Role of Rhythmic Oscillations in Neuronal Plasticity

大脑中不同类型的节律振荡与特定的睡眠和清醒阶段相关，也与唤醒程度相关。有人假设，其中一些节律可能是获得和巩固记忆所必需的，并影响精神状态，然而，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了节律性放电的产生和维持。在这些群体中，胆碱能神经元被认为是振荡活动的关键调节器。过去，胆碱能神经元的功能作用是通过免疫毒素消除胆碱能神经元来研究的，然而这种不可逆转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，我们将通过调节破伤风毒素轻链的表达来可逆地灭活小鼠大脑中的胆碱能神经元。这种毒素不会杀死神经元，但会通过分解突触短缩蛋白来阻止神经递质的分泌，突触短缩蛋白是突触小泡停靠所必需的。一旦毒素的表达被关闭，神经元应该会恢复它们的功能。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所起的作用。罗格斯大学的Buzsaki博士将对神经元活动进行活体记录和分析。 在过去的一年里，我们已经完成了可逆性胆碱能神经元基因失活方案的设计。该方案包括产生2个转基因小鼠品系。第一个品系将在胆碱能神经元中表达四环素反式激活因子。它将通过含有四环素反式激活剂基因的构建物靶向胆碱能位点而产生。第二条生产线将携带改良的非活性破伤风毒素，只有在多西环素从小鼠饮食中撤出后，这种毒素才能在大脑中被激活。 我们已经完成了小鼠胆碱能基因的克隆，产生了第一个表达四环素反式激活因子(TTA)的靶向构建体，并将TTA插入到胆碱能基因中创建了小鼠。 由于第二种结构含有一种改良型破伤风毒素，因此有必要验证计划中引入的改进型毒素不会干扰其活性。为了测试修改后的毒素的活性，我们构建了在毒素结构中携带相同修改的测试质粒，当它在大脑中激活后就会出现。我们还必须克隆Synaptobrevin的基因，它是毒素的底物。我们已经完成了该修饰毒素在细胞培养中的功能测试，证实其修饰后仍具有活性。这一确认使我们能够继续制造携带毒素的第二个结构。 在与Buzsaki博士的合作下，我们已经确定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，因此在记录来自转基因动物的记录之前，有必要对小鼠的大脑活动进行基本表征。

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.