A primary rodent triculture model to investigate the role of glia-neuron crosstalk in regulation of neuronal activity

Abstract

Neuroinflammation and hyperexcitability have been implicated in the pathogenesis of neurodegenerative disease, and new models are required to investigate the cellular crosstalk involved in these processes. We developed an approach to generate a quantitative and reproducible triculture system that is suitable for pharmacological studies. While primary rodent cells were previously grown in a coculture medium formulated to support only neurons and astrocytes, we now optimised a protocol to generate tricultures containing neurons, astrocytes and microglia by culturing in a medium designed to support all three cell types and adding exogenous microglia to cocultures. Immunocytochemistry was used to confirm the intended cell types were present. The percentage of ramified microglia in the tricultures decreases as the number of microglia present increases. Multi-electrode array (MEA) recordings indicate that microglia in the triculture model suppress neuronal activity in a dose-dependent manner. Neurons in both cocultures and tricultures were responsive to the potassium channel blocker 4-aminopyridine (4-AP) suggesting that neurons remained viable and functional in the triculture model. Furthermore, suppressed neuronal activity in tricultures correlated with decreased densities of dendritic spines and of the postsynaptic protein Homer1 along dendrites, indicative of a direct or indirect effect of microglia on synapse function. We thus present a functional triculture model, which, due to its more complete cellular composition, is a more relevant model than standard cocultures. The model can be used to probe glia-neuron interactions and subsequently aid the development of assays for drug discovery, using neuronal excitability as a functional endpoint.

Electrophysiological Activity of Primary Cortical Neuron-Glia Mixed Cultures

“…Additionally, we observed that the spike frequency decreases at a more rapid rate than the number of active channels, suggesting that changes in neural activity can be detected prior to changes in neuron health. This observation can be paralleled to the findings from the recent study that showed a decrease in electrophysiological activity with increasing microglia density in a dose-response manner [54], where higher microglia numbers in the culture lead to increased microglia reactivity, partially mimicking increased reactivity due to the LPS treatment here. This demonstrates that monitoring extracellular recordings from MEAs is a powerful method to non-invasively study the effect of neuroinflammation in vitro, especially when combined with our previously described tri-culture of neurons, astrocytes, and microglia that more faithfully represents the in vivo neuroinflammatory response.…”

“…Additionally, we saw some evidence that the tri-culture had an increase in spike frequency at DIV 21 as only the tri-culture showed a significant increase in spike frequency between DIV 7 and DIV 21 ( p = 0.0076 vs p = 0.51 for the co-culture), but once again there was no significant difference in spike frequency between the tri- and co-cultures at DIV 21 ( Figure 1d ). A recent study using a similar tri-culture showed a reversed trend, with microglia appearing to reduce spike frequency and other electrophysiological features [54]. However, in that paper, the amount of microglia was increased to 15-25% of the total cell population, which is more than double the number of microglia found in our tri-culture model [32] and the proportions found in vivo [55].…”

“…The copyright holder for this preprint this version posted December 29, 2022. ; https://doi.org/10.1101/2022.12.29.522240 doi: bioRxiv preprint density in a dose-response manner [54], where higher microglia numbers in the culture lead to increased microglia reactivity, partially mimicking increased reactivity due to the LPS treatment here. This demonstrates that monitoring extracellular recordings from MEAs is a powerful method to non-invasively study the effect of neuroinflammation in vitro, especially when combined with our previously described tri-culture of neurons, astrocytes, and microglia that more faithfully represents the in vivo neuroinflammatory response.…”