Role of Rhythmic Oscillations in Neuronal Plasticity

大脑中不同类型的节律振荡与睡眠和清醒的特定阶段有关，也与觉醒的程度有关。据推测，这些节律中的一些可能是获取和巩固记忆所必需的，并影响精神状态，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。有多个神经元群参与节律放电的产生和维持。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剂。过去，人们通过免疫毒素清除胆碱能神经元来研究胆碱能神经元的功能作用；然而，这种神经元的不可逆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，可逆地灭活小鼠大脑中的胆碱能神经元。这种毒素不杀死神经元，但通过切断突触短缩蛋白来阻止神经递质的分泌，而突触短缩蛋白是突触囊泡对接所必需的。一旦毒素的表达被关闭，神经元就会恢复它们的功能。我们将利用系统的可逆性来测试节律振荡在记忆形成、巩固和检索的不同阶段的作用。罗格斯大学的Buzsaki博士将进行神经元活动的体内记录和分析。

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.