November 17, 2015

NSCI323 10A: Molecular Underpinnings of LTP

NMDARs are required for induction of LTP, but not for expression of
LTPs.
- APV (AP-5): NMDA blocker. It is a competitive antagonist for
glutamate so that glut can no longer bind and activate the receptor.
- Two recordings done before and after in different conditions, with or
without the drug.
o The arrow represents plasticity/induction protocol (a strong and
fast stimulation that you give after a baseline stimulation).
o The gray box represents drug administration.
o In both experiments, we measured the response after the
protocol.
- Results:
o LEFT graph: if you apply the drug prior to the protocol, then there
is NO LTP. That implies that you need NMDAR to produce this
potentiation and synaptic transmission.
o RIGHT graph: Instead of applying the drug before the protocol,
they applied the drug after the protocol. They found that the LTP
was not affected: they got a normal LTP.
o Conclusion: NMDAR are important in the events that lead to the
increase in synaptic transmission, but not important in
maintaining it. Once the changes are established (ie the LTP
produced), you no longer need NMDARs.
No NMDARs, no LTP induction in the hippocampus, or amygdala or
many many places in the brain.
- NMDAR blockage reduces LTPs: When applying APV, the amplitude of
the LTP was reduced. However, after letting the APV wash out, another
stimulation was able to activate another LTP of its original magnitude.
Random note from last class:
- Reasons why the amygdala is better for studying memory and learning
than the hippocampus:
o Hippocampus is involved in many complex phenomena (including
memory and learning). Therefore its more difficult to study in
animals.
o The amygdala is involved in implicit memory that involves few
synapses (and is therefore simple). So studying memory the
amygdala in rodents is much easier than studying the
hippocamp.
Blocking amygdala NMDARs in vivo reduced fear conditioning:
- In an experiment, APV is applied locally in the rats amygdala
before pairing the shock with the tone.
- The graph on the slide shows how often the rat freezes when hearing
the tone. Under normal circumstances the rat should eventually learn

that the tone and the electrical shock occur one after the other (~70%
freezing rate). The same occurred when the mouse was just injected
with CSF. However, when injected with the solution containing the drug
(APV, NMDAR blocker), the rat froze less often, implying that the rat
was not conditioning to the fear, as would a normal rat. The rat is
unable to pair the tone to the shock. It is unable to learn.
- This experiment demonstrates a causal relationship between LTP and
learning behavior.
- If, on the other hand, APV is administered after you train the animal (ie
the rat pairs the tone with the shock), the drug does not seem to alter
the learning that had already happened. The animal reacts the exact
same as it would have if the drug werent administered in the first
place.
Recall: NMDARs let calcium in.
- Recall that NMDAR requires glut and the postsynaptic cell to be depol.
o Depol causes the Mg to leave and therefore the channel is
unblocked, and ions are let thru.
o The Ca2+ entry is what leads to the sequence of events that
leads to more functional AMPAR at the synapse and better
communication of information.
- Graph: at resting potential, the membrane voltage across the NMDA
stays at rest. However during postsynaptic depolarization, when the
criteria for the NMDAR to open are all met (depol and glut), the current
across the NMDA depolarizes due to the inflow of Ca and Na ions. And
the inflow of Ca is what causes the LTP (learning and memory).
Ca2+ binds to calmodulin and activates CaM-KII.
- Once Ca enters, it binds to Calmodulin. Once Ca is bound, calmodulin
undergoes a conformational change. This new shape enables it to
bind to other proteins that do not have Ca binding sites. A
primary target is CaMKII.
- Calmodulin interacts with CaMKII.
o CaMKII is tightly linked to LTP, which is why we will focus on II,
but there are other CaMKs as well.
- The CaMKII subunit consists of two domains. The regulatory
domain contains a phosphorylation site that is covered when
the kinase is inactive. Ca-calmodulin comes in, binds to CaMKII
and activates the kinase. This exposes both the catalytic domain and
the phosphorylation site.
- Phosphorylation of this will also keep the kinase in an active
state even when the second messenger dissociates from the
regulatory domain.
LTP stimulation protocol promotes CaMKII autophosphorylation.
- Once Ca2+ enters, binds to calmodulin, and reveals the
phosphorylation site on CaMKII, CaMKII phosphorylates itself and keeps
itself going for a long time.
- To stop it/deactivate it, you need a phosphatase to dephosphorylate it.

If you add a CaM-KII blocker, you dont get as much phosphorylation of

CaMKII.
LTP Protocol promotes CaMKII-dependent phosphorylation of
AMPAR.
- Looking at phosphorylation of AMPAR.
- Recall AMPAR can be phosphorylated.
- Add CaMKII inhibitor no phosphorylation of CaMKII reduced
phosphorylation of AMPAR. This is evidence that one of the targets of
CaMKII is the AMPAR.
o Therefore the two targets of CaMKII: itself and AMPAR.
- When adding CaMKII Inhibitor, you see a decrease in the EPSP slope.
o See a decrease since CaMKII cannot phosphorylate AMPAR, and
less efficient information conductance (therefore EPSP slope
decrease).
- Adding an NMDA blocker decreases AMPAR phosphorylation too.
Local AMPA response on dendritic spines increase after LTP.
- Relevance of the following, I dunno:
o
o

Neuron put in caged glutamate solution (ie gluatamate cannot bind to its binding site on either the
AMPAR or NMDAR). The CAGEDness can be removed with light. Removal can therefore be done very
locally.
So you have glut everywhere, but are shining light on very localized areas only.

Red: lots of calcium and sodium entering the cell.

Swelling: structural changes that appear as a result of the LTP, in
addition to the electrical/chemical changes.

TAKE HOME:
- NMDARs are only active when 2 neurons fire together.
o But one presynaptic can both provide the glut and the
depol to the postsynaptic. So why is it necessary to have
2 neurons fire together? If come from the same neuron,
dont activate?
o NMDARs detect simultaneous glutamate release and
post-synaptic activity.
- NMDARs let Ca inside the post synaptic neurons to allow those
two neurons to wire together.
- Wiring occurs because of CaMKII-dependent phosphorylation
activity.
o The phosphorylation activity results in a bigger AMPA
response.
-