Atomic Force Microscopy as a tool to study synapse formation, axonal damage and repair
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Atomic Force Microscopy as a tool to study synapse formation, axonal damage and repair. Monserrat Lopez, Matt Rigby, Fernando Suarez, Peter Grutter Physics Department, McGill University Margaret H. Magdesian and David R. Colman (1949-2011) Montreal Neurological Inst. .

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Monserrat Lopez, Matt Rigby, Fernando Suarez, Peter Grutter

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Monserrat lopez matt rigby fernando suarez peter grutter

Atomic Force Microscopy as a tool to study synapse formation, axonal damage and repair

Monserrat Lopez, Matt Rigby, Fernando Suarez,

Peter Grutter

Physics Department, McGill University

Margaret H. Magdesian and

David R. Colman (1949-2011)

Montreal Neurological Inst.


Spm applied to nanoelectronics the grutter research group

Magnetic reversal

Molecular electronics

Quantum dots

Interfacing to living neurons

Biochemical sensors

MFM with in-situ field

UHV AFM/STM/FIM, AFM/STM/SEM

4K, 8T and 50mK, 16T AFM

AFM + SNOM + patch clamp + single photon fluorescence + TIRFM

Cantilevers and electrochemical AFM

SPM applied to nanoelectronics: the Grutter Research Group

www.physics.mcgill.ca/~peter


Monserrat lopez matt rigby fernando suarez peter grutter

axon

spine

Synapse formation is accompanied by change in mechanical properties

Left: Hippocampal neuron imaged with an AFM probe.

Right: Corresponding stiffness maps (bright is soft, dark is stiff).

Spines appeared soft relative to the dendrite shafts, where

stiff patches or fibers were identified (small arrows). Spine

shapes were irregular, often exhibiting small surface

protrusions (arrowheads). Axons were not observed in

close proximity to the soft spines.

Ben Smith et al.,

Biophys. J. 92, 1419 (2007)


Monserrat lopez matt rigby fernando suarez peter grutter

Studying Axonal Degeneration by AFM

  • Advantages

  • live imaging during gradual injury

  • precise control of injury parameters (positioning and force determinations in the sub-nanoscale)

Before

Compression

Recovery

Deformation

Increased deformation

Degeneration

Hippocampal DRG


Results

Results

Using the AFM as an imaging tool we can follow the morphological response of axons to injury.

Using the AFM in force spectroscopy mode to cause gradual damage to axons we can follow the response of different axonal components to injury. Hippocampal axons can support 100 ± 50 Pa for 10 minutes while DRG axons resist up to 500 ± 200 Pa for 30 minutes.

Axonal rupture - optical labeling of components allow determination of failure mechanism: axonal stiffness decrease due to disrupting microtubules and degeneration due to disruption of mitochondrial transport.

mitochondria

tip

Axon

Magdesian et al., 2012


Monserrat lopez matt rigby fernando suarez peter grutter

Forming a synapse with a functionalized bead attached to an AFM tip: controlling synapse location and time!

TEM and SEM indicate that structure of bead-induced synapse is identical to a natural synapse.

Attach bead to AFM tip!

Allows recruitment time of various (labelled) proteins to be quantified.

Allows extraction of functioning neuronal filaments!

Observation:

Rapid assembly of functional presynapticboutons triggered by adhesive contacts with Poly-D-Lysin coated beads attached to AFM tip (circle above).

Control: uncoated beads – no recruitment!

Stable synapes formed

Recruited!


Monserrat lopez matt rigby fernando suarez peter grutter

  • “Neurite” (S) formation observed upon pulling the PDL coated bead away from an axon. Neurons labeled with synaptophysin-GFP. We have observed “neurites” as long as 50um. These neurites contain tubulin,actin, bassoon and synaptophysin.

Using the AFM to repair axons

Images from Fernando S. Sanchez

unpublished


Monserrat lopez matt rigby fernando suarez peter grutter

Using the AFM to repair axons

Detector

Model

Laser

Cantilever

PDL-Bead

1

2

3

20 min. contact

Lift cantilever

20 min. contact

Lift cantilever


Monserrat lopez matt rigby fernando suarez peter grutter

Using the AFM to repair axons

2 3

2

1 2

1

20 min contact lift cantilever 20 min. contact lift cantilever

with axon 1 pulling “neurite” with axon 2

Problem: the neurite is not dettaching from the PDL-coated bead


Monserrat lopez matt rigby fernando suarez peter grutter

Using the AFM to repair axons

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