Now a new topic We go on to the output zone (and the input zone): Synaptic transmission 1

Input and output zones 2

Synaptic input Graded potential Gives rise to frequency-coded APs Then to graded secretion: output zone 3

Quick overview: Events at a chemical synapse 4

Quick overview: The postsynaptic membrane 5

Chemical transmission topics Presynaptic events: transmitter release Postsynaptic events: direct channel gating Postsynaptic events: indirect gating and ion channel modulation Integration of excitatory and inhibitory synaptic input Plasticity (long term potentiation and memory) 6

Chemical transmission: first evidence Loewi 1921: Two perfused frog hearts Stimulated vagus nerve to heart 1 Heart 1 slowed Fluid from heart 1 used to perfuse heart 2 Heart 2 slowed too Conclusion: nerves release a chemical substance that is responsible for the action of nerve impulses on the tissue Loewi called it “Vagusstoff” It was acetylcholine (ACh) 7

Neuromuscular junction: basic structure 8

Neuromuscular junction: basic structure 9

Neuromuscular junction: basic structure 10

Curare Paralyses muscle (useful arrow poison) Curarised muscle still contracts with electrical stimulus: ...no effect on the contractile apparatus Curare doesn’t stop release of ACh on nerve stimulation ...no effect on the presynaptic nerve Curare does block the effect of ACh applied to denervated muscle ...it affects the postsynaptic membrane at the NMJ So what happens if we apply a small amount of curare and record from the muscle fibre? 11

Endplate potential and muscle AP Curare AP Endplate potential (EPP) 12

Endplate potential and muscle AP Curare allows us to separate the muscle action potential from the subthreshold depolarisation that causes it: the endplate potential (EPP) It does this by reducing the EPP amplitude so that it is sometimes subthreshold AP Endplate potential (EPP) 13

Endplate potential is a local potential Exponential decline... 14

Endplate potential is a local potential ...so think of local circuit currents! Inward current at motor endplate 15

Acetylcholine (ACh) is released from motor nerve endings ACh produces an endplate potential Endplate potential is a local potential If big enough (suprathreshold) it will elicit an AP Normally it is always suprathreshold: it can be experimentally reduced by curare Today’s main topic: How is ACh released from motor nerve terminals? 16

How is ACh released? Presynaptic terminal Muscle fibre Presynaptic terminals contain vesicles These vesicles contain ACh that is released on depolarisation What’s the evidence? 17

Depolarisation and ACh release Depolarisation then fast freezing: we can visualise fusion between vesicle and membrane Depolarisation makes fusions more frequent 18

Depolarisation and ACh release Depolarisation depletes vesicle population of nerve terminal: Before After 15 min 20 Hz stimulation 19

Depolarisation and ACh release Of course... We need to verify it’s reversible! After 15 min 20 Hz stimulation 60 min after end of stimulation 20

The vesicles are “packets” of ACh Fatt & Katz (1952): Microelectrode recording at motor endplate Under these conditions we get MEPPs: spontaneous “miniature end plate potentials” How many ACh molecules are in a packet? 21

How many molecules in a packet of ACh? 22

How many molecules in a packet of ACh? MEPP can be reproduced exactly by iontophoresed ACh We can estimate exactly how many ACh molecules this needs because: - each ACh molecule has one positive charge - and we know how much current was used About 7000 ACh molecules are involved 23

EPP is made up of a sum of MEPPs Mg2+ applied to inhibit transmitter release Because transmitter release is inhibited, each stimulus now gives only small EPPs and many failures... 24

EPP is made up of a sum of MEPPs These small EPPs are made up of discrete steps Step size is ~1 mV: similar to spontaneous MEPPs Conclusion: the MEPP is the “building block” of the EPP 2 1 0 2 1 25

The MEPP is the “building block” of the EPP MEPP amplitude EPP amplitudes are multiples of this 26

Depolarisation and ACh release Depolarisation increases MEPP frequency Increasing depolarisation 27

Conclusion on vesicles and ACh Vesicles in the nerve terminal are “packets” of ACh They are released by depolarisation of the terminal Next: How does depolarisation cause transmitter release? - experimental evidence for the role of calcium 28

Role of calcium in ACh release How do we know Ca2+ entry is needed for transmitter release? Here’s the experiment: Neuromuscular junction perfused with Ca2+-free solution Recording in muscle fibre (postsynaptically) Stimulus applied to nerve Iontophoretic application of Ca2+ before or after stimulus 29

Role of calcium in ACh release No Ca2+ present Depolarising stimulus pulse (P) applied to nerve No EPP observed 30

Role of calcium in ACh release Same setup: Ca2+ applied (Ca) just before the depolarising pulse (P) Now we get an EPP 31

Role of calcium in ACh release Same setup again Now Ca2+ (Ca) is applied after the depolarising pulse (P) No EPP observed So: Ca2+ entry is necessary for ACh release Ca2+ has to be applied before the depolarising pulse, i.e. Ca2+ must be present during depolarisation of the nerve So depolarisation-induced Ca2+ entry is involved: this means voltage-gated Ca2+ channels 32

Calcium alone is sufficient for ACh release New preparation: squid giant synapse 33

Calcium alone is sufficient for ACh release Squid giant synapse Nitrophen - “caged” Ca2+ Nitrophen releases Ca2+ in response to a UV flash Response to nitrophen is an EPSP (excitatory post-synaptic potential) EPSP after nitrophen EPSP after nerve stimulation So no depolarisation is needed: Ca2+ alone is enough 34

How does calcium cause ACh release? 35

How does a vesicle release ACh? The moment of exocytosis 36

How does a vesicle release ACh? Exocytosis and recycling 37

How does a vesicle release ACh? Measuring exocytosis and recycling: capacitance measurement Membrane capacitance is proportional to membrane area Recycling 38

Reading for today’s lecture: Purves et al Chapter 5 (pages 80-95) Nicholls et al chapter 9 (up to top of page 158 and pages 160-162) Nicholls et al chapter 11 Kandel et al chapters 10 (page 182 onwards) and 14 Next lecture: Postsynaptic events at synapses and the neuromuscular junction: ligand-gated ion channels Purves et al chapter 5 (pages 96 - 107); chapter 6 (up to page 125) Nicholls et al chapters 3 & 9 - sections on ACh, glutamate, GABA and glycine receptor channels; chapter 13 - pages 244-247 Kandel et al chapter 11, chapter 12 (pages 212-214)