins. For a large-scale screening tool, rodent MEA-based cortical-neuron biosensors would be very costly but chick forebrain neurons (FBNs) are abundant, cost-effective, and easy to dissect. However, chick FBNs have a lifespan of ~14 days in vitro and their spontaneous spike activity (SSA) has been difficult to develop and detect. We used a high-density neuron-glia co-culture on an MEA to prolong chick FBN lifetime to 3 months with lifetime-long SSA. A remarkable embryonic age-dependency in the culture's morphology, lifespan, and most features of SSA signal was discovered. Our results show the feasibility of developing a chick FBN-MEA biosensor and also establish a new electrophysiological platform for functional study of an in vitro neuronal network.

Establishment of a long-term chick forebrain neuronal culture on a microelectrode array platform

“…In addition, in our previous study 10 when M199 + was used, E8, E10, and E12 cultures exhibited a remarkable embryonic age-dependent morphology. At the same cell plating density, the older the embryonic age, the lesser the cell aggregation, and the faster the culture became confluent.…”

“…A threshold of −7 times the standard deviation (SD) of the mean noise amplitude was set for spike detection in this study. Bursts were defined using the three criteria described in our previous paper 10 and detected using NeuroMEA, a MatLab-based program developed in our lab. NeuroMEA also created raster plots, which are commonly used with MEA data to present temporal and spatial features of SSA.…”

“…Results of dose-response experiments with selected classic neuroactive agents were reported earlier. 7 This paper deals with how long and how stable C-FBN-Cs lived in this condition, i.e., three weeks early development succeeded by repeated intervention of dose-response experiments. Some key results of the dose-response experiments are mentioned or summarized in the sections on Results and Discussion when necessary.…”

“…7 This novel biosensor system advanced our understanding about the functional features of cortical neuronal networks in vitro in two aspects. 1) Based on early patch clamp data on synapse formation and function in chick FBN culture (C-FBN-C), 8 we found that the synapse formation and function in vitro is much faster than in vivo, and there is a critical, narrow time window for the rapid early development of synapses.…”

“…9 Based on this important finding, we provided a first line of in vitro functional data that support this finding: in comparison to rodent counterpart, the features of the spontaneous spiking activity (SSA) from chick FBN biosensor showed remarkable functional similarities in spatial and temporal firing pattern, tissue specificity in comparison to SSA pattern from spinal cord neurons, and responsiveness to selected classic neuroactive agents in terms of dose ranges used and EC 50 (concentration that results in 50% of maximum response) for each agent. 7 The selected classic neuroactive agents include tetrodotoxin, a specific voltage-gated sodium channel blocker; verapamil, a specific voltage-gated L-type calcium channel blocker; Mg 2+ , a NMDA channel blocker; NMDA, the prototype agonist of NMDA channels; APV, a specific NMDA channel antagonist; bicuculline, a specific GABA A type channel antagonist; and musimol, a specific GABA A channel agonist. Because of the shared drug responsiveness between mammals and birds, its application may be potentially extended as an alternative approach to rodent counterpart in neurotoxin-related testing in known shared receptor domains, especially for a large-scale screening of environmental neurotoxic chemicals, in terms of convenience and cost-effectiveness.…”