2013 Fiscal Year Final Research Report

Imaging of presynaptic activity in cultured cerebellar granule neurons

Research Project

Project/Area Number	23700387
Research Category	Grant-in-Aid for Young Scientists (B)
Allocation Type	Multi-year Fund
Research Field	Neuroscience in general
Research Institution	Keio University
Principal Investigator	IBATA KEIJI 慶應義塾大学, 医学部, 助教 (30462659)
Project Period (FY)	2011 – 2013
Keywords	イメージング / 膜輸送
Research Abstract	To understand the cerebellar neuronal circuit, it is important to measure the presynaptic activities of granule cells when animals are moving or learning. In my research, I developed the reporter proteins for presynaptic activity using luciferase. Because luminescence of luciferase is product of chemical reaction, it does not need excitation light, which may sometimes hurt the cells or make autofluorescence from tissue and cell. Therefore using luminescence should give a better signal to noise ratio. I used Gaussia luciferase, which was pH sensitive. I performed the random mutagenesis to obtain bright luminescence. To visualize presynaptic activity, VAMP2 presynaptic protein was used. As a result, I obtained mutant Gluc (mGluc), which showed about 10 times brighter than wild type Gluc. The VAMP2 mGluc transfected neuron showed strong luminescence after neuronal depolarization, indicated this VAMP2-mGluc presynaptic reporter protein can be used to monitor presynaptic activity.

(3 results)