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Cell and Molecular Neuroscience > Jorgensen Material > Flashcards

Jorgensen Material Flashcards

1
Q

How can gap junctions inhibit neurotransmission?

A

Gap Junctions can be closed, gated,

Rectifying gap junctions pass current in only 1 direction

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2
Q

How did Katz demonstrate that evoked responses were composed of many quanta?

A

Poisson distribution

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3
Q

Bennet Experiment

A

Gap Junctions!

Injected membrane impermeant dye into cell. Saw that it passed into other cells. These must have been connected.

These gap junctions have a pore that allows molecules <1k Da. (calcium (40 DA) or NT (150 Da) but not GFP)

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4
Q

Electrical Synapse Summary

A
  1. Direct coupling of cytoplasm (electrically/dye coupled)
  2. attenuates signal (100 mV AP reduced to 1 mV current)
  3. often short duration (width of 1 AP, linked to membrane potential timecourse)
  4. mimic ionic conditions of the cell (inhibitiory/excitatory)
  5. bidirectional information flow (unless rectifying)
  6. can be gated (pH, Ca2+)
  7. generally not plastic
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5
Q

Eccles, Katz, Kuffler

1941-1942

A

Stimulated motor neuron, caused muscle to fire AP.

  1. Curare blocked muscle AP, therefore proving that muscle AP was result of ACh
  2. Eserine blocks ACh esterase and prolongs depolarization. This prolongs presence of ACh and increases muscle response

TAKEAWAY: acetylcholine mediates fast neurotransmission at neuromuscular junction

Frog NMJ

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6
Q

Brooks, Combs, ECCLES

1952

A

Inhibitory synapse in spinal cord. When this inhibitory synapse is stimulated, a hyperpolarization is recorded, instead of dissipated current, indicating that the CNS worked by chemical signaling and not electric

TAKEAWAY: inhibitory postsynaptic potentials kill the “spark” hypothesis (neurotransmitter glycine)

cat spinal cord

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7
Q

Fatt, Katz

1950,1952

A

Recording at endplate (synapse) showed an end plate potential, then AP in muscle.

Spontaneous endplate potentials (minis) were observed w/out stimulation OF UNIFORM SIZE.

Step 1 of quantal hypothesis

Frog NMJ

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8
Q

Del Castio, Katz & Katz,Miledi

1954, 1967 respectively

A

In absence of APs and under low calcium, evoked (subthreshold) endplate potentials (minis) are observed of UNITARY SIZE.

Relies on Poisson Statistics

Frog NMJ

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9
Q

Boyd, Martin

1956

A

Stimulated @ low calcium concentrations, resulted in release of multiple quanta. Fits Poisson statistics and confrims Katz!

Cat NMJ

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10
Q

Heuser, Reese

1979

A

Freeze Slam!

Stimulate NMJ as platform falls. Stimulus is timed so that the set up will freeze while vessicles are fusing

frog NMJ

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11
Q

What is needed to load vessicles?

A

ATP creates a proton gradient

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12
Q

3 predictions of vessicle hypothesis

A
  1. synaptic vessicles fuse w/ membrane
  2. synaptic vessicles contain neurotransmitters
  3. neurotransmitter in vessicle is required for neurotransmission
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13
Q

quantal content

A

number of quanta that contriubte to an evoked response

quantal content = (evoked response/ mini size) = #SV/APs

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14
Q

Hodgkin and Huxley demonstrated that conduction
along axons is via ______

A

Action Potentials

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15
Q

Eccles and Kuffler demonstrated that synaptic
transmission is via

A

Diffusable Neurotransmitters

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16
Q

Katz demonstrated that neurotransmitter release is
quantal, via synaptic

A

Vessicle Function

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17
Q

how does an action potential translate into
neurotransmitter release?

A

Calcium

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18
Q

Fatt, Katz and Katz Miledi

1952 and 1965

A

Post Synaptic potentials depend on extracellular calcium

frog NMJ

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19
Q

Katz, Miledi and Fatt, Katz + Cowan

A

synaptic delay = time from presynaptic action potential invasion of bouton to beginning of postsynaptic potential

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20
Q

Speed of AP in mylinated neuron

A

150 mm / ms

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21
Q

Speed of AP in unmylinated Neuron

A

10 mm / ms

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22
Q

Length (in time) of Synaptic Delay

A

.5 - 3 ms (1 ms)

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23
Q

Velocity of an AP

A

~25 mm / ms
(60 mph)

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24
Q

width (time course) of an AP

A

1 ms

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25
Q

Range in width (time course) of AP

A

100 us - 1 ms

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26
Q

Time for AP to travel 1 mm axon at 10 mm / ms

A

100 us

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27
Q

Time length of synaptic delay

A

(.5 - 3 ms)
~ 1 ms (velocity of 3 in/hr)

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28
Q

Time it takes for NT to diffuse across synaptic cleft?

A

1 us

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29
Q

How long would it take NT to diffuse the length of a 1 mm axon?

A

30 mins

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30
Q

What is the rate limiting step, that causes synaptic delay?

A

Activation of calcium channel

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31
Q

Length of time it takes to open calcium channel

A

300 us

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32
Q

length of time it takes membranes to fuse

A

150 us

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33
Q

Size of synaptic cleft

A

20-50 nm

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34
Q

Time it takes for NTs to diffuse across cleft

A

1-5 us

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35
Q

Length of time it takes to open post-synaptic ligand gated channels

A

150 us

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36
Q

Typical intracellular sodium concentration

A

10 mM
(5-20)

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37
Q

Typical Extracellular sodium concentration

A

140 mM
(130-160)

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38
Q

Typical intracellular potassium concentration

A

140 mM
(130-160)

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39
Q

Typical extracellular concentration of potassium

A

5 mM
(4-8)

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40
Q

Typical intracellular calcium conentration

A

50 nM
(.05 - 1 uM)

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41
Q

Typical extracellular calcium concentration

A

2 mM
(1.2-4)

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42
Q

Rahamimoff

A

high extracellular calcium required for exocytosis
calcium sensor has a low affinity for calcium (lots of calcium required to keep the sensor occupied with calcium)

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43
Q

Dodge and Rahamimoff

1967

A

exocytosis responds cooperatively with calcium
the curve is sigmoidal demonstrating cooperativity among calcium binding sites
suggests cooperative interactions among 4 calciums
hill coefficient = 4

44
Q

Non-cooperative binding

A

1 binding site.
When plotted linearly, it forms a rectangular hyperbola with an assymptote of

45
Q

Cooperarative Calcium

A
46
Q

Llinas, Steinberg, Walton 1981

A

neurotransmitter release relies on membrane depolarization and Ca++, but not Na+ or K+. Voltage gated Ca channels in presnatpci cell

Blocked sodium/potassium channels pharmacologically.

he’s wrong. only calcium is super important

47
Q

Tsien - FURA

A

Fluorescent way to tag calcium ions. Bound and unbound calcium are excited at slightly different wavelngths (340 and 380), respectively. However they emit at the same savelength. So you can monitor the eratio of bound/unbound calcium

48
Q

Llinas, Steinberg, Walton 1981 Traces

A

he’s wrong

49
Q

LLinas Right/Wrong

A
  • Right:
  • Ca channels are voltage gated
  • NT release depends on presynaticpic influx of calcium
    *Wrong
    concludes ca current is driving rate of NT release
    Hill coefficient = 1
    there is a residual-voltage deendence of vesicle fusion, indpenednet of calcium
50
Q

Calcium Conclusions

A

presynaptic calcium concentration, not flux drives vesicle fusion

the hill coefficient = 4,

4 calciums ions ‘cooperate’ to fuse a vesicle

there is no voltage-dependence of vesicle fusion
it’s just calcium.

51
Q

Aequorin

A

Aequorin converts calcium binding to blue light. Blue light stimulates GFP, causing a green-light emission.

52
Q

Takeaways of Fura/Aquorin

A

calcium levels are high in microdomains. internal calcium buffers prevent broad spreading of calcium.

53
Q

length (in time) of synaptic delay?

A

500 us

54
Q

What determines calcium influx?

A
  1. kinetics of calcium channel.
    i. slow activation kinetics
    ii.high temperature sensitivity
  2. driving force of calcium by membrane potential
    (reported reversal potential of calcium 40-70 mV)
    i. low driving force of peak of AP
    ii. high driving force as Vm repolaririzes
55
Q

Model of calcium channels and influx

A

@ peak AP: low Ca current. Only 1/4 of channels are open. low driving fCa driving force awhen Vm = +30.

@ peak Ca Current: Vm nearly totally repolarizied. nearly 1/2 of all Ca channels are open. high Ca driving force

56
Q

(Dis/)Advantages of Calyx of Held

A

Advantages:
Large (presynaptic bouton easily clamped)
Accurate. Large EPSPs visible when postsynaptic membrane clamped
reliable
dynamic (100 Hz stim)
Strong 600 active zones (site for release)

Disadvantage:
weird synapse

57
Q

Borst Saakman, 1999

A

Step action potential: fewere Ca channels open. Ca current reduced during repolarization. Hardly any postsynaptic response.

1 ms plateau AP: all Ca channels open, but no driving force.Ca current much larger during repolarziation than control

broad: same Ca channels open. increased duration of Ca influx during repolarization. larger postsynaptic current

58
Q

Sabatini, Regher

A

Ca channels open quickly at body temperatrues for mammals

59
Q

Fain (caged Ca)

A

Ca caged. Cage broken by photolysis.

independent of external calcium concentration
independent of voltage-gated calcium channel opening
independent of diffusion of calcium to synaptic vesicle

60
Q

Schneggenburger Neher 2000

A

Calcium affinity not so low. = high affinity calcium sensor half maximal is 9uM free calcium in the Calyx of Held.

calcium is essential, not membrane voltage

COOPERATIVE
At least 4 calcium ions act cooperatively to drive vesicle
fusion

61
Q

new consensus: calcium required for neurotransmitter release

A

25 uM

62
Q

Ca influx steps

A

step 1: open the calcium channel by depolarizing membrane
step 2: drive calcium into cell by hyperpolarizing membrane

63
Q

(dis/)Advantages of accute hippocampus slice

A

Advantages: accessible, connectivity intact
Diadvantages: dead cells on surface. Acute transfections are not possible

64
Q

4 hippocampal preps

A
  1. acute slice
  2. organotypic slice
  3. dissociated culture
  4. autaptic islands
65
Q

(Dis/)Advantages of organotypic hippocampal culture

A

Advantages: useful for transfections, connectivity in tact.
Disadvantages: monolayer of cells, cells change connectivity. changes in cell nature

66
Q

(Dis/)Advantages of dissociated hippocampal neuron culture

A

useful for mutants: physiology. microscopy: transfections, synapses visualized.
disadvantages - connectivity random, changes in nature of cells
(for example, AChRs expressed at high levels in pyramidal neurons)

67
Q

(Dis/)Advantages of autaptic hippocampal neurons

A

advantage - more normal cell, compared to NMJ or Calyx
disadvantages - no architecture, not even a monolayer of cells, no normal connectivity, changes in cell nature (for example, AChRs expressed at high levels in pyramidal neurons)

68
Q

Allen, Stevens Experiment

A

SYNAPSES ARE UNRELIABLE minimal stimulation protocol in acute hippocampal slices. multiple axons that synapsed to one neuron were stimulated at lower and lower values to see how reliable synapses were.

69
Q

Allen, Stevens Results

A

SYNAPSES ARE UNRELIABLE.

70
Q

MK801

A

MK801 irreversibly blocks NMDA channels

71
Q

Malinow

A

Malinow showed that the NMDA channels didnt open every time, meaning that the synapses didn’t fire and realease NT everytime they receied an AP stimulation.

Discovers 2 classes of snapses. One that fires 37% of the time. one that fires 6% of the time

72
Q

Size of synaptic bouton

A

1 um

73
Q

size of synaptic vesicle

A

40 nm

74
Q

size of Kcsa potassium channel

A

20 nm

75
Q

size of single protein

A

5 nm
300 AA

76
Q

size of protein complex

A

12 nm

77
Q

Size of plasma membrane

A

4 nm

78
Q

NSF

A

N-ethylmaleimide sensitive factor.

Named because the activity is blocked by the drug

79
Q

SNAPS (a,b)

A

soluble NSF attachment proteins.

Named based on discovery method (binds to NSF)

80
Q

SNARE

A

SNAP receptor.

Named based on discovery of binding NSF via a/b SNAP

81
Q

v-SNARE

A

vesicle SNARE (R-SNARE). Transmembrane protein in the vesicle which confers specificity. Called ‘synaptobrevin’ or ‘VAMP’ at the synapse

82
Q

t-SNARE

A

brane protein in the target membrane which confers specificity. Called ‘syntaxin’ and ‘SNAP-25) at the synapse.

83
Q

SNARE complex

A

ultra-stable trimeric complex composed of V-SNARE (synaptobrevin), T-SNAREs (syntaxin and SNAP-25)

84
Q

specialized SNAREs mediate fusion at each trafficking
step in the cell

A
85
Q

Söllner et al

A

the NSF ATPase binds SNARE proteins

86
Q

Correct SNARES hypothesis

A

SNAREs dock vesicles
and
SNAREs fuse vesicles

87
Q

Broadie

A

Syntaxin and synaptobrevin are required for evoked release of neurotransmitter upon calcium influx

syntaxin is required for fusion

88
Q

When does syntaxin act?

A

acts during fusion

89
Q

when does NSF act?

A

after fusion

90
Q

Purfiying neuronal snares

A
91
Q

FRET precursor vessicles

A
92
Q

FRET Theory

A
93
Q

FRET results

A

need both v and t snares to fuse vesicle.

preinculbation increases fluorescence.

94
Q

SNARE model

A
95
Q
A
96
Q

Conceptual evolution of synaptic transmission

A
97
Q

Brose

A

calcium-dependent binding of membranes by synaptotagmin
10 uM calcium EC50 for lipid binding
same for vesicle fusion

98
Q

Sudhof

A

mouse synaptotagmin 1 mutants lack fast exocytosis, therefore, ynaptotagmin is required for rapid SV fusion

99
Q

T. Nishiki and G. Augustine 2004

A

synaptotagmin I accelerates release but is
not essential for release

100
Q

Burgalossi Jung Meyer Jockusch Jahn Taschenberger O’Connor Nishiki Takahashi Brose Rhee. 2010. Neuron.
also for Syt2: Young and Neher 2009. Neuron.

A

suggests synaptotagmin required for tethering to PM for rapid exocytosis

101
Q

Redundant synaptotagmyn

A

Eric now thinks this is wrong.

102
Q

Di Antonio, Schwarz 1994

A

first genetic assay that indicated that synaptotagmyn may inhibit exocytosis. More minis seen from syn knockout.

103
Q

Chicka, Hui, Liu, Chapman 2008

A

synaptotagmin-1 inhibits exocytosis in hippocampal
neurons in a genetic assay in mouse

synaptotagmin-1 inhibits exocytosis in SNAREs biochemical liposome fusion assay

104
Q

Huntwork Littleton 2007

A

complexin holds vessicles at ready point.

flies lacking complexin exhibit a high frequency of
spontaneous miniature currents

in fly complexin mutants, miniature currents are
spontaneous fusions – independent of calcium

105
Q

Jorgensen Journal Club

A

paper 2 CiVDS. think about experiments that argued about voltage sensitive voltage vesicle fusion. REMEMBER THE EXPERIMENTS THAT KNOCKED OUT THIS IDEA AND PROVED IT WAS ONLY CALCIUM.

Cell and Molecular Neuroscience (15 decks)

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