In vivo Fluorescence Imaging of Extracellular ATP in the Mouse Cerebral Cortex with a Hybrid-type Optical Sensor

Abstract

Adenosine 5'-triphosphate (ATP) works as an extracellular signaling molecule for cells in the brain, such as neurons and glia. Cellular communication via release of ATP is involved in a range of processes required for normal brain functions, and aberrant communication is associated with brain disorders. To investigate the mechanisms underlying these cellular processes, various techniques have been developed for the measurement of extracellular ATP. To monitor the dynamics of extracellular ATP signaling with high spatiotemporal resolution, we recently developed a hybrid-type ATP optical sensor (ATPOS) that enables in vivo fluorescence imaging of extracellular ATP dynamics in the brain. ATPOS is synthesized by labeling an ATP-binding protein, Bacillus FoF1-ATP synthase ε subunit, with a smallmolecular fluorescent dye Cy3. Injection of ATPOS into the cerebral cortex of living mice enables visualization of the wave-like propagation of extracellular ATP release in response to electrical stimulation. The protocol described here should be useful for visualizing ATP signaling in diverse processes involved in intercellular communication in the brain.

Quantitative modeling of regular retinal microglia distribution

“…Finally, we observed the extracellular ATP distribution and microglia using ATPOS 20 , 21 (Fig. 6 C–E).…”

“…We observed the extracellular ATP concentration with ATPOS fluorescent sensor 20 , 21 . Dissected P5 retina cups were washed in pH 7.4 HEPES buffer solution (HBS.…”

“…Dissected P5 retina cups were washed in pH 7.4 HEPES buffer solution (HBS. 25 mM HEPES, 125 mM NaCl, 4 mM KCl, 2 mM CaCl and 1 mM MgCl ), and incubated for 5 minutes at room temperature in 100 µl of 0.33 µM neuronal-surface-targeted ATPOS, a molecular complex of ATPOS with BoNT/C-Hc and Alexa Fluor 488 (Alexa488) labeled-streptavidin 20 , 21 with 1/100 IB4-DyLight649 (DL-1208-5, Vector Laboratories). Then, the retina cups were washed with HBS + 0.1% BSA three times and transferred on the slide glass of a glass-bottom dish (3911-035-IN, Iwaki Glass).…”

Quantitative modeling of regular retinal microglia distribution

“…We observed the extracellular ATP concentration with ATPOS fluorescent sensor [20,21]. Dissected P5 retina cups were washed in pH 7.…”

“…Finally, we observed the extracellular ATP distribution and microglia using ATPOS [20,21] (Fig. 6C-E).…”