OWLS APPLICATION NOTES NO-014

DETECTING CELL PHYSIOLOGICAL CHANGES IN RESPONSE TO LIGHT-INDUCED ION CHANNEL OPENING

Using OPTICAL WAVEGUIDE LIGHTMODE SPECTROSCOPY (OWLS) detection

Radial glia-like neural stem cells expressing ChR2(H134) channels

THEORETICAL BACKGROUND
Light-gated ion channels1 allow modifying transmembrane ion fluxes and consequently, t h e i n t r a c e l l u l a r d i s t r i b u t i o n o f i o n s wi t h o u t applying electric fields or introducing stimulating electrodes. Channelrhodopsyn 2 (Chr2(H134); www. optogenetics.org) is a Na+ and Ca2+permeable cation channel, which opens in response to illumination at wavelengths of 460-490 nm. The influx of Na+ 2+ and Ca depolarizes the cells and results in an increase of the intracellular Ca-concentration. Increased intracellular Ca-levels promote secretion of cellular material and can increase cell motility. These events can be recorded by OW LS assays detecting c hanges i n the overall material deposition by cells. The presence of functional light-operated c a t i o n c h a n n e l s i n C h R 2 e xp r e s s i n g c e l l s wa s proved by path-clamp techniques2,3 using either short light pulses or phasic (t 500 ms) illumination. Material deposition caus ed by motility or sec retion by living cells was detectable regardless of illumination or the presence of ChR2. ChR2e xp r e s s i n g c e l l s , h o w e v e r , p r o d u c e d s i g n i f i c a n t l y larger amount of deposited material in response to illumination indicating that light-evoked ion-fluxes increased cell motility and/or secretion.

O WL S D E T E C T I O N O F L I G H T I N D U C E D
ION CHANNEL OPENING The cells were grown on the surface of OW 2400 sensors coated wi t h AK-c(RGDfC) synthetic adhesive peptide2. Illumination did not increase the temperature inside the cuvette indicating that changes in material deposition were not due to heat changes. Cuvette temperature and the amount of deposited material (absorbed mass) produced by control cells are s h o wn . References 1. Zhang F, Aravanis A.M, Adamantidis A, de LeceaL, Deisseroth K. 2. C o n f l u e n t c u l t u r e s o n s e n s o r c h i p s we r e inserted into a tem perature controll ed OW LS c u v e t t e a n d s e r u m - f r e e c u l t u r e m e d i u m wa s added. Polarized 473 nm light (0.35 mW /mm2) was introduced into the cuvette by an optical fiber through a central inlet. OW LS signals were recorded from both ChR2 e xp r e s s i n g a n d c o n t r o l c u l t u r e s wi t h r e p e a t e d illumination over 180 240 minutes, at 37 oC. Illumination did not cause any detectable cell decay or cell detachment from the sensor surface.
2007. Circuit-breakers: optical technologies for probing neural signals and systems Nat.Rev.Neurosci. 8: 577-581 Marko K, Ligeti M, Mez G, Mihala K, Kutnyinszky E, Kiss E, Hudecz F, Madarasz E. A novel synthetic peptide polymer with cyclic RGD motifs supports serum-free attachment of anchorage-dependent cells. Bioconjugate Chemistry 2008 19, 1757-1766. Kroly Mark, Tmea Khidi, Nra Hdinger, Mrta Jelitai, Gbor Mez, Emlia Madarsz. Stem cells isolated by selective adhesion from distinct brain regions, PlosOne; 2011 6 : e28538 Vrs, J. J. Ramsden, G. Csucs, I. Szendr, S.M. De Paul, M. Textor, N. D. Spencer, Biomaterials 2002, 23, 3699-3710

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Measurements were made at Inst. of Experimental Medicine of Hung.Acad.Sci. by I. Szkcs,T.Andrssy, T.Khidi, E. Madarsz

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