Regulation of Signal Strength by Presynaptic Mechanisms

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

2. Background / Theory

3. Background / Theory The Presynaptic Specialization

4. Background / Theory Presynaptic Characteristics of Interest

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

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

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

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

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

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

12. (At Least) Two Modes of Fusion

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

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

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

38. 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 simple release Amperometry complex release dopamine ventral midbrain m Staal, Mosharov, Sulzer (Nat.Neurosci. 2004)

42. 16 ms Receptors Differ in Sensitivity to the Timecourse of Release of Neurotransmitter Quanta Concentrationtimecourse(normalized) 100 ms 100 pA NMDAR 100 ms 200 pA GABAAR AMPAR 10 ms 50 pA

43. Regulation Implications for Synaptic Transmission

44. Regulation Implications for Development of Neural Networks

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

46. During Development of Excitatory Signaling in Hippocampal Circuits, Transmission Changes from a Slow, “Silent” Type, to a Fast, Functional Type Mature: “Functional Transmission” Immature: Silent Transmission 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 Isynaptic 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.