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

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? After Synaptic NMDA Receptor Activation

  • 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 After Synaptic NMDA Receptor Activation

  • Tag with Green Fluorescent Protein

    • Functional?

    • Distributed normally?


How was the ampar tagged
How was the AMPAR tagged? After Synaptic NMDA Receptor Activation

  • Viral infection with recombinant GluR1-GFP

  • Plain GFP fluoresces internally

NonpermeabilizedPermeabilized


Was the tagged ampar functional hek 293 cells
Was the tagged AMPAR functional? After Synaptic NMDA Receptor ActivationHEK 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? After Synaptic NMDA Receptor ActivationCA1 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? After Synaptic NMDA Receptor Activation

  • Present in synaptic region

    • Colocalized with presynaptic marker (synapsin 1)and GluR2


Was the tagged ampar normally distributed1
Was the tagged AMPAR normally distributed? After Synaptic NMDA Receptor Activation

  • 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? After Synaptic NMDA Receptor Activation

  • Intracellular; not extracellular

  • Inserts in membrane


Visualizing ampar recap
Visualizing AMPAR After Synaptic NMDA Receptor ActivationRecap

  • 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 After Synaptic NMDA Receptor Activation

    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 After Synaptic NMDA Receptor Activation

    (a) “Empty” spine

    (b) “Active” spine


    Glur1 gfp insertion into spines1
    GluR1-GFP Insertion into Spines After Synaptic NMDA Receptor Activation

    • 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 After Synaptic NMDA Receptor Activation

    • R50% is brightness decay vs. distance

    • Smaller radius indicative of clustering


    Clustering linked to more surface glur1 gfp
    Clustering linked to more After Synaptic NMDA Receptor Activationsurface 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 After Synaptic NMDA Receptor Activation

    dependent?


    Nmdar dependent
    NMDAR Dependent After Synaptic NMDA Receptor Activation


    Nmdar dependent1
    NMDAR Dependent After Synaptic NMDA Receptor Activation

    • APV blocks clustering

    • APV prevents increased fluorescence


    RECAP After Synaptic NMDA Receptor Activation

    Do AMPARs redistribute

    to synapses due to tetanus?


    Yes! After Synaptic NMDA Receptor Activation


    Do AMPARs redistribute After Synaptic NMDA Receptor Activation

    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 After Synaptic NMDA Receptor Activation

    • 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


    Thank you for enduring us on sun god
    Thank you for enduring After Synaptic NMDA Receptor Activationus on Sun God!


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