1. Regulation of Signal Strength by Presynaptic Mechanisms 9.013 / 7.68: Core Class Sheng Lectures Presynaptic Mechanisms Nathan Wilson

2. Background / Theory

3. The Presynaptic Specialization

4. Background / Theory Presynaptic Characteristics of Interest

5. Non- Uniform Volume Non- Uniform Filling Non- Uniform Flux Non- Uniform Alignment of Release Variable Number of Vesicles Fusing Possible Explanations for Variability in a Synapse’s Quantal Amplitude

6. Non- Uniform Volume Non- Uniform Filling Non- Uniform Flux Non- Uniform Alignment of Release Variable Number of Vesicles Fusing Possible Explanations for Variability in a Synapse’s Quantal Amplitude

7. Non- Uniform Volume Non- Uniform Filling Non- Uniform Flux Non- Uniform Alignment of Release Variable Number of Vesicles Fusing Possible Explanations for Variability in a Synapse’s Quantal Amplitude

8. Non- Uniform Volume Non- Uniform Filling Non- Uniform Flux Non- Uniform Alignment of Release Variable Number of Vesicles Fusing Possible Explanations for Variability in a Synapse’s Quantal Amplitude

9. Non- Uniform Volume Non- Uniform Filling Non- Uniform Flux Non- Uniform Alignment of Release Variable Number of Vesicles Fusing Possible Explanations for Variability in a Synapse’s Quantal Amplitude

10. Non- Uniform Volume Non- Uniform Filling Non- Uniform Flux Non- Uniform Alignment of Release Variable Number of Vesicles Fusing Possible Explanations for Variability in a Synapse’s Quantal Amplitude

12. (At Least) Two Modes of Fusion

13. Non- Uniform Volume Non- Uniform Filling Non- Uniform Flux Non- Uniform Alignment of Release Variable Number of Vesicles Fusing Possible Explanations for Variability in a Synapse’s Quantal Amplitude

14. Clever Methods Method 1: Capacitance (Direct Sensing of Transmitter Release)

15. Capacitance Technique

16. 2 s Capacitance Reveals Different “Fusion Signatures”

17. Klyachko and Jackson, Nature, 2002

18. Clever Methods Method 2: Amperometery (Direct Sensing of Transmitter Release)

19. Westerink, Neurotoxicity, 2004

20. Measurement of Fusion Modes via Amperometry Burgoyne and Barclay, Trends in Neurosci., 2003

22. Clever Methods Method 3: FM Imaging (Fluorescent Staining of Active Vesicles)

23. B C D A 10 min stimulation FM 1-43 1AP F1F1 image ADVASEP-7 1 load1 unload 480AP 30AP 2 load F2F2 480AP 2 unload 10 min 1  M a b c d 1 2 4 3 2 1 AP 30 AP low high 0 1 120 160

24. Aravanis et al., Nature, 2003

25. Aravanis et al., Nature, 2003

26. Clever Methods Method 4: Synaptophluorins (pH-Sensitive Visualization of Fusion Events)

27. Gandhi and Stevens, Nature, 2003

28. Regulation Molecular Regulation of Fusion Process

29. Regulation of the Fusion Pore The rate of fusion pore expansion is regulated by: –Intracellular Ca2+

30. Elhamdani et al., Neuron, 2001

31. Regulation of the Fusion Pore The rate of fusion pore expansion is regulated by: –Intracellular Ca2+

32. Regulation of the Fusion Pore The rate of fusion pore expansion is regulated by: –Intracellular Ca2+ –Phorbol esters (e.g. PMA, via PKC pathway)

33. Graham et al., Biochimie, 2000

34. Regulation of the Fusion Pore The rate of fusion pore expansion is regulated by: –Intracellular Ca2+ –Phorbol esters (e.g. PMA, via PKC pathway)

35. Regulation of the Fusion Pore The rate of fusion pore expansion is regulated by: –Intracellular Ca2+ –Phorbol esters (e.g. PMA, via PKC pathway) Fusion pore open time is regulated by –synaptotagmin I/IV –dynamin

36. Regulation of the Fusion Pore The rate of fusion pore expansion is regulated by: –Intracellular Ca2+ –Phorbol esters (e.g. PMA, via PKC pathway) Fusion pore open time is regulated by –synaptotagmin I/IV –dynamin Shift of the mode of exocytosis to “kiss-and-run” by: –High extracellular Ca2+ –Staurosporine (kinase inhibitor) (?) –Phorbol esters (e.g., PMA, PKC activator) –Munc-13

37. Fusion pore can be regulated (A) Spikes from control and phorbol ester (PMA)-treated cells; (B) spikes from control non- transfected and from Munc18(R39)-expressing cells; Catecholamines Carbon Fibre Amperometry Chromaffin Cells Fisher et al., Science, 2001

39. Regulation Molecular Regulation of Fusion Process

40. Regulation Implications for Synaptic Transmission

41. Quantal size is regulated by the fusion pore in small vesicles of dopaminergic neurons  Staal, Mosharov, Sulzer (Nat.Neurosci. 2004) simple release complex release Amperometry dopamine ventral midbrain

42. NMDAR GABA A R AMPAR Concentration timecourse (normalized) 100 ms 100 pA 100 ms 200 pA 10 ms 50 pA 16 ms Receptors Differ in Sensitivity to the Timecourse of Release of Neurotransmitter Quanta

43. Regulation Implications for Synaptic Transmission

44. Regulation Implications for Development of Neural Networks

45. Immature: Silent Transmission Mature: Functional During Development of Neural Networks, Transmission Changes from “Silent” Type to Functional Synaptic Release Causes NMDA Receptor-Mediated Current Synaptic Release Causes (NMDA + AMPA) Receptor-Mediated Current

46. Immature: Silent Transmission Mature: “Functional Transmission” During Development of Excitatory Signaling in Hippocampal Circuits, Transmission Changes from a Slow, “Silent” Type, to a Fast, Functional Type Synaptic Release Causes NMDA Receptor-Mediated Current Synaptic Release Causes (NMDA + AMPA) Receptor-Mediated Current What’s Different?

47. Summary of presynaptic neurotransmitter release maturation I synaptic FM1-43 = =

49. Early glutamatergic synapses exhibit “silent” transmission consisting mainly of NMDA currents.

50. Later in development, NMDA currents are joined by AMPA currents.

53. Regulation Implications for Development of Neural Networks

54. What is the Fusion Pore?

57. Summary

58. Non- Uniform Volume Non- Uniform Filling Non- Uniform Flux Non- Uniform Alignment of Release Variable Number of Vesicles Fusing Possible Explanations for Variability in a Synapse’s Quantal Amplitude

59. Regulation of Signal Strength by Presynaptic Mechanisms 9.013 / 7.68: Core Class Sheng Lectures Presynaptic Mechanisms Nathan Wilson