Presynapse Formation Assay Using Presynapse Organizer Beads and “Neuron Ball” Culture
| Autor: | Yukio Sasaki, Renoma Takeda, Shumaia Parvin |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
General Immunology and Microbiology biology Chemistry General Chemical Engineering General Neuroscience Vesicular glutamate transporter 1 Synaptic vesicle General Biochemistry Genetics and Molecular Biology Presynapse Cell biology 03 medical and health sciences 030104 developmental biology 0302 clinical medicine medicine.anatomical_structure Excitatory synapse medicine Excitatory postsynaptic potential Biological neural network biology.protein Neuron Active zone 030217 neurology & neurosurgery |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| Popis: | During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in appropriate order by transport from cell bodies and/or local translation in immature synapses. However, it is not fully understood how synaptic proteins accumulate in synapses in proper order. Here, we present a novel method to analyze presynaptic formation by using the combination of neuron ball culture with beads to induce presynapse formation. Neuron balls that is neuronal cell aggregates provide axonal sheets far from cell bodies and dendrites, so that weak fluorescent signals of presynapses can be detected by avoiding overwhelming signals of cell bodies. As beads to trigger presynapse formation, we use beads conjugated with leucine-rich repeat transmembrane neuronal 2 (LRRTM2), an excitatory presynaptic organizer. Using this method, we demonstrated that vesicular glutamate transporter 1 (vGlut1), a synaptic vesicle protein, accumulated in presynapses faster than Munc18-1, an active zone protein. Munc18-1 accumulated translation-dependently in presynapse even after removing cell bodies. This finding indicates the Munc18-1 accumulation by local translation in axons, not transport from cell bodies. In conclusion, this method is suitable to analyze accumulation of synaptic proteins in presynapses and source of synaptic proteins. As neuron ball culture is simple and it is not necessary to use special apparatus, this method could be applicable to other experimental platforms. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|