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

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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


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

  • Tag with Green Fluorescent Protein

    • Functional?

    • Distributed normally?


How was the AMPAR tagged?

  • Viral infection with recombinant GluR1-GFP

  • Plain GFP fluoresces internally

NonpermeabilizedPermeabilized


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

  • Infection of CA1 neurons with GluR1-GFP

    • Infected cell

    • Uninfected cell

  • Greater rectification in infected

  • Displayed LTP


Was the tagged AMPAR normally distributed?

  • Present in synaptic region

    • Colocalized with presynaptic marker (synapsin 1)and GluR2


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 distributed?

  • Intracellular; not extracellular

  • Inserts in membrane


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


  • 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

    (a) “Empty” spine

    (b) “Active” spine


    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

    • R50% is brightness decay vs. distance

    • Smaller radius indicative of clustering


    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


    NMDAR

    dependent?


    NMDAR Dependent


    NMDAR Dependent

    • APV blocks clustering

    • APV prevents increased fluorescence


    RECAP

    Do AMPARs redistribute

    to synapses due to tetanus?


    Yes!


    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

    • 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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