In vivo imaging of Ca²⁺ dynamics in lacrimal gland of Yellow Cameleon-Nano transgenic mice.

Abstract

Purpose : Intracellular calcium (Ca²⁺) signal via choline muscarinic stimuli in lacrimal gland (LG) acinar cell plays an important role in tear secretion. Mechanisms of Ca²⁺ dynamics in LG has been investigated only with isolated LG acinar cells. In this study, we verified the usefulness of mouse lines ubiquitously expressing Yellow Cameleon (YC)-Nano 15, a calcium-sensing protein, as a tool for investigating Ca²⁺ dynamics in LG in living animal.

Methods : Eight-weeks-old female YC-Nano 15 transgenic mice were used. Mice were placed in a prone position, and their LG were exposed by incision of the skin on the left temporal side of the head under anesthesia. In vivo FRET ratio imaging was performed by two-photon microscopy of LG in order to visualize Ca²⁺ dynamics. The LG was excited at 840 nm, and the emission fluorescence of CFP and YFP were detected at 460-500 nm and 520-560 nm, respectively. The images were acquired approximately 1 frame/sec. To calculate the FRET ratio, the fluorescence images of each emission wavelength were analyzed by the software FluoView FV1200MPE. The changes in FRET ratio after intravenous injection of bethanechol, cholinergic agonist, were evaluated at a dosage from 0.05 to 0.5 mg/kg.

Results : YC-Nano 15 probe was expressed in LG acinar cells of YC-Nano 15 transgenic mice, not in wiled type mice. Bethanechol dose-dependently increased in the peak and duration of the FRET ratio, and pre-treatment with atropine, cholinergic antagonist, obviously suppressed these alterations.

Conclusions : These results suggested that YC-Nano 15 transgenic mouse is possibly useful for understanding the relationship between Ca²⁺ dynamics in LG and tear secretion in living animal.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.