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Bi/CNS 150 Lecture 5

Wednesday, October 9, 2013 Revised after lecture 10/9/13

Presynaptic transmitter release

Henry Lester’s “office” hours Mon, 1:15-2 PM, Fri 1:15-2 outside the Red Door

Chapters 9, 12 (co-written by T. Sudhof, one of this week’s Nobel Prize awardees)

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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

[neurotransmitter]

openclosed

chemical transmission atsynapses:

electric field

openclosed

electrical transmission inaxons:

Past lectures:V-gated Na+ channelsV-gated K+ channelsToday: V-gated Ca2+ channels

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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

4Figure 9-1

Many basic principles of chemical transmissionanddevelopmental neurosciencewere discovered at the neuromuscular junction (nerve-muscle synapse); acetylcholine is the transmitter.

0.3 µm

Fine structure of the NMJ

Figure 9-1 5

ACh receptors

Incl. acetylcholinesterase

Life cycle of a synaptic vesicle

Figure 12-10 6

Caught by flash-freezing,

invented at Caltech ~ 50 yr ago

A. Van Harreveld

Presynaptic terminal

postsynaptic cell

Like Figure 12-7 7

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

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SynaptophysinSynaptotagmin (the Ca2+ sensor)Snares (residents of either the vesicle [v-snare]

or the target membrane [t-snare])VAMP (also called synaptobrevin), a v-snareSyntaxin, 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)

Proteins associated with synaptic vesicles, slide 2

MaryKennedy’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).”

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NeurotransmitterandATP

(3,000 to 10,000 molecules of each)

Transporter #2: Proton-coupled neurotransmitter transporter

cytosol

Transporter #1: ATP-driven proton pump

H+

cytosol

~ isotonic!

How synaptic vesicles fill from the cytosolvesicle interior

vesicle interior

See Figure 13-1A

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Transporter # 3. Na+-coupled cell membrane neurotransmitter transporters:

Antidepressants (“SSRIs” = serotonin-selectivereuptake inhibitors):Prozac, Zoloft, Paxil, Celexa, Luvox

Drugs of abuse: MDMA

Attention-deficit disorder medications:

Ritalin, Dexedrine, Adderall

Drugs of abuse: cocaine amphetamine

Na+-coupledcell membrane serotonintransporter

Na+-coupledcell membrane dopamine transporter

NH

HO NH3+

HO

HO

H2C

CH2

NH3+

cytosol

outside

Presynapticterminals

Trademarks:

From Lecture #1

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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docked vesicle

voltage-gated Ca2+ 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 impulseNa+ and K+ channels

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voltage-gated Ca2+ channel

Electricity, then chemistry triggers synaptic vesicle fusion

Ca2+

docked vesicle

neurotransmitter

See 1st part of Chapter 12

We’ll show a more complete animation in a few minutes

nerve impulseNa+ and K+ channels

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fused vesicle

Ca2+

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 Ca2+ channels produce some depolarization, but their main function: to introduce the intracellular messenger Ca2+

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.

http://stke.sciencemag.org/content/vol2004/issue264/images/data/re19/DC2/slowtrack2.swf

Animation of “full collapse fusion”:

Synaptotagmin is the calcium sensor

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Like Figure 12-13

II. Peripheral membrane proteinsA. 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 releaseA. 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

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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/vol2004/issue264/images/data/re19/DC2/newFasttrack2.swf

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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 [Ca2+] and transmitter release

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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 EPSP’s can be modulated by prolonging the action potential.

Figure 12-1

mV

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measured postsynaptic response

1 ms 5

mV

-60

+60

large“synaptic potential” leads to muscle action potential

subthreshold synaptic events(revealed in low Ca2+)

stimulus to presynaptic motor axon, producing action potential

Electrophysiological analysis of

quantal synaptic transmission(slide 1)

V

(Figure 12-6, Box 12-1)

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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 responsestimulus to presynaptic motor axon, producing action potential

V

(Figure 12-6, Box 12-1)

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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)

Analysis of Quantal Synaptic Transmission

00.10.20.30.40.50.60.70.80.9

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1 2 3 4 5 6Amplitude of Postsynaptic Response (mV)

Fra

ctio

n o

f O

bse

rvat

ion

s

Stimulated

Spontaneous

0 1 2 3 4 5

(Figure 12-6, Box 12-1)

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nNpnpn

NnP

1)(

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

,0 pN and As

(Figure 12-6, Box 12-1)

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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)

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fused vesicle adds capacitance

C

E

G

Na+ K+ Cl-inside

outside

C

inside

outside

A more direct electrical measurement of quantal release:Measuring the presynaptic capacitance increase due to vesicle fusion

See Figure 12-8

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To measure the conductances, we set IC = CdV/dt = 0, but G/dt 0.

To measure capacitance, we set IC = CdV/dt 0, but G/dt = 0.

C

E

G

Na+ K+ Cl-

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

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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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End of Lecture 5

Reminder: Henry Lester’s “office” hoursMon, 1:15-2 PM, Fri, 1:15-2 PMoutside the Red Door