Optical Control of Ligand-Gated Ion Channels

In the vibrant field of optogenetics, optics and genetic targeting are combined to commandeer cellular functions, such as the neuronal action potential, by optically stimulating light-sensitive ion channels expressed in the cell membrane. One broadly applicable manifestation of this approach are covalently attached photochromic tethered ligands (PTLs) that allow activating ligand-gated ion channels with outstanding spatial and temporal resolution. Here, we describe all steps towards the successful development and application of PTL-gated ion channels in cell lines and primary cells. The basis for these experiments forms a combination of molecular modeling, genetic engineering, cell culture, and electrophysiology. The light-gated glutamate receptor (LiGluR), which consists of the PTL-functionalized GluK2 receptor, serves as a model

Monitoring Calcium Levels With Genetically Encoded Indicators

Calcium indicators are widely used to monitor activity in living neuronal tissue because of the tight relation between action potential firing and increases in the intracellular calcium concentration. Here, we describe the use of genetically encoded calcium indicators (GECIs) of the latest generation for monitoring calcium levels in the mammalian brain. We discuss how to choose the sensor for a given experiment, how to introduce the sensor into the cells of interest and how to estimate the sensitivity of the sensor in situ and in vivo. Finally, we illustrate the application of these sensors for high resolution in vivo imaging of sensory-driven neuronal activity