This problem becomes increasingly significant when imaging in the noisy in vivo condition and when imaging small structures, such as dendritic spines. In these conditions, high-affinity calcium dyes remain, with all their limitations, the indicators of choice. Fortunately, calcium indicators with different properties can

often be easily used complementarily in an experimental series. The new developments will certainly add up to our ability of deciphering the highly complex mechanisms of neuronal signaling in the intact nervous system. The loading of calcium indicators into neurons depends on the type of calcium indicator, the biological preparation, and the specific scientific question. Figure 3A illustrates the three most widely used approaches for dye loading of individual Lapatinib neurons. In the early imaging experiments, chemical calcium dyes were delivered through sharp microelectrodes both in vitro (Jaffe et al., 1992) and in vivo (Svoboda et al., 1997) (Figure 3A, left panel). In more recent years, dye delivery through whole-cell patch-clamp micropipettes became the standard selleck products procedure for single-cell dye loading for many applications (Figure 3A, middle panel) (Eilers and Konnerth, 2009 and Margrie et al., 2002). A particularly useful variant of this method involves

in vivo whole-cell recordings that are performed under visual guidance using two-photon imaging by applying the “shadow patching” technique (Jia et al., 2011 and Kitamura et al., 2008). This approach can be combined with the targeting of genetically identified cells expressing a fluorescent marker protein (Margrie et al., 2003). Other attractive and relatively easy-to-use single-cell approaches are the targeted electroporation (Judkewitz et al., 2009, Kitamura et al., 2008 and Nevian and Helmchen, 2007) or single-cell bolus loading (Helmchen et al., 1996). After approaching the soma of the target neuron with a micropipette in the electroporation experiments (Figure 3A, right panel), a few current pulses of appropriate polarity mediate

dye delivery to the cell. This approach relies on two distinct mechanisms (for review, see De Vry et al., 2010). First, the electrical current disrupts the integrity of the cellular plasma membrane for a short period of time causing the transient formation of pores through which the dye molecules diffuse into the heptaminol cell. Second, the current “pushes” the charged indicator molecules out of the pipette into the cell of interest. Importantly, this approach can be used for chemical calcium indicators as well as for DNA encoding for GECIs. A limitation of this method is that, because of the absence of the recording whole-cell microelectrode, the functional status of the neurons is not entirely clear. This can be overcome by combining electroporation of single cells with the cell-attached recordings involving the use of a second, fresh micropipette (Chen et al.