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1. The Role of EphA7 in Synapse Formation during Cortical Development Sigma Xi Student Virtual Research Showcase March 13 - 23 2013Marie-Sophie van der GoesGeorgetown UniversitySenior, Neurobiology 2. Background 2 3. The formation of synapses is mediated bysignaling molecules Signaling molecules in the post-synaptic densityJohnston et al, 2009 Feng & Zhang, 2009 3 4. Eph-Ephrins are a receptor-ligand family of signaling molecules between neuronsSignaling molecules in the post-synaptic density Alexandra Russo, 2011Feng and Zhang, 2009 4 5. Steps in the shaping of post-synapticprotrusions in layers IV-V of the cortexAlexandra Russo, 20115 6. Structure and Function of EphA-EphrinA Noren And Pasquale 2004Egea and Klein 20076 7. Complementary expression pattern of EphA7 and EphrinA5 in the mouse cortex E 15.5 P0 Miller et al 2006 7 8. Question: What role plays EphA7 receptor inthe formation of the synapses in layers IV-Vof the cortex?EphA7 signaling ?Hypothesis:EphA7 mediates signalingearly in development toregulate protrusion ofpotential post-synaptic sites4 9. Experimental Procedure 1) Ex Utero Electroporation (Mouse ) 2) Primary Cortical Cell Culture3) DIV 14 - 18 - 21: Single whole-cell recordings9 10. Experimental Procedure (Continued)4) Selection for Regular Spiking Neurons5) AMPA mediated mEPSCs with acute treatment with TTX+BMR10 11. What is a mEPSC ?Pharmacologicalisolation ofAMPA(excitatory)receptorsAnderen & Soleng, 1999 12. Minis allow to measure Synaptic StrengthParameters of Measurement:-Frequency(# synaptic sites)-Amplitude(# receptors) Slide credit: Bridget Queenan 9 13. Results (a): In vitro Recordings show a slowed maturation of the synapses of A7-/- cortical neurons 14. Results(b): Spine analysis shows similar deficiencies in A7-/- neurons at DIV 18GFPPSD 95 MergeWTEphA7 -/-14 15. Next Research Step: More Background15 16. The FL form is expressed early in development, the TR later Carrie Leonard (2012) 17. Structure of two alternative splice forms ofEphA7 ReceptorFL: Full Length sequenceTR: the protein lacks theintracellular catalyticdomain Miller et al, 200617 18. EphrinA5-EphA7-FL mediates repulsive signalingbetween the cells, EphrinA5-EphA7-TR adhesive FL A7TR A7 Red: EphA7-expressing cells Green: Bed of EphrinA5-expressing cellsHolmberg et al., 200018 19. Question: What is the specific role of each formof EphA7 receptor in the formation of theappropriate synapses in Layers IV-V of the ? cortex? ?A7FL signaling A7TR signaling 19 20. Hypothesized mechanism of action of the twoalternative splice forms of EphA7 receptorEphA7FLEphA7TR 21. Experimental Procedure1) In Utero Electroporation: Genetic Manipulation inE 15.5 A7-/-: a) dsRed+A7FL b) dsRed+A7TR 22. Experimental Procedure (Continued)2) Slice recording 3) Biocytin injection of untransfected neurons 4) Analysis of AMPA-mediated mEPSCs22 23. Preliminary Results from In vivo Experiments *WTA7 -/-A7 -/- + TRA7 rescue Significance across age Significance within age*Note: Technical difficulties have prevented data collection forFL-A7 rescue experiments and increase in the N. The lack ofnormal distribution and the low N prevent test with Two-WayANOVA. Preliminary T-tests are shown. 24. Preliminary conclusions from in vivo experiments - In WT synapses get stronger after P14 in terms of active sites, but not in terms of post-synaptic receptors - The average number of active sites is larger in the TR rescue than in WT and in KO at P14-16 - The average strength of the post-synaptic sites is larger in the TR rescue than in WT at P12-13 and P14-16 and also than in KO at P14-16 -The strength of post-synaptic sites increases in KO from P14-16 to P17-19 24 25. Future Directions-Add experimental group/ increase N (KO, A7FL and A7TR Rescues)-Perform Immunohistochemical Analysis-Correlate cell function with cell morphology (Confocal Microscopy) P13P16 26. References-Ciossek, T., Lerch, M. M. and Ullrich, A. (1995). Cloning, characterization, anddifferential expression of MDK2 and MDK5, two novel receptor tyrosine kinases of theeck/eph family. Oncogene 11, 2085-2095.-Ciossek, T., Lerch, M. M. and Ullrich, A. (1999) Identification of alternatively splicedmRNAs encoding variants of MDK1, a novel receptor tyrosine kinase expressed in themurine nervous system. Oncogene 10(1): 97-108-Noren and Pasquale, E. (2003) Eph receptor-ephrin bidirectional signals that target Rasand Rhoproteins. J. Cellular Signaling,16 (2004) 655666-Goel A., Lee H. K. (2007). Persistence of experience-induced homeostatic synapticplasticity through adulthood in superficial layers of mouse visual cortex. J. Neurosci. 27,66926700. doi: 10.1523/JNEUROSCI.5038-06.2007.-Holmberg J, Clarke DL, Frisen J (2000) Regulation of repulsion versus adhesion bydifferent splice forms of an Eph receptor. Nature 408:203206.-Miller K, Kolk SM, Donoghue MJ (2006) EphA7-ephrin-A5 signaling in mousesomatosensory cortex: developmental restriction of molecular domains and postnatalmaintenance of functional compartments. J Comp Neurol 496:627642. 12 27. Acknowledgements- Stefano Vicini, PhD-Maria Donoghue, PhD-John Partridge, PhD-Rupa Lalchandani-Bridget Queenan-Meredith Clifford, PhD-Xiumei Zhao-Carrie Leonard-Wardah Athar-Chao Chen-Ruixi Luo13