Spira’s lectures 2008

1. Spira’s lectures 2008

2. Synapses asymmetric structure, presynaptic containing vesicles and active zone, gap post synaptic membrane -postsynaptic density (PSD) structural proteins that hold and collect receptors and signaling machinery. The two membrane are held in register by CAMs and the ECM Variability in synaptic structures- number of vesicles, PSD , size shape for example spines that form restricted isolated compartment

4. Synapses are formed from hundreds of different proteins in a highly orchestrated way. • What induce synapse formation? • How do synapses assemble? • How are synapses maintained?

5. Synapse formation Neurons acquire the competence to form synapse as part of a developmental program hippocampal neurons E18 form synapses while under the same conditions E16 will form silent synapses no matter how long you wait. Time Space Intrinsic factors Extrinsic factors

6. lasting 1–2 h, during which vesicles and synaptic proteins accumulate within the presynaptic bouton and at the cytoplasmic face of the opposing postsynaptic membrane

7. Placement • Synapse assembly requires that the plasma membrane of the pre and post synaptic cells are placed in close proximity this is executed by the: • Cadherins • Ig superfamily ( a large number of molecules among them the neuroligin neurexin)

10. Reciprocal interactions • In vertebrates cadherins were shown to stabilize synapses but are not necessary for the induction. • Neuroligin a members of the Ig super family of CAMs (associated with the PSD) induce presynaptic functional active zone. • Neuroligin (post synaptic ) neurexin (presynaptic).

12. Synapse induction requires bi-directional signaling during axo-dendritic contact • More then one inducer drive the pre and post synaptic differentiation • Different induces drive the assembly of different synapses

13. The cellular way by which a synapse is assembled- Two scenarios • Proteins produced in the soma arrive stochastically and independently to the site of synapse formation and subsequently assemble the structure • The molecular component are packaged in the cell body and arrive in a group to rapidly assemble the

15. But • Presynaptic varicosities are formed in the absence of post synaptic cells??!! • Ready to go presynaptic elements!! • Increase the probability of synapses to be formed

16. Structural and functional maturation • Increase in the number of SVs • Increase PSD • Dendritic filopodia into mushrooms and stubby spines • Reduced spine mobility • Reduced sensitivity to actin perturbing drugs • N-type voltage gated calcium channels to P/Q type • NMDA receptores • Release probability • GABA is an excitatory transmitter becomes inhibitory by changes in the ionic driving forces (co-transporters of K and Cl)

17. Activity dependent synapse formation • The stage in which GABA serves as an excitatory transmitter is thought to serve growth and synaptogenesis at early developmental stages. • Dose activity play an essential role in development????

18. Activity dependent ? • Synapses are formed in the absence of activity. • But post synaptically silent synapses become active following stimulation- silent synapses • Also presynaptic silent synapses become active by different mechanisms

19. Two Alternatives For Synapse Formation: Step-By-Step or Preformed Packets. Ziv and Garner, 2004

20. EM micrograph of a synapse

21. Synapses: The Structure Dillon andGoda, 2005

23. Questions • What are the rates at which synaptic proteins are lost from and reincorporated into individual synaptic structures? • What happened to the molecules that are lost? Are they reused? Degraded? • Are they distributed among other synaptic structures? • Are they processed in a use dependent manner? • To what degree protein losses are compensated by local protein synthesis or somatic resources?

24. How to study these questions? Fluorescence recovery after photo bleaching FRAP. Photo activation. Time laps confocal microscopy

25. Synapsin • A marker of presynaptic terminals • Is known to disperses in response to stimulation! Therefore, to prevent spontaneous activity the glutamate receptors were blockade. • Few boutons were bleached (to about 30%) by high intensity 488 laser light. And then the fluorescent recovery was monitored. • To make sure that the imaging site is synaptic and that the FRAP does not damage the endocytotic/exocytotic machinery FM4-64 was loaded and unloaded.

26. Are the proteins that compose the presynaptic apparatus stationary, kept in place? And if not what is the turn over rate? Does activity affect the rates? Rate of reincorporation of synapsin We see the recovery but what is the source of synapsin????

27. Distribution of synapsin 1 to other synapses? Or degradation of the molecule? • To that end PA synapsin is used • PA GFP is not seen before activation therefore co expressed cyan fluorescence protein (CFP) .

28. PA-GFP Synapsin at 0 time the varicosity was activated

29. A post synaptic protein • ProSAP2 a protein composing the PSD of glutaminergic synapses. The Master organizer of the post synaptic cytoarchitecture

31. Figure 5. Synaptic Activity Accelerates GFP:ProSAP2 Loss and Reincorporation Rates (A) Mean recovery time course for all photobleached puncta in three separate experiments identical to that shown in Figure 4. Data shown are mean standard deviation for all photobleached puncta. (B) The data were fit according to a model that assumed two GFP:ProSAP2 pools with different recovery kinetics as described in the text. (C) Extrapolation of recovery time courses using the recovery rates and relative pool sizes that provided the best fit to the data.

32. Redistribution of ProSAP2 among nearby dendritic spines • Activation of about 10 spines (15 mm)

34. We showed re assimilation of synaptic proteins • However synaptic proteins have a defined half life time. • Protein synthesis must take place • Local??? • Soma??????

35. Cell body activated for synapsin

36. prosap

37. to remember • Exchange of synaptic proteins at a rate that is by far slower than diffusion. • Is not effected by protein synthesis inhibitors, protein degradation by the proteasome system • Is accelerated by stimulation • And greatly exceed the rates of protein replenishment by the cell body

38. More over • Synaptic vesicles may move from one synaptic terminal to others • Likewise actin, mitochondria. • It is reasonable that since the rate of supply of new proteins is so slow that Sharing of resources is an economical solution for relatively rapid changes • Remember that activity accelerate recovery. • Is it possible that activity create conditions to capture local exchangeable resources??????? • Sharing resources by synapses independent on the cell body - a point to think about in terms of our society

39. lasting 1–2 h, during which vesicles and synaptic proteins accumulate within the presynaptic bouton and at the cytoplasmic face of the opposing postsynaptic membrane