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 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  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  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  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  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  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  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. AMPA Receptor Distribution  Experimental (GluR1- GFP)  88% Dendritic Shaft (a)  9% Dendritic Shaft Surface (b)  2% Spines (c)  0.4% PSD (d)  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)

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

17. 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  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

18. First control Demonstrate GluR1-GFP is functional

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

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

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

22. Changes in AMPAR distribution: clustering in dendritic shaft

23. NMDAR activation required for redistribution of AMPAR

24. 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  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

25. Any Questions? Thank you! Any Questions? Thank you!

26. GluR1 Delivery to SpinesClustering of GluR1 in dendritic shaft