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

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

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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

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Abstract

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

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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

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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

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

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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

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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

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Second Control Expression of GluR1-GFP in
dissociated neurons is targeted to synapses.
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GluR1 Expression in organotypic hippocampal slice
culture is primarily intracellular
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AMPA Receptor Distribution

• 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)

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Changes in AMPAR distributionspine delivery

• Empty Spines
• Before 200 AU
• After tetanus 1737 AU
• Active Spines
• Before 1023 AU
• After tetanus 2210 AU

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Changes in AMPAR distributionclustering in
dendritic shaft
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NMDAR activation required for redistribution of
AMPAR
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(No Transcript)
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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

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First control Demonstrate GluR1-GFP is
functional
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Second Control GluR1-GFP expressed at synapses
and dendritic tree
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GluR1 Expression in organotypic hippocampal slice
culture is primarily intracellular
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Changes in AMPAR distributionspine delivery

• Empty Spines
• Before 200 AU
• After tetanus 1737 AU
• Active Spines
• Before 1023 AU
• After tetanus 2210 AU

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Changes in AMPAR distributionclustering in
dendritic shaft
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NMDAR activation required for redistribution of
AMPAR
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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

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• Any Questions?
• Thank you!

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GluR1 Delivery to Spines
Clustering of GluR1 in dendritic shaft