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
Rapid Spine Delivery and Redistribution of AMPA Receptors After Synaptic NMDA
Receptor ActivationSong-Hai Shi, Yasunori Hayashi, Ronald S. Petralia, Shahid H. Zaman,
Robert J. Wenthold, Karel Svoboda, Roberto Malinow11 June 1999
Song-Hai Shi, Yasunori Hayashi, Ronald S. Petralia, Shahid H. Zaman, Robert J. Wenthold, Karel Svoboda, Roberto Malinow
11 June 1999
Group 4Group 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
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
AbstractAbstract
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: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: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 First Control:
Kidney (HEK)293 cells show that GluR1-GFP is functional
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HEK TransfectionHEK 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 NeuronsViral 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
ImmunostainingImmunostaining
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.
Second Control: Expression of GluR1-GFP in dissociated neurons is
targeted to synapses.
GluR1 Expression in organotypic hippocampal slice culture is primarily intracellular
GluR1 Expression in organotypic hippocampal slice culture is primarily intracellular
AMPA Receptor DistributionAMPA 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)
Control (Endogenous GluR1) 71% Dendritic Shaft
(a) 20% Dendritic Shaft
Surface (b) 8% Spines (c) 3% PSD (d)
Changes in AMPAR distribution:spine delivery
Changes in AMPAR distribution:spine delivery
Empty Spines: Before:
200 AU After tetanus:
1737 AU
Active Spines: Before:
1023 AU After tetanus:
2210 AU
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
Changes in AMPAR distribution:clustering in dendritic shaft
NMDAR activation required for redistribution of AMPAR
NMDAR activation required for redistribution of AMPAR
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What has been proven?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 First control
Demonstrate GluR1-GFP is functional
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Second Control GluR1-GFP expressed at synapses and dendritic tree
Second Control GluR1-GFP expressed at synapses and dendritic tree
GluR1 Expression in organotypic hippocampal slice culture is primarily intracellular
GluR1 Expression in organotypic hippocampal slice culture is primarily intracellular
Changes in AMPAR distribution:spine delivery
Changes in AMPAR distribution:spine delivery
Empty Spines: Before:
200 AU After tetanus:
1737 AU
Active Spines: Before:
1023 AU After tetanus:
2210 AU
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
Changes in AMPAR distribution:clustering in dendritic shaft
NMDAR activation required for redistribution of AMPAR
NMDAR activation required for redistribution of AMPAR
Critique and Further ExperimentsCritique 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 Spines Clustering of GluR1 in dendritic shaft
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