Jorgensen Material Flashcards
How can gap junctions inhibit neurotransmission?
Gap Junctions can be closed, gated,
Rectifying gap junctions pass current in only 1 direction
How did Katz demonstrate that evoked responses were composed of many quanta?
Poisson distribution
Bennet Experiment
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)
Electrical Synapse Summary
- Direct coupling of cytoplasm (electrically/dye coupled)
- attenuates signal (100 mV AP reduced to 1 mV current)
- often short duration (width of 1 AP, linked to membrane potential timecourse)
- mimic ionic conditions of the cell (inhibitiory/excitatory)
- bidirectional information flow (unless rectifying)
- can be gated (pH, Ca2+)
- generally not plastic
Eccles, Katz, Kuffler
1941-1942
Stimulated motor neuron, caused muscle to fire AP.
- Curare blocked muscle AP, therefore proving that muscle AP was result of ACh
- 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
Brooks, Combs, ECCLES
1952
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
Fatt, Katz
1950,1952
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
Del Castio, Katz & Katz,Miledi
1954, 1967 respectively
In absence of APs and under low calcium, evoked (subthreshold) endplate potentials (minis) are observed of UNITARY SIZE.
Relies on Poisson Statistics
Frog NMJ
Boyd, Martin
1956
Stimulated @ low calcium concentrations, resulted in release of multiple quanta. Fits Poisson statistics and confrims Katz!
Cat NMJ
Heuser, Reese
1979
Freeze Slam!
Stimulate NMJ as platform falls. Stimulus is timed so that the set up will freeze while vessicles are fusing
frog NMJ
What is needed to load vessicles?
ATP creates a proton gradient
3 predictions of vessicle hypothesis
- synaptic vessicles fuse w/ membrane
- synaptic vessicles contain neurotransmitters
- neurotransmitter in vessicle is required for neurotransmission
quantal content
number of quanta that contriubte to an evoked response
quantal content = (evoked response/ mini size) = #SV/APs
Hodgkin and Huxley demonstrated that conduction
along axons is via ______
Action Potentials
Eccles and Kuffler demonstrated that synaptic
transmission is via
Diffusable Neurotransmitters
Katz demonstrated that neurotransmitter release is
quantal, via synaptic
Vessicle Function
how does an action potential translate into
neurotransmitter release?
Calcium
Fatt, Katz and Katz Miledi
1952 and 1965
Post Synaptic potentials depend on extracellular calcium
frog NMJ
Katz, Miledi and Fatt, Katz + Cowan
synaptic delay = time from presynaptic action potential invasion of bouton to beginning of postsynaptic potential
Speed of AP in mylinated neuron
150 mm / ms
Speed of AP in unmylinated Neuron
10 mm / ms
Length (in time) of Synaptic Delay
.5 - 3 ms (1 ms)
Velocity of an AP
~25 mm / ms
(60 mph)
width (time course) of an AP
1 ms
Range in width (time course) of AP
100 us - 1 ms
Time for AP to travel 1 mm axon at 10 mm / ms
100 us
Time length of synaptic delay
(.5 - 3 ms)
~ 1 ms (velocity of 3 in/hr)
Time it takes for NT to diffuse across synaptic cleft?
1 us
How long would it take NT to diffuse the length of a 1 mm axon?
30 mins
What is the rate limiting step, that causes synaptic delay?
Activation of calcium channel
Length of time it takes to open calcium channel
300 us
length of time it takes membranes to fuse
150 us
Size of synaptic cleft
20-50 nm
Time it takes for NTs to diffuse across cleft
1-5 us
Length of time it takes to open post-synaptic ligand gated channels
150 us
Typical intracellular sodium concentration
10 mM
(5-20)
Typical Extracellular sodium concentration
140 mM
(130-160)
Typical intracellular potassium concentration
140 mM
(130-160)
Typical extracellular concentration of potassium
5 mM
(4-8)
Typical intracellular calcium conentration
50 nM
(.05 - 1 uM)
Typical extracellular calcium concentration
2 mM
(1.2-4)
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)
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
Non-cooperative binding
1 binding site.
When plotted linearly, it forms a rectangular hyperbola with an assymptote of
Cooperarative Calcium
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.
he’s wrong. only calcium is super important
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
Llinas, Steinberg, Walton 1981 Traces
he’s wrong
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
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.
Aequorin
Aequorin converts calcium binding to blue light. Blue light stimulates GFP, causing a green-light emission.
Takeaways of Fura/Aquorin
calcium levels are high in microdomains. internal calcium buffers prevent broad spreading of calcium.
length (in time) of synaptic delay?
500 us
What determines calcium influx?
- kinetics of calcium channel.
i. slow activation kinetics
ii.high temperature sensitivity - 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
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
(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
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
Sabatini, Regher
Ca channels open quickly at body temperatrues for mammals
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
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
new consensus: calcium required for neurotransmitter release
25 uM
Ca influx steps
step 1: open the calcium channel by depolarizing membrane
step 2: drive calcium into cell by hyperpolarizing membrane
(dis/)Advantages of accute hippocampus slice
Advantages: accessible, connectivity intact
Diadvantages: dead cells on surface. Acute transfections are not possible
4 hippocampal preps
- acute slice
- organotypic slice
- dissociated culture
- autaptic islands
(Dis/)Advantages of organotypic hippocampal culture
Advantages: useful for transfections, connectivity in tact.
Disadvantages: monolayer of cells, cells change connectivity. changes in cell nature
(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)
(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)
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.
Allen, Stevens Results
SYNAPSES ARE UNRELIABLE.
MK801
MK801 irreversibly blocks NMDA channels
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
Size of synaptic bouton
1 um
size of synaptic vesicle
40 nm
size of Kcsa potassium channel
20 nm
size of single protein
5 nm
300 AA
size of protein complex
12 nm
Size of plasma membrane
4 nm
NSF
N-ethylmaleimide sensitive factor.
Named because the activity is blocked by the drug
SNAPS (a,b)
soluble NSF attachment proteins.
Named based on discovery method (binds to NSF)
SNARE
SNAP receptor.
Named based on discovery of binding NSF via a/b SNAP
v-SNARE
vesicle SNARE (R-SNARE). Transmembrane protein in the vesicle which confers specificity. Called ‘synaptobrevin’ or ‘VAMP’ at the synapse
t-SNARE
brane protein in the target membrane which confers specificity. Called ‘syntaxin’ and ‘SNAP-25) at the synapse.
SNARE complex
ultra-stable trimeric complex composed of V-SNARE (synaptobrevin), T-SNAREs (syntaxin and SNAP-25)
specialized SNAREs mediate fusion at each trafficking
step in the cell
Söllner et al
the NSF ATPase binds SNARE proteins
Correct SNARES hypothesis
SNAREs dock vesicles
and
SNAREs fuse vesicles
Broadie
Syntaxin and synaptobrevin are required for evoked release of neurotransmitter upon calcium influx
syntaxin is required for fusion
When does syntaxin act?
acts during fusion
when does NSF act?
after fusion
Purfiying neuronal snares
FRET precursor vessicles
FRET Theory
FRET results
need both v and t snares to fuse vesicle.
preinculbation increases fluorescence.
SNARE model
Conceptual evolution of synaptic transmission
Brose
calcium-dependent binding of membranes by synaptotagmin
10 uM calcium EC50 for lipid binding
same for vesicle fusion
Sudhof
mouse synaptotagmin 1 mutants lack fast exocytosis, therefore, ynaptotagmin is required for rapid SV fusion
T. Nishiki and G. Augustine 2004
synaptotagmin I accelerates release but is
not essential for release
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
Redundant synaptotagmyn
Eric now thinks this is wrong.
Di Antonio, Schwarz 1994
first genetic assay that indicated that synaptotagmyn may inhibit exocytosis. More minis seen from syn knockout.
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
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
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.
