LTP expression Flashcards
Malinow et al 1989
LTP doesnt require ongoing activity of CaMKII.
Inhibitor of the enzyme CaMKII, and instead of applying the inhibitor in the inductive phase of LTP, add it after the establishment of the LTP.
If the enzyme was responsible for continually phsophorylaiton of the recepor and keeping the conductance high and the rephosphorylation of the receptor is needed for the sustanance of the conductance, then the inhibition of the enzyme should lead to the LTP to be stopped and the conductance should come back down to baseline.
But this does not happen and there is no difference in the LTP. Blocks induction but not expression of LTP.
Other second messenger systems…
PKC: Malinow (1989)
PKA: Frey (1993)
ERK: Winder et al (1999)
Tyrosine Kinase: O’Dell (1991)
Why so many other second messenger systems?
Since that time many other enzymes were also implicated. Everything that you apply to a brain slice, somehow caused some imapct on the LTP. People even started to think about methylation etc.
If anything impacts the synaptic transmission, there is an impact in LTP as LTP is simply an extension of synaptic transmission itself
Ling et al 2005
PKMzeta is a an atypical protein kinase C, is necessary and sufficient for LTP maintainance.
If you use a typical protein kinase C inhibitor and apply it to the synapses interested in, then there is no difference in the LTP expression.
If you use a pharmacological inhibitor for the atypical protein kinase C isoform PKMzeta, it is able to inhibit the LTP expression. For the first time there is evidence for the inhibition of a phosphorylating enzyme of compromising the expression of LTP.
This came out of the Sacktor group - who was also able to develop a new inhibitor for the PKMZ - he could also administer this to animals and see if this affected the behaviour of the rodent. This was reported in the subsequent paper from the group.
Volk et al 2013
PKMZ is not required for hippocampal synaptic plasticity, learning and memory.
Full genetic deletion of PKMZ does not compromise LTP. The consequence of this is to completely crush the Sacktor hypothesis.
Pre and post synaptic loci
Presynaptic locus - this could easily chnge and support the LTP
There could be change in the transmitter release and other factors.
There could be varying degrees of interation with the pre and post syanptic loci - where the pre could be inducing the LTP but the post is expressing it or some combination fo that
Emptage et al 2003
Calcium enters the postsynaptic cells through the NMDAR. So if you could watch the calcium flux occur though the NMDAR in response to a stimulus, then you could measure how often the glutamate was interacting with the NMDAR and measure how often transmitter was beign released to cause that interaction to happen.
– single stimuli
Take brain slice, load a pyramidal neurone with a calcium indicator, focus using microscope on a small zone of the cell which is being imapcted by the presynaptic transmitter release, look for calcium rises at each spine at a time and image it before and after the induction of LTP
So you have the ability to measure how frequent the spine becomes activated before and after LTP for a given stimulus. Is there any change?
The probability goes up - LTP increases Pr
Siegelbaum??
Schuman & Madison 1991
When LTP expression was thought to be post syanptic, we didnt need to solve this problem, as this meant that all the things needed are in the post synaptic cell. How do you now signal from the post to the pre to tell them that the post syanptic cell has been activated?
retrograde messenger?
NO forms the all critical retrograde messenger - they dont suggest that AMPAR and NMDAR and the CaMKII are not important but they just added a few steps involving the NO.
NO activates the cGMP pathways and that modulate sthe performance of transmitter release follwing.
Lots of people since have provided more evidence that the retrograde NO transmission is important. There is also a wide range of suggestions for the type of retrograde messenger that is involved in this process such as arachadonic acid, CO etc, but only NO stood the test of time.
Isaac, Nicoll & Malenka 1995
Silent synapses - returns us back to the post synaptic expression of LTP
In the hippocampus, there are many axons interacting with cells. They patched an electrode amongst the axons and turned down the stimulus intensity of the stimulating electrode such that he would only provide enough stimulus to have an AP generated in one of the axons, and not those in the close proximity.
Now you can interogate what is going on at one synapse and not at the others. They turned down the stimulus response so effectively that everything was turned off. The voltage at which they were patching from -60mV to depolarise to +30mV, this is the current at which the current through the NMDAR would be at the maximum. There are now currents present - not AMPAR mediated but NMDAR mediated currents.
This can be further proved by blocking the NMDAR with the AP5.
The hypothesis is that there were synapses which had only the NMDAr and not the AMPARs, so they were functionally silent at the resting membrane potential and if you depolarise the cell, the receptors would become apparent.
They then argued that if youinduce LTP, AMPARs would miraculously appear at the site of the NMDAR mediated cells are present. This could mean that they are produced or expressed at the surface of the cell, or covalently modified and activated? etc. so that they can conduct a current
You could check this by seeing if NMDAR only synapses ever occured in the brain. This can be done with IHC or electron microscopy, where you can tag the receptor to see if there are any synapses had only the NMDARs
Saktor 2016
Came up with another theory agaist the Volk et al 2013 paper, saying that the PKMZ is being replaced by another PKMdelta/lambda.
We dont know this is entirely true but we dont know if this is true.
The Huganir group is in the late stages of producing a zeta and lambda douple KO animal and if this succeeds in proving that the zeta is not needed then Sacktor group is in trouble.
Zakarenko et al 2001
Use of FM styryl dye labels in synaptic vesicles.
FM1-43 unloading is accelerated after induction of LTP
Flurescence dye is poured onto the cells, and then the tissue is stimulated, and when endo and exo cytosis is happening, the dye is taken up by the vesicle membrane. The dye on the surface is washed off.
Stimulating the cell again causes the dye to be released. You can measure the rate at which the dye is being released before the LTP is induced at the synapse and after th LTP is induced. If more transmitter is being released after the LTP is induced, then you see that the dye is released much quicker - this could suggest that the presynaptic cell is involved in the LTP process.
This could mean that more transmitter is being released or the probability of any one vesicle emptying the vesicle contents to the outside of the cell is increasing. The distinction cannot be made form this experiment only.
Huganir lab - Liao et al. 1999
You could check this by seeing if NMDAR only synapses ever occured in the brain. This can be done with IHC or electron microscopy, where you can tag the receptor to see if there are any synapses had only the NMDARs
They generated flurescent antibodies one of which was for NMDARs and the other for AMPARs, co label them on neurones in culture, they showed that there were many synapses where there were only NMDARs but none where there is AMPARs only but plenty with both. So they provided circumstantial evidence for the NMDAR only hypothesis.
How can you test this functionally? -
Shi et al. (1999)
How can you test this presence of NMDAR only synapses functionally? - Use of GFP - if this way if the AMPARs are travelling to the membrane of the synapse upon stimulation then you can see the GFP being activated and coming out of the cell - so you can watch the AMPARs migrating to the cell membrane
This is what happens in the results of the paper. Absence of any green, but after the induction of LTP there is a green colour at the site consistant of the idea that there is a migraiton of the AMPARs to the surface of the cell.
There appears to be some structural re-arrangement but it does not conclusively prove if this si due to the movement of the GFP or something else.
AMPA receptor subunits are inserted into dendritic spines following LTP inducing stimuli
Regulated insertion of AMPA receptors may maintain LTP
There is a constant replenishment of a replccement receptor into the cell membrane (constitutive insertion by GluR2/3 and the activity induced GluR1/2)
Jia et al. 1996
k/o mice lacking GluR2 (constitutive insertion) show no transmission deficit
Granger et al. Nature 2013
LTP requires a reserve pool of glutamate receptors independent of subunit type
If you were to artficially manipulate the cell so that it expresses kainate receptors postsynaptically, you can generate LTP in a way that is previously described at silent synapses.
