In vivo two-photon calcium imaging using multicell bolus loading of fluorescent indicators

Imaging the activity of neuronal populations allows the simultaneous mapping of spontaneous and evoked responses of numerous neurons and is critical for determining the function
of neuronal circuits. This chapter describes a simple and effective method called “multicell bolus loading” that has been developed to deliver a calcium indicator in vivo to a large population of cortical neurons. Cells are stained by a brief injection of a membrane-permeant Ca2+-indicator dye into the extracellular space. The injected dye diffuses into the cells of interest, where it is hydrolyzed by intracellular esterases. The activity-dependent calcium signals from multiple cells can then be imaged with two-photon microscopy at single-cell resolution. Calcium imaging of neuronal circuits was first developed in slices, in which it was shown that calcium transients correspond to action potentials. This method has been successfully applied to in vivo imaging of calcium signals during spontaneous or evoked activity in various brain regions, including the somatosensory cortex, the visual cortex, the olfactory bulb, the spinal cord, the cerebellum, and the hippocampus. The same protocol can be used in different species and at different developmental ages, for example, in zebrafish larvae, mice, rats, cats, ferrets, and monkeys.