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Multi-user two-photon microscope facility for advanced neuron imaging in-vivo and in-vitro

English title Multi-user two-photon microscope facility for advanced neuron imaging in-vivo and in-vitro
Applicant Schneggenburger Ralf
Number 139219
Funding scheme R'EQUIP
Research institution Laboratoire de mécanismes synaptiques EPFL - SV - BMI - LSYM
Institution of higher education EPF Lausanne - EPFL
Main discipline Neurophysiology and Brain Research
Start/End 01.07.2012 - 30.06.2013
Approved amount 320'000.00
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Lay Summary (English)

Lead
Lay summary

In the brain, nerve cells exchange information relevant for sensory perception, movement control, and higher brain functions at highly specific contact sites, the synapses. During sensory perception and sensorimotor integration, complex networks of neurons are activated by the incoming sensory information, interact with each other at multiple layers of synapses, and eventually initiate motor action. However, the action of specific neurons in these circuits is only incompletely understood. In this project co-funded by the Swiss National Science Foundation, two-photon (2P) fluorescence excitation microscopes will be acquired, and further optimized for use in the Brain Mind Institute of the École Polytechnique Fédérale in Lausanne.

In a first project, the signalling pathways that are active during the growth of nerve terminals and during activity-dependent plasticity will be imaged. Nerve terminals of the axon of a sending neuron connect with the cell body or the dendrite of a receiving neuron, and establish synapses. However, our knowledge of the signalling pathways which specify the size and therefore the signalling strength of a nerve terminal is limited. Here, proteins of a candidate signalling pathway will be labelled with a donor and an acceptor fluorophore based on green fluorescent protein (GFP) variants, and genetically engineered into nerve terminals. Fluorescence resonance energy transfer between the pair of the donor and acceptor flourophores will then be imaged in a 2P-fluorescence lifetime imaging approach. This will allow us to visualize the activation of specific signalling pathways during the growth of nerve terminals, and during activity-dependent plasticity. The focus of these studies will be on the so-called Rho-GTPases, which are known to signal to the cytoskeleton and regulate cell shape changes in cells. Specifically, we will investigate whether Rho-GTPases are activated during presynaptic plasticity and downstream of extracellular signals that act on the growing nerve terminal.

In a second project, neuronal activity in the cortical and subcortical areas of living mice performing tasks of sensory-motor integration will be imaged in a 2P-microscope. The activity of nerve cells will be read-out in the form of Ca2+ signals, detected by Ca2+ sensing proteins genetically engineered into specific nerve cell populations. In order to maximize the observation depth into the light-scattering brain tissue, the 2P excitation and the detection of fluorescence emission will be optimized. Together, this new equipment acquired with the help of the Swiss National Science Foundation is expected to significantly enhance our knowledge about signalling pathways activated during synaptic plasticity and nerve terminal growth, and about the role of specific nerve cell populations during sensory processing and motor integration in the living mouse brain.

Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Collaboration

Group / person Country
Types of collaboration
Peter Scheiffele/Biozentrum der Universität Basel Switzerland (Europe)
- Exchange of personnel
Prof. Fritjof Helmchen/University of Zurich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Dr. Gergely Katona, Femtonics Hungary (Europe)
- Industry/business/other use-inspired collaboration
Prof. Ryohei Yasuda/Howard Hughes Medical Institute, Duke University Medical Center United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results

Associated projects

Number Title Start Funding scheme
127440 Transcriptional mechanisms of circuit formation and synapse specification 01.01.2010 Sinergia
131089 Synaptic Mechanisms of Sensory Perception and Associative Learning 01.04.2010 Project funding (Div. I-III)
122496 Molecular determinants of Ca2+-dependent transmitter release and its modulation by protein kinase C at the calyx of Held 01.10.2008 Project funding (Div. I-III)
127289 Structure, Function and Plasticity of the Barrel Cortex 01.01.2010 Sinergia

Abstract

Multiphoton imaging has provided fundamental new insights into brain function. Neural tissue is highly scattering and the inherent optical sectioning afforded by two-photon excitation provides the only current technique applicable to high resolution imaging of living brain tissue. Here, we seek funding to build new two-photon microscopes for advanced imaging applications in collaborative research projects at the Brain Mind Institute of the Ecole Polytechnique Federale de Lausanne (EPFL). One multiphoton setup will be dedicated to in vitro fluorescence lifetime imaging (FLIM) for measurement of intracellular signalling pathways using genetically-encoded fluorescence resonance energy transfer (FRET) probes. This approach promises to significantly further our understanding of signalling pathways activated during synaptic plasticity and synapse development and the dynamics of neuron-glia metabolic coupling. The other multiphoton setup will be used to develop technology for deep tissue in vivo calcium imaging at video rate. Neuronal and astrocytic calcium signals will be imaged throughout the depth of the neocortex and in the dorsal striatum by optimization of two-photon excitation; construction of microendoscopes; and improvements in fluorescence detection. These novel multiphoton microscope approaches will be used in collaborative projects involving several different laboratories of the Brain Mind Institute and the EPFL, significantly enhancing multi-disciplinary neuroscience research at the EPFL through collaborative development of an advanced multiphoton imaging platform.
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