A study of cellular calcium dynamics in culture using fluorescence microscopy – A statistical and mathematical approach

Calcium in its ionic form is very dynamic, especially in excitable cells such as muscle and brain cells, moving from the high concentration exterior of the cell to much lower concentrations inside the cell, where calcium is used as a second messenger. In brain cells, and neurons especially, calcium is a key signaling ion involved in memory and learning with excitatory neurotransmitters such as glutamate turning neurons on. Glutamate excites the neurons in part by causing large and dynamic changes in the intracellular calcium concentration. While these dynamics are essential for normal signaling in the brain, excessive and sustained elevations in neuronal intracellular calcium are related to neuronal injury including long-term neurodegenerative processes. Helping to regulate these dynamics in the brain are the glial cells known as astrocytes. Astrocytes aids glutamate transporters, and in this way, diminish the time that neurons are exposed to glutamate, thus also shaping the calcium dynamics in neurons. Here we describe an in vitro cell culture system composed of rat brain cortical neurons with different densities of astrocytes which we have used to statistically and mathematically analyze the intracellular calcium dynamics in individual neurons. With the proposed applied statistical and mathematical tools we now provide a system for predicting: 1) whether the order of repeated glutamate stimulation alters neuronal intracellular calcium dynamics and 2) how the presence of different densities of astrocyte modulates neuronal brain dynamics. We anticipate that this combined experimental/analytical approach will also have utility in understanding various brain diseases such as brain tumors