Figure 4.

Uncaging IP₃ in astrocytes produces an intracellular Ca²⁺ wave that does not propagate into neighboring cells. a, In a field of astrocytes bulk-loaded with Ca²⁺ green 1-AM ester dye, a single astrocyte marked by the UV light spot (cell 1) was patch clamped with pipettes containing Ca²⁺ dye OGB-1, caged IP₃, and 1% neurobiotin. Neurobiotin was included in the intracellular solution to show that gap junction coupling was not inhibited by any components of the intracellular solution used in the experiments. b, Cells 3 and 6 adjacent to the patched cell 1 responded to a perfusion of 50 μm tACPD with a Ca²⁺ elevation but did not increase Ca²⁺ after the robust IP₃ uncaging responses produced in cell 1 (arrows). Even a prolonged Ca²⁺ increase in cell 1 produced by a train of UV light pulses (10 pulses at 0.3 Hz, lightning bolt) did not produce any Ca²⁺ increase in cells 3 and 6. c, The astrocyte patched in a was extensively coupled to other astrocytes in the slice. Astrocytes (angled arrows) were clearly evident by the perinuclear labeling by streptavidin Alexa 488; a vertical arrow points to a blood vessel. The original patched cell could not be identified in part because of the vast number of coupled astrocytes, but also because Ca²⁺ indicator dyes fix poorly by aldehydes. These results were reproduced in eight different slices; never did a Ca²⁺ increase evoked by IP₃ uncaging propagate to an adjacent cell.