Combining calcium imaging with optogenetics in hippocampal primary cultures

Calcium imaging and optogenetics are two powerful tools in neuroscience research. Their combination can give great insights on the working of neural networks and circuits and can complement electrophysiological data. The present investigation shows that it is possible to analyse neuronal networks with optogenetic tools in vitro, where light is used to open and close ionic channels that have conduction properties identical to native channels. In this project we have opted for the optogenetic approach of Photoisomerizable Tethered Ligands (PTL). By tethering synthetic photoisomerizable compounds (PTLs, also called photoswitches) to engineered native proteins (channels or receptors), we gain the possibility of controlling them with light. In our case, these engineered proteins were ionotropic kainate-type glutamate receptors, called LiGluK2. In rat primary hippocampal cultures, we combined the optical stimulation of LiGluK2 with optical sensing by means of calcium imaging. Localized activation of LiGluK2 with confined illumination allowed us to control single neurons and analyse the effect of their activation on small neural networks. Different stimulation protocols were applied, and we found transfected neurons to have mainly three different kinds of responses in terms of calcium transients: single peaks, single prolonged calcium transients, multiple peaks. This activity affected surrounding neurons with variability, which can be ascribed to the physiological properties of the neurons involved and to the random connectivity of the analysed neural networks