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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. Song-Hai Shi, Yasunori Hayashi, Ronald S. Petralia, Shahid H. Zaman, Robert J. Wenthold, Karel Svoboda, Roberto Malinow 11 June 1999. Group 4.

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

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  1. Rapid Spine Delivery and Redistribution of AMPA Receptors After Synaptic NMDA Receptor Activation Song-Hai Shi, Yasunori Hayashi, Ronald S. Petralia, Shahid H. Zaman, Robert J. Wenthold, Karel Svoboda, Roberto Malinow 11 June 1999

  2. Group 4 • Nickisa Hodgson, Ben Kelley, Pablo Inzunza, My Hanh Huynh, Aria Jafari, Riley Landreth, Francis Hwee, Jessica Hoffman, Teresa Kim, David Kee, Anna Karstens, Amanda Hodge, Lindsay King, Wen-Hsin Jiang

  3. Abstract • Tetanus induces two changes: • Delivery of GluR1 to spines • Clustering of GluR1 in the dendritic shaft. • Postsynaptic trafficking requires NMDA receptor activation

  4. What was known: • Excitatory synaptic transmission is mediated by AMPA and NMDA-glutamate receptors • Repetitive synaptic activity activates NMDAR and triggers LTP, expressed as an increase in AMPAR function

  5. What was not known: • Molecular basis for activity-induced changes in AMPAR function • Possible reasons: • Changes in channel conductance • Delivery of AMPAR to synapses • Hypothesis: Increase in number of AMPAR at synapses may occur rapidly during NMDAR dependent synaptic plasticity

  6. First Control: Kidney (HEK)293 cells show that GluR1-GFP is functional

  7. HEK Transfection • First tagged GluR1 at the N-terminus with GFP • Plasmid-based mammalian expression vector with lipofectin to transfect GluR1-GFP • Immunoblot to verify that GluR1-GFP is expressed • Advantages to HEK (Human Embryonic Kidney) cells • Easy to culture and transfect • HEK cells would only display transfected channel electrophysiology

  8. Viral Infection of Neurons • Introduce GluR1-GFP into neurons via Sindbis Viral Expression System • Follows characteristic viral life cycle to insert DNA into targeted cell • High efficiency • Following incorporation, neurons were observed to have normal passive membrane properties

  9. Immunostaining • Fix cells with Paraformaldehyde (PFA) in Phosphate Buffered Solution (PBS) • Allows for detection of surface epitopes • Treat with Triton-X in PBS • Allows for detection of intracellular epitopes • Follow with blocking solution, primary, and secondary antibody, conjugated with fluorescent particle or gold • Immunostaining also detects colocalization • GFP and red flourescence overlay and diplay a yellow signal

  10. Second Control: Expression of GluR1-GFP in dissociated neurons is targeted to synapses.

  11. GluR1 Expression in organotypic hippocampal slice culture is primarily intracellular

  12. Experimental (GluR1-GFP) 88% Dendritic Shaft (a) 9% Dendritic Shaft Surface (b) 2% Spines (c) 0.4% PSD (d) Control (Endogenous GluR1) 71% Dendritic Shaft (a) 20% Dendritic Shaft Surface (b) 8% Spines (c) 3% PSD (d) AMPA Receptor Distribution

  13. Changes in AMPAR distribution:spine delivery • Empty Spines: • Before: 200 AU • After tetanus: 1737 AU • Active Spines: • Before: 1023 AU • After tetanus: 2210 AU

  14. Changes in AMPAR distribution:clustering in dendritic shaft

  15. NMDAR activation required for redistribution of AMPAR

  16. What has been proven? • GluR1-GFP is functional • Before tetanus, GluR1-GFP is localized in the dendritic tree • After tetanus, GluR1-GFP clustering in dendritic shaft and delivery to spine are observed • Spine delivery and clustering of tagged AMPA requires NMDA activation • Data suggests redistribution is involved in the increase in synaptic transmission • There is link between receptor recruitment and activity-induced forms of plasticity • Clusters may represent a structural modification serving as a long-lasting memory mechanism

  17. First control Demonstrate GluR1-GFP is functional

  18. Second Control GluR1-GFP expressed at synapses and dendritic tree

  19. GluR1 Expression in organotypic hippocampal slice culture is primarily intracellular

  20. Changes in AMPAR distribution:spine delivery • Empty Spines: • Before: 200 AU • After tetanus: 1737 AU • Active Spines: • Before: 1023 AU • After tetanus: 2210 AU

  21. Changes in AMPAR distribution:clustering in dendritic shaft

  22. NMDAR activation required for redistribution of AMPAR

  23. Critique and Further Experiments • Demonstrate AMPAR insertion into the membrane. • More electrophysiological experiments to support hypothesis • Use a Universal GFP tag for all GluR subunits (GluR1-GluR4) • Experiment did not rule out possibility of an increase in AMPAR conductance

  24. Any Questions? Thank you!

  25. GluR1 Delivery to Spines Clustering of GluR1 in dendritic shaft

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