1. 1 Bi/CNS 150 Lecture 6 Friday, October 9, 2015 Revised after lecture 10/9/13 Presynaptic transmitter release Henry Lester’s has to skip “office” hours today Chapters 9, 12 (co-written by T. Sudhof, a 2013 Nobel Prize awardee)

2. 2 Proof of chemical synaptic transmission, 1921 Vagus nerve runs from the head to the heart Spontaneous heartbeats in both hearts are stopped by stimuli to the “upstream” vagus smoked drum The diffusible substance: acetylcholine acting on muscarinic ACh receptors

3. [neurotransmitter] open closed chemical transmission at synapses: electric field open closed electrical transmission in axons: Past lectures: V-gated Na + channels V-gated K + channels Today: V-gated Ca 2+ channels 3 Friday: ACh-gated excitatory cation (Na + / K + / Ca 2+ ) channels & GABA- and glycine-gated inhibitory anion (Cl - channels Next week: Glutamate-gated excitatory (Na + / K + / Ca 2+ ) channels

4. 4 Figure 9-1 Many basic principles of chemical transmission and developmental neuroscience were discovered at the neuromuscular junction (nerve-muscle synapse); acetylcholine is the transmitter.

5. 0.3 µm Fine structure of the NMJ Figure 9-1 5 ACh receptors Incl. acetylcholinesterase

6. Life cycle of a synaptic vesicle Figure 12-10 6

7. Caught by flash-freezing, invented at Caltech ~ 50 yr ago A. Van Harreveld Presynaptic terminal postsynaptic cell Like Figure 12-7 7

8. A. Homogenize brain in isotonic sucrose. B. Isolate synaptosomes (cut-off nerve terminals) by differential and sucrose gradient centrifugation C. Lyse synaptosomes in hypotonic solution to release vesicles. D.Isolate vesicles by glass bead column chromatography. Vesicles can be isolated from brain tissue by cell biological methods Proteins associated with synaptic vesicles, slide 1 8

9. Synaptophysin Synaptotagmin (the Ca 2+ sensor) Snares (residents of either the vesicle [v-snare] or the target membrane [t-snare]) VAMP (also called synaptobrevin), a v-snare Syntaxin, a t-snare that also associates with Ca 2+ channels SNAP-25, a t-snare (~25 kD) ATP-driven proton pump creates concentration gradient that drives neurotransmitter uptake against concentration gradient (one of three transporters that function in transmitter release) Proteins associated with synaptic vesicles, slide 2 Mary Kennedy’s work Lecture 1 asked, “Could cells utilize plasma membrane H + fluxes?” “Probably not. There are not enough protons to make a bulk flow, required for robustly maintaining the ion concentration gradients. (but some very small organelles (~ 0.1  m) and bacteria do indeed store energy as H + gradients).” 9

10. 10 Neurotransmitter and ATP (3,000 to 10,000 molecules of each) Transporter #2: Proton-coupled neurotransmitter transporter cytosol Transporter #1: ATP-driven proton pump H+H+ cytosol ~ isotonic! How synaptic vesicles fill from the cytosol vesicle interior See Figure 13-1A

11. 11 Transporter # 3. Na + -coupled cell membrane neurotransmitter transporters: Antidepressants (“SSRIs” = serotonin-selective reuptake inhibitors): Prozac, Zoloft, Paxil, Celexa, Luvox Drugs of abuse: MDMA Attention-deficit disorder medications: Ritalin, Dexedrine, Adderall Drugs of abuse: cocaine amphetamine Na + -coupled cell membrane serotonin transporter Na + -coupled cell membrane dopamine transporter cytosol outside Presynaptic terminals Trademarks: From Lecture #1 See Figure 13-1B, C

12. 12 From a previous recent lecture Atomic-scale structure of (bacterial) Na + channels (2011, 2012) As of fall 2013, there are no crystal structures of voltage- gated Ca 2+ channels. From the similarities in sequence, we expect the secondary and tertiary structures to resemble those of K + and Na + channels. A voltage-gated Na+ channel can be changed to a voltage- gated Ca 2+ channel by mutating... just 2 out of 1800 amino acids See Table 12-1

13. 13 docked vesicle voltage-gated Ca 2+ channel neurotransmitter Electricity, then chemistry triggers synaptic vesicle fusion See 1st part of Chapter 12 We’ll show a more complete animation in a few minutes nerve impulse Na + and K + channels

14. 14 voltage-gated Ca 2+ channel Electricity, then chemistry triggers synaptic vesicle fusion Ca 2+ docked vesicle neurotransmitter See 1st part of Chapter 12 We’ll show a more complete animation in a few minutes nerve impulse Na + and K + channels

15. 15 fused vesicle Ca 2+ neurotransmitter Electricity, then chemistry triggers synaptic vesicle fusion See 1st part of Chapter 12 We’ll show a more complete animation in a few minutes 1. The Na + channels have produced the voltage change (depolarization); the K + channels have rendered it brief (~ 1 ms) 2. The Ca 2+ channels produce some depolarization, but their main function: to introduce the intracellular messenger Ca 2+

16. Synaptotagmin has as many as 40 Ca 2+ -binding sites. Perhaps binding of more Ca 2+ increases the rate of fusion and/or pushes the vesicle toward the “slow track” and full fusion. http://stke.sciencemag.org/content/vol2004/issue264/images/data/re19/DC2/slowtrack2.swf Animation of “full collapse fusion”: Synaptotagmin is the calcium sensor 16 Like Figure 12-13

17. II.Peripheral membrane proteins A.Synapsins anchor vesicles to cytoskeleton. B.Rab 3A is a GTPase perhaps involved in vesicle trafficking III.Soluble proteins that participate in vesicle fusion and release A.SM proteins  Munc-18-1 binds to the N-terminus of syntaxin and participates in vesicle docking and priming.  Munc-13 - essential for all forms of synaptic vesicle fusion, participates in vesicle priming. B.Complexins interact with SNARE complex and stabilize SNARE complex. C.NSF and its associated proteins are needed for SNARE recovery. Other proteins that act on synaptic vesicles 17

18. An alternative form of Ca 2+ -dependent vesicle fusion, termed fast tracking, or “kiss and run” predominates at low frequency stimulation Animation: http://stke.sciencemag.org/content/vol2004/issue264/images/data/re19/DC2/newFasttrack2.swf 18

19. 19 Transmitter release depends strongly on extracellular Ca concentration HAL’s first paper, Nature 1970 Experiments at the squid giant synapse, which excites the giant axon (See Figs. 12-1, 12-2, 12-3) Cooperative processes cause nonlinear relation between [Ca 2+ ] and transmitter release

20. 20 Timing of synaptic events “Synaptic delay”, between the peak of the action potential and the start of transmitter release, is ~ 0.5 ms. Delay between the peak of the Ca 2+ current and the beginning of the EPSP is ~ 0.2 ms (more at lower temperature). Most of the “synaptic delay” is caused in opening of Ca 2+ channels during the action potential. The size and timing of the EPSP’s can be modulated by prolonging the action potential. Figure 12-1 mV

21. 21 measured postsynaptic response 1 ms 5 mV -60 +60 large “synaptic potential” leads to muscle action potential subthreshold synaptic events (revealed in low Ca 2+ ) stimulus to presynaptic motor axon, producing action potential Electrophysiological analysis of quantal synaptic transmission (slide 1) V (Figure 12-6, Box 12-1)

22. 22 repeated identical stimuli to the presynaptic neuron...... yield variable postsynaptic responses! 5 mV 5 ms Electrophysiological analysis of quantal synaptic transmission (slide 2) measured postsynaptic response stimulus to presynaptic motor axon, producing action potential V (Figure 12-6, Box 12-1)

23. 23 no stimulus; spontaneous “miniature” postsynaptic potentials repeated stimuli to presynaptic neuron 5 mV 50 - 1000 channels (differs among types of synapse). This is induced by the transmitter in a single vesicle. Electrophysiological analysis of quantal synaptic transmission (slide 3) 0 1 2 3 4 5 (Figure 12-6, Box 12-1)

24. 24 N vesicles per terminal (3 in this example) p probability of release per vesicle what is the probability P of releasing n vesicles? (n = 2 for this action potential) N and p sometimes change during memory, learning, and drug addiction Electrophysiological analysis of quantal synaptic transmission (slide 4): Binomial statistics of vesicle release binomial distribution becomes Poisson distribution (Figure 12-6, Box 12-1)

25. 25 1.Stimulated postsynaptic potentials (psp’s) have variable amplitudes 2.Spontaneous “miniature” postsynaptic potentials occur with only modest amplitude variability. 3.The amplitudes of the stimulated psp’s are integral multiples of the spontaneous “miniature” psp’s Electrophysiological analysis of quantal synaptic transmission (slide 5): Summary of the classical evidence: (Figure 12-6, Box 12-1)

26. 26 fused vesicle adds capacitance C E G Na + K+K+ Cl - inside outside CC inside outside A more direct electrical measurement of quantal release: Measuring the presynaptic capacitance increase due to vesicle fusion See Figure 12-8

27. 27 To measure the conductances, we set I C = CdV/dt = 0, but  G/dt  0. To measure capacitance, we set I C = CdV/dt  0, but  G/dt = 0. C E G Na + K+K+ Cl - CC Measuring the presynaptic capacitance increase due to vesicle fusion  C ~ 1 femtofarad = 1 fF = 10 -15 F Phys1 reminders, as usual See Figure 12-8

28. 28 On a time scale of seconds, Signaling at synapses occurs via 2 classes of mechanisms Discussed today 1. Chemical signaling is the dominant form in mammalian nervous systems. A. A chemical transmitter is secreted by the presynaptic terminal and diffuses within the gap or “cleft”, binding with specialized receptors in the membrane of the postsynaptic cell. B. The bound transmitter receptor can electrically excite or inhibit the postsynaptic cell. It sometimes also “modulates” the action of other transmitters. Not discussed today: 2. Electrical signaling results when current generated in one cell spreads to an adjacent cell through low resistance channels called “gap junctions” (see pages 178 – 185)

29. 29 End of Lecture 6 Henry Lester needs to skip “office” hours today Usually: Mon, 1:15-2 PM, Fri, 1:15-2 PM outside the Red Door