Role of Rhythmic Oscillations in Neuronal Plasticity

节律振荡在神经元可塑性中的作用

• 批准号: 6982744
• 负责人: Alexei Morozov
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6982744
• 关键词: acetylcholine; brain electrical activity; cholinergic receptors; cognition; drug withdrawal; gene targeting; genetically modified animals; laboratory mouse; molecular cloning; neural plasticity; neural transmission; neurons; neuroregulation; neurotransmitter antagonist; tetanus toxin; tetracyclines; tissue /cell culture

Various types of rhythmic oscillations in the brain are associated with specific stages of sleep and wakefulness and also correlate with degree of arousal. It is hypothesized that some of those rhythms may be required for the acquisition and consolidation of memories and affect mental state, however the direct proofs of this hypothesis are still absent. One way to test the role of these oscillations is to interfere with the function of neurons producing those oscillations. There are multiple neuronal populations involved in generation and maintenance of rhythmic firing. Among these groups, cholinergic neurons are considered the key modulators of the oscillatory activities. In the past, the functional role of cholinergic neurons has been studied by the elimination of these neurons with immunotoxins, however this irreversible elimination of neurons brings about irreversible changes compromising interpretation of behavioral experiments. To directly test role of oscillations in learning, memory and mood, we will reversibly inactivate cholinergic neurons in the mouse brain using regulated expression of the light chain of tetanus toxin. This toxin does not kill neurons, but prevent secretion of neurotransmitter by cleaving synaptobrevin, which is required for docking of synaptic vesicles. Once the expression of the toxin is turned off, neurons should recover their functions. We will test the role of rhythmic oscillations at different stages of memory formation, consolidation and retrieval taking advantage of the reversibility of the system. In vivo recording and analysis of neuronal activity will be performed by Dr. Buzsaki at Rutgers University. During the past fiscal year we have completed the design of the scheme for reversible genetic inactivation of cholinergic neurons. The scheme includes generation of 2 lines of genetically modified mice. The first line will express tetracycline transactivator in the cholinergic neurons. It will be produced by targeting cholinergic locus with the construct harboring a gene for tetracycline transactivator. The second line will carry modified inactive tetanus toxin, which could only be activated only in the brain following a withdrawal of doxycycline from mouse diet. We have completed cloning of the mouse cholinergic locus, generation of the first targeting construct for the expression of tetracycline transactivator (tTA) and creation of mice with the insertion of tTA into the cholinergic locus . Since the second construct harbors a modified tetanus toxin, it was necessary to verify that the planned modification introduced into the toxin does not interfere with its activity. To test the activity of modified toxin, we have constructed testing plasmids carrying the same modifications in the toxin structure, which will appear following its activation in the brain. We also had to clone a gene for synaptobrevin, a substrate for the toxin. We have completed functional testing of this modified toxin in cell culture confirming that it retains activity after modification. This confirmation allows us to proceed with making the second construct carrying the toxin. In collaboration with Dr. Buzsaki, we have characterized rhythmic activities in the brain of mouse strains used for gene targeting. Since most of recordings from rodent brains were done in rats, it was necessary to carry basic characterization of the mouse brain activities before recording from genetically modified animals.

大脑中各种类型的节律振荡与睡眠和清醒的特定阶段有关，也与唤醒程度有关。有人假设，其中一些节奏可能是获得和巩固记忆所必需的，并影响心理状态，但这一假设的直接证据仍然缺乏。测试这些振荡作用的一种方法是干扰产生这些振荡的神经元的功能。有多个神经元群体参与节律性放电的产生和维持。其中，胆碱能神经元被认为是振荡活动的关键调节器。在过去，胆碱能神经元的功能作用已经通过用免疫毒素消除这些神经元来研究，然而，这种不可逆的神经元消除带来了不可逆的变化，损害了行为实验的解释。为了直接测试振荡在学习、记忆和情绪中的作用，我们将使用破伤风毒素轻链的调节表达可逆地抑制小鼠脑中的胆碱能神经元。这种毒素不杀死神经元，但通过裂解突触泡蛋白来阻止神经递质的分泌，这是突触囊泡对接所必需的。一旦毒素的表达被关闭，神经元应该恢复它们的功能。我们将利用系统的可逆性来测试节律振荡在记忆形成、巩固和提取的不同阶段的作用。神经元活动的体内记录和分析将由罗格斯大学的Buzsaki博士进行。 在过去的财政年度，我们已经完成了可逆的遗传失活胆碱能神经元的方案的设计。该方案包括产生2系遗传修饰小鼠。第一条线将在胆碱能神经元中表达四环素反式激活因子。它将通过用含有四环素反式激活因子基因的构建体靶向胆碱能位点来产生。第二条线将携带经过修饰的无活性破伤风毒素，该毒素仅在小鼠饮食中停用强力霉素后才能在大脑中被激活。 我们已经完成了小鼠胆碱能基因座的克隆，产生的第一个靶向构建四环素反式激活因子（tTA）的表达和创建的小鼠与tTA插入到胆碱能基因座。 由于第二种构建体含有修饰的破伤风毒素，因此有必要验证引入毒素的计划修饰不会干扰其活性。为了测试修饰的毒素的活性，我们构建了在毒素结构中携带相同修饰的测试质粒，这些修饰将在其在脑中激活后出现。我们还必须克隆小突触泡蛋白的基因，它是毒素的底物。我们已经在细胞培养中完成了这种修饰毒素的功能测试，证实它在修饰后保留了活性。这一确认使我们能够继续制造携带毒素的第二个构建体。 在与Buzsaki博士的合作中，我们描述了用于基因靶向的小鼠品系大脑中的节律活动。由于大多数啮齿动物大脑的记录都是在大鼠身上进行的，因此在记录转基因动物之前，有必要对小鼠大脑活动进行基本表征。