Atomic Force Microscopy as a tool to study

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

SPM applied to nanoelectronics: the Grutter Research Group • Magnetic reversal MFM with in-situ field • Molecular electronics UHV AFM/STM/FIM, AFM/STM/SEM 4 K, 8 T and 50 m. K, 16 T AFM + SNOM + patch clamp + single photon fluorescence + TIRFM • Quantum dots • Interfacing to living neurons • Biochemical sensors Cantilevers and electrochemical AFM www. physics. mcgill. ca/~peter

Synapse formation is accompanied by change in mechanical properties 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, 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)

Studying Axonal Degeneration by AFM Advantages Before • precise control of injury parameters (positioning and force determinations in the sub-nanoscale) Compression Hippocampal DRG • live imaging during gradual injury Recovery Deformation Increased deformation Degeneration

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

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! Recruited! Stable synapes formed 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!

Using the AFM to repair axons “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 50 um. These neurites contain tubulin, actin, bassoon and synaptophysin. Images from Fernando S. Sanchez unpublished

Using the AFM to repair axons Detector r se La Model Can tile ver PDL-Bead 1 20 min. contact Lift cantilever 2 3 20 min. contact Lift cantilever

Using the AFM to repair axons 1 2 2 2 3 1 20 min contact with axon 1 lift cantilever pulling “neurite” 20 min. contact with axon 2 Problem: the neurite is not dettaching from the PDL-coated bead lift cantilever

Using the AFM to repair axons 090626