Brenowitz Stephan D, Regehr Wade G
Cold Spring Harb Protoc. 2012 Apr 1;2012(4):465-71. doi: 10.1101/pdb.prot068551.
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.
葡聚糖偶联钙指示剂能稳定地保留在神经元内。因此，它们非常适合在生理温度下测量突触前钙。此外，葡聚糖指示剂可用于在体内标记神经元及其突触前终扣。这使得人们能够测量那些无法稳定加载其他类型指示剂的投射纤维突触前终扣中的钙。本实验方案描述了一种用葡聚糖偶联指示剂对小脑进行攀缘纤维投射体内加载的技术，以便随后在脑片中进行突触前钙成像。该技术适用于许多能制备脑片的物种的投射纤维研究。使用立体定位装置将葡聚糖指示剂注入下橄榄核。1 - 3天后，制备小脑切片，并在生理温度下测量标记的攀缘纤维中的突触前钙瞬变。该实验方案还讨论了使用葡聚糖偶联指示剂测量突触前钙的重要注意事项。
