2. Long-term potentiation
LTP is a persistent strengthening of synapses based on recent patterns of activity and major cellular mechanisms that
underlies learning and memory.
The late phases of LTP are thought to communicate via the extracellular signal-regulated kinase (ERK)
Early LTP (less than 1 h) had initially been explained either by presynaptic increases in glutamate release or by direct mo
dification of postsynaptic AMPAR
Early LTP mechanism
Late phases of LTP mechanism
Introduction
3. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR):
An ionotropic transmembrane receptor for glutamate
Mediates fast synaptic transmission in the central nervous system
AMPARs are composed of four types of subunits, designated as GluA1,
GluA2, GluA3, and GluA4
Most AMPARs are heterotetrameric, consisting of symmetric 'dimer of
dimers' of GluA2 and either GluA1, GluA3 or GluA4
6. FRAP: Fluorescence Recovery After Photobleaching
Determining the kinetics of diffusion through tissue or cells.
It is capable of quantifying the two dimensional lateral diffusion
This technique is very useful in biological studies of cell membrane diffusion and protein binding
Methods
7. Question
What is the role of AMPAR surface diffusion in synaptic plasticity and memory?
Materials
Organotypic hippocampal slice / Acute slice from wild type and Gra2-/- mice
Crosslinking surface AMPAR : bAP::SEP::GluA1/2
Methods
FRAP (Fluorescence Recovery After Photobleaching), Whole-cell patch clamp, Context/Cue-dependent
Fear Conditioning
Answer
Postsynaptic AMPAR is critical for the expression on hippocampal LTP and learning through surface
diffusion
8. To confirm the technique to crosslink surface AMPAR
Purpose
Method
FRAP(fluorescence recovery after photobleaching)
Results
Crosslinking of AMPAR surface diffusion is worked well.
Conclusion
9. In wild-type mice (Gria2+/+)- hippocampus neuron express hetero-tetrameric AMPARs composed of the GluA
1 and GluA2 subunits, which have a linear current–voltage (I–V) relationship
in the absence of GluA2 (Gria2−/−) AMPAR currents are inwardly rectifying
To identify the function of AMPAR surface diffusion in basal synaptic transmission
Purpose
Method
Whole-cell patch clamp, Glu uncaging
Results
10. To identify the function of AMPAR surface diffusion in synaptic plasticity
Purpose
Method
Whole-cell patch clamp (HFS-induced LTP)
Results
12. Results
To confirm AMPAR surface diffusion is an important trafficking step for endogenous AMPARs using antibody in synaptic plasticity
Purpose
Method
FRAP (from cultured neuron) / Field recording (HFS-induced LTP from hippocampal brain slice and in vivo recording)
14. Results
To confirm the function of AMPAR surface crosslinking in hippocampal memory
Purpose
Method
Contextual-dependent fear conditioning
Foot shock: 3 shocks (0.6mA for 2s with 60s ISI)
16. Discussion
Synaptic potentiation is followed by replenishment of AMPARs through exocytosis and surface diffusion
of extra synaptic AMPARs
Synaptic potentiation(especially early LTP) & Learning can be manipulated by modulating surface
diffusion not basal transmission
Postsynaptic AMPARs are critical for the expression on hippocampal LTP and learning through surface
diffusion
Editor's Notes
However, the exact mechanism through which synapses can rapidly recruit new AMPARs during early LTP remains unknown.
A widely accepted model of long-term potentiation (LTP) suggests that AMPA receptors (AMPARs) laterally diffuse within the plasma membrane and are trapped at synapses by slot proteins at the postsynaptic membrane. Before LTP induction (left), AMPARs are localized to postsynaptic density protein 95 (PSD95)‑containing nanodomains that are anchored to the actin cytoskeleton in spines. The AMPARs are retained within these domains through interactions between transmembrane AMPAR regulatory proteins (TARPs) and PSD95, but they are continually repositioned within the PSD because of constitutive actin filament dynamics. Extrasynaptic AMPARs laterally diffuse within the membrane, and the number of surface-expressed AMPARs is maintained by constitutive recycling in the spine and dendrite. Induction of LTP increases the AMPAR-binding capacity of the slot proteins, potentially through phosphorylation (denoted by the P) of the TARP stargazin, enhancing its binding to PSD95. This results in the entrapment of laterally diffusing AMPARs and potentiation of the synapse. In addition, net AMPAR exocytosis replenishes the pool of extrasynaptic receptors.
Author developed manipulations that crosslink surface AMPARs, thereby preventing their diffusion on the cell membrane. First, they created constructs to autonomously express recombinant biotin-tethered AMPAR subunits 1 and 2 (bAP::SEP::GluA1 and 2, where AP (acceptor protein) indicates the biotin acceptor tag, bAP indicates the biotinylated AP tag when co-expressed with the biotin ligase BirA, SEP is super-ecliptic phluorin and GluA1 encodes subunit 1 of the AMPAR, also known as Gria1), which we could surface crosslink by tetrameric biotin-binding proteins (BBPs, approximately 60 kDa, Fig. 1a, b
In wild-type mice (Gria2+/+), principal neurons of the hippocampus predominantly express hetero-tetrameric AMPARs composed of the GluA1 and GluA2 subunits, which have a linear current–voltage (I–V) relationship (Fig. 1e, grey). By contrast, AMPAR currents in the absence of GluA2 (Gria2−/−) are inwardly rectifying
Purpose 얘기하고, Method 얘기하고
Like figure 1, they did FRAP to see the effect of GluA2 IgG on AMPAR surface diffusion. As you can see, control and fragment antigen binding(Fab) shows normal level of Fluorescence recovery kinetics compared to figure 1. But GluA2 IgG shows low level of it. And it means this antibody impairs AMPAR surface diffusion.
To measure the synaptic plasticity in hippocampus, they infuse the antibody to hippocampal CA1 and recording field EPSP and Paired pulse ratio.
(Data 읽고). But Paired pulse ratio shows little difference(Meaning: presynaptic component of synaptic potentiation is same)
And they also check (Data 읽기, s는 spontaneous; AP event도 있을 수 있음) it shows any other basal synaptic transmissions are normal level in all conditions.
Figure B shows no stable synaptic potentiation following HFS or pHFS. (figure C data 설명) It also shows GluA2 Antibody completely abolish eLTP in slice.
Then they confirmed endogenous AMPAR diffusion is an important trafficking step through in vivo recording. It also showed consistent data with acute slice LTP recording. Figure E, F, G shows each normalized fEPSP slope. As you can see, both control conditions show potentiated slope but not GluA2 IgG condition. And fEPSP level is also consistent with this data.
So they concluded antibody crosslink of endogenous GluA2 attenuates LTP of CA1 field EPSPs acute slice and in vivo.
Until now, they confirmed LTP change in dorsal hippocampus. This region is well known as a critical region of learning and memorize of contextual fear memories. So they confirmed (Purpose)
For this behavior test, they infused antibody in the same region where they injected before.
Figure C shows no difference of fear conditioning between before and after antibody injection
Moreover, In cue-conditioned fear learning test which is well-known hippocampal independent memory shows established cue-conditioned fear learning and memory and no significant difference among all antibody conditions.
So based on these results, authors concluded infusion of crosslinking anti-GluA2 IgG, blocking AMPAR surface impairs hippocampal dependent learning task.
