Use of Randomized Submaximal Glutamate Stimulus to Interpret Glial Effects on Neuronal Calcium Dynamics

Abstract

Glutamate (GLU) binding to neurons can cause dynamic changes in intracellular calcium. We tested effects of a 3-group submaximal glutamate stimulus (250, 500 and 750 nanomolar GLU in randomized orders) on neurons in culture, and measured intracellular calcium dynamics in cultures high and low in glia at 8 and 9 days in vitro. Gliadepleted cultures responded to increasing GLU with synchronized dynamics, leading to a greater "area under the curve" (AUC) for intracellular calcium over time. The AUC determined if the neuron would respond dynamically to the next addition of glutamate. This observation was not displayed within cultures high in glia, where AUC returned to baseline with every GLU addition, regardless of order of addition. Furthermore, the 3-group stimulus resulted in decreasing average AUC, regardless of order. In contrast, for cultures depleted of glia, the deciding factor of a responding cell to dynamically respond to GLU additions depended on the ability of the cell to distribute the calcium load (AUC) of the prior addition. Determining how neurons respond and behave such as in the presence of functional or dysfunctional glia, may help our understanding of signal processing in the brain.

Negative Feedback Role of Astrocytes in Shaping Excitation in Brain Cell Co-cultures

“…After 5 days in vitro , one half (+ F side) of the cell culture plate was treated with 4 μM of the anti-mitotic 5-Fluoro-2-deoxyuridine (FdU) (Sigma-Aldrich) to isolate neurons in cell cultures by depleting glial cells. The remaining half (−F side) was supplemented with warmed NCM ( Hui et al, 2016 ; Kelly et al, 2016 ).…”

“…Ionomycin was used as a positive control (event 4) for the experiments, which was added at frame 200. The same protocol was used for imaging of other cell types ( DeCoster et al, 2002 ; Kelly et al, 2016 ).…”

“…Astrocytes in the cultures were identified on the basis of [Ca 2+ ] i responses to KCl and ATP stimulations. In separate experiments, DAPI staining and Diffquik staining was performed to understand the networks of ROIs in cultures ( Kelly et al, 2016 ). Additionally, DAPI was used for cell counting and for calculating nuclear area and the nuclear area factor (NAF) ( DeCoster, 2007 ), comparing cultures with high and low (−) feedback.…”

Novel Drugs for Neurodisorders

“…Investigation in this study reveals that determined how neurons respond and behave in presence of functional or dysfunctional glia; in turn helps us to understand the signal processing in the brain [1].…”