New Modes of GPCR Signalling

Calcium Signaling in Astrocytes Induced by Photostimulation with
Femtosecond Laser
Yuan Zhao, Yuan Zhang, Wei Zhou, Shaoqun Zeng*
Britton Chance Center for Biomedical Photonics
Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and
Technology,Wuhan, 430074, China
Astrocytes have been proved to actively contribute to brain functions, communicating
with neurons and other brain cells. However, conventional stimulation approaches are
hard to be feasibly utilized to precisely activate astrocytes for delicate investigations.
Here, we developed non-invasive photostimulation with high accuracy to evoke calcium
(Ca 2+ ) signaling in astrocytes. The near infrared (800 nm) femtosecond laser was
focused onto the cell membrane of an astrocyte. Ca 2+ elevation was immediately evoked
in the stimulated cell and subsequently in surrounding cells, forming a radial Ca 2+ wave
throughout the network. Localized transient photoporation was demonstrated to be
induced on the cell membrane by laser irradiation. Extracellular Ca 2+ entry into the
cytoplasm through this tiny poration was necessary for astrocytic Ca 2+ signaling.
Repetitive photostimulation was actually performed on one cell with well-controlled
laser intensity and targeting, resulting in similar responses. Further, different patterns of
Ca 2+ elevation in the stimulated astrocyte were observed by varying femtosecond laser
power. Generally, the amplitude of Ca 2+ response in the stimulated cell was increased
with enhanced stimulating laser power, and concomitantly, the Ca 2+ wave was widened.
Therefore, noncontact photostimulation of astrocytes with femtosecond laser is
demonstrated to be non-disruptive, reproducible, and with high spatiotemporal precision.
Photogenerated Ca 2+ response follows a femtosecond laser-power-dependent manner, so
that distinct Ca 2+ signaling ranges can be feasibly provided for specific studies. This
versatile tool is thus promisingly efficient and helpful for resolving problems that
conventional stimulations are difficult to touch, especially for in vivo studies.
Key Words: astrocyte, Ca 2+ signaling, photostimulation, femtosecond laser,
photoporation
*Corresponding author : Email: sqzeng@mail.hust.edu.cn