Dynamics and Homeostasis of Glycinergic Synapses

描述（由申请人提供）：突触可塑性在神经回路的功能中起着重要作用。然而，如果增强机制加强有效的突触，那么正反馈可能会导致整个输入的响应增益饱和。如果没有稳态机制来降低突触强度，同时保持平衡的兴奋和抑制，神经网络将停止运作。此外，突触稳态可能在兴奋和抑制平衡改变的疾病中起病因作用，例如自闭症和癫痫。斑马鱼模型提供了一个无与伦比的机会，询问神经元是否在体内进行重新缩放，因为它的光学方法的透明度和丰富的分子遗传和生理工具可用。在这个建议中，我检查体内抑制性甘氨酸能突触蛋白的稳态，并测试神经元活动，昼夜节律和行为睡眠/觉醒状态在突触重新缩放调节中的可能作用。甘氨酸在脊髓中起主要的抑制作用，很少有研究涉及甘氨酸能突触的稳态调节。目的1旨在确定脊髓运动神经元如何改变甘氨酸能突触蛋白在白天和晚上的积累和清除的相对速率。这些速率将在表达与甘氨酸能蛋白融合的可转化蛋白质dendra的脊髓运动神经元中通过量化白天和夜晚期间转化蛋白质的去除速率和非转化蛋白质的积累来测量。目标2试图从生理学上测试甘氨酸能突触强度在白天和黑夜之间的变化。CatCH-eYFP通道视紫红质变体将在脊髓中的一类甘氨酸能中间神经元中特异性表达，并用于直接测量突触后运动神经元中的突触后抑制电流的平均振幅。目标3将确定前两个目标中观察到的调节模式是否通过昼夜节律、睡眠/觉醒状态或神经元活动进行调节。为了确定昼夜节律是否调节突触重新缩放，将在白天和晚上睡眠剥夺期间测量dendra标记的突触蛋白的动力学（如在Aim 1中）。与先前目标中观察到的调节模式相同的调节模式将表明昼夜节律起调节作用，因为神经元活动和清醒状态都已被睡眠剥夺改变。如果不是这种情况，神经元活动的作用将通过扰动单个运动神经元的活动和测量突触动力学来测试。内向整流钾通道将在运动神经元中表达以降低活性，并且CatCH构建体表达以增加活性。缺乏活动的影响可能表明其他睡眠相关变化在该过程中的作用，例如神经调节水平的变化。这些实验将提供有关甘氨酸能突触中作为行为状态的函数的重新缩放的数据，并增加我们对重新缩放机制如何有助于活体动物神经回路的功能和稳定性的理解。

DESCRIPTION (provided by applicant): Synaptic plasticity plays a fundamental role in the function of neural circuits. However, if potentiation mechanisms reinforce effective synapses, then positive feedback could lead to a saturation of response gain across inputs. Without homeostatic mechanisms to down-scale synaptic strengths while maintaining balanced excitation and inhibition, neural networks would cease functioning. Additionally, synaptic homeostasis may play an etiological role in disorders where the balance of excitation and inhibition is altered, such as in autism and epilepsy. The zebrafish model offers an unparalleled opportunity to ask whether neurons perform re- scaling in vivo because of its transparency for optical approaches and the wealth of molecular-genetic and physiological tools available. In this proposal, I examine homeostasis of inhibitory glycinergic synaptic proteins in vivo and test the possible roles of neuronal activity, circadian rhythms, and behavioral sleep/wake states in the regulation of synaptic re-scaling. Glycine plays the dominant inhibitory role in the spinal cord and few studies have addressed homeostatic regulation of glycinergic synapses. Aim 1 seeks to determine how spinal cord motoneurons alter the relative rates of accumulation and removal of synaptic proteins from glycinergic synapses during the day and night. These rates will be measured in spinal motoneurons expressing the convertible protein dendra fused with glycinergic proteins by quantifying the rate of removal of converted protein and accumulation of non-converted protein during day and night. Aim 2 seeks to physiologically test changes in the strength of glycinergic synapses between day and night. The CatCH-eYFP channelrhodopsin variant will be expressed specifically in a class of glycinergic interneurons in the spinal cord an used to directly measure the mean amplitude of postsynaptic inhibitory currents in postsynaptic motoneurons. Aim 3 will determine whether the patterns of regulation observed in the first two Aims are regulated through circadian rhythms, sleep/wake states, or neuronal activity. In order to determine if circadian rhythms regulate synaptic re-scaling, the dynamics of dendra-tagged synaptic proteins will be measured (as in Aim 1) during the day and during sleep deprivation at night. A pattern of regulation identical to that observed in the previous Aims, would suggest that circadian rhythms play a regulatory role, because both neuronal activity and wake state have been altered by sleep deprivation. If this is not the case, the role of neuronal activity will be tested by perturbing the activity of single motoneurons and measuring synaptic dynamics. An inward rectifying potassium channel will be expressed in motoneurons to reduce activity and the CatCH construct expressed to increase activity. A lack of effect of activity would suggest a possible role for other sleep related changes in the process, such as changes in neuromodulatory levels. These experiments will provide data on re-scaling in the glycinergic synapse as a function of behavioral state and increase our understanding of how re-scaling mechanisms contribute to the function and stability of neural circuits in a living animal.