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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
Range in width (time course) of AP
100 us - 1 ms
26
Time for AP to travel 1 mm axon at 10 mm / ms
100 us
27
Time length of synaptic delay
(.5 - 3 ms) ~ 1 ms (velocity of 3 in/hr)
28
Time it takes for NT to diffuse across synaptic cleft?
1 us
29
How long would it take NT to diffuse the length of a 1 mm axon?
30 mins
30
What is the rate limiting step, that causes synaptic delay?
Activation of calcium channel
31
Length of time it takes to open calcium channel
300 us
32
length of time it takes membranes to fuse
150 us
33
Size of synaptic cleft
20-50 nm
34
Time it takes for NTs to diffuse across cleft
1-5 us
35
Length of time it takes to open post-synaptic ligand gated channels
150 us
36
Typical intracellular sodium concentration
10 mM (5-20)
37
Typical Extracellular sodium concentration
140 mM (130-160)
38
Typical intracellular potassium concentration
140 mM (130-160)
39
Typical extracellular concentration of potassium
5 mM (4-8)
40
Typical intracellular calcium conentration
50 nM (.05 - 1 uM)
41
Typical extracellular calcium concentration
2 mM (1.2-4)
42
Rahamimoff
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)
43
Dodge and Rahamimoff | 1967
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
Non-cooperative binding
1 binding site. When plotted linearly, it forms a rectangular hyperbola with an assymptote of
45
Cooperarative Calcium
46
Llinas, Steinberg, Walton 1981
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. ## Footnote he's wrong. only calcium is super important
47
Tsien - FURA
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
Llinas, Steinberg, Walton 1981 Traces
## Footnote he's wrong
49
LLinas Right/Wrong
* 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
Calcium Conclusions
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
Aequorin
Aequorin converts calcium binding to blue light. Blue light stimulates GFP, causing a green-light emission.
52
Takeaways of Fura/Aquorin
calcium levels are high in microdomains. internal calcium buffers prevent broad spreading of calcium.
53
length (in time) of synaptic delay?
500 us
54
What determines calcium influx?
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
Model of calcium channels and influx
@ 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
(Dis/)Advantages of Calyx of Held
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
Borst Saakman, 1999
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
Sabatini, Regher
Ca channels open quickly at body temperatrues for mammals
59
Fain (caged Ca)
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
Schneggenburger Neher 2000
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
new consensus: calcium required for neurotransmitter release
25 uM
62
Ca influx steps
step 1: open the calcium channel by depolarizing membrane step 2: drive calcium into cell by hyperpolarizing membrane
63
(dis/)Advantages of accute hippocampus slice
Advantages: accessible, connectivity intact Diadvantages: dead cells on surface. Acute transfections are not possible
64
4 hippocampal preps
1. acute slice 2. organotypic slice 3. dissociated culture 4. autaptic islands
65
(Dis/)Advantages of organotypic hippocampal culture
Advantages: useful for transfections, connectivity in tact. Disadvantages: monolayer of cells, cells change connectivity. changes in cell nature
66
(Dis/)Advantages of dissociated hippocampal neuron culture
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
(Dis/)Advantages of autaptic hippocampal neurons
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
Allen, Stevens Experiment
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
Allen, Stevens Results
SYNAPSES ARE UNRELIABLE.
70
MK801
MK801 irreversibly blocks NMDA channels
71
Malinow
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
Size of synaptic bouton
1 um
73
size of synaptic vesicle
40 nm
74
size of Kcsa potassium channel
20 nm
75
size of single protein
5 nm 300 AA
76
size of protein complex
12 nm
77
Size of plasma membrane
4 nm
78
NSF
N-ethylmaleimide sensitive factor. Named because the activity is blocked by the drug
79
SNAPS (a,b)
soluble NSF attachment proteins. Named based on discovery method (binds to NSF)
80
SNARE
SNAP receptor. Named based on discovery of binding NSF via a/b SNAP
81
v-SNARE
vesicle SNARE (R-SNARE). Transmembrane protein in the vesicle which confers specificity. Called ‘synaptobrevin’ or ‘VAMP’ at the synapse
82
t-SNARE
brane protein in the target membrane which confers specificity. Called ‘syntaxin’ and ‘SNAP-25) at the synapse.
83
SNARE complex
ultra-stable trimeric complex composed of V-SNARE (synaptobrevin), T-SNAREs (syntaxin and SNAP-25)
84
specialized SNAREs mediate fusion at each trafficking step in the cell
85
Söllner et al
the NSF ATPase binds SNARE proteins
86
Correct SNARES hypothesis
SNAREs dock vesicles and SNAREs fuse vesicles
87
Broadie
Syntaxin and synaptobrevin are required for evoked release of neurotransmitter upon calcium influx syntaxin is required for fusion
88
When does syntaxin act?
acts during fusion
89
when does NSF act?
after fusion
90
Purfiying neuronal snares
91
FRET precursor vessicles
92
FRET Theory
93
FRET results
need both v and t snares to fuse vesicle. preinculbation increases fluorescence.
94
SNARE model
95
96
Conceptual evolution of synaptic transmission
97
Brose
calcium-dependent binding of membranes by synaptotagmin 10 uM calcium EC50 for lipid binding same for vesicle fusion
98
Sudhof
mouse synaptotagmin 1 mutants lack fast exocytosis, therefore, ynaptotagmin is required for rapid SV fusion
99
T. Nishiki and G. Augustine 2004
synaptotagmin I accelerates release but is not essential for release
100
Burgalossi Jung Meyer Jockusch Jahn Taschenberger O'Connor Nishiki Takahashi Brose Rhee. 2010. Neuron. also for Syt2: Young and Neher 2009. Neuron.
suggests synaptotagmin required for tethering to PM for rapid exocytosis
101
Redundant synaptotagmyn
## Footnote Eric now thinks this is wrong.
102
Di Antonio, Schwarz 1994
first genetic assay that indicated that synaptotagmyn may inhibit exocytosis. More minis seen from syn knockout.
103
Chicka, Hui, Liu, Chapman 2008
synaptotagmin-1 inhibits exocytosis in hippocampal neurons in a genetic assay in mouse synaptotagmin-1 inhibits exocytosis in SNAREs biochemical liposome fusion assay
104
Huntwork Littleton 2007
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
Jorgensen Journal Club
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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