Confocal imaging and local photolysis of caged compounds: dual probes of synaptic function.
Chemical signals generated at synapses are highly limited in both spatial range and time course, so that experiments studying such signals must measure and manipulate them in both these dimensions. We describe an optical system that combines confocal laser scanning microscopy, to measure such signals, with focal photolysis of caged compounds. This system can elevate neurotransmitter and second messenger levels in femtoliter volumes of single dendrites within a millisecond. The method is readily combined with whole-cell patch-clamp measurements of electrical signals in brain slices. In cerebellar Purkinje cells, photolysis of caged IP3 causes spatially restricted intracellular release of Ca2+, and photolysis of a caged Ca2+ compound locally opens Ca(2+)-dependent K+ channels. Furthermore, localized photolysis of the caged neurotransmitter GABA transiently activates GABA receptors. The use of focal uncaging can yield new information about the spatial range of signaling actions at synapses.
Wang
S S
SS
Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Augustine
G J
GJ
D000818:Animals; D001921:Brain / Q000502:physiology*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D003911:Dextrans; D005452:Fluoresceins; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D018613:Microscopy, Confocal; D018377:Neurotransmitter Agents / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010782:Photolysis; D015221:Potassium Channels / Q000502:physiology; D011689:Purkinje Cells / Q000502:physiology*; D051381:Rats; D015290:Second Messenger Systems; D015398:Signal Transduction; D013569:Synapses / Q000502:physiology*; D014466:Ultraviolet Rays; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D003911:Dextrans; D005452:Fluoresceins; D018377:Neurotransmitter Agents; D015221:Potassium Channels; C052526:fluorescein-dextran; D005680:gamma-Aminobutyric Acid; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium