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Localization Microscopy in Neuroscience: Understanding Neuronal Structure to the Nano Level Symposium

Satellite Event at NEUROSCIENCE 2018, San Diego, CA

Symposium & Reception

Saturday, November 3rd

6:30 PM – 10 PM

Manchester Grand Hyatt, Harbor Ballroom ABC

Hosted bar & appetizers 

Program

"Activity-dependent plasticity of tripartite synapses monitored with super-resolution microscopy"

Dr. Janosch Heller
Institute of Neurology, University College London

Astrocytes play an active role in shaping and maintaining neuronal circuits through an intimate relationship with nearby synapses. Whilst the molecular signal exchange between astroglia and synapses occurs in a highly heterogeneous microenvironment on the nanoscale, the spatial subcellular distribution of the underlying molecular machineries remains poorly understood. We have therefore employed single molecule localisation microscopy to visualise the 3D positions of neurotransmitter receptors and transporters in astrocytes and neurons in fixed cultured cells and brain slices. Moreover, through multi-colour imaging, we assessed the positional relationship between synapses and astroglial receptors and transporters in different conditions compatible with long-term synaptic potentiation or depression.

 

"Unraveling spatial organization of synaptic protein with multiple color 3D STORM microscopy and quantitative distance analysis with Icy SODA plugin"
Dr. Lydia Danglot
INSERM

Elucidating molecular organization during synapse construction requires to precisely localize single or aggregated molecules and to analyze quantitatively their spatial distributions.We investigated the distribution of synaptic proteins with multi-color 3D STORM microcopy on primary hippocampal neurons and brain slices. To unravel fine distance and molecular arrangement we developed a new user friendly plugin called SODA for Statistical Object Distance Analysis. SODA uses micro- and nano-scopy to significantly improve standard colocalisation analysis and is freely available in ICY [1]. Based on Ripley’s function our method considers both the geometry of the cell and the densities of molecules to provide colored maps of isolated and statistically coupled molecules.

We statistically characterized spatial organization of thousands of synapses. As a proof of concept, we imaged presynaptic glutamatergic terminals with 3D-STORM microscopy and analysed the coupling between more than 180,000 localizations of vesicular Glutamate Transporter (VGLUT) and Synapsin molecules inside synaptic boutons. We evaluated with SODA that each Synapsin or VGLUT localization is at a mean distance of 52 ± 0,04 nm. These results demonstrate that SODA is a versatile and effective tool to statistically map large data sets of multi-color molecular assemblies with high spatial resolution.

We are now using new super-resolution membrane probes called MemBright [2] to investigate subcellular localization of synaptic proteins with a reference membrane probe. These probes are compatible with long-term live cell imaging and immunostaining. We will show how MemBright can be used to unravel plasma membrane structure at the nanoscale level since it can unravel the dendritic spines neck in 3D STORM. Moreover, we developed new protocole to preserve lipid membrane localization after gentle permeabilization. Using 3D multicolor dSTORM in combination with immunostaining we could thus revealed en-passant synapse displaying endogenous glutamate receptors clustered at the axonal-dendritic contact site.

 

"Live multi-color super-resolution microscopy" 
Dr. Erik Jorgensen
University of Utah, HHMI

In our continuing efforts to understand rapid membrane and protein dynamics at synapses we are developing live super-resolution techniques capable of distinguishing multiple fluorescent tags simultaneously in a living specimen. There are several problems posed by live super-resolution imaging: organelles must be densely labeled, dyes must be membrane permeable, the dyes must blink and have long off-times, and the dyes must also be bright enough for accurate localization. Two applications will be described: first, 3-color imaging of calcium channels at synapses in the nematode C. elegans, and second, organelle movement in hippocampal growth cones and at synapses. The goal will be to detect the movement of synaptic vesicle proteins during exocytosis and during ultrafast endocytosis.

When?

Saturday, November 3rd 

6:30 PM | Symposium talks commence. A series of 20 minute presentations with Q&A.

8:00 PM | Reception begins with light fare and open bar for socializing.

10:00 PM | Planned evening comes to close.

Where?

Manchester Grand Hyatt, Harbor Ballroom ABC

Count me in!

Please RSVP. Limited seating available.