Figure 4.

Perivascular astrocytic endfeet and processes generate spatially heterogeneous [Ca²⁺]_i signals. (A) Laser scanning confocal microscopy image series of a fluo-5F AM loaded endfoot and perivascular processes within a cortical brain slice. In the image taken at the beginning of EFS (0 s), the location of the endfoot (dotted circle) and perivascular processes are outlined. The location of the arteriole is also shown, part of which was underneath the endfoot. In this example, EFS induced a [Ca²⁺]_i wave in both perivascular processes, as well as a wave in the endfoot that traveled perpendicular to the waves in the processes. The direction of the [Ca²⁺]_i waves is denoted by the arrows. Many areas of spatially localized [Ca²⁺]_i increase were found, as well as a subregion of elevated [Ca²⁺]_i within the endfoot that was maintained for an extended period of time after EFS. See Video 2 (available at http://www.jgp.org/cgi/content/full/jgp.200609650/DC1). These experiments were performed using the Ca²⁺-sensitive dye fluo-5F, which has a slightly lower affinity for Ca²⁺ than fluo-4 (K _d = 388 nM vs. 215 nM for fluo-4 at 35°C), to aid in the detection of spatially localized regions of high [Ca²⁺]_i. (B and C) Regions of spatially localized [Ca²⁺]_i increase within a single endfoot in response to EFS. In this example (representative of at least six experiments), in which the images were cropped to include only the endfoot and immediately adjacent regions of the brain slice, three regions of Ca²⁺ release were found within the endfoot, as denoted by the arrows. These regions acted as initiation sites for the global endfoot [Ca²⁺]_i increase. As seen in the traces in C, each region of interest, identified by the boxes in the 0 sec image, responded with distinct release kinetics.