| Abstract No.: | A-B1075 |
| Country: | Canada |
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| Title: | GLUTAMATE-DEPENDENT RAPID REMODELLING OF RADIAL GLIA IN XENOPUS LAEVIS |
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| Authors/Affiliations: | 2 Marc Tremblay*; 2 Vincent Fugère; 1 Bruno A. N. Travençolo; 1 Luciano da Fontoura Costa; 2 Edward Ruthazer;
1 Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Brazil; 2 Montreal Neurological Institute, McGill University, QC, Canada
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| Content: | Objectives: Glial cells have been reported to undergo rapid structural rearrangement and to modulate synaptic activity and synaptogenesis. However most of this work has been carried out in vitro. In the amphibian brain, radial glia are functionally comparable to astrocytes in mammals. Using single-cell live imaging in intact Xenopus laevis tadpoles, we measured the rates of dynamic remodelling of radial glial processes extending within the optic tectal neuropil and quantified the effect of neural activity blockade in modulating their dynamics.
Materials and Methods: Two-photon microscopy of radial glia in vivo was performed using an Olympus FV300 confocal custom converted for multiphoton imaging using a MaiTai Ti:sapphire femtosecond pulsed laser at 910nm. Stage 46 albino Xenopus laevis tadpoles were imaged 24 to 48 hours after electroporation in the optic tectum of plasmid encoding farnesylated-enhanced green fluorescent protein (f-EGFP). At the beginning of the imaging procedure, animals were paralyzed by injection with 1mM D-tubocurare. Following 30 minutes of baseline imaging, animals were treated for at least 40 min with blockers of glutatmatergic transmission (GYKI, MK801 or control), followed by a second 30 minute imaging sequence. 7 images were taken in each 30 min interval, exactly one image every 5 min. For analysis, maximum intensity projections of two-photon z-series stacks over time were aligned by translation and rotation using the Stackreg macro for ImageJ. Glial motility was analyzed using custom Matlab programs that performed sequential image edge detection followed by fixed radius dilation and then measured the change in added and lost pixels between sequential time points. The motility index of a cell was defined as the mean change in pixel overlap normalized by total number labeled pixels over all time points.
Results: Radial glia exhibit rapid extension and retraction of long, fine filopodial processes that are present at greatest density within the tectal neuropil region where retinal ganglion cell (RGC) terminals contact tectal cell dendrites. We therefore set out to test whether synaptic transmission between the RGC axons and their postsynaptic targets also affected the filopodial dynamics of radial glia. It was found that application of MK801 (an antagonist of the glutamate n-methyl-d-aspartate receptor; NMDAr), or GYKI (an antagonist of glutamate α-amino-3-hydroxy-5-methylisoazol-4-propionate receptor; AMPAr), greatly attenuated radial glia dynamics. Compared to control cells, motility index values of animals acutely exposed to MK801 or GYKI were reduced by 39% and 36% respectively.
Conclusion: We have demonstrated that radial glia in the tectum of living Xenopus tadpoles are highly structurally dynamic and that their dynamics are glutamate-dependent.
E Ruthazer is supported by grants from the CIHR, NARSAD, and the EJLB Foundation. B. A. N. Travençolo is grateful to FAPESP (07/02938-5) for financial support. L. da F. Costa thanks FAPESP (05/00587-5) and CNPq (301303/06-1) for financial support.
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