Articles by Rumelo Amor in JoVE
In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal Adekunle T. Bademosi1, Elsa Lauwers2, Rumelo Amor3, Patrik Verstreken2, Bruno van Swinderen3, Frédéric A. Meunier1 1Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, 2VIB Centre for Brain and Disease Research, KU Leuven Department of Neurosciences, Leuven Institute for Neurodegenerative Disease (LIND), 3Queensland Brain Institute, The University of Queensland Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.
Other articles by Rumelo Amor on PubMed
Spontaneous Activity in the Zebrafish Tectum Reorganizes over Development and Is Influenced by Visual Experience Current Biology : CB. | Pubmed ID: 28781054 Spontaneous patterns of activity in the developing visual system may play an important role in shaping the brain for function. During the period 4-9 dpf (days post-fertilization), larval zebrafish learn to hunt prey, a behavior that is critically dependent on the optic tectum. However, how spontaneous activity develops in the tectum over this period and the effect of visual experience are unknown. Here we performed two-photon calcium imaging of GCaMP6s zebrafish larvae at all days from 4 to 9 dpf. Using recently developed graph theoretic techniques, we found significant changes in both single-cell and population activity characteristics over development. In particular, we identified days 5-6 as a critical moment in the reorganization of the underlying functional network. Altering visual experience early in development altered the statistics of tectal activity, and dark rearing also caused a long-lasting deficit in the ability to capture prey. Thus, tectal development is shaped by both intrinsic factors and visual experience.
Visualizing Endocytic Recycling and Trafficking in Live Neurons by Subdiffractional Tracking of Internalized Molecules Nature Protocols. | Pubmed ID: 29189775 Our understanding of endocytic pathway dynamics is restricted by the diffraction limit of light microscopy. Although super-resolution techniques can overcome this issue, highly crowded cellular environments, such as nerve terminals, can also dramatically limit the tracking of multiple endocytic vesicles such as synaptic vesicles (SVs), which in turn restricts the analytical dissection of their discrete diffusional and transport states. We recently introduced a pulse-chase technique for subdiffractional tracking of internalized molecules (sdTIM) that allows the visualization of fluorescently tagged molecules trapped in individual signaling endosomes and SVs in presynapses or axons with 30- to 50-nm localization precision. We originally developed this approach for tracking single molecules of botulinum neurotoxin type A, which undergoes activity-dependent internalization and retrograde transport in autophagosomes. This method was then adapted to localize the signaling endosomes containing cholera toxin subunit-B that undergo retrograde transport in axons and to track SVs in the crowded environment of hippocampal presynapses. We describe (i) the construction of a custom-made microfluidic device that enables control over neuronal orientation; (ii) the 3D printing of a perfusion system for sdTIM experiments performed on glass-bottom dishes; (iii) the dissection, culturing and transfection of hippocampal neurons in microfluidic devices; and (iv) guidance on how to perform the pulse-chase experiments and data analysis. In addition, we describe the use of single-molecule-tracking analytical tools to reveal the average and the heterogeneous single-molecule mobility behaviors. We also discuss alternative reagents and equipment that can, in principle, be used for sdTIM experiments and describe how to adapt sdTIM to image nanocluster formation and/or tubulation in early endosomes during sorting events. The procedures described in this protocol take ∼1 week.