Dynamics and Homeostasis of Glycinergic Synapses

描述(申请人提供)：突触可塑性在神经回路的功能中起着基本的作用。然而，如果增强机制加强了有效的突触，那么正反馈可能会导致跨输入的反应增益饱和。如果没有动态平衡机制来降低突触强度，同时保持兴奋和抑制的平衡，神经网络就会停止运作。此外，突触内稳态可能在兴奋和抑制平衡改变的疾病中发挥病因学作用，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来研究神经元是否在体内进行重新缩放，因为它对光学方法以及丰富的分子遗传学和生理工具可用。在这项建议中，我在体内研究了抑制性甘氨酸能突触蛋白的动态平衡，并测试了神经元活动、昼夜节律和行为睡眠/清醒状态在调节突触重新调节中的可能作用。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动态平衡调节。目的1试图确定脊髓运动神经元如何改变白天和夜间甘氨酸能突触中突触蛋白的积累和移除的相对速度。这些速率将在表达可转换蛋白Dendra与甘氨酸能蛋白融合的脊髓运动神经元中通过量化白天和夜间转换蛋白的移除和非转换蛋白的积累来测量。目的2试图从生理学角度测试甘氨酸能突触在白天和夜间的强度变化。CATCH-EYFP通道视紫红质变异体将在脊髓中的一类甘氨酸能中间神经元中特异表达，并用于直接测量突触后运动神经元的突触后抑制电流的平均幅度。目标3将确定在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来调节。为了确定昼夜节律是否调节突触的重新缩放，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态(如目标1所示)。与之前观察到的相同的调节模式表明，昼夜节律起着调节作用，因为睡眠剥夺改变了神经元的活动和觉醒状态。如果情况并非如此，将通过干扰单个运动神经元的活动和测量突触动力学来测试神经元活动的作用。内向整流钾通道将在运动神经元中表达以降低活性，而CATCH结构将表达以增强活性。缺乏活动的影响表明，在这个过程中，其他与睡眠相关的变化可能也起到了作用，比如神经调节水平的变化。这些实验将提供关于甘氨酸能突触作为行为状态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.