Circuit Specializations of Cerebellar Molecular Layer Interneurons

项目摘要 小脑控制依赖于预测的行为，包括运动学习，眼球运动， 平衡和认知情感功能。小脑皮质将苔藓纤维（MF）输入转换为 浦肯野细胞（PC）输出。至关重要的是要描绘出这个电路的元素和它们的连接， 电路特性对功能和疾病的影响。为了提高我们对小脑的理解， 皮质，这项建议旨在描绘最丰富的类型的中间神经元的电路特性， 小脑，分子层中间神经元（MLI）。MLI抑制PC和其他MLI以控制 小脑皮质然而，单核RNA测序最近确定了两种分子上不同的类型， MLI，MLI 1型（MLI 1）和MLI 2型（MLI 2）。有趣的是，MLI1表达连接蛋白36，而MLI2表达连接蛋白36。 没有这表明MLI1可能是相互偶联的间隙连接，这可能促进MLI1的表达。 同步发射，使MLI1非常适合控制PC输出的时序。的电路特性 MLI1和MLI2是未知的。在这里，我将使用切片电生理学来阐明MLI1和MLI2的连接性， 小鼠中的连续电子显微镜（EM）重建。本建议的第一个目的是确定 使用脑切片中的成对全细胞记录的MLI1和MLI2的输出。首先，我将描述MLI1和 MLI2突触连接到PC上。我还将描述MLI1和MLI2对ephaptic的贡献， 抑制，这是由MLI特化（称为pinceaux）附近的大细胞外信号引起的， 包围PC轴突起始段。最后，我将描述突触连接和电耦合 在MLI1和MLI2之间。该建议的第二个目的是表征超微结构电路 并生成MLI1和MLI2突触和肝突触的综合图谱 使用大规模EM重建的连通性。电生理学的互补方法 表征和EM重建将提供功能特性的完整画面， 解剖学上的联系初步研究结果表明，MLI1和MLI2靶向不同的细胞，MLI1 主要抑制PC，而MLI2主要抑制其他MLI。根据这些初步研究， 工作模型是，MLI1非常适合于控制PC输出的定时，而MLI2促进PC 通过抑制PC的兴奋性。完成拟议的工作将确定这些电路的属性 元件，如果初步结果得到确认，将有必要修改电路图的 小脑皮质这些实验也将导致未来的研究，将确定在体内发射 MLI1和MLI2的性质，并确定MLI1和MLI2如何有助于处理，学习和 行为这些研究有望阐明小脑计算的机制， 神经障碍和神经精神疾病。

Project Summary The cerebellum controls behaviors that depend on prediction, including motor learning, eye movements, balance, and cognitive-affective functions. The cerebellar cortex transforms mossy fiber (MF) inputs into Purkinje cell (PC) outputs. It is vital to delineate the elements of this circuit and their connectivity to relate circuit properties to function and disease. To improve our understanding of transformations in the cerebellar cortex, this proposal seeks to delineate the circuit properties of the most abundant type of interneuron in the cerebellum, the molecular layer interneuron (MLI). MLIs inhibit PCs and other MLIs to control the output of the cerebellar cortex. However, single nucleus RNA sequencing recently identified two molecularly distinct types of MLIs, MLI type 1 (MLI1) and MLI type 2 (MLI2). Intriguingly, MLI1s express connexin 36, whereas MLI2s do not. This suggests that MLI1s might be gap junction coupled with each other, which could promote synchronous firing and make MLI1s well-suited to controlling the timing of PC outputs. The circuit properties of MLI1 and MLI2 are not known. Here I will clarify MLI1 and MLI2 connectivity using slice electrophysiology and serial electron microscopy (EM) reconstructions in mice. The first aim of this proposal is to determine the outputs of MLI1 and MLI2 using paired whole-cell recordings in brain slice. First, I will characterize MLI1 and MLI2 synaptic connections onto PCs. I will also characterize the contributions of MLI1 and MLI2 to ephaptic inhibition, which arises from large extracellular signals near MLI specializations known as pinceaux that surround PC axon initial segments. Lastly, I will characterize synaptic connections and electrical coupling between MLI1 and MLI2. The second aim of this proposal is to characterize the ultrastructural circuit specializations of MLI1 and MLI2 and generate a comprehensive map of MLI1 and MLI2 synaptic and ephaptic connectivity using large-scale EM reconstructions. The complementary approaches of electrophysiological characterization and EM reconstructions will provide a complete picture of the functional properties and the anatomical connectivity. Preliminary findings suggest that MLI1 and MLI2 target different cells, with MLI1s mainly inhibiting PCs, whereas MLI2s primarily inhibit other MLIs. Based on these preliminary studies, my working model is that MLI1s are well suited to controlling the timing of PC outputs, whereas MLI2s promote PC excitability by disinhibiting PCs. Completion of the proposed work will define the properties of these circuit elements, and if preliminary results are confirmed, it will be necessary to revise the circuit diagram of the cerebellar cortex. These experiments will also lead to future studies that will determine the in vivo firing properties of MLI1 and MLI2 and determine how MLI1 and MLI2 contribute to processing, learning and behavior. These studies promise to shed light on mechanisms of cerebellar computations that are relevant to neurological disorders and neuropsychiatric diseases.