RYAN, Timothy, Synapses, Weill Cornell, April 3, 2014
<p class="MsoNormal" >RYAN, Timothy, Synapses, Weill Cornell, April 3, 2014</p>
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This is a noon conference at Stfd in the Neurosci Instit program;
in Clark Aud.</p>
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he discusses relationship btwn metabolic state and cognitive state;</p>
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if you don't eat for 48 hours, it's guaranteed to affect your cognitive state;</p>
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video clip:
shows action potentials (using archea-rhodopsin fluorescence)
(amazing: looks exactly like an intra cell electrode recording...
tracks in real time;</p>
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also shows video clip of intracell calcium;</p>
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what are the metabolic needs for brain func. that are specifrically related to neuron function?</p>
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neurons: excitable tissue: must maintain gradients (and consume ATP )</p>
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how big is the safety factor for ATP (in terms of reserve)</p>
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key Qs:</p>
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how does electrical activ impact nerve terminal ATP levels etc.</p>
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we use this reporter system:</p>
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Photonus Pyrallis luciferase:</p>
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Luciferin... gives off a photon as ATP is consumed
he shows luceriferase chem. as ATP is converted to AMP + CO2 + PPi.</p>
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2 hurdles to use luciferase as a quantitative tool to measure ATP...</p>
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bathed in luceiferin; single neuon has luciferase in it...</p>
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at 5 to 10 mins... you see lots of luminescense in dendrites all the way out to terminals...
(but only want to see it at synap boutons)</p>
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solutions: add a synaptic "targeting seq to enrich concentration at synapse.</p>
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mCherry labeled at synapses...</p>
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we can put ATP into nerve terminals</p>
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we can see a nice titration curve of
x axis ATP conc. vs luminescence (avg presynap L/F from 150 neurons</p>
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there are about 1M ATP molecules per nerve terminal...</p>
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we can keep rat neurons alive for a few weeks in vitro... can measure one single neuron...</p>
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now how abt chronic incubation in TTX (tetrodo) ; how does that change ATP</p>
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answer: NO! how to interpret?</p>
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either:
1 )activity does not impose change on ATP OR
2) is lack of activty also decr ATP synth. (YES)</p>
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lifetime of ATP is only abt 2 minutes (ie must continually synthesize it)</p>
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Activity DOES drive large ATP consumption! (but must keep up by incr synthesis)</p>
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if you block glycolysis, then you see decr ATP and same with blocking F1 atp-ase.</p>
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(he jokes... you do NOT need to breathe if you are not firing aciton potentials)</p>
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ie if you block synth of ATP... not problem... ATP level does not change BUT
if you have firing, then ATP falls rapidly unless you replete it.</p>
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so... how does the synapse "know" to make more ATP?</p>
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possib:
1) feedback (reacting to dips in ATP)? (no)</p>
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2) feed fwd (predicting that you will need more)</p>
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How to spend a million ATP molecules...</p>
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block mitochondrial metab with oligomycin...</p>
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conclusion action potentials are a small piece of energetic burden at synapses.</p>
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how about removing synaptic vesicle cycle? (does it consume ATP?)</p>
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he shows the synaptic vesicle cycle (from 2009 diagram)</p>
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In the absence of the vesicle cycle ATP levels rise !!! (we are building up more ATP)</p>
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Conclusion: <b>VESICLE CYCLE is the primary determinant of ATP consumption
</b> (much more than simply firing at terminal)</p>
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what happens when ATP levels falls...</p>
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yes... endocytosis drops when ATP levels fall (when u block glycolysis)
and also when you block acidification of vesicles.</p>
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result: <b>Exo-endocyctosis is a big hog of ATP</b>.</p>
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1 neurons worth of nerve terminal consume abt 109 ATPs in an active state.</p>
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</p>
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Understanding the levers ofpower at synapses.</p>
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n * Pv = primed vesicles (docked vesicles) times prob of "full" occupancy
of the calcium sensors.</p>
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calcium channels in presynaptic terminal</p>
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<b>calcium channels are the most important thing to work on</b> at
presynaptic terminals of synapses
(in terms of fast synap transmission)</p>
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he shows Voltage gated calcium channels...
from Hoppa in Nature 2012.</p>
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shows alpha 1a calcium channels..
concl<b>: calcium channels are tightly regulated</b> ;</p>
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target of Neurontin and Lyrica (annual sales of abt 4B dolalrs);</p>
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alpha 2 delta is found at all brain synapses (subseq found that that is
where those drugs bind but fxn is unknown)</p>
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when his lab incr alpha 2 delta it gave rise ot 3X more synap calcium channels</p>
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what is alpha 2 deltas role in modulating syanp function? ...</p>
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can take away from with shRNA (block it) and effect goes away.</p>
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simply incr the density of calcium channels is prob suffic to incr exocytosis.</p>
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expt to show coupling btwn calcium channels and exocytosis...</p>
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how to measure the calcium influx at synap boutons?</p>
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use calcium dye with VAMP-mCherry...</p>
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use archeae-rhosopsin (used by circuit crackers... well known at Stfd Karl D)</p>
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Krahl Nat Methods 2012</p>
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it is a voltage sensitive fluor protein!!!!</p>
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shows Arch fused to GFP...</p>
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but Arch emits much much less light than GFP molec (about 1/1000)</p>
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but you can actually see the spikes! (v large change in fluor assoc with voltage) (from -65 mV to + 40 mV during action potential)</p>
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it does not bleach.</p>
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he shows back propagating dendritic spine waveform (origin. in the soma)</p>
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and forward propag to synap bouton waveform.</p>
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what are th waveforms assoc with alpha2 delta expression...</p>
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it has a huge effect on amplitude and duration of calcium entry.</p>
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potassium channels sculpt the action pots.</p>
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but. different Kvs control AP shape...
shows expts using TEA and dendrotoxin (DTX) that block K+ channels.</p>
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can change peak and the width (the overshoot of AP over zero)</p>
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conclusion alpha2delta changes calcium channels density and hence
"fusogenciity" of vesicles (Pv = probability of exocytosis)</p>
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New principesl</p>
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alpha 2 delta levels are rate limiting in determ presynap calc channel abundance</p>
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presynap AP is not a simple all or non switch.
It is plastic and the peak is closely matched to peak of Ca channel I-V curve.</p>
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comment: how to change efficacy of channels:
change conc, location, kinetics, voltage gated character.</p>
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really want to map out the locations of the channels... (quotes expert Rick Aldrich)</p>
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