Presynaptic imaging of projection fibers by in vivo injection of dextran-conjugated calcium indicators.

Dextran-conjugated calcium indicators are stably retained within neurons. As a result, they are well suited to measuring presynaptic calcium at physiological temperatures. In addition, dextran indicators can be used to label neurons and their presynaptic boutons in vivo. This has allowed measurements of calcium in the presynaptic boutons of projection fibers that cannot be stably loaded with other types of indicators. This protocol describes a technique for in vivo loading of the climbing fiber projection to the cerebellum with dextran-conjugated indicators for subsequent presynaptic calcium imaging in brain slices. This technique is applicable to studies of projection fibers in many species from which brain slices can be prepared. The dextran indicator is injected into the inferior olive using a stereotaxic device. After a period of 1-3 d, cerebellar slices are prepared and presynaptic calcium transients are measured at physiological temperature in labeled climbing fibers. The protocol also discusses important considerations for using dextran-conjugated indicators to measure presynaptic calcium.