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Presentacion curso inb 2012

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Presentacion curso inb 2012

  1. 1. “Ligandos Wnt como neurotrofinas, plasticidad sináptica modulada por ellos” Dr. Jorge Parodi Universidad Católica Temuco
  2. 2. Bubnik 2007 TRENDS
  3. 3. Schulte 2010, Phar- Review
  4. 4. Janda et al 2012 Science
  5. 5. Nusse and Varmus 2012 EMBO
  6. 6. Janda et al 2012 Science
  7. 7. Nusse and Varmus 2012 EMBO
  8. 8. Inestrosa and Arenas, 2009, Nat Rev Neurosci
  9. 9. Farias et al., 2009, J Biol Chem Cuitino et al., 2010, J Neurosci
  10. 10. In collaboration with Dr. Francisco José Muñoz (Universidad Pompeau Fabra, Barcelona) and Dr. Juan Pablo García-Huidobro (P. Universidad Católica de Chile, CARE)Technical Support: Juan Godoy and Inés Poblete
  11. 11. Condition Reversal potential Control -53 ± 7 mV Wnt-5a -56 ± 4 mV TEA -55 ± 2 mV
  12. 12. 1500 Control Kv 3.2 1500 pA HEK Control Kv 3.2 pA 1 nM SNAP Wnt-5a 10 nM 7-NI 100 nM 1000 nM 1000 1000 500 500 mV mV 0 0-150 -100 -50 0 50 100 150 -150 -100 -50 0 50 100 150 -500 -500 1500 1500 pA pA Kv 3.2 control Kv 3.2 control Wnt 1:100 SNAP 100 nM Wnt 1.25 SNAP 25 nM Wnt 1:10 SNAP 1 µM 1000 1000 500 500 mV mV 0 0-150 -100 -50 0 50 100 150 -150 -100 -50 0 50 100 150 -500 -500Neuron
  13. 13. 800 * Wnt-5aMembrane resistence (MΩ ) 600 * 400 * ¿Afecta la actividad 200 miniatura? 0 Control Wnt-5a sFRP 7-NI Go 6976
  14. 14. Control AMPA AMPA 100 * * Amplitude miniture current (pA) Control Wnt-5aWnt-5a AMPA 80 60 40Control NMDA NMDA 20Wnt-5a NMDA Control 0 Control Wnt-5a Wnt-5a + sFRP Formyl-6aa Wnt-5a 100 Control * Amplitude miniture current (pA) 15 min 80 * 60 40 20 0 Control AMPA NMDA
  15. 15. 3,0m NMD A frequ en cy (p A ) 2,5 ** 2,0 1,5 1,0 0,5 0,0 Control Wnt-5a Wnt-5a + 7-NI 60 ** m NMD A am p litud e (pA ) 50 40 30 20 10 0 Control Wnt-5a Wnt-5a + 7-NI
  16. 16. Plasticidad, efectosmas complejos del ligando Wnt
  17. 17. 2 5 fV (%of Control)fEPSP s lop e % o f C on tro l (R .U.) 4 0 -20 0 20 40 60 80 100 120 140 X Axis Title 3 2 1 0 -20 0 20 40 60 80 100 120 140 160 Time (min)
  18. 18. 4 3,5 ControlfEPSP s lop e % of C on trol (R .U.) fEPSP s lo pe % o f C o n trol (R .U.) Wnt 5a ACSF 3,0 2,5 2 2,0 Wnt 5a 40 min 1,5 Wnt 5a 20 min Control 1,0 0 -60 -40 -20 0 20 40 60 80 100 120 -40 -20 0 20 40 60 80 100 120 Time (min) Time (min)
  19. 19. 5 5 Control Medium 4 Control 4 train TBS fEPSP s lo p e (R .U.) sFRP 3 4 2fEPSP s lop e (R .U.) 1 0 -40 -20 0 20 40 60 Time (min) 3 2 1 0 -40 -20 0 20 40 60 Time (min)
  20. 20. 5 Wnt-5a 2 train TBS Wnt-5a/sFRP 4 sFRPfEPSP s lope (R .U.) 3 2 1 0 -40 -20 0 20 40 60 Time (min)
  21. 21. 5 5 Control 2 train Control 7NI 7NI 0.5 µ M 4 4 7NI 1 µ M fEPSP s lop e (R .U.)fEPSP s lo pe (R .U.) 3 3 2 2 1 1 0 0 -20 0 20 40 60 -40 -20 0 20 40 60 Time (min) Time (min) 5 Wnt-5a 2 train Wnt-5a 2 train/7-NI 4 fEPS P s lope (R .U.) 3 2 1 0 -20 0 20 40 60 Time (min)
  22. 22. 3.020 mV pulse amplitude (µ A) 2.5 10 mM Calcium 2.0 1.5 1.0 0.5 0.0 Control Wnt-5a Control Wnt-5a
  23. 23. Serum BAPTA 20 mV 0 mVBAPTA/Serum BAPTA/Wnt-5a 20 mV 0 mVBAPTA/Wnt-5a
  24. 24. 120Viabilidad % of control 100 80 60 40 Control 20 Wnt-5a 0 0 1 2 3 4 Days 1000 Control Wnt-5a 800 ATP current (nA) 600 400 200 0 Acute 24 hrs
  25. 25. 20 Control Wnt-5aMem b rane potential (m V ) 10 0 24 hrs 48 hrs 72 hrs -10 -20 -30 -40 -50 200 mV 0 -200 -150 -100 -50 0 50 100 -200 Control -400 Wnt-5a 500 nA 24 hrs Wnt-5a -600 -800 -1000 nA
  26. 26. Control Prog. Wnt-5a Wnt-5a 120 Medium 100 80 % GVBD 60 40 Control Medium Prog. 20 Prog Wnt-5a 0 Control 0 5 10 15 20 25 Hours of Washed
  27. 27. AgradecimientosDr. Nibaldo InestrosaDr. Fernando Romero, UFRODr. Ataulfo Martinez-Torres UNAMFelipe Serrano
  28. 28. 2.0 Case Control Serum Depleted 1.5 ACSF fEPSP Amplitude (relative units) 1.0 0.5 Serum 0 -10 0 10 20 30 Time (min) 2.0 1.5 0.5 mV 0.5 mV * 10 ms 50 ms(relative units) Facilitation Index Amplitude 1.5 1.0 R2-R1/R1 fEPSP 1.0 0.5 0.5 0 ACSF Control Case Depleted 0 Serum Control Case
  29. 29. 2.0 ACSF Case CSF fEPSP Amplitude Depleted (relative units) 1.5 1.0 0.5 CSF 0 - 0 10 20 30 10 Time (min) 2.0 mV 0.5 1.5fEPSP Amplitude mV 0.5 * 10 ms (relative units) Facilitation Index 1.5 50 ms 1.0 R2-R1/R1 1.0 0.5 0.5 0 ACSF Case Depleted 0 Control CSF CSF
  30. 30. 8.5 0.025 µg/ml 0.25 µg/ml Anti-P 2.5 µg/ml ACSF fEPSP Amplitude 6.0 (relative units) 3.5 1.0 0 -10 0 10 20 30 40 50 Time 8.5 (min) 0.5 mV * 10 msfEPSP Amplitude (relative units) 6.0 3.5 * 1.0 0 ACSF 0.025 0.25 2.5 µg/ml µg/ml µg/ml Anti-P
  31. 31. 4.0 Anti-P (0.25 µg/ml) Anti-P + APV (50 µM) ACSF 3.0fEPSP Amplitude (relative units) 2.0 1.0 0 -10 0 10 20 30 40 50 Time (min) mV 0.5 4.0 * 10 ms fEPSP Amplitude (relative units) 3.0 2.0 1.0 0 ACSF Anti-P Anti-P + d-APV
  32. 32. 3 KO mouse ControlfEPSP amplitude (relative units) 2 1 0 -20 -10 0 10 20 30 Time (min)

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