Synaptic Transmision: Presynaptic Events

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Synaptic TransmisionPresynaptic Events

• Molecular Mechanisms of Release
• Quantal Theory of Neurotransmitter Release
• Neurotransmitter is released in vesicular amounts
• Role of Calcium in Neurotransmitter Release
• Depolarization causes calcium influx
• Calcium in microdomain triggers vesicle fusion

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Overview of Synaptic Transmission

• Synthesis of neurotransmitter
• Packaging of neurotransmitter into vesicles
• Transport to synaptic terminal
• Preparation of vesicle for release
• Release of vesicle contents in response to
  stimulus
• Binding of neurotransmitter to postsynaptic
  receptors
• Channel opening to produce electrical signal

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Storage of Neurotransmitter

• Storage is in synaptic vesicles
• Purpose
• Protect from enzyme degradation
• Ready for release
• Types of vesicles
• Small, synaptic vesicles (50 nm diameter)
  triggered by single AP
• Large, dense core vesicles (100 nm) released by
  burst firing or repetitive stimulation

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Uptake of Transmitter into Vesicles

• Vacuolar proton pump
• ATPase
• Pumps protons into lumen of vesicles
• Proton gradient is energy source for
• Transmitter transporters
• 12 membrane spanning regions (except GABA which
  has 10)
• Four distinct types e.g. Monoamines, Glutamate
• Vary in dependence on
• pH gradient
• Potential gradient

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Pools of Vesicles

• Stages of Release involve different pools
• Reserve Pool
• Vesicles connected to each other and to actin
  cytoskeleton by thin cross links
• Readily releasable pool
• Closely associated with pre-synaptic plasma
  membrane (active zone)
• Two types
• Docked
• Fusion competent

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Vesicles at Neuromuscular Junction
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Reserve Pool Cross Linking Molecules

• Synapsin I
• Binds both actin and vesicles
• Injection of synapsin I decreases transmitter
  release
• Calmodulin-dependent protein kinase II
• CamKII
• Negatively regulates binding properties of
  synapsin I
• Injection of CamKII increases transmitter release

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Stages of Vesicle Release

• Docking
• Movement of vesicle from reserve pool to tight
  association with plasma membrane
• Priming
• Reactions that convert vesicle to form that can
  fuse in response to action potential
• Fusion
• Local elevation of calcium concentration
  stimulates vesicle to fuse with membrane
• Estimated to require 200 microseconds (from time
  of action potential)

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Stages of Vesicle Release
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Pools of Synaptic VesiclesTime for Replenishment
Time to reload vesicle
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Vesicle Docking and Fusion

• All membrane trafficking steps within a cell use
  a similar set of proteins
• Vesicle fusion is the same at synaptic terminal
  and at Golgi
• Similar proteins and process occurs all
  eukaryotic cells mammals, vertebrates, even
  yeast
• Other than requirement for calcium to trigger the
  event

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SNARE complex

• Group of proteins involved in docking/priming
• Synaptobrevin
• Synaptic vesicle protein (vSNARE)
• Also known as VAMP
• Single Transmembrane Segment
• Syntaxin
• Plasma membrane protein (tSNARE)
• Single Transmembrane Segment
• SNAP-25
• Synaptosomal associated protein of size 25 kDa
• Anchored to plasma membrane by palmityl chains

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Role of SNARE complex in Release

• Clostridial toxins are proteases that block
  release of neurotransmitter
• Clostridium botulinum (botulism) cleaves various
  parts of different snare proteins
• Clostridium tetani (tetanus) cleaves
  synaptobrevin
• Exact role and timing of SNARE formation is
  unknown
• What prevents SNARE complexes from forming
  continually?

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Pre-SNARE complex

• Synaptophysin binds Synaptobrevin
• Four membrane spanning segments
• Homo-oligomer
• Also binds cholesterol
• Prevents SNARE complex formation
• Munc-18 binds Syntaxin
• Prevents syntaxin from binding to synaptobrevin
• Regulates formation of fusion complexes
• unc-18 is invertebrate analog to Munc-18
• Both munc-18 and unc-18 are forms of sec

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SNARE complex

• Spontaneous assembly into three-protein SNARE
  complex
• Munc-18 and synaptophysin unbind
• DOC2 (vesicle) and Mint (Plasma) are unlocking
  proteins
• Synaptobrevin, syntaxin and SNAP-25 form complex
• The ATPase NSF becomes part of the SNARE complex
• NSF binds to SNARE via a-SNAP

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Formation of SNARE complex
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SNARE complex

• Once SNARE complex is formed
• Synaptotagmin associates with SNARE
• Calcium sensitive
• Syntaxin molecules bind calcium channels
• P/Q or N type most common
• Site of largest calcium concentration
• Calcium channels seen near fusion pores
• SNARE complex itself is not sensitive to calcium

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Fusion

• Binding of synaptotagmin I to calcium induces
  conformation change
• Two C2 calcium binding domains
• Fusion pore is formed first
• Reversible - may open and close
• Fusion pore expands to produce full fusion
• Synaptotagmin I mutation does not prevent
  neurotransmitter release
• Asynchronous release still occurs
• Multiple variants of synaptotagmin involved in
  both asynchronous and synchronous release

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SynaptotagminDifferential but convergent
functions of Ca2 binding to synaptotagmin-1 C2
domains mediate neurotransmitter releasePNAS
2009 10616469-16474 Shin, Xu, Rizo,
Südhofhttp//www.pnas.org/content/106/38/16469.fu
ll

• Neurotransmitter release is triggered by Ca
  binding to a presynaptic Ca sensor that induces
  synaptic vesicle exocytosis with a high degree of
  Ca cooperativity. Synaptotagmin-1 (Syt1) and two
  of its homologs, synaptotagmin-2 and -9, are the
  primary Ca sensors for synaptic vesicle
  exocytosis, Syt1 and its homologs are vesicle
  proteins that are composed of a short
  intravesicular sequence, a single transmembrane
  region, a variable linker sequence, and two
  conserved C2 domains that bind Ca. Both interact
  with and bend phospholipid membranes as a
  function of Ca. Mutation of Syt1 in flies
  impaired neurotransmitter release. Deletion of
  Syt1 in mice blocked fast synchronous release
  without decreasing asynchonous release, and
  without altering synaptic vesicle exocytosis
  induced by Ca-independent mechanisms.

• Point mutations that selectively alter the Ca
  affinity of Syt1 without changing its structure
  or Ca-triggering function demonstrated that
  changing the apparent Ca affinity of Syt1 for
  either its phospholipid interactions, or its
  SNARE binding, altered the apparent Ca affinity
  of release correspondingly. These mutations not
  only formally established the function of Syt1 as
  a Ca sensor in release, but also demonstrated
  that this function involves both phospholipid and
  SNARE protein binding.The same mutations not only
  alter evoked release, but also spontaneous mini
  release, consistent with the notion that
  spontaneous release is induced by local Ca fluxes
  which activate Syt1.

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NSF

• N-ethylmaleimide Sensitive Factor
• ATPase
• Activity requires association with
• Soluble NSF accessory Proteins (a-SNAP)
• Unrelated to SNAP-25
• SNAPs wrap around SNARE complex
• Several NSF molecules assemble at end of complex
• Provide energy for fusion
• NSF hydrolysis of ATP causes SNARE to disassemble
• Unknown whether ATP hydrolysis / disassembly
  occurs prior to or after fusion

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Fusion
synapto- brevin
munc18
also SNAP25
syntaxin
Role of synaptotagmin not illustrated ATP
hydrolysis may occur after fusion to SNARE
recycling
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Other Preparatory (Docking or Priming) Proteins

• Rab3 is a GTP binding protein involved in
  neurotransmitter release
• Activity is via interactions with
• Rabphilin
• RIM1a
• Rabphilin and RIM1a both contain two C2 domains
• Rabphilin binds calcium and phospholipids (and
  Rab3)
• RIM1a binds phopholipids (and Rab3), not calcium
• Piccolo, Bassoon, and Complexin

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Rab3/Rabphilin/RIM

• Rab3 is a GTP binding protein involved in
  neurotransmitter release
• Rab3-GTP binds to synaptic vesicles
• GTP is hydrolyzed during or after vesicle fusion
• Rab3-GDP dissociates from vesicles
• Rab3-GDP binds to GDP dissociation inhibitor
  (GDI)
• GDI promotes exchange of GDP for GTP and
  re-association with vesicle.
• Mutation produces only small changes in synaptic
  properties

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Fusion - Rab3Exact Role Unknown
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Fusion Variations

• Vesicle may not open long enough to completely
  discharge neurotransmitter
• FM flourescent dyes used to stain synaptic
  vesicles
• FMI-43 dissociates slower than FM2-10
• Complete discharge is long enough for both dyes
  to completely dissociate (no difference in
  destaining rate)
• Brief vesicle opening allows FM2-10 destaining,
  but not FMI-43 destaining
• In cultured 1 day old neurons at RT, different
  destaining rates were noted

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Fusion Variations

• Kiss and Stay
• Vesicle remains associated (docked) with active
  zone membrane
• Vesicle quickly recharged with neurotransmitter
  (1 sec)
• Used at slow stimulation frequencies

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Fusion Variations

• Kiss and Run
• Vesicle is removed from membrane via fast
  endocytosis pathway, and re-filled with
  neurotransmitter (30-40 sec)
• Used at slow stimulation frequencies

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Fusion Variations

• Endosomal recycling
• Vesicle is removed from membrane via slow,
  clathrin dependent endocytosis pathway (minutes)
• Used at high stimulation frequencies

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Vesicle Recycling

• Clathrin coat
• Complex of clathrin molecules
• Three heavy chains
• Three light chains
• Resembles chicken wire
• Each link is "three-legged" protein
• Dynamin
• Motor protein that promotes rapid retrieval
  GTPase
• Activity modulated by calcium-regulated
  phosphorylation
• Shortens the open duration of the fusion pore
  (kiss-stay)

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Vesicle Recycling

• Process
• Binding of adaptor proteins
• AP2 binds to synaptotagmin
• AP180
• Adaptor proteins bind clathrin
• Dynamin forms collar of protein at neck of bud
• Allows bud to pinch off of membrane
• Vesicle moves to and fuses with early endosome

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Reserve Pool Synapsin CamKII
SNARES Syntaxin Synaptobrevin SNAP25
Adaptor Proteins Clathrin Synaptotagmin Dynamin
Rab3/RIM SNAP/NSF
Synaptotagmin Calcium channels
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Quantal Hypothesis

• Neurotransmitter is released in vesicles
• Size of EPP or EPSP is proportional to number of
  vesicles released

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Quantal Release

• Each vesicle of neurotransmitter molecule is
  referred to as a packet or quanta
• Vesicles are fairly uniform in size and number of
  molecules
• Neurotransmitter is released in discrete quanta,
  1,2, ... n vesicles
• Vesicle exocytosis coincides with
  neurotransmitter release
• Action potentials (depolarization) accelerates
  the rate of vesicle release

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Vesicles Fuse after Nerve Stimulation
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Quantal Release

• Binding of neurotransmitter to post-synaptic
  membrane generates electrical signal
• Signal is proportional to amount of
  neurotransmitter
• Depolarization in response to 1 quanta called
  mini
• Spontaneous release of single vesicles occurs
• Single quanta depolarization of NMJ called
  miniEPP
• Stimulation in low calcium causes small
  depolarizations
• Amplitude is integer factor of mEPP / mEPSP
• Histogram of amplitude is multi-modal

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Quantal Release at NMJ
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Spontaneous Release in Thalamus
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Evoked Release in Thalamus
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Standard Katz Model to Analyze Release

• Every quantum produces same electrical signal in
  postsynaptic cell
• Response to 1 quantum quantal size, Q
• Vesicle release is probabilistic
• Average number of quanta released, m, is product
  of available quanta, n, times average release
  probability, p m np
• Action potential increases probability of release
• Post-synaptic response quantal size number of
  quanta EPSPQmQnp

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Binomial Distribution

• Probability of observing x quanta released is
  given by binomial distribution
• Probability of release p
• Probability of not releasing q 1-p
• Number of available quanta n
• Identical to probability of obtaining x heads
  when tossing a coin n times (p0.5)

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Poisson Distribution

• If probability of release is small and number of
  releasable quanta is large, probability of
  release is approximated using Poisson distribution

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Quantal Parameters

• Number of releasable quanta, n
• Not the number of vesicles in terminal
• Not the number of docked vesicles
• Possibly the number of active zones (release
  sites)
• Dense bar on cytoplasmic face of membrane
• Rows of intramembranous particles (channels)
• Pyramidal axons have one active zone per axon
  terminal
• NMJ has many active zones
• Possibly influenced by the number of fusion
  competent vesicles

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Quantal Parameters

• Probability of release, p, at one site is product
  of
• The number of vesicles in the immediately
  releasable pool
• Probability that one vesicle will be released by
  AP
• Controversy whether a release site can release
  more than one vesicle
• Determining whether n, p or q changes can help
  determine molecular mechanisms of plasticity

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Assumptions of Standard Katz Model

• Quantal Uniformity
• Uniform vesicle size
• Unlikely given different types of fusion
• Uniform post-synaptic receptor properties at each
  release site
• Independent and identical release probability
• Probability of release identical at each site
• Probability of release at one site not affected
  by release at other site
• Recent observations suggest this isn't true

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Assumptions of Standard Katz Model

• Low Noise
• Good recording conditions
• Post-synaptic ion channel noise is low
• Minimal variation due to stochastic channel
  opening
• Low noise if number of channels, k, activated at
  synapse large or probability of opening is large
• True at NMJ
• Not true at most central synapses (k lt 100)

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Assumptions of Standard Katz Model

• Stationarity
• No change in p or n over time
• Linear summation of PSCs
• Multiple release sites (ngt1)
• Some CNS synapses have only 1 release site
• Multiple quanta do not saturate receptors
• Not always true
• Multiple synapses at end of axon terminal
• Different location of synapses on dendritic tree
• Amplitude modified by electrotonic properties

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Quantal Analysis

• Purpose
• Calculate size of quanta, Q, and mean number of
  quanta, m, released by action potential
• Analyze mechanisms of synaptic plasticity
• Analysis of mini's
• Requires low variance in amplitude distribution
  of spontaneous PSPs, PSCs or EPPs, EPCs
• Low recording noise to avoid missed detections
• Requires validity of assumptions of Katz Model

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Quantal Analysis

• Cause of different types of changes to mini's
• Frequency of events (np)
• Usually pre-synaptic, change in release
  probability
• Could be insertion of AMPA receptors into
  "silent" synapses
• Quantal size (Q)
• Usually post-synaptic, receptor properties

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Quantal Analysis

• Evoked Release
• Usually performed under low calcium conditions to
  decrease probability of release
• Calculate Q from mini analysis, then calculate m
• mean evoked amplitude / mean mini amplitude
• Method of Failures

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Calcium

• Evidence that calcium is the trigger for release
• If extracellular calcium is removed, no release
• If extracellular calcium is increased, release is
  facilitated
• Injection of calcium into axon terminal (flash
  uncaging) evokes release
• Calcium indicator dyes show an increase in
  calcium concentration
• Occurs first at active zones

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Calcium

• Evidence that calcium is the trigger for release
• Activation of ICa evokes release
• Block INa with TTX and IK with TEA
• Prevents action potentials, leaves ICa
• Depolarization, with either step or action
  potential shape, activates ICa
• Spatial distribution of calcium channels matches
  sites of transmitter release
• N type and P/Q type most prevalent

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Calcium channels co-localized with fusion pores
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Calcium

• Features of calcium release
• Requires multiple calcium ions binding
  (cooperative)
• Release proportional to Ca3 to 4
• Occurs rapidly following calcium entry
• Onset of release in response to depolarization is
  "slow" due to time for calcium channel activation
• Onset of release in response to repolarization is
  within 200 microseconds
• No calcium entry if depolarization to 50 mV
• Calcium channels activate
• Large, rapid calcium entry on repolarization

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Release proportional to Ca3 to 4
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Onset of Release is Rapid
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Calcium

• Calcium microdomains
• Calcium buffers and mitochondria slow the
  diffusion of calcium
• Ca is high within microdomain around channel
• 100 mM at distance of 10 nm
• 10 mM at distance of 50 nm
• Docked vesicles are surrounded by up to 10
  channels within distance of 50 nm
• Calcium near vesicle may reach 100-200 mM
• Briefly during channel opening
• Buffering and diffusion return calcium to resting
  levels soon after channel closure

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Calcium

• Slow acting buffers (EGTA) have no effect on
  release
• Fast acting buffers (BAPTA) can impede release
• On-rate is 5x108 /M/s
• Calcium binding must have similar on-rate
• Rapid termination of transmitter release implies
  off-rate is fast, 1000/s
• Affinity of vesicle binding is low, 9-140 mM

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Summary - Important Points

• How neurotransmitters are packaged into vesicles
• Three different pools of vesicles
• Three steps of release
• docking
• priming
• fusion

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Summary

• Molecules involved in transmitter release
• SNARE complex
• Capping proteins
• NSF and SNAP proteins
• Synaptotagmin
• Three types of molecules involved in endocytosis

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Summary

• Standard Katz Model and assumptions
• Calculate probability of release using either
  Poisson or Binomial
• Know when Poisson is appropriate model
• Calculate m using method of failures
• Evidence that calcium is the trigger for release