TWO-PHOTON IMAGING OF ASTROCYTES USING CALCIUM SENSITIVE FLUORESCENT PROTEINS IN VIVO DURING EPILEPTIC EVENTS
Abstract number :
1.085
Submission category :
Year :
2003
Submission ID :
3891
Source :
www.aesnet.org
Presentation date :
12/6/2003 12:00:00 AM
Published date :
Dec 1, 2003, 06:00 AM
Authors :
Hajime Hirase, Lifen Qian, Judy Creso, Malaika Singleton, Peter Bartho, Ian Creese, Gyorgy Buzsaki Center for Molecular and Behavioral Neuroscience, Rutgers University, Newark, NJ
Cytosolic calcium concentration can usually be observed using chemical calcium probes such as calcium-green, fura-2, or fluo-4, in vitro. These dye are hard to be introduced for in vivo imaging study of adult animals. We used a virus to introduce calcium sensitive fluorescent proteins into glial cells to perform imaging studies during control and epileptic conditions.
Recombinant adenoviruses (serotype 5) with CMV promoter was made to express calcium sensitive fluorescent protein YC6.1 and GCaMP1.6. When infected a mouse brain by intra-parenchymal injection, predominantly astrocytes and endothelial expressed the protein. Using a two-photon microscope, fluorescent cells were imaged in vivo, together with unit and field recording from the proximity of the infected area.
Glial cells that expressed FRET based YC6.1 protein did not yield notable change in signal, possibly due to its temperature instability at body temperature. Glial cells that expressed CaGMP1.6 had spontaneous fluctuation in fluorescent intensity peaking as high as 70% increase from the basal intensity during interictal periodes induced by GABA-A antagonist bicuculline. The high fluorescent state lasting tens of seconds occurred typically a few times in the course of 10 minute imaging period. The change in fluorescence, presumed to reflect cytosolic calcium change, is similar to spontaneous calcium oscillations observed in vitro and did not have apparent correlate with local field potential nor multi-unit activity.
Our experimentation suggests that the circularly permutated green fluorescent protein can be used as a potential calcium probe in vivo, as well as observing spontaneous glial calcium oscillation is a good protocol to test calcium indicators in vivo. Spontaneous calcium oscillation in glial cells are observed more often in interictal periods induced by bicuculline. The spontaneous calcium oscillation in glial cell may have some important functions such as local regulation of blood flow and metabolism.
[Supported by: American Epilepsy Society
NIH (NS34994, NS43157; MH54671).
MS was supported by NIGMS/MBRS.]