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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