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F. Muller, D. Kaschuba, T. Gensch, M. Mank, O. Griesbeck; Expression of the Genetically Encoded Calcium Sensor TN-L15 in Retinal Ganglion Cells of Transgenic Mice. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2804.
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A variety of neuronal processes are induced or accompanied by changes in the intracellular calcium concentration. Calcium-dependent fluorophores are widely used to monitor intracellular calcium, however, loading of neuronal populations with such dyes in a cell-type specific way is difficult to achieve. Here we employ a transgenic approach to express the genetically encoded fluorescent calcium indicator protein TN-L15 in ganglion cell populations of the mouse retina.
TN-L15 was expressed under the control of the thy-1 promotor in two different transgenic mouse lines. TN-L15 is a ratiometric sensor that consists of the calcium-binding protein troponin C and a pair of fluorescent proteins (donor and acceptor) engineered for fluorescence resonance energy transfer (FRET). Isolated retinae of transgenic mice were flat-mounted and imaged using two-photon laser scanning microscopy. Retinal ganglion cell types were identified using appropriate immunohistochemical markers and confocal microscopy.
TN-L15 was found in the majority of ganglion cells. Only few ganglion cell types lacked expression of TN-L15. Transient increases in intracellular calcium concentration as response to high extracellular potassium or stimulation of glutamate receptors by different agonists were readily observed as a decrease in donor fluorescence accompanied by an increase in acceptor fluorescence, indicating FRET.
Genetically encoded calcium sensor proteins can be stably expressed in ganglion cells of transgenic mice. These mouse lines will be helpful to study calcium signals, pharmacological receptor profiles, and synaptic excitation in ganglion cell populations in vitro.
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