Investigating CNS synaptogenesis at single-synapse resolution by combining reverse genetics with correlative light and electron microscopy
| Autor: | Olivier Urwyler, Derya Ayaz, Pieter Baatsen, Saskia Lippens, Milan Petrovic, Christopher J. Guérin, Azadeh Izadifar, Anna Kremer, Dietmar Schmucker, Dan Dascenco, Haihuai He |
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| Rok vydání: | 2015 |
| Předmět: |
Central Nervous System
Cell type Synaptogenesis Biology Immunohistochemistry Synaptic vesicle Reverse Genetics Sensory neuron Cell biology Synapse Imaging Three-Dimensional medicine.anatomical_structure Synapses Organelle Microscopy Electron Scanning medicine Ultrastructure Animals Drosophila RNA Interference Cytoskeleton Mechanoreceptors Molecular Biology Developmental Biology |
| Zdroj: | Development. |
| ISSN: | 1477-9129 0950-1991 |
| Popis: | Determining direct synaptic connections of specific neurons in the central nervous system (CNS) is a major technical challenge in neuroscience. As a corollary, molecular pathways controlling developmental synaptogenesis in vivo remain difficult to address. Here, we present genetic tools for efficient and versatile labeling of organelles, cytoskeletal components and proteins at single-neuron and single-synapse resolution in Drosophila mechanosensory (ms) neurons. We extended the imaging analysis to the ultrastructural level by developing a protocol for correlative light and 3D electron microscopy (3D CLEM). We show that in ms neurons, synaptic puncta revealed by genetically encoded markers serve as a reliable indicator of individual active zones. Block-face scanning electron microscopy analysis of ms axons revealed T-bar-shaped dense bodies and other characteristic ultrastructural features of CNS synapses. For a mechanistic analysis, we directly combined the single-neuron labeling approach with cell-specific gene disruption techniques. In proof-of-principle experiments we found evidence for a highly similar requirement for the scaffolding molecule Liprin-α and its interactors Lar and DSyd-1 (RhoGAP100F) in synaptic vesicle recruitment. This suggests that these important synapse regulators might serve a shared role at presynaptic sites within the CNS. In principle, our CLEM approach is broadly applicable to the developmental and ultrastructural analysis of any cell type that can be targeted with genetically encoded markers. |
| Databáze: | OpenAIRE |
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