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

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

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

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

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

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

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

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

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

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

GluR1 Expression in organotypic hippocampal slice culture is primarily intracellular

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)

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

Changes in AMPAR distribution: clustering in dendritic shaft

NMDAR activation required for redistribution of AMPAR

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

First control Demonstrate GluR1-GFP is functional

Second Control GluR1-GFP expressed at synapses and dendritic tree

GluR1 Expression in organotypic hippocampal slice culture is primarily intracellular

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

Changes in AMPAR distribution: clustering in dendritic shaft

NMDAR activation required for redistribution of AMPAR

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

Any Questions? Thank you! Any Questions? Thank you!

GluR1 Delivery to SpinesClustering of GluR1 in dendritic shaft