An optogenetic approach to exploring climbing fiber connections in the cerebellum

描述（由申请人提供）：小脑的实验性损伤和钝力创伤导致行为异常，表明该大脑区域在控制平稳协调运动和运动记忆方面起着重要作用（Fine, Ionita, & Lohr, 2002）。具体来说，研究人员认为小脑会评估意图和行动之间的差异，然后调整运动输出以纠正这些差异，从而产生期望的、平滑的运动行为。实验表明，这些纠正是由小脑皮层和深部核突触连接强度的动态变化引起的。此外，这些变化可能是由来自两个特定途径的信号的关联或一致检测驱动的，一个是通过苔藓纤维（MF，携带感觉信息），另一个是通过攀爬纤维（CF，表明运动命令中的差异或错误）。起源于下橄榄的攀爬纤维提供了一种独特而强大的输入，在小脑皮层的浦肯野细胞（PCs）的唯一输出中产生了一个“复杂的spike”。这种输入，当与平行纤维（PF；苔藓纤维中继）激活配对时，会减少pc的体感接受野（Jvrntell & Ekerot, 2002）。感受野的这种变化是由于PF-PC突触的一个子集的抑制，这种机制被认为是去除产生不良运动行为的感觉信号。类似的实验也表明，CFs驱动突触连接到pc的分子层抑制性中间神经元（MLI）的感受野的关联变化。然而，CF-MLI连接的性质尚不清楚，驱动联想可塑性的机制也不清楚，这种可塑性导致了接受野的变化。目前小脑知识的缺乏是由于无法在不激活其他神经元类型的邻近轴突的情况下可靠地刺激CFs。为了克服这一技术挑战，一种新的光遗传方法已经被开发出来，可以对分离的CFs进行强大的刺激。本研究的第一个目的是通过系统地探索强刺激CF所需的光刺激和病毒注射参数，进一步证实表达Channelrhodopsin 2的光刺激CF的可靠性和特异性的初步结果。使用这种技术，我建议描述CF- mli传输的性质以及控制CF驱动的平行光纤和mli之间的联想可塑性的机制和规则。这将通过选择性CF光刺激下急性切片MLIs的全细胞膜片钳记录来完成。这些实验将首次证明光遗传学技术在探索小脑皮层方面的有效性。最终，这些实验的结果将允许更好地预测小脑皮层如何评估和纠正意图和行动之间的差异。

DESCRIPTION (provided by applicant): Experimental lesions and blunt force traumas to the cerebellum result in behavioral abnormalities that indicate this brain region plays an important role in controlling smooth coordinated movement and motor memory (Fine, Ionita, & Lohr, 2002). Specifically, researchers believe the cerebellum evaluates the disparities between intention and action, and then adjusts the motor output to correct for these disparities in order to generate a desired, smooth-motor behavior. Experiments suggest these corrections arise from dynamic changes in the strength of synaptic connections in both the cerebellar cortex and deep nuclei. In addition, these changes are likely driven by the association or coincident detection of signals from two specific pathways, one by way of the mossy fibers (MF, carrying sensory information) and the other by way of the climbing fibers (CF, indicating a disparity or error in the motor command). Originating in the inferior olive the climbing fiber delivers a unique and powerful input that generates a "complex spike" in the sole output of the cerebellar cortex, the Purkinje cells (PCs). This input, when paired with parallel fiber (PF; mossy fiber relay) activation decreases the somatosensory receptive fields of PCs (Jvrntell & Ekerot, 2002). This change in receptive field is due to the depression of a subset of PF-PC synapses, a mechanism believed to remove sensory signals producing undesired motor behaviors. Similar experiments also demonstrate CFs drive associative changes in the receptive fields of molecular layer inhibitory interneurons (MLI) that synapse onto PCs. However, the nature of the CF-MLI connection remains unclear, nor are the mechanisms driving the associative plasticity that result in receptive field changes known. This deficiency in the current state of cerebellar knowledge is the result of an inability to reliably stimulate CFs without activating neighboring axons from other neuron types. To overcome this technical challenge, a novel optogenetic approach has been developed to allow robust stimulation of isolated CFs. The first aim of this proposal will further confirm preliminary results demonstrating the reliability and specificity of photostimulating CFs expressing Channelrhodopsin 2 by systematically exploring the optical stimulation and viral injection parameters necessary for robust CF stimulation. Using this technique, I propose to describe both the nature of CF-MLI transmission as well as the mechanisms and rules governing the CF- driven associative plasticity between parallel fibers and MLIs. This will be accomplished through whole-cell patch clamp recordings from MLIs in acute slices during selective CF photostimulation. These experiments will be the first of their kind to illustrate the effectiveness of optogenetic techniques in exploring the cerebellar cortex. In the end results from these experiments will allow for better predictions of how the cerebellar cortex evaluates and corrects for disparities between intention and action. PUBLIC HEALTH RELEVANCE: Cerebellar dysfunction, which usually results from disease, blunt force trauma or the effects of aging, often presents itself as deficits in motor control and/or motor memory. The goal of this proposal is to examine the basic cellular properties and mechanisms governing motor control and memory in the cerebellar cortex using a novel technique to stimulate neurons in the brain. With a more informed knowledge base of cerebellar function at the cellular level researchers will be better prepared to design therapies to cure or alleviate the symptoms of cerebellar dysfunctions.