An optogenetic approach to exploring climbing fiber connections in the cerebellum

描述(申请人提供)：实验性损伤和对小脑的钝力损伤导致行为异常，表明该大脑区域在控制平稳协调运动和运动记忆方面发挥重要作用(Fine，Ionita，&Lohr，2002)。具体地说，研究人员认为，小脑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，从而产生所需的、流畅的运动行为。实验表明，这些修正源于小脑皮质和深层核团突触连接强度的动态变化。此外，这些变化很可能是由来自两个特定路径的信号的关联或一致检测所驱动的，一个通过苔藓纤维(MF，携带感觉信息)，另一个通过攀爬纤维(CF，表明运动指令中的差异或错误)。从下橄榄发出的攀爬纤维提供了一种独特而强大的输入，在小脑皮质的唯一输出--浦肯野细胞(PC)--中产生一个“复杂的尖峰”。当这种输入与平行纤维(PF；苔藓纤维中继器)一起激活时，PC的躯体感觉感受野就会减少(Jvrntell&Ekerot，2002)。感受野的这种变化是由于PF-PC突触的子集被抑制，这是一种被认为可以移除产生不希望看到的运动行为的感觉信号的机制。类似的实验还表明，CFS可以驱动分子层抑制性中间神经元(MLI)突触到PC的感受场的关联性变化。然而，Cf-MLI连接的性质仍然不清楚，也不知道驱动联合可塑性导致感受野变化的机制。目前小脑知识状态的不足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的结果。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，以允许对分离的CFs进行强有力的刺激。这项建议的第一个目标将通过系统地探索光刺激和病毒注射参数来进一步证实初步结果，证明光刺激表达通道视紫红质2的CFs的可靠性和特异性。利用这一技术，我建议既描述CF-MLI传递的本质，也描述平行纤维和MLI之间由CF驱动的缔合塑性的机制和规律。这将通过选择性CF光刺激期间急性切片中的MLI的全细胞膜片钳记录来实现。这些实验将是此类实验中第一次展示光遗传技术在探索小脑皮质方面的有效性。最终，这些实验的结果将能够更好地预测小脑皮质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.