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

1. 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

2. 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?

3. Visualizing AMPAR • Tag with Green Fluorescent Protein • Functional? • Distributed normally?

4. How was the AMPAR tagged? • Viral infection with recombinant GluR1-GFP • Plain GFP fluoresces internally NonpermeabilizedPermeabilized

5. Was the tagged AMPAR functional?HEK 293 Cells • Infected cells rectified nominally • GluR2 co-transfected cells did not rectify • Suggesting hetero and homo-oligomerization

6. 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

7. Was the tagged AMPAR normally distributed? • Present in synaptic region • Colocalized with presynaptic marker (synapsin 1)and GluR2

8. 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

9. Was the tagged AMPAR normally distributed? • Intracellular; not extracellular • Inserts in membrane

10. 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

11. 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

12. GluR1-GFP Insertion into Spines (a) “Empty” spine (b) “Active” spine

13. 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

14. Clustering throughout dendrite • R50% is brightness decay vs. distance • Smaller radius indicative of clustering

15. 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

16. NMDAR dependent?

17. NMDAR Dependent

18. NMDAR Dependent • APV blocks clustering • APV prevents increased fluorescence

19. RECAP Do AMPARs redistribute to synapses due to tetanus?

20. Yes!

21. 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

22. 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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