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Rapid Spine Delivery and Redistribution of AMPA Receptors After Synaptic NMDA Receptor Activation. AUTHORS: Song-Hai Shi, Yasunori Hayashi, Ronald S. Petralia, Shahid H. Zaman, Robert J. Wenthold, Karel Svoboda, Roberto Malinow. PRESENTING: Kayla Giang Julianne Huang  Ryan Ferrell.

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rapid spine delivery and redistribution of ampa receptors after synaptic nmda receptor activation

Rapid Spine Delivery and Redistribution of AMPA Receptors After Synaptic NMDA Receptor Activation

AUTHORS:

Song-Hai Shi, Yasunori Hayashi, Ronald S. Petralia, Shahid H. Zaman, Robert J. Wenthold, Karel Svoboda, Roberto Malinow

PRESENTING:

Kayla Giang

Julianne Huang 

Ryan Ferrell

ltp pre or post synaptic
LTP: Pre- or Post- Synaptic?
  • Tetanus → → AMPAR/NMDAR→ → LTP
  • Post-synaptic AMPAR changes:
    • Increase in number at spine
      • Some spines lack AMPAR
        • Z. Nusser et al. – Cell Type and Pathway Dependence of Synaptic AMPA Receptor Number and Variability in the Hippocampus
      • Activation/insertion
    • Efficiency increases

Do AMPARs redistribute

to synapses due to tetanus?

visualizing ampar
Visualizing AMPAR
  • Tag with Green Fluorescent Protein
    • Functional?
    • Distributed normally?
how was the ampar tagged
How was the AMPAR tagged?
  • Viral infection with recombinant GluR1-GFP
  • Plain GFP fluoresces internally

NonpermeabilizedPermeabilized

was the tagged ampar functional hek 293 cells
Was the tagged AMPAR functional?HEK 293 Cells
  • Infected cells rectified nominally
  • GluR2 co-transfected cells did not rectify
    • Suggesting hetero and homo-oligomerization
was the tagged ampar functional ca1 pyramidal neurons
Was the tagged AMPAR functional?CA1 Pyramidal Neurons
  • Infection of CA1 neurons with GluR1-GFP
    • Infected cell
    • Uninfected cell
  • Greater rectification in infected
  • Displayed LTP
was the tagged ampar normally distributed
Was the tagged AMPAR normally distributed?
  • Present in synaptic region
    • Colocalized with presynaptic marker (synapsin 1)and GluR2
was the tagged ampar normally distributed1
Was the tagged AMPAR normally distributed?
  • 3x more GluR1-GFP than endogenous (over expression due to viral infection)
  • Less than endogenous in spine and on PSD by immunogold electron microscopy
was the tagged ampar normally distributed2
Was the tagged AMPAR normally distributed?
  • Intracellular; not extracellular
  • Inserts in membrane
visualizing ampar recap
Visualizing AMPAR Recap
  • How was the AMPAR tagged?
      • GFP to GluR1
  • Was it functional?
      • Rectifying/endogenous current indicative of homo/hetero oligomerization
      • Delivered to the surface
      • LTP
  • Was the tagged AMPAR normally distributed?
      • Colocalized with GluR2 and synapse region
      • Intracellular; not extracellular
      • Some on membrane
slide11

Do AMPARs redistribute

to synapses due to tetanus?

  • GluR1-GFP inserted into dendritic spines
    • In to “active” and “empty”
    • Density of “active” spines increased
  • GluR1-GFP clustered throughout dendrite
    • NMDAR dependent
    • Clustering linked to more surface GluR1-GFP
glur1 gfp insertion into spines
GluR1-GFP Insertion into Spines

(a) “Empty” spine

(b) “Active” spine

glur1 gfp insertion into spines1
GluR1-GFP Insertion into Spines
  • Fluorescence increased
    • “Active” spines

1023 ± 101 AU → 2210 ± 245 AU

    • “Empty” spines

200 ± 43 AU → 1737 ± 235 AU

      • Likely not generated after tetanic stimulation (length)
      • Density of spines increased
clustering throughout dendrite
Clustering throughout dendrite
  • R50% is brightness decay vs. distance
  • Smaller radius indicative of clustering
clustering linked to more surface glur1 gfp
Clustering linked to more surface GluR1-GFP
  • Smaller R(%) ratio means greater clustering
  • Surface Fraction =

Surface GluR1-GFP

Intracellular GluR1-GFP

More surface AMPAR

  • Unaffected synapses did not show more surface AMPAR

More clustering

slide16
NMDAR

dependent?

nmdar dependent1
NMDAR Dependent
  • APV blocks clustering
  • APV prevents increased fluorescence
slide19

RECAP

Do AMPARs redistribute

to synapses due to tetanus?

slide21

Do AMPARs redistribute

to synapses due to tetanus?

  • AMPAR redistribution with tetanus
    • Delivered to PSD
    • Clustering in dendritic compartment
    • Density of spines increased
  • AMPAR clustering blocked with APV
    • Thus, NMDAR Dependent
future experiments
Future experiments
  • What is the pathway that causes insertion and clustering?
    • Calcium buffers of different intensities
    • CaMKII – constant activation
    • Knock-out regions likely to interact with proteins
  • Are GluR2-4 trafficked as well?
    • Label each subunit to track trafficking and insertion
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