Monserrat Lopez, Matt Rigby, Fernando Suarez, Peter Grutter

Monserrat Lopez, Matt Rigby, Fernando Suarez, Peter Grutter

Physics Department, McGill University

Margaret H. Magdesian andDavid R. Colman (1949-2011) Montreal Neurological Inst.

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

and repair

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

www.physics.mcgill.ca/~peter

Synapse formation is accompanied by change in mechanical properties

Ben Smith et al., Biophys. J. 92, 1419 (2007)

axon

spine

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, wherestiff patches or fibers were identified (small arrows). Spineshapes were irregular, often exhibiting small surfaceprotrusions (arrowheads). Axons were not observed inclose proximity to the soft spines.

Studying Axonal Degeneration by AFM

Before

Compression

Recovery

Deformation

Increased deformation

Degeneration

Hipp

ocam

pal

DRG

Advantages

•live imaging during gradual injury

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

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.

Magdesian et al., 2012

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.

tipmitochondria

Axon

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

quantified.2. Allows extraction of functioning neuronal filaments!

Observation: Rapid assembly of functional presynaptic boutons triggered by adhesive contacts with Poly-D-Lysin coated beads attached to AFM tip (circle above). Control: uncoated beads – no recruitment!

Recruited! Stable synapes formed

“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. Sanchezunpublished

1

2

3

Detector

Cantilever

Laser

PDL-Bead

20 min. contact

Lift cantilever

20 min. contact

Lift cantilever

Using the AFM to repair axons

Model

1

1 2 2 2 3

Using the AFM to repair axons

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

090626

Using the AFM to repair axons