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: 7312902
• 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/7312902
• 关键词: Role; Rhythmic; Oscillations; Neuronal; Plasticity

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 previous years, 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. During the last fiscal year we have completed a targeting construct for making mouse line expressing tetanus toxin under control of tet-O-driven promoter. The construct will be sent to NIMH knockout core facility for generating the mice.

大脑中各种类型的节奏振荡与睡眠和清醒的特定阶段有关，并且与唤醒程度相关。假设这些节奏可能是获得和巩固记忆并影响精神状态所必需的，但是这种假设的直接证明仍然没有。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。有许多神经元种群在节奏射击的产生和维持中涉及。在这些组中，胆碱能神经元被认为是振荡活动的关键调节剂。过去，通过消除了具有免疫毒素的这些神经元研究了胆碱能神经元的功能作用。但是，这种不可逆转的消除神经元带来了不可逆转的变化，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tanus毒素轻链的调节表达在小鼠脑中反抗胆碱能神经元。这种毒素不会杀死神经元，而是通过裂解突触纤维来防止神经递质的分泌，这是突触囊泡对接所必需的。一旦关闭毒素的表达，神经元就应恢复其功能。我们将利用系统的可逆性来测试节奏振荡在记忆形成，整合和检索不同阶段的作用。罗格斯大学的Buzsaki博士将对神经元活动进行体内记录和分析。 在过去的几年中，我们完成了胆碱能神经元可逆遗传失活方案的设计。该方案包括生成2行转基因的小鼠。第一行将在胆碱能神经元中表达四环素反式激活剂。它将通过靶向胆碱能基因座，其构建构建基因具有四环素反式激活剂的基因。第二行将携带改良的非活性破伤风毒素，仅在小鼠饮食中戒断强力霉素后，只能在大脑中激活。我们已经完成了小鼠胆碱能基因座的克隆，生成了四环素反式激活器（TTA）表达的第一个靶向构建体以及将TTA插入胆碱能基因座的小鼠的创建。由于第二构建体具有修饰的破伤风毒素，因此有必要验证引入毒素中的计划修饰不会干扰其活性。为了测试改性毒素的活性，我们已经构建了测试质粒在毒素结构中携带相同修饰的质粒，在大脑中激活后将出现相同的修饰。我们还必须克隆一个基因进行突触，这是毒素的底物。我们已经完成了这种修饰的毒素在细胞培养中的功能测试，证实了它在修饰后保留活性。 在上一个财政年度，我们完成了一个靶向构建体，以使小鼠线在控制Tet-O驱动启动子的控制下表达破伤风毒素。该结构将被发送到NIMH敲除核心设施以生成小鼠。