Neuronal Network Imaging in Acute Slices Using Ca2+ Sensitive Bioluminescent Reporter

Genetically encoded indicators are valuable tools to study intracellular signaling cascades in real time using fluorescent or bioluminescent imaging techniques. Imaging of Ca²⁺ indicators is widely used to record transient intracellular Ca²⁺ increases associated with bioelectrical activity. The natural bioluminescent Ca²⁺ sensor aequorin has been historically the first Ca²⁺ indicator used to address biological questions. Aequorin imaging offers several advantages over fluorescent reporters: it is virtually devoid of background signal; it does not require light excitation and interferes little with intracellular processes. Genetically encoded sensors such as aequorin are commonly used in dissociated cultured cells; however it becomes more challenging to express them in differentiated intact specimen such as brain tissue. Here we describe a method to express a GFP-aequorin (GA) fusion protein in pyramidal cells of neocortical acute slices using recombinant Sindbis virus. This technique allows expressing GA in several hundreds of neurons on the same slice and to perform the bioluminescence recording of Ca²⁺ transients in single neurons or multiple neurons simultaneously.