Monserrat Lopez, Matt Rigby, Fernando Suarez, Peter Grutter Physics Department, McGill University Margaret H. Magdesian and David R. Colman ( ) 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 MFM with in-situ field UHV AFM/STM/FIM, AFM/STM/SEM UHV AFM/STM/FIM, AFM/STM/SEM 4K, 8T and 50mK, 16T AFM 4K, 8T and 50mK, 16T AFM AFM + SNOM + patch clamp + single photon fluorescence + TIRFM AFM + SNOM + patch clamp + single photon fluorescence + TIRFM Cantilevers and electrochemical AFM Cantilevers and electrochemical AFM

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, 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.

Studying Axonal Degeneration by AFM Before Compression Recovery Deformation Increased deformation Degeneration Hippocampal 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. tip mitochondria Axon

Anna Lisa Lucido, Fernando Suarez Sanchez, Peter Thostrup, Adam V. Kwiatkowski, Sergio Leal-Ortiz, Gopakumar Gopalakrishnan, Dalinda Liazoghli, Wiam Belkaid, R. Bruce Lennox, Peter Grutter, Craig C. Garner and David R. Colman Rapid Assembly of Functional Presynaptic Boutons Triggered by Adhesive Contacts Journal of Neuroscience 29, (2009) 250 nm 1  m 250 nm SEM TEM

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. Sanchez unpublished

Detector Cantilever Laser PDL-Bead 20 min. contact Lift cantilever 20 min. contact Lift cantilever Using the AFM to repair axons Model

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

Using the AFM to repair axons