| 研究実績の概要 |
Project 1: For these experiments we virally infected noradrenaline neurons in the locus coeruleus (LC) with archaerhodopsin-T (ArchT, a green light-activated outward proton pump used to inhibit neural activity) and used laser light to inactivate LC neuron terminals in the lateral amygdala (LA) during the unconditioned stimulus. Different adenoviral serotypes were injected to check for viral expression in the LC, and we tested various incubation times to verify terminal expression in the LA for light manipulation. Only the AAV/CAG-FLEX-GFP/ArchT vectors in serotype AAV9 infected LC neurons of TH-Cre rats robustly and at least 8 weeks of incubation was required for terminal expression. We used this approach to optogenetically inhibit locus coeruleus (LC) input terminals to the lateral amygdala (LA), a site of fear memory storage. Surprisingly this had no effect on fear conditioning, but expression of opsins in the LC terminals in LA was low and could explain this result.
Project 2: We examined whether CRTC1 translocation in LA neurons is necessary for fear learning. To do this we virally overexpressed a dominant negative form of CRTC1, which limits the transcriptional activity of CRTC1,or a short hairpin RNA (shRNA), to knockdown CRTC1, specifically in LA neurons. We found that viral knockdown or overexpression of a dominant negative form of CRTC1 impairs fear memory consolidation without affecting the initial learning. This was consistent with our previous results showing translocation of CRTC1 with fear learning.
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