Electrophysiological Activity of Primary Cortical Neuron-Glia Mixed Cultures

Background We have previously described a tri-culture of neurons, astrocytes, and microglia that accurately mimics the in vivo neuroinflammatory response (both neurotoxic and neuroprotective) to a wide range of neuroinflammatory stimuli. Electrophysiological activity is an essential measure of neuronal function, which is influenced by neuroinflammation. Microelectrode array (MEA) technology is a versatile tool to non-invasively study in vitro neural networks by simultaneously recording electrophysiological activity from multiple sites. In this study, we used extracellular recordings to determine the effect of microglia on neural network formation and stability in primary cortical cultures and monitor the changes in neural activity in response to neuroinflammatory stimuli. Methods Primary neonatal rat cortical tri-cultures of neurons, astrocytes, and microglia or co-cultures of neurons and astrocytes were cultured on custom MEAs and the neural activity was monitored for 21 days in vitro to assess culture maturation and network formation. Quantification of synaptic puncta and averaged spike waveforms were used to determine the difference in excitatory to inhibitory neuron ratio (E/I ratio) of the neurons in tri- and co-cultures. The electrophysiological response to lipopolysaccharide (LPS) treatment of both culture types were compared. Results The tri- and co-culture showed minimal difference in electrophysiological markers of neural network formation and stability with the exception of a significant increase in spike frequency in the tri-culture at later timepoints (DIV 17 and 21). Additionally, there was no significant difference in the density of either post-synaptic or excitatory pre-synaptic puncta between the culture types. However, characterization of the average spike waveforms revealed that the tri-culture had an E/I ratio much closer to that found in the rat cortex. Finally, only the tri-culture displayed a significant decrease in both the number of active channels and spike frequency following LPS exposure. Conclusions This study demonstrates that the microglia in the tri-culture do not disrupt neural network formation and stability as quantified using extracellular recordings and may be a better representation of the in vivo cortex due to the closer E/I ratio than more traditional isolated neuron and neuron-astrocyte co-cultures. Additionally, the tri-culture is better able to mimic the neuroinflammatory response to LPS, which was quantified via changes in neural electrophysiological activity.