ノルアドレナリンによる恐怖学習・記憶調節の神経回路および分子メカニズム

去甲肾上腺素调节恐惧学习记忆的神经回路及分子机制

• 批准号: 13F03209
• 负责人: JOHANSEN JOSHUA
• 金额: $ 1.47万
• 依托单位: The Institute of Physical and Chemical Research
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2013
• 资助国家: 日本
• 起止时间: 2013-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13F03209/
• 关键词: Fear; amygdala; CRTC1; noradrenaline; optogenetics; animal behavioral testing; fear learning & memory; synaptic plasticity; locus coeruleus; lateral amygdala

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.

项目 1：在这些实验中，我们用古视紫红质-T（ArchT，一种绿光激活的外向质子泵，用于抑制神经活动）病毒感染蓝斑 (LC) 中的去甲肾上腺素神经元，并在无条件刺激期间使用激光灭活外侧杏仁核 (LA) 中的 LC 神经元末梢。注射不同的腺病毒血清型以检查 LC 中的病毒表达，并且我们测试了不同的孵育时间以验证 LA 中的末端表达以进行光操作。只有血清型AAV9中的AAV/CAG-FLEX-GFP/ArchT载体能够强烈感染TH-Cre大鼠的LC神经元，并且最终表达需要至少8周的孵育。我们使用这种方法来光遗传学抑制蓝斑（LC）输入端子到外侧杏仁核（LA），这是恐惧记忆存储的部位。令人惊讶的是，这对恐惧条件反射没有影响，但 LA LC 末端视蛋白的表达较低，这可以解释这一结果。项目 2：我们检查了 LA 神经元中的 CRTC1 易位对于恐惧学习是否是必要的。为此，我们通过病毒过度表达了 CRTC1 的显性失活形式，这限制了 CRTC1 或短发夹 RNA (shRNA) 的转录活性，从而敲低 CRTC1，特别是在 LA 神经元中。我们发现，CRTC1 显性阴性形式的病毒敲除或过度表达会损害恐惧记忆巩固，但不会影响初始学习。这与我们之前显示 CRTC1 易位与恐惧学习的结果一致。