Bi/CNS 150 Lecture 5

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Henry Lesters office hours Mon, 115-2 PM, Fri
115-2 outside the Red Door
Bi/CNS 150 Lecture 5 Wednesday, October 9, 2013
Revised after lecture 10/9/13 Presynaptic
transmitter release
Chapters 9, 12 (co-written by T. Sudhof, one of
this weeks Nobel Prize awardees)
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Proof of chemical synaptic transmission, 1921
Vagus nerve runs from the head to the heart
The diffusible substance acetylcholine acting
on muscarinic ACh receptors
Spontaneous heartbeats in both hearts are stopped
by stimuli to the upstream vagus
smoked drum
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Past lectures V-gated Na channels V-gated K
channels Today V-gated Ca2 channels
Friday ACh-gated excitatory cation (Na / K /
Ca2) channels GABA- and glycine-gated
inhibitory anion (Cl- channels Next week
Glutamate-gated excitatory (Na / K / Ca2)
channels
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Many basic principles of chemical
transmission and developmental neuroscience were
discovered at the neuromuscular junction
(nerve-muscle synapse) acetylcholine is the
transmitter.
Figure 9-1
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Fine structure of the NMJ
Incl. acetylcholinesterase
ACh receptors
Figure 9-1
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Life cycle of a synaptic vesicle
Figure 12-10
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Caught by flash-freezing, invented at Caltech
50 yr ago A. Van Harreveld
Like Figure 12-7
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Proteins associated with synaptic vesicles, slide
1
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Proteins associated with synaptic vesicles, slide
2

• Synaptophysin
• Synaptotagmin (the Ca2 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
  Ca2 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)

Mary Kennedys 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
mm) and bacteria do indeed store energy as H
gradients).
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vesicle interior
How synaptic vesicles fill from the cytosol
Transporter 1 ATP-driven proton pump
cytosol
Transporter 2 Proton-coupled neurotransmitter
transporter
Neurotransmitter and ATP (3,000 to 10,000
molecules of each)
cytosol
vesicle interior
isotonic!
See Figure 13-1A
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From Lecture 1
Transporter 3. Na-coupled cell membrane
neurotransmitter transporters
Antidepressants (SSRIs serotonin-selective r
euptake inhibitors) Prozac, Zoloft, Paxil,
Celexa, Luvox Drugs of abuse MDMA
Attention-deficit disorder medications Ritalin,
Dexedrine, Adderall Drugs of abuse
cocaine amphetamine
Presynaptic terminals
Na-coupled cell membrane dopamine transporter
Na-coupled cell membrane serotonin transporter
cytosol outside
See Figure 13-1B, C
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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 Ca2 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 Ca2 channel by
mutating . . . just 2 out of 1800 amino acids
See Table 12-1
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Electricity, then chemistry triggers synaptic
vesicle fusion
docked vesicle
neurotransmitter
voltage-gated Ca2 channel
Well show a more complete animation in a few
minutes
See 1st part of Chapter 12
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Electricity, then chemistry triggers synaptic
vesicle fusion
docked vesicle
neurotransmitter
Ca2
voltage-gated Ca2 channel
Well show a more complete animation in a few
minutes
See 1st part of Chapter 12
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Electricity, then chemistry triggers synaptic
vesicle fusion
1. The Na channels have produced the voltage
change (depolarization) the K channels have
rendered it brief ( 1 ms)
fused vesicle
Ca2
2. The Ca2 channels produce some
depolarization, but their main function to
introduce the intracellular messenger Ca2
neurotransmitter
Well show a more complete animation in a few
minutes
See 1st part of Chapter 12
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Synaptotagmin is the calcium sensor
Synaptotagmin has as many as 40 Ca2-binding
sites. Perhaps binding of more Ca2 increases
the rate of fusion and/or pushes the vesicle
toward the slow track and full fusion.
Like Figure 12-13
Animation of full collapse fusion
http//stke.sciencemag.org/content/vol2004/issue26
4/images/data/re19/DC2/slowtrack2.swf
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Other proteins that act on synaptic vesicles

• Peripheral membrane proteins
• Synapsins anchor vesicles to cytoskeleton.
• Rab 3A is a GTPase perhaps involved in vesicle
  trafficking

• Soluble proteins that participate in vesicle
  fusion and release
• 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.
• Complexins interact with SNARE complex and
  stabilize SNARE complex.
• NSF and its associated proteins are needed for
  SNARE recovery.

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An alternative form of Ca2-dependent vesicle
fusion, termed fast tracking, or kiss and run
predominates at low frequency stimulation
Animation http//stke.sciencemag.org/content/vol2
004/issue264/images/data/re19/DC2/newFasttrack2.sw
f
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Transmitter release depends strongly on
extracellular Ca concentration
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 Ca2 and transmitter release
HALs first paper, Nature 1970
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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 Ca2 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 Ca2 channels during the action potential.
The size and timing of the EPSPs can be
modulated by prolonging the action potential.
Figure 12-1
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stimulus to presynaptic motor axon, producing
action potential
measured postsynaptic response
V
large synaptic potential leads to muscle
action potential
Electrophysiological analysis of quantal
synaptic transmission (slide 1)
(Figure 12-6, Box 12-1)
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stimulus to presynaptic motor axon, producing
action potential
repeated identical stimuli to the presynaptic
neuron . . .
measured postsynaptic response
V
Electrophysiological analysis of quantal
synaptic transmission (slide 2)
. . . yield variable postsynaptic responses!
(Figure 12-6, Box 12-1)
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Electrophysiological analysis of quantal
synaptic transmission (slide 3)
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.
(Figure 12-6, Box 12-1)
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Electrophysiological analysis of quantal synaptic
transmission (slide 4) Binomial statistics of
vesicle release
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)
binomial distribution becomes Poisson distribution
N and p sometimes change during memory, learning,
and drug addiction
(Figure 12-6, Box 12-1)
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Electrophysiological analysis of quantal synaptic
transmission (slide 5) Summary of the classical
evidence
1. Stimulated postsynaptic potentials (psps)
have variable amplitudes 2. Spontaneous
miniature postsynaptic potentials occur with
only modest amplitude variability. 3. The
amplitudes of the stimulated psps are integral
multiples of the spontaneous miniature psps
(Figure 12-6, Box 12-1)
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A more direct electrical measurement of quantal
release Measuring the presynaptic capacitance
increase due to vesicle fusion
fused vesicle adds capacitance
inside
outside
See Figure 12-8
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Measuring the presynaptic capacitance increase
due to vesicle fusion
DC 1 femtofarad 1 fF 10-15 F
To measure the conductances, we set IC CdV/dt
0, but DG/dt ? 0. To measure capacitance, we set
IC CdV/dt ? 0, but DG/dt 0.
Phys1 reminders, as usual
See Figure 12-8
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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)
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Reminder Henry Lesters office hours Mon,
115-2 PM, Fri, 115-2 PM outside the Red Door
End of Lecture 5