1. Brain Repair

2. Introduction For successful regeneration after axotomy to take place, the injured neuron has to recall a blueprint and prepare a Gant-chart to efficiently execute a new bioengineering project in which a new machine ( a motile constructions- the GC) is to be assembled instead of an old (stable element) in the middle of a busy town. As an engineering project that is to be effectively executed, the injured neuron has to simultaneously take care of many processes: starting with handing-out the information (crisis, its location and severity), recruit participating partners, recruit resources, coordinate among the different participants, prepare the construction site (demolish exciting structure, paving roads for supply, mobilizing materials...) executing in an orchestrated manner the new construction and deciding when the project is terminated......!!!!!!!!!!!!!

7. Growth cone formation after Axotomy The neurons demonstrate high capacity to regenerate and self assemble a competent GC

8. Dynamics of membrane seal formation after axotomy Electrophysiological criteria / Dye exclusion Free intracellular calcium concentration The most critical event - Crisis management by the formation of a membrane seal over the cut end to regain homeostasis

10. Calcium

11. Elevation of the free intracellular Ca 2+ concentration following Axotomy – Mag fura-2 ratio imaging 20 images 1 sec apart (20sec)

12. Not only that the Ca concentration/membrane seal formation are temporally correlated they are also causally related. If the calcium concentration is not elevated a seal will not be formed

14. Calcium

18. Bifurcated cells from the Buccal ganglia of Aplysia were loaded with the [Ca+2]i indicator Flou-4 and thereafter one of their branches was transected. Axotomy induces a burst of action potentials and the simultaneous rise of [Ca+2]i in both branches (16.8 sec). While the calcium levels in the non-axotomyzed branch recovers to the base level within tens of seconds the [Ca+2]i in the transected branch remains high for minutes. Colors in the graph correspond to the areas marked on the 14.4sec All reviews tells us that -axotomy-induced retrograde train of action potentials.... Is that the first long-distance signal that initiate growth signal????

19. What are the signals that tells the neuron that it has been injured? Fast first signal Calcium (????); Decreased trafficking of retrograde trophic factors to the soma (NGF); New injury induced signals generated at the site of injury signals (importin families of nuclear transport system) are actively transported ( molecular motors).

20. ERK, PKA, PKC, PLA2, Calpain The inhibition of one cascade is sufficient to impair regeneration after axotomy. I will concentrate on calpain Calcium

21. bis(CBZ-Alanyl-Alanine amine) Rhodamine 110 is nonfluorogenic whereas its cleavage products are highly fluorescent. Both the substrate and the cleavage products are membrane permeable. The fluorescent product accumulate at the site of the substrate cleavage for as long as the rate of proteolytic activity exceeds the rate of the product removal by diffusion out of the cell.

24. Control- cross sections 38-58 min after axotomy substrate level 3µm above the substrate

26. spectrin microtubules actin Preparing the “construction site” by removal of preexisting structures And then contribute to the restructuring Ca and calpain

29. / triton

31. EGFP-α tubulin- 3µm above substrate 100 images 6 sec apart (10min) EGFP-Actin-substrate level 72 images 10 sec apart (12min)

32. EGFP-Actin-substrate level 23 images 15 sec apart (~6min)

34. Mechanism of vesicle traps formation A word about the forces that reconfigure the MTs polar orientation and maintain it, and a word about the distorted distribution of subcellular resources in the distal axonal segment

35. Membrane supply ? ? ?

41. EGFP-Eb3 in the cell body 80 images 4 sec apart (5min)

42. Eb3 in Aplysia Axon 30 images 6 sec apart (3 min) Cell body EGFP-EB3

43. 180 images 6 sec apart (18.5 min) Axotomy- Proximal side Cell body EGFP-EB3

45. EB3 RH237 EB3+RH237 180 images 6 sec apart 180 images 6 sec apart (18.5 min) Axotomy- Proximal side

46. + - Vesicles transport on MTs

47. EB3 SNAP25 MEGRE 20 images 23 sec apart (7 min 40 sec)

48. Internalized plasma membrane is retrogradlly transported SR101 75min after wash 60 images 5 sec apart (5 min) Cell body

49. SR101 EB3 SR101 MEGRE 60 images 7 sec apart (7 min)

50. SNAP25 RH237 SR101 SNAP25 SR101

53. Vesicles fuse with the plasma membrane SynpH

55. SynpH ext 488nm Acidification of the ASW Normal ASW Ext 405 nm merge