Use of Optogenetics to Assess the Role of Climbing Fibers in Motor Learning

描述（由申请人提供）：候选人对大脑如何学习有长期的兴趣，并在她的博士论文中研究了鸣禽的鸣叫学习。候选人决定关注小鼠前庭-眼反射（VOR）中小脑依赖的运动学习，原因有三个：电路相对简单，学习容易量化，小鼠适合分子遗传操作。在这个奖项中，她将学习系统神经科学家研究回路功能的常用方法之一：刺激。此外，她将学习使用一种刺激神经元活动的新方法：光遗传学。光遗传学将使她能够对不同的小脑回路元素对清醒行为动物的VOR运动学习的具体贡献进行详细分析。这些分析以前是不可行的；因此，她将成为她研究领域的先驱。在这个提议中，她将验证长期存在的假设，即攀爬纤维为运动学习提供了关键的指导性信号。她的短期目标是获得终身教职。她的长期目标是将小脑回路中每个元素的功能映射到运动学习中。候选人将在斯坦福大学神经生物学系雷蒙德博士的实验室进行拟议的实验。Raymond博士是她的导师，是体内电生理记录方面的专家，他拥有实验室空间、设备和资源来支持候选人在获奖期间的研究。Karl deisserth博士是一位共同导师，他是光遗传学的先驱之一，也在斯坦福大学。他将为候选人提供提案中光遗传学部分的材料和建议。斯坦福大学的神经科学社区为智力刺激和讨论提供了许多场所，如研讨会、期刊俱乐部、全系月度会议、年度静修以及联合实验室会议。此外，雷蒙德博士每周举行实验室和个人会议。医学院和斯坦福社区提供各种课程和研讨会，以促进科学家的职业发展。任何熟练动作的执行都需要精确校准该动作的幅度和时间。这种运动学习在很大程度上依赖于小脑的正常功能。然而，小脑如何计算运动学习还没有得到很好的理解。向小脑输入的攀爬纤维被认为为运动学习提供了一个关键的指导信号。然而，攀爬纤维的活动并不是小脑中唯一可用的指示信号。本研究旨在确定在学习诱导过程中，攀爬纤维活动可以可靠地预测运动学习的哪些成分（即振幅、时间或两者都有），哪些成分可以被攀爬纤维活动诱导，以及攀爬纤维指导信号的性质。这些实验将在小鼠中进行，并将使用前庭-眼反射（VOR）中的运动学习作为学习范例。VOR是一种反射性眼球运动，稳定视网膜中的图像运动。为了实现本研究的目标，在运动学习的诱导过程中，将首先在体内记录攀爬纤维的活动，使用训练范式来诱导VOR的振幅、时间或两者的学习变化。攀爬纤维活动将与运动学习中不同成分的学习变化相关。其次，利用光遗传学的方法来激活和/或抑制攀爬纤维，模拟在每个训练模式中记录的攀爬纤维活动模式，以确定运动学习的哪些部分是由攀爬纤维充分驱动的。光遗传刺激将取代正常行为训练中通过攀爬纤维传递的驱动VOR运动学习的错误信号。最后，在体内同时记录和光遗传刺激攀爬纤维活动将提供攀爬纤维活动的不同特征与运动学习之间的因果关系。这项提议的亮点是利用光遗传学，或光活化蛋白的基因靶向表达，来操纵攀爬纤维的活性。与其他刺激方法不同的是，光遗传学可以在毫秒级的时间内精确和可靠地实现攀爬纤维激活的区域特异性。本研究的结果和对运动学习的见解将有助于指导小脑患者新疗法的合理设计。神经系统疾病的诊断是在行为水平，而大多数治疗方法是在分子水平。要弥合行为水平和分子水平之间的差距，需要了解连接这两个水平的神经回路功能。研究足以驱动运动学习的神经元活动的模式和特征将为运动功能障碍的治疗开辟新的途径。此外，由于小脑还参与其他功能，如认知（语言）、注意力（自闭症）和时间感知，因此本研究得出的小脑功能的基本原理可以推广到这些其他小脑功能。

DESCRIPTION (provided by applicant): The candidate has a longstanding interest in how the brain learns, and studied song learning in songbirds for her doctoral thesis. The candidate decided to focus on the cerebellum-dependent motor learning in the vestibulo-ocular reflex (VOR) in mice for three reasons: the circuit is relatively simple, learning is easily quantifiable, and mice lend themselves well to molecular-genetic manipulation. In this award, she will learn one of the general methods employed by systems neuroscientists to study circuit function: stimulation. Moreover, she will be learning to use a novel method of stimulating neuronal activity: optogenetics. Optogenetics will allow her to perform detailed analyses of the specific contributions of different cerebellar circuit elements to VOR motor learning in awake, behaving animals. These analyses were previously not feasible; she will thus be a pioneer in her field of research. In this proposal, she will test the long-standing hypothesis that climbing fibers provide a key instructive signal for motor learning. Her short-term goal is to obtain a tenure-track faculty position. Her long-term goal is to map the function of each element of the cerebellar circuit to motor learning. The candidate will be conducting the proposed experiments in Dr. Raymond's laboratory in the Dept. of Neurobiology at Stanford. Dr. Raymond is her Mentor, an expert in in vivo electrophysiological recordings, who has the laboratory space, equipment, and resources to support the candidate's research during the award. Dr. Karl Deisseroth, a co-Mentor, is one of the pioneers of optogenetics, and is also at Stanford. He will provide the candidate with materials and advice for the optogenetics portion of the proposal. The Neuroscience community at Stanford provides many venues for intellectual stimulation and discourse, such as seminars, journal clubs, department-wide monthly meetings, annual retreat, as well as joint lab meetings. In addition, Dr. Raymond holds weekly lab and individual meetings. The School of Medicine and the Stanford community offer various courses and workshops that enhance the career development of scientists. Research Project The execution of any skilled movement requires accurate calibration of the amplitude and timing of that movement. This motor learning is heavily dependent on the normal function of the cerebellum. However, how the cerebellum computes motor learning is not well understood. The climbing fiber input to the cerebellum has been thought to provide a key instructive signal for motor learning. Climbing fiber activity, however, is not the only available instructive signal in the cerebellum. This proposal aims to determine which components (i.e., amplitude, timing, or both) of motor learning can be reliably predicted by the climbing fiber activity during induction of learning, which components can be induced by climbing fiber activity, and the nature of the climbing fiber instructive signal. These experiments will be conducted in mice and will use motor learning in the vestibulo-ocular reflex (VOR) as the learning paradigm. VOR is a reflexive eye movement that stabilizes image motion in the retina. To achieve the goals in this proposal, the climbing fiber activity will first be recorded in vivo during induction of motor learning using training paradigms that induce learned changes in the amplitude, timing, or both of the VOR. The climbing fiber activity will be correlated with the learned changes in the different components of motor learning. Second, the climbing fibers will be activated and/or inhibited using optogenetics, mimicking the patterns of climbing fiber activity recorded during each training paradigm, to determine which components of motor learning are sufficiently driven by climbing fibers. The optogenetic stimulation will replace the error signal that drives VOR motor learning, which is transmitted via climbing fibers in normal behavioral training. Lastly, simultaneous recording and optogenetic stimulation of climbing fiber activity in vivo will provide a causal link between different features of climbing fiber activity and motor learning. The highlight of this proposal is the use of optogenetics, or genetically targeted expression of light-activated proteins, to manipulate climbing fiber activity. Unlike other methods of stimulation, optogenetics allows regional specificity of climbing fiber activation with good precision and reliability in a millisecond-time scale. The results and insights on motor learning that will be obtained from this proposal will help guide rational designs of new therapy for cerebellar patients. Neurological disorders are diagnosed at the behavioral level, while most of the therapies employed are at the molecular level. What is needed to bridge the gap between the behavioral and molecular levels is an understanding of the circuit function, which links both levels. Investigating the patterns and features of neuronal activity sufficient to drive motor learning will open new avenues of treatments for motor dysfunction. Moreover, because the cerebellum is involved in other functions such as cognition (language), attention (autism), and perception of timing, the underlying principles of cerebellar function derived from this study can be generalized to these other cerebellar functions. PUBLIC HEALTH RELEVANCE: Neurological disorders are often diagnosed at the behavioral level, while most therapies employed are at the molecular level. What is needed to bridge the gap between the behavioral and molecular levels is an understanding of the function of the circuit which links both levels. Investigating the patterns and features of neuronal activity sufficient to drive motor learning will open new avenues of treatments for motor dysfunction, which can be generalized to other cerebellar functions such as cognition (language), attention (autism), and perception of timing.